Research Collection

Doctoral Thesis

Ecological significance of ploidy level of native and invasive populations of Solidago gigantea

Author(s): Schlaepfer, Daniel Rodolphe

Publication Date: 2008

Permanent Link: https://doi.org/10.3929/ethz-a-005691046

Rights / License: In Copyright - Non-Commercial Use Permitted

This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use.

ETH Library Diss. ETH No. 17677 %COLOGICALSIGNIFICANCEOFPLOIDYLEVELOFNATIVEANDINVASIVEPOPULATIONSOF Ecological$ISS%4(.O significance of ploidy level of native and invasive populations of %COLOGICALSIGNIFICANCEOFPLOIDYLEVELOF NATIVEANDINVASIVEPOPULATIONSOFSolidago gigantea 3OLIDAGOGIGANTEA 3OLIDAGOGIGANTEA $ANIEL23CHLAEPFER

$ANIEL2ODOLPHE3CHLAEPFER 

Daniel Rodolphe Schlaepfer 2008

Diss. ETH No. 17677

Ec o l o g i c a l Sign i ficance o f Pl o i d y Le v e l o f Na t i v e a n d In v a s i v e Po p u l a t i o n s o f So l i d a g o g i g a n t e a

A dissertation submitted to

ETH ZURICH

for the degree of

Doctor of Sciences

presented by

Daniel Rodolphe Schlaepfer

Dipl. Umwelt-Natw. ETH born 17th January 1977 citizen of Montreux VD and Rehetobel AR

accepted on the recommendation of

Prof. Peter J. Edwards, examiner Dr. Regula Billeter, co-examiner Prof. Heinz Müller-Schärer, co-examiner

2008

Co n t e n t s

Summary 1

Zusammenfassung 3

General introduction 7

Chapter 1 25 Cytogeography of Solidago gigantea (Asteraceae) and its invasive ploidy level

Chapter 2 65 Habitat differentiation between native ploidy levels and invasive tetraploids of Solidago gigantea (Asteraceae)

Chapter 3 87 Ploidy-level precise common garden with native and invasive Solidago gigantea (Asteraceae): differences in clonal growth and nutrient responses indicate colonization success of tetraploids

Chapter 4 113 Phylogeography of native ploidy levels and invasive tetraploids of Solidago gigantea (Asteraceae)

General conclusions 145

Acknowledgements 153

Curriculum Vitae 155

Summary

Su m m a r y

1. Invasive alien species can be valuable model systems to gain new insights into evolutionary, biogeographic and ecological processes. Recent research has stressed the importance of genetic change and variation in making populations of some plant species invasive, but the significance of polyploidy needs further research. Although profound consequences of polyploidy for gene expression, physiology and ecology have been described in detail, it is not understood why polyploidy is particularly common among invasive alien plant species. The adoption of a comparative biogeographic approach, comparing populations from the invasive with those from the native range, can reveal factors influencing the invasion success. However, inadequate knowledge of the ecology of a species in its native range often impedes progress towards understanding why it becomes invasive. This project uses Solidago gigantea Aiton (Asteraceae) as model species to investigate these issues; in its native range in North America, the species occurs in three ploidy levels—diploids, tetraploids, and hexaploids—and it is invasive in a wide range of habitats over large parts of Europe. By studying the ecology and genetics of ploidy levels in the native and the invasive range, using a comparative biogeographic approach, I aimed to improve our understanding of the significance that polyploidy plays in biological invasions. 2. The distribution of ploidy levels of S. gigantea in North America, Europe and East Asia was mapped based on locations with ploidy level data gathered from field surveys, collaborators and a literature review. North American populations on the eastern side of the Appalachian Mountains and also in southern Ontario and Québec are diploid. Tetraploids are found in eastern North America as far west as Missouri; hexaploids occur in mid-western North America, westwards from Manitoba to the Rocky Mountains. Strong geographic separation among ploidy levels was found, and populations with mixed ploidy were rare. In Europe and East Asia, only tetraploid plants were detected. Further comparisons between native and introduced populations need to account for ploidy level. 3. In a field survey, habitat conditions of populations ofS. gigantea in the native and invasive ranges were measured and environmental envelopes were constructed based upon a dataset of locations of known ploidy level. At a more local scale, the most consistent difference between diploids and tetraploids was that diploids were calcifuge whereas tetraploids were indifferent to soil calcium. An incompatible habitat template is unlikely to be the main reason why diploids do not occur in the invasive range, although some differences were found between habitat conditions in the invasive and the native range. More relevant is the fact that tetraploids of S. gigantea were successful colonizers in North America, extending their range well beyond that

1 Summary occupied by their diploid progenitors. Therefore, a pre-disposition for successful spreading is proposed to be linked to the tetraploid status. 4. In a common garden experiment, diploid and tetraploid S. gigantea were grown under high and low nutrient and calcium treatments. The results revealed large differences in life history between ploidy levels but less so between native and invasive tetraploids. Diploid plants had higher specific leaf area and leaf nutrient concentrations than native tetraploids, but tetraploids produced many more shoots and rhizomes. Diploids grew less well when calcium was present, whereas tetraploids were not affected, reflecting the known habitat differences of the ploidy levels in the field. European plants were less likely to flower and produced smaller capitulescences than North American tetraploids, but biomass production, and numbers of shoots and rhizomes did not differ. These results suggest that the same traits that made tetraploids successful in North America, e.g. higher colonization ability and wider habitat tolerance than diploids, may have predisposed tetraploids to becoming successful invaders. 5. For a phylogeographic analysis, parts of chloroplast intergenic spacer DNA from native and invasive populations of S. gigantea were sequenced. Inheritance of chloroplasts was confirmed to be maternal. In North America, significant differentiation among regions, private haplotypes, and isolation by distance were detected. The haplotype network was shallow, with one tetraploid-only star-shaped structure that was successful in colonization. The data suggest that tetraploids have formed several times in North America. Haplotype number and diversity were lower in European populations than in the native range, and evidence was found that four haplotypes were introduced to Europe from two source areas, New England and the Southern Appalachian Mts. Despite lowered diversity in the invasive range, the process of invasive spread is comparable to the spread of successful tetraploid lineages in the native range. 6. This project is the first to investigate the ecology and genetics of ploidy levels in the native range of S. gigantea and to consider how ploidy level influences the invasion potential of the species. I conclude that the ploidy levels differ markedly in habitat requirements, niche width and the distribution of genetic diversity. However, the most important difference is in the development of the rhizome system and hence presumably in the ability of ploidy levels to colonize and spread. I argue that the invasive potential of S. gigantea was probably not acquired through adaptation after introduction but through the formation of polyploids in the native range. Overall, this project has furnished evidence that polyploidy in S. gigantea does matter ecologically; and it seems probable that polyploidy also plays a significant role in biological invasions more generally.

2 Zusammenfassung

Zusammenfassung

1. Invasive gebietsfremde Arten können ausgezeichnete Studienobjekte sein, um neue Erkenntnisse über evolutive, biogeographische und ökologische Prozesse zu gewinnen. Insbesondere wurde kürzlich erkannt, wie wichtig genetische Veränderung und Variation sind, um die Ursachen von biologischen Invasionsprozessen besser zu verstehen, jedoch ist mehr Forschung über die Bedeutung von Polyploidie nötig. Unklar ist zum Beispiel, weshalb Polyploidie so häufig in invasiven gebietsfremden Pflanzenarten vorkommt, obwohl verschiedentlich beschrieben worden ist, dass Polyploidie die Genexpression, die Physiologie und die Ökologie von Pflanzen stark verändern kann. Gerade um solche Faktoren zu bestimmen, die den Erfolg von biologischen Invasionen ursächlich beeinflussen, ist der vergleichende biogeographische Ansatz geeignet. Mangelndes Wissen über die Ökologie von invasiven Arten in ihrem ursprünglichen Gebiet verhindert jedoch häufig ein besseres Verständnis von Invasionsprozessen. Dieses Projekt wählte die Art Solidago gigantea Aiton (Asteraceae) als Untersuchungsobjekt. Diese Art kommt in ihrem ursprünglichen Verbreitungsgebiet in Nordamerika in drei Ploidiestufen vor, nämlich als diploide, tetraploide und hexaploide Individuen. In Europa ist sie invasiv in verschiedenen Habitaten und weitverbreitet. Durch Untersuchungen über die Ökologie und die Genetik der verschiedener Ploidiestufen dieser Art in einem vergleichenden biogeographischer Ansatz versuchte ich, unser Verständnis für die Bedeutung zu vertiefen, die die Polyploidie in biologischen Invasionsprozessen hat. 2. Die Verbreitung der Ploidiestufen von S. gigantea wurde sowohl in Nordamerika als auch in Europa und Ost-Asien kartiert. Diese Kartierung basiert auf Fundorten, an welchen die Ploidiestufe der Population bestimmt wurde. In Nordamerika sind die Populationen östlich der Appalachen, im südlichen Ontario und Québec diploid. Tetraploide Populationen kommen im östlichen Nordamerika, westlich bis nach Missouri vor. Hexaploide Populationen wurden im mittleren Westen gefunden, zwischen Manitoba und den Rocky Mountains. Somit sind die Ploidiestufen geographisch und auch auf der Skala der Population stark getrennt, da fast keine gemischten Populationen gefunden wurden. In Europa und Ost-Asien wurden ausschliesslich tetraploide Individuen gefunden. Dies zeigt, dass zukünftige Vergleiche zwischen Populationen aus dem ursprünglichem und dem eingeführten Gebiet immer die Ploidiestufen berücksichtigen sollten. 3. In einer Felduntersuchung wurden die Habitatansprüche von S. gigantea Populationen sowohl im ursprünglichen als auch im eingeführten Gebiet untersucht. Zusätzlich wurden Umwelt-Parameter über das allgemeine Vorkommen von S. gigantea anhand einer Datenbank von Fundorten mit bekannter Ploidiestufe berechnet. Auf lokaler Skala, unterscheiden sich die

3 Zusammenfassung diploiden Populationen von den Tetraploiden derart, dass die ersteren Kalk meidend während die letzteren indifferent gegenüber Kalk im Boden sind. Die Habitatsansprüche von diploiden Populationen scheinen kein Grund zu sein, dass sie in Europa nicht vorkommen, obwohl gewisse Unterschiede zwischen ursprünglichen und eingeführten Populationen gefunden wurden. Viel wichtiger als diese Unterschiede scheint die Tatsache, dass tetraploide Populationen bereits in Nordamerika erfolgreich ein grosses neues Gebiet kolonisieren konnten, welches weit grösser ist als das ihrer diploiden Vorfahren. Deshalb scheint eine Veranlagung zu erfolgreicher Ausbreitung mit dem tetraploiden Zustand einherzugehen. 4. In einem Gartenexperiment wuchsen diploide und tetraploide S. gigantea unter niedrigen und hohen Nährstoff- und Calcium-Behandlungen in Töpfen. Grosse Unterschiede im Lebenszyklus wurden zwischen den Ploidiestufen, aber nicht zwischen Populationen aus dem ursprünglichen und aus dem eingeführten Gebiet gefunden. Diploide Pflanzen hatten eine grössere spezifische Blattfläche und mehr Blattnährstoffe als Tetraploide, doch die tetraploiden Pflanzen produzierten signifikant mehr Rhizome und Stengel. Wenn Calcium vorhanden war, wuchsen diploide Pflanzen weniger gut, während die Tetraploiden nicht beeinflußt wurden. Dieses Resultat bestätigt somit festgestellte Unterschiede im Habitat von der Felduntersuchung. Eingeführte Pflanzen blühten weniger oft und produzierten weniger Blütenbiomasse als Tetraploide aus Nordamerika, jedoch bestanden für Biomasse-Produktion und Anzahl an Stengel und Rhizome keine Unterschiede. Die Resultate bestätigen die Vermutung, dass tetraploide S. gigantea eine Veranlagung zur erfolgreichen Ausbreitung haben. Dank dieser Eigenschaften, wie hohes Ausbreitungspotential und breite Habitatstoleranz, konnten sich tetraploide S. gigantea nicht nur in Nordamerika erfolgreich ausbreiten, sondern auch im eingeführten Gebiet. 5. Für eine phylogeographische Analyse wurden von Individuen von S. gigantea sowohl vom ursprünglichen als auch vom eingeführten Gebiet, DNA aus nicht-kodierenden Abschnitten des Chloroplasten-Genoms sequenziert. Zusätzlich wurde bestätigt, dass die Vererbung von Chloroplasten über die mütterliche Linie stattfindet. In Nordamerika, konnten signifikante Unterschiede zwischen den Regionen, regions- und populationsspezifische Haplotypen und ein Zusammenhang zwischen geographischer Distanz und genetischer Ähnlichkeit nachgewiesen werden. Das Netzwerk der Haplotypen war nur schwach hierarchisch ausgebildet. Eine sternähnliche Struktur nur aus tetraploiden Haplotypen wurde nachgewiesen; diese Struktur war erfolgreich in der Ausbreitung. Tetraploide S. gigantea wurden mehrfach gebildet. In Europa waren die Anzahl der Haplotypen und die genetische Vielfalt geringer als in ursprünglichen Gebiet. Die Resultate weisen darauf hin, dass vier Haplotypen aus zwei Ursprungsregionen, nämlich Neu-England und südliche Appalachen, nach Europa eingeführt worden waren. Doch trotz einer geringeren genetischen Vielfalt im eingeführten Gebiet, sind die Prozesse der Ausbreitung in Europa mit denen der Ausbreitung der erfolgreichen Haplotypen in Nordamerika vergleichbar.

4 Zusammenfassung

6. Dieses Projekt ist das erste, welches die Ökologie und Genetik der verschiedenen Ploidiestufen von S. gigantea im ursprünglichen Gebiet untersuchte und welches erforschte, wie die Ploidiestufen das Invasionspotential der Art beeinflussen. Ich folgere, dass die Ploidiestufen sich zum Teil beträchtlich unterscheiden in Habitatsansprüchen, Habitatstoleranz, Lebenszyklen und der Verteilung von genetischer Vielfalt. Der jedoch bedeutendste Unterschied liegt in der Ausprägung des Rhizomsystems, und somit vermutlich im Potential zur Kolonisierung und Ausbreitung. Ich argumentiere, dass das Invasionspotential von S. gigantea vermutlich nicht nach der Einführung nach Europa durch Adaption erworben wurde, sondern durch Bildung von höheren Ploidiestufen. Insgesamt hat dieses Projekt Hinweise gefunden, dass Ploidiestufen von S. gigantea von ökologischer Bedeutung sind und dass somit Ploidiestufen generell eine bedeutende Rolle in Invasionsprozessen spielen können.

5

General introduction

Ge n e r a l In t r o d u c t i o n

Research on invasive alien species can provide valuable insights into fundamental evolutionary and ecological processes (Sax et al., 2007). Recently, there has been considerable interest in the role of genetic factors such as local adaptation and hybridization in promoting biological invasions (Lee, 2002; Cox, 2004; Suarez & Tsutsui, 2008). In particular, polyploidy has been stressed as a factor influencing the success of invasive alien plants (Müller-Schärer et al., 2004). Despite research efforts into invasive species in their introduced range, often little is known about the abundance, community interactions and ecosystem impacts of these species in their native range (Hierro et al., 2005). However, such knowledge is crucial for understanding why certain species become invasive, and for predicting future spread. However, comparing populations from the entire native range to populations from the invasive range does not necessarily yield the answers to the questions asked (Dlugosch & Parker, 2008). For instance, if the aim is to detect genetic change between native and invasive populations, e.g. polyploidization or hybridization events, and to determine when any change occurred relative to the time of introduction (Facon et al., 2006), then it is essential to compare introduced populations with the original source population(s) (Dlugosch & Parker, 2008); otherwise, genetic change could be attributed to adaptation in the new range, whereas it actually occurred but was undetected in the native range. This project concerns the significance of polyploidy in plant invasions, using Solidago gigantea Aiton (Asteraceae) as a model species. One aim of the work described here was to elucidate aspects of the ecology of the species in its native range, in order to provide an adequate biogeographic comparison with introduced populations. Fulfilling this aim also required an assessment of the ecological significance of polyploidy in both native and invasive populations.

Wh a t a r e i n v a s i v e a l i e n s p e ci e s a n d w h a t c a n w e l e a r n f r o m t h e m ?

In the literature, the term ‘invasive species’ is often used in different ways despite several attempts to unify the terminology (e.g. Richardson et al., 2000; Colautti & MacIsaac, 7 General introduction

2004). In this thesis, a species is called an invasive alien when, having been accidentally or deliberately introduced into a new geographic area by humans, it spreads from the point(s) of introduction. Invasive alien species are a major and increasing concern for ecosystem management worldwide. They alter ecosystem functioning such as hydrology, fire regimes or nutrient cycling (Chapin et al., 2000) and threaten biodiversity (Wilcove et al., 1998). Invasive alien species also affect human health, for instances through pathogen infections, direct toxicity or allergenic potential, (Vitousek et al., 1997) and economic activity because they lower yield of agriculture or forestry and because they cause high costs of control (Pimentel et al., 2005; Colautti et al., 2006). In addition, once established in a new area, they may be almost impossible to eradicate (Simberloff, 2003). To address management challenges associated with invasive alien species, international policy instruments have been developed, e.g. Article 8h of the Convention on Biological Diversity (CBD) from the United Nations (UN), and the Global Invasive Species Programme (GISP) was founded (McNeely et al., 2001). To address gaps in scientific understanding of biological invasions, i.e. species invasiveness, habitat invasibility, invasion management, a Scientific Committee on Problems of the Environment (SCOPE) program was initiated which has promoted considerable research activity (Drake et al., 1989). However, despite having advanced ecological and evolutionary understanding of species, community interactions and ecosystems (Sax et al., 2007), no general mechanisms have yet been found to explain and predict why some species become invasive (Dietz & Edwards, 2006; Mitchell et al., 2006). Different hypotheses have been put forward to explain such controlling mechanisms in biological invasions (see compilations, e.g. in Hierro et al., 2005; Dietz & Edwards, 2006; Mitchell et al., 2006). Mechanisms explaining invasion success have distinguished between intrinsic species traits (invasiveness) and extrinsic community traits (invasibility) (Richardson & Pysek, 2006; McKenney et al., 2007); however, recent research understands biological invasions as a combination of interactions between species, communities (Shea & Chesson, 2002) and transport vectors (e.g. propagule pressure) that cannot be appreciated only through characteristics from either one (Facon et al., 2006; Richardson & Pysek, 2006). It has also been increasingly realised that all invasions pass through a series of stages—transport, colonization, establishment and spread—and success at each of these stages is controlled by filters that are similar for all biological invasions (Theoharides & Dukes, 2007). Charles Darwin (1859, cited by Ludsin & Wolfe, 2001; Mitchell et al., 2006) was one of the first to take interest in the processes of biological invasions and to initiate an ecological understanding governing invasions. His ideas on community assembly and ecological niches (though of course he did not use these terms) provoked the idea of what is now called the ‘naturalization hypothesis’ (Proches et al., 2008)—that introduced species are less likely to become invasive if they encounter closely related established species (Daehler, 2001). Because mechanisms influencing phylogenetic patterns operate on different taxonomic and spatial

8 General introduction scales, refinements of this hypothesis have been proposed (Mitchell et al., 2006; Proches et al., 2008). In 1958, the publication of Charles Elton’s book on the ecology of invasion stimulated new interest in the topic which increasingly became a new focal area of research in ecology (Myers & Bazely, 2003) and prompted various conservation initiatives (Davis, 2005). Elton proposed the ‘biodiversity-invasibility’ hypothesis (Levine & D’Antonio, 1999), i.e. that species-rich communities are less likely to be invaded than species-poor communities. For instance, species-poor communities could be more likely to have empty niches, to include fewer suitable competitors or to reduce available resources less strongly, all of which could promote invasions (Collins et al., 2007). However, empirical studies showed both positive and negative correlations between community diversity and invasibility (Levine & D’Antonio, 1999; Collins et al., 2007; Maron & Marler, 2007). Generally, release from community interactions that regulate growth and competition in the native range has been seen as a powerful mechanism to explain biological invasions. For instance, the ‘enemy-release’ hypothesis proposes that the lack of enemies in the introduced range is related to the abundance and success of invasive populations (Colautti et al., 2004). This hypothesis was refined through the ‘resource-enemy release’ hypothesis, which postulates that the alien species most likely to profit from enemy release are those adapted to conditions of high resource availability (Blumenthal, 2005; Blumenthal, 2006). Another idea, the ‘novel weapon’ hypothesis, proposes that resident species are ‘naïve’ to novel ‘allelopathic’ chemicals of invasive species, (Callaway & Aschehoug, 2000) thus creating new community interactions that give an advantage to the introduced species (Callaway & Ridenour, 2004). Another hypothesis also postulates differing community interactions in the native and introduced ranges: in this case, it is argued that high invasibility is likely to result if antagonistic species are specialists while mutualists and facilitators are generalists (Sax et al., 2007). In these circumstances, an introduced species may escape from negative species-specific interactions, e.g. pathogenic soil biota, while benefiting from new, positive interactions with generalist soil biota (Klironomos, 2002). In addition to these hypotheses about ‘external’ factors influencing invasions, research is realising the importance of genetic factors for invasion processes (Lee, 2002). Through the introduction process itself bottlenecks may be created and genetic drift and inbreeding in small founder populations become apparent, even though the importance of those disadvantages may have been overrated (Dlugosch & Parker, 2008). Opportunities for intra-specific hybridization through mixing of populations that were isolated in their native ranges or for inter-specific hybridization with species in the invasive range could lead to evolution of invasiveness (Ellstrand & Schierenbeck, 2000; Suarez & Tsutsui, 2008). Introduced species face strongly altered selection pressures and may evolve rapidly in areas of introduction (Sakai et al., 2001; Bossdorf et al., 2005); for instance, if an introduced species is released from enemies, it may reallocate resources from defence to vegetative growth, thereby increasing its competitive

9 General introduction ability (EICA hypothesis, Blossey & Notzold, 1995). Therefore, the genetic makeup of invasive species, e.g. genetic diversity and distribution, genetic change, hybridization and polyploidy, are fields of intensive research effort (Suarez & Tsutsui, 2008). In ecological terms, three main factors contribute to a species’ growth rate in a given community, i.e. biotic interactions, abiotic environment and resources. Since all species must be able to increase in abundance when rare, i.e. pass the ‘invasion criterion’, all species have the potential for ‘invasions’ in a community context (Thompson et al., 1995; Crawley et al., 1996; Shea & Chesson, 2002; Silvertown, 2005). The community sets up an ‘establishment’ threshold that any new arrival must somehow overcome in order to capture niche opportunities (Shea & Chesson, 2002; Facon et al., 2006). These mechanisms follow the same fundamental ecological rules for both native and introduced species. Therefore, at a fundamental level, invasion ecology asks similar questions to those of more classical fields of ecology (Callaway & Maron, 2006). Despite these parallels, and the potential reciprocal benefits for both fields, there has been a tendency to see invasion ecology as a separate discipline that investigates phenomena distinct from those influencing native species (Davis et al., 2001). Nevertheless, many insights into ecological and evolutionary processes could be gained on structuring of communities, ecosystem processes, speciation, adaptation and biogeographic distributions (Lodge, 1993; Levin, 2003; Callaway & Maron, 2006; Sax et al., 2007). In particular, invasive alien species are ideal model organisms to study questions related to evolutionary change and adaptation, and how genetic variation influences ecosystem processes (Lee, 2002; Cox, 2004; Sax et al., 2007; Suarez & Tsutsui, 2008).

Wh y i s p o l y p l o i d y i m p o r t a n t ?

Polyploidy—the condition when each cell nucleus contains more than two complete sets of chromosomes—plays an important role in plant evolution. At least 2–4% of angiosperm speciation events are associated with polyploidy (Otto & Whitton, 2000) and as many as 70% of angiosperm species show signs of polyploidy in their history (Masterson, 1994). Polyploidization can have profound consequences for gene expression (Osborn et al., 2003); for instance, compared to the diploid progenitors about 5% of studied genes of allopolyploid Tragopogon miscellus are silenced, and another 4% show novel expression (Soltis et al., 2004). Furthermore, polyploidization offers the potential for novel physiological (Soltis et al., 2003) and ecological behaviours in plants (Lumaret, 1988; Müller, 1989). Differences in pollination mechanisms, for example, have been found between the ploidy levels of Heuchera grossulariifolia (Segraves & Thompson, 1999; Nuismer & Cunningham, 2005); and enemy-ploidy interactions have been described for insect gallmakers of Solidago altissima

10 General introduction

(Halverson et al., 2008a) and for herbivores of several other species (Nuismer & Thompson, 2001; Thompson et al., 2004; Münzbergova, 2006). Polyploidization also brings about strong, though not necessarily complete, reproductive isolation from the diploid progenitors, and therefore represents a barrier to gene flow (Husband & Sabara, 2004). The polyploidization event is often the first stage in the development of a new polyploid species; the second is the establishment and development of a viable population (Ramsey & Schemske, 2002). Newly formed polyploids are likely to face several obstacles to successful establishment and spread (Fowler & Levin, 1984). Because they originate within diploid populations, they face competition both between and within ploidy levels. And because triploids are likely to have a very low fitness, they also face limited opportunities for pollination, which is usually frequency-dependent by ploidy level. As a result, the rarer ploidy level experiences a larger loss of offspring, a phenomenon referred to as the ‘minority cytotype disadvantage’ (Levin, 1975; Felber, 1991). These constraints, and also chances, of polyploid establishment have been investigated in a number of experimental and theoretical studies (e.g. Rodriguez, 1996a, b; Husband, 2004; Li et al., 2004; Rausch & Morgan, 2005). These suggest that after polyploid formation two outcomes are most probable: either one ploidy level becomes extinct as a result of competition, stochastic loss or minority cytotype disadvantage, or both ploidy levels persist as a result of ecological differentiation. However, the fact that polyploids are so common indicates that establishment does occur (Soltis, 2005), leading some authors to conclude that this step may be less constrained than is usually assumed. For example, Baack (2005) has proposed that if seeds and pollen are dispersed only locally co-existence may be possible even without habitat differentiation. Establishment and spread both of invasive alien species and of polyploids seem to be governed by similar processes. Firstly, newly formed polyploids (Husband & Sabara, 2004) and newly introduced species both experience genetic isolation and strong selection pressures. Under these conditions, factors such as selfing rates and genetic diversity become important. Secondly, some of the effects of isolation may be alleviated, in the one case by the recurrent formation of polyploids and in the other by multiple introductions (Petit, 2004). Thirdly, establishment success of both polyploids (Johnson et al., 2003) and alien species (Maron et al., 2004) may be associated with adaptation to new habitats, so that plants interact in novel ways with their biotic and abiotic environment. For example, alien populations lose interactions from their native range and gain new interactions in the introduced range (Mitchell et al., 2006), while polyploids may interact with pollinators (Segraves & Thompson, 1999), enemies (Soltis et al., 2003 and references therein) and competitors (Maceira et al., 1993) differently from their diploid progenitors. In view of these similarities, it is perhaps not surprising that polyploids are more frequent among invasive alien plants than among angiosperms in general. This tendency has been shown, for example, for the ‘world’s worst weeds’ (Brown & Marshall, 1981), for important world weeds and British garden weeds (Bennett et al., 1998), for the flora of France (Verlaque et al., 2002) and for the flora of Singapore (Pandit et al., 2006). While the

11 General introduction phenomenon of biological invasions cannot be explained by polyploidy alone (Ehrendorfer, 1980; Barrett & Richardson, 1986; Roy, 1990), these comparisons do suggest that research into polyploid establishment will give insights into the success of some alien species. Knowledge of the mechanisms of polyploid establishment in the native range can help in understanding the role of polyploidy in invasive alien populations (Kubatova et al., 2008). For example, an analysis of the geographic distribution of ploidy levels can show whether the ploidy levels coexist or are spatially separated, and may reveal contact zones where ploidy levels interact with each other (Halverson et al., 2008b). Data on mixed-ploidy populations and the frequency of triploids can yield information on the rates of formation and establishment of new polyploids (Husband, 2004). And information on the relative fitness and life-history of the different ploidy levels, their degree of ecological differentiation and their pattern of spread will be important in understanding the mechanisms of success of polyploid invasive alien species.

Wh a t d o w e k n o w a b o u t So l i d a g o g i g a n t e a ?

Solidago gigantea Aiton (Asteraceae) is a convenient model species for studying the ecological significance of polyploidy in biological invasions. This is because it is both an invader ofa wide range of habitats over large parts of Europe (Guzikowa & Maycock, 1986; Botta-Dukat & Dancza, 2001; Weber & Jakobs, 2005) and also occurs in three ploidy levels—diploids, tetraploids, and hexaploids—over a wide geographic range native in North America. It is a 0.5–2.5 m tall herb with annual shoots. Reproduction is guaranteed both sexually by partially flowering shoots that are obligatory outcrossing and pollinated by a wide range ofinsects (Melville & Morton, 1982) and clonally by perennial rhizomes. In Europe, it was introduced in 1758 as an ornamental plant (Weber & Schmid, 1993). Solidago gigantea is also naturalised in northern Japan, Russia and various other parts of the world. Little is known about the ecology of the different ploidy levels of S. gigantea in its native range in North America (but see, Melville & Morton, 1982; Kruse & Groninger, 2003; Kercher & Zedler, 2004; Abrahamson et al., 2005). Nevertheless, a substantial dataset on chromosome numbers has been accumulated (e.g. Beaudry, 1970; Melville & Morton, 1982; Morton, 1984; Semple et al., 1984; Semple & Cook, 2006), and there has been much work to elucidate the taxonomic relationships of this ‘difficult’ group (e.g. Beaudry, 1970; Morton, 1984; Nesom, 1993; Zhang, 1996; Semple & Cook, 2006). On the other hand, in Europe, S. gigantea has received considerable research attention as a model species for investigating plant invasions and their impact on site conditions (Weber & Jakobs, 2005). Some early studies elucidated community assemblages and tested management practices for improved control (Voser-Huber, 1983), while others used S. gigantea as a system

12 General introduction to study clonal growth in plants (Schmid & Bazzaz, 1987, 1992). The historical spread of S. gigantea in Europe has been reconstructed using herbarium vouchers (Weber, 1998) and potential geographic and climatic limits of range expansion have been determined (Weber, 2001). It is known that invasive alien species can alter soil nutrient composition and cycling for their advantage (Ehrenfeld, 2003), and this aspect of invasion ecology has also been investigated for S. gigantea. However, the results for invasive populations of S. gigantea are mixed. Total nutrient concentrations under invaded and non-invaded sites did not differ in a lakeshore wetland (Güsewell et al., 2005) and nutrient-poor sites (Eggenschwiler, 2007), but other studies found increased concentrations of available phosphorus at invaded sites (Vanderhoeven et al., 2005; Chapuis-Lardy et al., 2006; Vanderhoeven et al., 2006). An increase of available phosphorus in topsoils invaded by S. gigantea could be explained by enhanced turnover rates, potentially driven by an excessive seasonal increase of phosphorus stocks of rhizomes in the autumn (Herr et al., 2007). However, this increase cannot be explained by phosphorus concentrations in the biomass of invasive S. gigantea as these are either the same as in co-occuring native vegetation (Güsewell et al., 2005) or lower (Chapuis-Lardy et al., 2006). The hypothesis of enemy release has also been evoked to explain invasion success of S. gigantea. Because a low level of mostly generalist insects has been observed on invasive populations of S. gigantea (Bopp, 1997), it is listed together with S. canadensis as one of the 20 most promising species for biological control in Europe (Sheppard et al., 2006). Although introduced S. gigantea experiences a release from herbivores (Hahn, 2005), rates of pathogen attack are comparable with those on native species in the same habitats. Furthermore, S. gigantea was among the first invasive alien species where rapid evolutionary change after introduction was detected (Weber, 1994), for instance in the form of a latitudinal cline in introduced populations (Weber & Schmid, 1998). However, human activities may also have affected the genetic stock of S. gigantea in Europe, since the species was originally introduced for its ornamental value (Weber, 1994). And because S. gigantea was distributed and released on a large scale by horticuralists and bee-keepers (Meier, 1958; Voser-Huber, 1983), propagule pressure which is known to be increased by horticultural trade and price (Dehnen-Schmutz et al., 2007), was certainly another factor influencing its successful invasion. To substantiate theories of enemy release and genetic change, studies are needed that compare native with invasive populations of S. gigantea. For instance, whereas leaf traits and nutrient concentration do not differ between populations from the native and the invasive range when grown in a common garden, genetic change was identified in clonal growth (Güsewell et al., 2006; Meyer & Hull-Sanders, 2007). Based on observations that S. gigantea grows taller, denser and suffers less from herbivores in the invasive range than in its native range (Jakobs et al., 2004), the hypothesis of evolution of increased competitive ability was investigated (Clare, 2003; Jakobs, 2004). In one study, invasive populations of S. gigantea showed lower levels of some secondary defences compared to native populations (Johnson et al., 2007), but no such differences were found in another (Hull-Sanders et al., 2007). While a generalist caterpillar

13 General introduction grew better on invasive populations of S. gigantea than on native populations, growth of three specialist herbivores was not affected (Hull-Sanders et al., 2007). Also, invasive populations of S. gigantea were more susceptible to some leaf pathogens than native populations (Meyer et al., 2005). Interestingly, compensation of insect herbivory was comparable between native and invasive populations, though different strategies were involved (Meyer & Hull-Sanders, 2007). Overall, it seems probable that enemy release has been a factor influencing the invasion success of S. gigantea in Europe and also that some genetic change has occurred. However, results for the evolution of increased competitive ability for S. gigantea are mixed and overall unconvincing (Güsewell et al., 2006; Meyer & Hull-Sanders, 2007). None of the studies that compared native with invasive populations, however, controlled for ploidy level, despite the strong influence that polyploidy can have upon the ecological and growth characteristics of plants.

Ou t l i n e o f t h e t h e s i s

This thesis adopts a comparative biogeographic approach to understand the significance that polyploidy plays in biological invasions. Solidago gigantea serves here as model system because it combines three morphological indistinguishable ploidy levels in its native range in North America (Semple & Cook, 2006) and is an aggressive invader of a wide range of habitats including those with established vegetation in areas of introduction (Weber & Jakobs, 2005). With new insights into large- and small-scale distribution patterns of ploidy levels (chapter 1), habitat differentiation between ploidy levels (chapter 2), life-history traits of ploidy levels (chapter 3) and the genetic diversity and migration patterns (chapter 4), this project elucidates aspects of the ecology of S. gigantea in its native range as a basis for understanding its invasive potential. Thus, by combining field surveys and studies with experimental and genetic approaches, this thesis aims to provide a sound evaluation of the ecological significance of ploidy levels of S. gigantea, both in the native range and as an alien species.

In chapter 1—Cytogeography of Solidago gigantea (Asteraceae) and its invasive ploidy level—patterns of distribution of native ploidy levels (diploids, tetraploids and hexaploids) in North America are compared, and the ploidy level(s) of populations that became invasive in introduced areas of Europe and Asia are determined. By determining size of distribution, location of contact zones and degree of spatial segregation of ploidy levels in the native range, I aim to investigate the importance of geographic arrangement and interactions of ploidy levels in regards to introductions.

14 General introduction

In chapter 2—Habitat differentiation between native ploidy levels and invasive tetraploids of Solidago gigantea (Asteraceae)—by means of a field survey and by constructing environmental envelopes I investigate how the ploidy levels in the native range differ in their habitat associations and compare these to the habitats occupied by tetraploids in the invasive range in Europe. I aim to test the hypotheses that ecological differentiation explains the geographic separation between ploidy levels in North America and that ecological preferences (habitat templates) explain why no diploids have been found in Europe. I further investigate whether an ecological (niche) shift has occurred between North American and European tetraploids.

In chapter 3—Ploidy-level precise common garden with native and invasive Solidago gigantea: differences in clonal growth and nutrient responses indicate colonization success of tetraploids—a comparative common garden experiment with diploid and tetraploid S. gigantea of both the native and invasive range is set up with a nutrient and a soil calcium treatment. I aim to establish growth performance, life history and treatment response differences between native diploids and tetraploids that could help explaining observed habitat differentiation and geographic separation. Further, I compare invasive tetraploids with the corresponding native tetraploids to evaluate pre-disposition of invasive traits in S. gigantea.

In chapter 4—Phylogeography of native ploidy levels and invasive tetraploids of Solidago gigantea—with a phylogeographic approach, I elucidate migration history of diploid and tetraploid populations of S. gigantea in North America, and estimate the number of polyploidization events that must have occurred to produce the observed variation. I also compare the recent spread of S. gigantea in Europe with the pattern of colonization in the native range and identify likely source areas of the introduced haplotype lineages. The aim is to investigate the significance of ploidy level as a factor affecting the spread of S. gigantea, both in its native range and as an alien species in Europe.

Li t e r a t u r e

Abrahamson, W.G., Dobley, K.B., Houseknecht, H.R. & Pecone, C.A. (2005) Ecological divergence among five co-occurring species of old-field goldenrods. Plant Ecology, 177, 43-56

Baack, E.J. (2005) To succeed globally, disperse locally: effects of local pollen and seed dispersal on tetraploid establishment. Heredity, 94, 538-546

15 General introduction

Barrett, S.C.H. & Richardson, B.J. (1986) Genetic attributes of invading species. Ecology of biological invasions: an Australian perspective (ed. by R.H. Groves and J.J. Burdon), pp. 21-33. Australian Academy of Science, Canberra, Australia.

Beaudry, J.R. (1970) Studies on Solidago L. X. Les Solidago gigantea Ait. dans l’est de l’Amerique du Nord. Naturaliste Canadien, 97, 35-42

Bennett, M.D., Leitch, I.J. & Hanson, L. (1998) DNA amounts in two samples of angiosperm weeds. Annals of Botany, 82, 121-134

Blossey, B. & Notzold, R. (1995) Evolution of increased competitive ability in invasive nonindigenous plants - a hypothesis. Journal of Ecology, 83, 887-889

Blumenthal, D. (2005) Ecology - Interrelated causes of plant invasion. Science, 310, 243-244

Blumenthal, D.M. (2006) Interactions between resource availability and enemy release in plant invasion. Ecology Letters, 9, 887-895

Bopp (1997) Die amerikanische Goldrute Solidago canadensis L. und S. gigantea Ait. als Neophyten in Mitteleuropa: Besiedlung durch Insekten. Göttinger naturkundliche Schriften, 4, 181-206

Bossdorf, O., Auge, H., Lafuma, L., Rogers, W.E., Siemann, E. & Prati, D. (2005) Phenotypic and genetic differentiation between native and introduced plant populations. Oecologia, 144, 1-11

Botta-Dukat, Z. & Dancza, I. (2001) Effect of weather conditions on the growth of Solidago gigantea. Plant Invasions: Species Ecology and Ecosystem Management (ed. by G. Brundu, J. Brock, I. Camarda, L. Child and M. Wade), pp. 47-54. Backhuys Publishers, Leiden, The Netherlands.

Brown, A.H.D. & Marshall, D.S. (1981) Evolutionary changes accompanying colonization in plants. Evolution Today. Proceedings of 2nd international Congress of Systematics and Evolutionary Biology (ed. by G.G.E. Scudder and J.L. Reveal), pp. 351-363. Carnegie-Mellon University, Pittsburg.

Callaway, R.M. & Aschehoug, E.T. (2000) Invasive plants versus their new and old neighbors: A mechanism for exotic invasion. Science, 290, 521-523

Callaway, R.M. & Maron, J.L. (2006) What have exotic plant invasions taught us over the past 20 years? Trends in Ecology & Evolution, 21, 369-374

Callaway, R.M. & Ridenour, W.M. (2004) Novel weapons: invasive success and the evolution of increased competitive ability. Frontiers in Ecology and the Environment, 2, 436-443

Chapin, F.S., Zavaleta, E.S., Eviner, V.T., Naylor, R.L., Vitousek, P.M., Reynolds, H.L., Hooper, D.U., Lavorel, S., Sala, O.E., Hobbie, S.E., Mack, M.C. & Diaz, S. (2000) Consequences of changing biodiversity. Nature, 405, 234-242

Chapuis-Lardy, L., Vanderhoeven, S., Dassonville, N., Koutika, L.S. & Meerts, P. (2006) Effect of the exotic invasive plant Solidago gigantea on soil phosphorus status. Biology and Fertility of Soils, 42, 481-489

Clare, R. (2003) Evaluation of the evolution of increased competitive ability (EICA) hypothesis: insect performance on native and introduced populations of Solidago gigantea (Asteraceae). PhD thesis. University of Wisconsin-Milwaukee, Milwaukee.

16 General introduction

Colautti, R.I., Bailey, S.A., Van Overdijk, C.D.A., Amundsen, K. & Macisaac, H.J. (2006) Characterised and projected costs of nonindigenous species in Canada. Biological Invasions, 8, 45-59

Colautti, R.I. & MacIsaac, H.J. (2004) A neutral terminology to define ‘invasive’ species. Diversity and Distributions, 10, 135-141

Colautti, R.I., Ricciardi, A., Grigorovich, I.A. & MacIsaac, H.J. (2004) Is invasion success explained by the enemy release hypothesis? Ecology Letters, 7, 721-733

Collins, A.R., Jose, S., Daneshgar, P. & Ramsey, C.L. (2007) Elton’s hypothesis revisited: an experimental test using cogongrass. Biological Invasions, 9, 433-443

Cox, G.W. (2004) Alien species and evolution the evolutionary ecology of exotic plants, animals, microbes, and interacting native species. Island Press, Washington, D.C.

Crawley, M.J., Harvey, P.H. & Purvis, A. (1996) Comparative ecology of the native and alien floras of the British Isles. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences, 351, 1251-1259

Daehler, C.C. (2001) Darwin’s naturalization hypothesis revisited. American Naturalist, 158, 324-330

Davis, M.A. (2005) Invasion Biology 1958-2004: The pursuit of science and conservation. Conceptual ecology and invasions biology: reciprocal approaches to nature (ed. by M.W. Cadotte, S.M. Mcmahon and T. Fukami). Kluwer Publishers, London.

Davis, M.A., Thompson, K. & Grime, J.P. (2001) Charles S. Elton and the dissociation of invasion ecology from the rest of the ecology. Diversity and Distributions, 7, 97-102

Dehnen-Schmutz, K., Touza, J., Perrings, C. & Williamson, M. (2007) A century of the ornamental plant trade and its impact on invasion success. Diversity and Distributions, 13, 527-534

Dietz, H. & Edwards, P.J. (2006) Recognition that causal processes change during plant invasion helps explain conflicts in evidence. Ecology, 87, 1359-1367

Dlugosch, K.M. & Parker, I.M. (2008) Founding events in species invasions: genetic variation, adaptive evolution, and the role of multiple introductions. Molecular Ecology, 17, 431-449

Drake, J.A., Mooney, H.A., Di Castri, F., Groves, R.H., Kruger, F.J., Rejmanek, M. & Williamson, M. (1989) Biological invasions: A global perspective. SCOPE 37. John Wiley & Sons, Chichester, UK.

Eggenschwiler, H. (2007) Bedeutung von Boden-Pflanzen Interaktionen für die Invasivität vonSolidago gigantea. Master thesis, ETH Zurich, Zurich.

Ehrendorfer, F. (1980) Polyploidy and distribution. Polyploidy - Biological relevance (ed. by W.H. Lewis), pp. 45-60. Plenum Press, New York, London.

Ehrenfeld, J.G. (2003) Effects of exotic plant invasions on soil nutrient cycling processes. Ecosystems, 6, 503-523

Ellstrand, N.C. & Schierenbeck, K.A. (2000) Hybridization as a stimulus for the evolution of invasiveness in plants? Proceedings of the National Academy of Sciences of the United States of America, 97, 7043-7050

17 General introduction

Facon, B., Genton, B.J., Shykoff, J., Jarne, P., Estoup, A. & David, P. (2006) A general eco-evolutionary framework for understanding bioinvasions. Trends in Ecology & Evolution, 21, 130-135

Felber, F. (1991) Establishment of a tetraploid cytotype in a diploid population - effect of relative fitness of the cytotypes. Journal of Evolutionary Biology, 4, 195-207

Fowler, N.L. & Levin, D.A. (1984) Ecological constraints on the establishment of a novel polyploid in competition with its diploid progenitor. American Naturalist, 124, 703-711

Güsewell, S., Jakobs, G. & Weber, E. (2006) Native and introduced populations of Solidago gigantea differ in shoot production but not in leaf traits or litter decomposition. Functional Ecology, 20, 575-584

Güsewell, S., Zuberbühler, N. & Clerc, C. (2005) Distribution and functional traits of Solidago gigantea in a Swiss lakeshore wetland. Botanica Helvetica, 115, 63-75

Guzikowa, M. & Maycock, P.F. (1986) The Invasion and Expansion of 3 North-American Species of Goldenrod (Solidago canadensis L. s.l., Solidago gigantea Ait. and Solidago graminifolia (L.) Salisb.) in Poland. Acta Societatis Botanicorum Poloniae, 55, 367-384

Hahn, M. (2005) Pathogen infection and herbivory on the invasive plant species Solidago gigantea. Master thesis. ETH Zurich, Zurich.

Halverson, K., Heard, S.B., Nason, J.D. & Stireman, J.O. (2008a) Differential attack on diploid, tetraploid, and hexaploid Solidago altissima L. by five insect gallmakers. Oecologia, 154, 755-761

Halverson, K., Heard, S.B., Nason, J.D. & Stireman, J.O. (2008b) Origins, distribution, and local co- occurrence of polyploid cytotypes in Solidago altissima (Asteraceae). American Journal of Botany, 95, 50-58

Herr, C., Chapuis-Lardy, L., Dassonville, N., Vanderhoeven, S. & Meerts, P. (2007) Seasonal effect of the exotic invasive plant Solidago gigantea on soil pH and P fractions. Journal of Plant Nutrition and Soil Science-Zeitschrift für Pflanzenernährung und Bodenkunde, 170, 729-738

Hierro, J.L., Maron, J.L. & Callaway, R.M. (2005) A biogeographical approach to plant invasions: the importance of studying exotics in their introduced and native range. Journal of Ecology, 93, 5-15

Hull-Sanders, H.M., Clare, R., Johnson, R.H. & Meyer, G.A. (2007) Evaluation of the evolution of increased competitive ability (EICA) hypothesis: Loss of defense against generalist but not specialist herbivores. Journal of Chemical Ecology, 33, 781-799

Husband, B.C. (2004) The role of triploid hybrids in the evolutionary dynamics of mixed-ploidy populations. Biological Journal of the Linnean Society, 82, 537-546

Husband, B.C. & Sabara, H.A. (2004) Reproductive isolation between autotetraploids and their diploid progenitors in fireweed, Chamerion angustifolium (Onagraceae). New Phytologist, 161, 703-713

Jakobs, G. (2004) Evolution of increased competitive ability in the invasive perennial Solidago gigantea Aiton. Diss. 15536 ETH Zürich, Zürich.

Jakobs, G., Weber, E. & Edwards, P.J. (2004) Introduced plants of the invasive Solidago gigantea (Asteraceae) are larger and grow denser than conspecifics in the native range. Diversity and Distributions, 10, 11-19

18 General introduction

Johnson, M.T.J., Husband, B.C. & Burton, T.L. (2003) Habitat differentiation between diploid and tetraploid Galax urceolata (Diapensiaceae). International Journal of Plant Sciences, 164, 703- 710

Johnson, R.H., Hull-Sanders, H.M. & Meyer, G.A. (2007) Comparison of foliar terpenes between native and invasive Solidago gigantea. Biochemical Systematics and Ecology, 35, 821-830

Kercher, S.M. & Zedler, J.B. (2004) Flood tolerance in wetland angiosperms: a comparison of invasive and noninvasive species. Aquatic Botany, 80, 89-102

Klironomos, J.N. (2002) Feedback with soil biota contributes to plant rarity and invasiveness in communities. Nature, 417, 67-70

Kruse, B.S. & Groninger, J.W. (2003) Vegetative characteristics of recently reforested bottomlands in the lower Cache River Watershed, Illinois, USA. Restoration Ecology, 11, 273-280

Kubatova, B., Travnicek, P., Bastlova, D., Curn, V., Jarolimova, V. & Suda, J. (2008) DNA ploidy level variation in native and invasive populations of Lythrum salicaria at a large geographical scale. Journal of Biogeography, 35, 167-176

Lee, C.E. (2002) Evolutionary genetics of invasive species. Trends in Ecology & Evolution, 17, 386-391

Levin, D.A. (1975) Minority cytotype exclusion in local plant populations. Taxon, 24, 35-43

Levin, D.A. (2003) Ecological Speciation: Lessons From Invasive Species. Systematic Botany, 28, 643– 650

Levine, J.M. & D’Antonio, C.M. (1999) Elton revisited: a review of evidence linking diversity and invasibility. Oikos, 87, 15-26

Li, B.H., Xu, X.M. & Ridout, M.S. (2004) Modelling the establishment and spread of autotetraploid plants in a spatially heterogeneous environment. Journal of Evolutionary Biology, 17, 562-573

Lodge, D.M. (1993) Biological Invasions - Lessons for Ecology. Trends in Ecology & Evolution, 8, 133- 137

Ludsin, S.A. & Wolfe, A.D. (2001) Biological invasion theory: Darwin’s contributions from The Origin of Species. Bioscience, 51, 780-789

Lumaret, R. (1988) Adaptive strategies and ploidy levels. Acta Oecologica-Oecologia Plantarum, 9, 83-93

Maceira, N.O., Jacquard, P. & Lumaret, R. (1993) Competition between diploid and derivative autotetraploid Dactylis glomerata L. from Galicia - implications for the establishment of novel polyploid populations. New Phytologist, 124, 321-328

Maron, J. & Marler, M. (2007) Native plant diversity resists invasion at both low and high resource levels. Ecology, 88, 2651-2661

Maron, J.L., Vila, M., Bommarco, R., Elmendorf, S. & Beardsley, P. (2004) Rapid evolution of an invasive plant. Ecological Monographs, 74, 261-280

Masterson, J. (1994) Stomatal size in fossil plants - evidence for polyploidy in majority of angiosperms. Science, 264, 421-424

19 General introduction

McKenney, J.L., Cripps, M.G., Price, W.J., Hinz, H.L. & Schwarzlander, M. (2007) No difference in competitive ability between invasive North American and native European Lepidium draba populations. Plant Ecology, 193, 293-303

McNeely, J.A., Mooney, H.A., Neville, L.E., Schei, P.J. & Waage, J.K. (2001) A global strategy on invasive alien species. IUCN, Gland, Switzerland and Cambridge, UK.

Meier, K. (1958) Goldruten (Solidago)-Arten für die Verbesserung der Spätsommer- und Herbstpollentracht. Schweizerische Bienen-Zeitung, 81, 452-456

Melville, M.R. & Morton, J.K. (1982) A biosystematic study of the Solidago canadensis (Compositae) complex. 1. The Ontario populations. Canadian Journal of Botany - Revue Canadienne de Botanique, 60, 976-997

Meyer, G., Clare, R. & Weber, E. (2005) An experimental test of the evolution of increased competitive ability hypothesis in goldenrod, Solidago gigantea. Oecologia, 144, 299-307

Meyer, G.A. & Hull-Sanders, H.M. (2007) Altered patterns of growth, physiology and reproduction in invasive genotypes of Solidago gigantea (Asteraceae). Biological Invasions, in press. Available online 29 June 2007. doi:10.1007/s10530-007-9131-z

Mitchell, C.E., Agrawal, A.A., Bever, J.D., Gilbert, G.S., Hufbauer, R.A., Klironomos, J.N., Maron, J.L., Morris, W.F., Parker, I.M., Power, A.G., Seabloom, E.W., Torchin, M.E. & Vazquez, D.P. (2006) Biotic interactions and plant invasions. Ecology Letters, 9, 726-740

Morton, G.H. (1984) A practical treatment of the Solidago gigantea complex. Canadian Journal of Botany - Revue Canadienne de Botanique, 62, 1279-1282

Müller, H. (1989) Growth pattern of diploid and tetraploid spotted knapweed, Centaurea maculosa Lam. (Compositae), and effects of the root mining moth Agapeta zoegana (L.) (Lep.: Cochylidae). Weed Research, 29, 103-111

Müller-Schärer, H., Schaffner, U. & Steinger, T. (2004) Evolution in invasive plants: implications for biological control. TRENDS in Ecology & Evolution, 19, 417-422

Münzbergova, Z. (2006) Ploidy level interacts with population size and habitat conditions to determine the degree of herbivory damage in plant populations. Oikos, 115, 443-452

Myers, J.H. & Bazely, D. (2003) Ecology and control of introduced plants. Cambridge University Press, Cambridge.

Nesom, G.L. (1993) Taxonomic infrastructure of Solidago and Oligoneuron (Asteraceae: Astereae) and observations on their phylogenetic position. Phytologia, 75, 1-44

Nuismer, S.L. & Cunningham, B.M. (2005) Selection for phenotypic divergence between diploid and autotetraploid Heuchera grossulariifolia. Evolution, 59, 1928-1935

Nuismer, S.L. & Thompson, J.N. (2001) Plant polyploidy and non-uniform effects on insect herbivores. Proceedings of the Royal Society of London Series B-Biological Sciences, 268, 1937-1940

Osborn, T.C., Pires, J.C., Birchler, J.A., Auger, D.L., Chen, Z.J., Lee, H.S., Comai, L., Madlung, A., Doerge, R.W., Colot, V. & Martienssen, R.A. (2003) Understanding mechanisms of novel gene expression in polyploids. Trends in Genetics, 19, 141-147

20 General introduction

Otto, S.P. & Whitton, J. (2000) Polyploid incidence and evolution. Annual Review of Genetics, 34, 401- 437

Pandit, M.K., Tan, H.T.W. & Bisht, M.S. (2006) Polyploidy in invasive plant species of Singapore. Botanical Journal of the Linnean Society, 151, 395-403

Petit, R.J. (2004) Biological invasions at the gene level. Diversity and Distributions, 10, 159-165

Pimentel, D., Zuniga, R. & Morrison, D. (2005) Update on the environmental and economic costs associated with alien-invasive species in the United States. Ecological Economics, 52, 273-288

Proches, S., Wilson, J.R.U., Richardson, D.M. & Rejmanek, M. (2008) Searching for phylogenetic pattern in biological invasions. Global Ecology and Biogeography, 17, 5-10

Ramsey, J. & Schemske, D.W. (2002) Neopolyploidy in flowering plants.Annual Review of Ecology and Systematics, 33, 589-639

Rausch, J.H. & Morgan, M.T. (2005) The effect of self-fertilization, inbreeding depression, and population size on autopolyploid establishment. Evolution, 59, 1867-1875

Richardson, D.M. & Pysek, P. (2006) Plant invasions: merging the concepts of species invasiveness and community invasibility. Progress in Physical Geography, 30, 409-431

Richardson, D.M., Pysek, P., Rejmanek, M., Barbour, M.G., Dane, P.F. & West, C.J. (2000) Naturalization and invasion of alien plants: concepts and definitions. Diversity and Distributions, 6, 93-107

Rodriguez, D.J. (1996a) A model for the establishment of polyploidy in plants. American Naturalist, 147, 33-46

Rodriguez, D.J. (1996b) A model for the establishment of polyploidy in plants: Viable but infertile hybrids, iteroparity, and demographic stochasticity. Journal of Theoretical Biology, 180, 189-196

Roy, J. (1990) In search of the characteristics of plant invaders. Biological invasions in Europe and the Mediterranean basin (ed. by F. Di Castri, A.J. Hansen and M. Debussche), pp. 335-352. Kluwer Academic Publishers, Dordrecht.

Sakai, A.K., Allendorf, F.W., Holt, J.S., Lodge, D.M., Molofsky, J., With, K.A., Baughman, S., Cabin, R.J., Cohen, J.E., Ellstrand, N.C., McCauley, D.E., O’Neil, P., Parker, I.M., Thompson, J.N. & Weller, S.G. (2001) The Population Biology of Invasive Species. Annual Review of Ecology and Systematics, 32, 305-332

Sax, D.F., Stachowicz, J.J., Brown, J.H., Bruno, J.F., Dawson, M.N., Gaines, S.D., Grosberg, R.K., Hastings, A., Holt, R.D., Mayfield, M.M., O’Connor, M.I. & Rice, W.R. (2007) Ecological and evolutionary insights from species invasions. Trends in Ecology & Evolution, 22, 465-471

Schmid, B. & Bazzaz, F.A. (1987) Clonal Integration and Population Structure in Perennials Effects of Severing Rhizome Connections. Ecology (Washington D C), 68, 2016-2022

Schmid, B. & Bazzaz, F.A. (1992) Growth Responses of Rhizomatous Plants to Fertilizer Application and Interference. Oikos, 65, 13-24

Segraves, K.A. & Thompson, J.N. (1999) Plant polyploidy and pollination: floral traits and insect visits to diploid and tetraploid Heuchera grossulariifolia. Evolution, 53, 1114-1127

21 General introduction

Semple, J.C. & Cook, R.E. (2006) Solidago. Flora of North America North of Mexico (ed. by Flora of North America Editorial Committee), pp. 107-166. Oxford University Press, Oxford and New York.

Semple, J.C., Ringius, G.S., Leeder, C. & Morton, G. (1984) Chromosome numbers of Goldenrods, Euthamia and Solidago (Compositae, Astereae). II. Additional counts with comments on cytogeography. Brittonia, 36, 280-292

Shea, K. & Chesson, P. (2002) Community ecology theory as a framework for biological invasions. Trends in Ecology & Evolution, 17, 170-176

Sheppard, A.W., Shaw, R.H. & Sforza, R. (2006) Top 20 environmental weeds for classical biological control in Europe: a review of opportunities, regulations and other barriers to adoption. Weed Research, 46, 93-117

Silvertown, J.W. (2005) Demons in Eden the paradox of plant diversity. University of Chicago Press, Chicago.

Simberloff, D. (2003) How much information on population biology is needed to manage introduced species? Conservation Biology, 17, 83-92

Soltis, D.E., Soltis, P.S., Pires, J.C., Kovarik, A., Tate, J.A. & Mavrodiev, E. (2004) Recent and recurrent polyploidy in Tragopogon (Asteraceae): cytogenetic, genomic and genetic comparisons. Biological Journal of the Linnean Society, 82, 485-501

Soltis, D.E., Soltis, P.S. & Tate, J.A. (2003) Advances in the study of polyploidy since “Plant speciation”. New Phytologist, 161, 173-191

Soltis, P.S. (2005) Ancient and recent polyploidy in angiosperms. New Phytologist, 166, 5-8

Suarez, A.V. & Tsutsui, N.D. (2008) The evolutionary consequences of biological invasions. Molecular Ecology, 17, 351-360

Theoharides, K.A. & Dukes, J.S. (2007) Plant invasion across space and time: factors affecting nonindigenous species success during four stages of invasion. New Phytologist, 176, 256-273

Thompson, J.N., Nuismer, S.L. & Merg, K. (2004) Plant polyploidy and the evolutionary ecology of plant/animal interactions. Biological Journal of the Linnean Society, 82, 511-519

Thompson, K., Hodgson, J.G. & Rich, T.C.G. (1995) Native and alien invasive plants: More of the same? Ecography, 18, 390-402

Vanderhoeven, S., Dassonville, N., Chapuis-Lardy, L., Hayez, M. & Meerts, P. (2006) Impact of the invasive alien plant Solidago gigantea on primary productivity, plant nutrient content and soil mineral nutrient concentrations. Plant and Soil, 286, 259-268

Vanderhoeven, S., Dassonville, N. & Meerts, P. (2005) Increased topsoil mineral nutrient concentrations under exotic invasive plants in Belgium. Plant and Soil, 275, 169-179

Verlaque, R., Aboucaya, A. & Fridlender, A. (2002) Invasive alien flora of France: ecology, life-forms and polyploidy. Botanica Helvetica, 112, 121-136

Vitousek, P.M., D’Antonio, C.M., Loope, L.L., Rejmanek, M. & Westbrooks, R. (1997) Introduced species:

22 General introduction

A significant component of human-caused global change. New Zealand Journal of Ecology, 21, 1- 16

Voser-Huber, M.L. (1983) Studien an eingebürgerten Arten der Gattung Solidago L. Probleme mit S. gigantea, S. canadensis und S. graminifolia in Naturschutzgebieten des schweizerischen Mittellandes. Dissertationes Botanicae, 68, 97

Weber, E. (1994) Evolutionary trends in european neophytes: a case study of two Solidago species. Ph.D. dissertation. University of Basel, Basel, Switzerland.

Weber, E. (1998) The dynamics of plant invasions: a case study of three exotic goldenrod species (Solidago L.) in Europe. Journal of Biogeography, 25, 147-154

Weber, E. (2001) Current and potential ranges of three exotic goldenrods (Solidago) in Europe. Conservation Biology, 15, 122-128

Weber, E. & Jakobs, G. (2005) Biological flora of central Europe: Solidago gigantea Aiton. Flora - Morphology, Distribution, Functional Ecology of Plants, 200, 109-118

Weber, E. & Schmid, B. (1993) Das Neophytenproblem. Dissertationes Botanicae, 196, 209-227

Weber, E. & Schmid, B. (1998) Latitudinal population differentiation in two species of Solidago (Asteraceae) introduced into Europe. American Journal of Botany, 85, 1110-1121

Wilcove, D.S., Rothstein, D., Dubow, J., Phillips, A. & Losos, E. (1998) Quantifying threats to imperiled species in the United States. Bioscience, 48, 607-615

Zhang, J. (1996) A molecular biosystematic study of North American Solidago and related genera (Asteraceae: Astereae) based on chloroplast DNA RFLP Analysis. PhD thesis. University of Waterloo, Canada.

23

Ploidy level distribution

Ch a p t e r 1

Cytogeography of Solidago gigantea (Asteraceae) and its invasive ploidy level

Daniel R. Schlaepfer1, Peter J. Edwards1, John C. Semple2, Regula Billeter1

1Institute of Integrative Biology, Plant Ecology, ETH Zurich, Zürich, Switzerland 2Department of Biology, University of Waterloo, Waterloo, Canada

25 Chapter 1

Ab s t r a c t

Aim Newly formed polyploids experience problems of establishment and spread similar to those faced by newly introduced alien species. To help understand the significance that polyploidy plays in biological invasions, we mapped the distribution of ploidy levels in Solidago gigantea Aiton in its native range in North America and in Europe and East Asia where it is invasive. Methods We used flow cytometry to measure ploidy levels in a total of 834 plants from 149 locations. Together with data from contributors and a literature review we assembled ploidy level data for 336 locations. Cytogeographic maps from North America and Europe were prepared incorporating new and previously published ploidy level data. Results In the native range, we found diploid, tetraploid and hexaploid plants, and also one triploid and one pentaploid plant (2n = 3x and 2n = 5x are new reports for this species). There was a high degree of geographic separation among the ploidy levels, and populations with mixed ploidy were rare. However, four zones where plants of different ploidy could come into contact were identified. In Europe and East Asia only tetraploid plants were found. Main conclusions The present geographic separation in North America suggests that the ploidy levels are ecologically differentiated, though further investigations are needed to identify the nature of these differences. Alien populations appear to be exclusively tetraploid, but it is not clear whether this is because tetraploids were selectively introduced or diploids were unsuccessful. In any case, comparisons between native and introduced populations need to account for ploidy level.

Keywords: Polyploidy, tetraploid, establishment, geographic segregation, mixed-ploidy population, invasive alien species, flow cytometry.

26 Ploidy level distribution

In t r o d u c t i o n

Polyploidy plays an important role in plant evolution. At least 2–4% of angiosperm speciation events are associated with polyploidy (Otto & Whitton, 2000) and as many as 70% of angiosperm species show signs of polyploidy in their history (Masterson, 1994). Polyploidization brings about strong, though not necessarily complete, reproductive isolation from the diploid progenitors, and therefore represents a barrier to gene flow (Husband & Sabara, 2004). It also can have profound consequences for gene expression (Osborn et al., 2003; Soltis et al., 2004) and the physiological (Soltis et al., 2003) and ecological behaviour of plants (Lumaret, 1988; Thompson et al., 2004), as well as the distribution of genetic diversity (Soltis & Soltis, 2000). It is usually assumed that newly formed polyploids face severe obstacles to becoming established (Fowler & Levin, 1984). Not only are they in direct competition with their diploid progenitor(s) for resources, but since pollination is frequency-dependent on ploidy level, their chances of reproducing may be initially small (‘minority cytotype disadvantage’, Levin, 1975; Felber, 1991). Despite these disadvantages, polyploid taxa are common, which has prompted considerable research to identify what factors favour their establishment (e.g. Schönswetter et al., 2007; Halverson et al., 2008; Kao, 2008; Kubatova et al., 2008). Clearly, there can be no negative interactions with the diploid, if the polyploid does not grow in the same area as the diploid, and there are many examples of geographic separation—Lythrum salicaria (Kubatova et al., 2008), Ranunculus adoneus (Baack, 2004) and Chamerion angustifolium (Husband & Schemske, 1998). Possible adaptive mechanisms driving such geographic separation between ploidy levels could include ecological differentiation (Felber-Girard et al., 1996; Schönswetter et al., 2007), competitive and fitness advantages of polyploids (Novak et al., 1991; Maceira et al., 1993) and better colonising abilities of polyploids (De Wet, 1980; Soltis & Soltis, 2000; Brochmann et al., 2004). Non-adaptive explanations include the spatial structure of populations (Li et al., 2004; Baack, 2005) and stochastic effects (Rausch & Morgan, 2005). Interestingly, polyploids are more frequent among invasive alien plants than among angiosperms in general. This tendency has been shown, for example, for the ‘world’s worst weeds’ (Brown & Marshall, 1981), for important world weeds and British garden weeds (Bennett et al., 1998), for the flora of France (Verlaque et al., 2002) and for the flora of Singapore (Pandit et al., 2006). While the phenomenon of biological invasions cannot be explained by polyploidy alone (Ehrendorfer, 1980; Barrett & Richardson, 1986; Roy, 1990), a knowledge of the factors favouring polyploid establishment is likely to contribute to our understanding of why some alien plants become invasive. For species containing several ploidy levels in the native range, often only a high ploidy level is detected in the invasive range, as it is the case for Centaurea maculosa (Müller, 1989; Müller-Schärer et al., 2004) and Lythrum salicaria (Kubatova et al., 2008). And in a few cases, e.g. for hybrids involving Rorippa austriaca (Bleeker & Matthies, 2005) and for various Tragopogon spp. (Soltis et al., 2004), the role of polyploidy in conferring invasive behaviour has been well documented.

27 Chapter 1

Solidago gigantea Aiton (Asteraceae) is a convenient species to study aspects of both biological invasion and polyploid establishment. The species is native and widespread in North America. The basic chromosome number of the genus Solidago is x = 9 (Beaudry & Chabot, 1959; Semple et al., 1984), and S. gigantea occurs in three ploidy levels (Semple & Cook, 2006): diploids (2x), tetraploids (4x), and hexaploids (6x). It was introduced in 1758 to Europe where it became invasive (Weber & Schmid, 1993). It is also introduced on the islands of the Azores (Weber, 2003), in northern Japan (Fukuda, 1976; Anonymous, 2004), in Korea (Zerbe et al., 2004), in Ukraine (Mosyakin & Yavorska, 2002) and in Russia, e.g. in Kalinin district (Gusev, 1974; Malysheva, 1980) and Primorsky krai (Rudyka, 1995). Solidago gigantea belongs to the subsection Triplinerviae, together with S. canadensis, S. altissima, S. lepida and others (Nesom, 1993; Semple & Cook, 2006). It is a 0.5–2.5 m tall herb with annual, partially sexual shoots and persistent, clonal rhizomes. The species is an obligate outbreeder and is pollinated by a wide range of insects (Melville & Morton, 1982; Voser-Huber, 1983; Semple et al., 1999). In the past, three species (sometimes also referred to as subspecies or as varieties) differing in ploidy level, S. gigantea Ait., S. serotina Ait., S. shinnersii (Beaudry) Beaudry, and one form, S. serotina forma huntingdonensis Beaudry were recognised. These taxa were thought to be distinguishable by hairiness and size of leaves (Beaudry & Chabot, 1959; Beaudry, 1963, 1970, 1974). However, later work concluded that these morphological characters do not consistently separate the ploidy levels, and therefore no reliable infraspecific categories could be made (Melville & Morton, 1982; Morton, 1984; Semple et al., 1984). Taxonomic difficulties are also known from the closely related S. canadensis complex and are attributed to convergence or hybridisation between higher ploidy levels (Semple & Cook, 2006). To improve our understanding of the role of polyploidy in plant invasions, we investigated mechanisms of polyploid establishment by examining the distribution of ploidy levels in S. gigantea. Our objectives in this work were (i) to describe the cytogeography of S. gigantea in the native range, (ii) to determine the extent of spatial segregation of ploidy levels at the population level and at a regional scale and to detect possible contact zones, (iii) to measure the frequency of mixed-ploidy populations and of triploid plants, and (iv) to determine ploidy levels of invasive populations in Europe.

28 Ploidy level distribution

Ma t e r i a l s a n d Me t h o d s

Field survey

Details of the 149 sampling locations are given in Appendix S1 in Supplementary Material. About half of the samples were collected for us from 69 locations in Europe (Austria, Croatia, Czech Republic, Denmark, Germany, Hungary, Italy, Netherlands, Poland, Switzerland, United Kingdom), one location from USA, one from Russia and three locations from Japan. Subsequently, in autumn 2005 we visited five regions in North America (58 locations; diploids and tetraploids from areas of possible contact zones in the southern Appalachian Mts., in New England and in southern Ontario; hexaploids from South Dakota and from southern Manitoba) and three regions in Europe (20 locations; tetraploids from northern Italy, from Belgium and from southern Sweden). Populations, one per geographic location, were separated by at least 3 km, except for two tetraploid locations in Ontario that were 800 m apart. To reduce the risk of resampling the same clone, seed families or, at a few locations, rhizomes were collected from individual shoots at least 2 m apart. If possible, five plants from different seed families (or rhizomes) per sampled location were grown in a climate chamber to determine ploidy level. Vouchers from these plants were deposited in the herbarium Z+ZT (Zurich, Switzerland).

Ploidy level determination

Ploidy levels were determined by flow cytometry, having first established a relationship between relative DNA content (determined by flow cytometry) and chromosome number (chromosome counts in root tip squashes) for a subsample of the material. This method has been used for a large number of plant species, and has proved to be a reliable way of inferring ploidy levels (e.g. Dart et al., 2004; Keeler, 2004; Dolezel et al., 2007). Chromosome numbers were counted in root-tips obtained from cultivated plants with the aceto-lactopropionic squash method (Dyer, 1963; Baltisberger, 1991). The root-tips were treated with a 0.05% colchicine solution at 4°C for one hour before they were fixed with an ethanol:glacial acetic acid solution (3:1) at 4°C for 24 hours. They were stained with an orcein- lactopropionic acid solution at 4°C for 24 hours before they were macerated by boiling over a tea candle and mounted on a microscope slide. Per ploidy level, one plant having a low and a second plant having a high DNA index were used to calibrate the flow cytometry data with the number of chromosomes. The chromosomes from the triploid and the pentaploid plant were also counted. Relative DNA content (DNA index) was calculated as the relative fluorescence of the sample divided by the relative fluorescence of the standard. Relative fluorescence was measured for

29 Chapter 1 each sample plant with a flow cytometer PA-I (Partec GmbH, Münster, Germany) at UV light of a mercury arc lamp. A staining solution based on DAPI (4,6-diamidino-2-phenylindole; CyStain UV Ploidy, Partec GmbH, Münster, Germany) was used to stain cell nuclei, of which 2000 were recorded per sample. Only samples producing a histogram peak with a low coefficient of variation (< 5%) were retained. Lycopersicon esculentum Mill. cv. Gardener’s Delight (source of seeds: Royal Botanical Gardens, Kew, United Kingdom) was used as an external reference because it has a similar DNA content (2C DNA = 2.05 pg, prime estimate in Bennett & Leitch, 2004) to that of S. gigantea (as recommended in Johnston et al., 1999). The external reference was checked after every five samples. Tests with refrigerated or frozen (-80°C) leaves produced either broad peaks with high levels of debris or sharp peaks that shifted rapidly to smaller values. From this, we concluded that to obtain reliable results it was essential to use fresh material. The linearity of the flow cytometer was checked using red blood cells of trout that also form doublets and triplets (DNA control UV, Partec GmbH). The flow cytometer showed trends within dates and among dates. The trend within dates could be accounted for with the reference standard; however, variation among dates could not be completely eliminated, neither by using a reference standard nor with three S. gigantea control plants, these being measured at the beginning and end of each measurement series. The measurement error was estimated as the maximal DNA index difference between measurements of the same plant (i) on different days (0.22 for a tetraploid) and (ii) on the same day (0.09 for a tetraploid). The resolution in our measurements was high enough to distinguish clearly the different ploidy levels (Tab. 1). Although locations differed in relative DNA content and there were indications of genomic downsizing in tetraploids and hexaploids (data not shown), these differences were smaller or similar than the measurement error, and were therefore not further considered.

Literature review

We searched for published information on chromosome numbers in relation to location in ISI- index journals, in journals from the institute library and in cytological indices (Index of Plant Chromosome Numbers, IPCN; International Organization of Plant Biosystematics (IOPB) chromosome number reports). Reports with the same locality description were assumed to be from the same population. Localities were georeferenced with digital maps and gazetteers (Appendix S3 in Supplementary Material). Resulting distribution maps were checked for outliers.

30 Ploidy level distribution

Data analysis

Spatial segregation of diploid and tetraploid populations in North America was tested separately for each contact zone with Fisher’s exact test (Baack, 2004). For each diploid and tetraploid population, we determined the ploidy level of the nearest location at least 1 km apart. Mixed- ploidy populations were counted twice, one time as being of one ploidy level with the other one as neighbour, the second time vice versa. Under random distribution (spatial mix of populations), we expect the proportion of populations of one ploidy level with a population of the same ploidy level as neighbour to be equal to the proportion of populations of this ploidy level in our sample. The probability of finding mixed-diploid-tetraploid populations among the single-diploid or single-tetraploid populations for each contact zone in North America was calculated using the binomial distribution (Baack, 2004). If the probabilities of sampling plants of different ploidy levels from a population are independent, then the frequency of mixed populations is the product of the diploid and tetraploid frequencies. All statistics were calculated with SPSS 13.0.0 (SPPS Inc., Chicago, USA).

Re s u l t s

The DNA index values of the various ploidy levels did not overlap, and ploidy level could be unambiguously determined using the flow cytometer results (n = 834 plants from 149 populations; Tab. 1). All plants were diploid, tetraploid or hexaploid, except for one triploid and one pentaploid plant, both found in one North American population. In addition to our data, we found 187 published chromosome counts associated with a specific location (Tab. 2), most of these from North America (156). By combining the published and new data, we were able to prepare cytogeographic maps for North America and for Europe (Figs 1–2) based on a total of 336 locations (Appendices S1 and S2 in Supplementary Material).

Ploidy levels in North America

All three ploidy levels occurred in North America. In our survey, diploids and tetraploids were found in Ontario, New England and in the southern Appalachian Mountains, while hexaploids were found in Manitoba and South Dakota. For most of their ranges, the ploidy levels were spatially segregated (Fig. 1). However, we identified four possible contact zones between ploidy levels (Fig. 1(a)-(c)). One of these—between tetraploid and hexaploid populations—was located

31 Chapter 1 2x 2x + 4x km + 4x 2x + 4x 200 2x 2x 4x km 100 km 200 4x 200 0 B 100 100 4x 4x 0 A 0 2x C 2x 4x ) re tu 4x ra 2x te Li dy ( oi d id pl loi lo d d- ap oi ap tr pl levels mixe di te hex rs oidy te + 6x Pl me lo a) 4x Ki 000 w dat 1’ ne dy ( oi d id pl loi lo d d- ap oi ap tr pl levels mixe di te hex nd 6x 500 ge oidy Le Pl 250 USA da 0 na Ca

32 Ploidy level distribution

Figure 1 Cytogeography of Solidago gigantea Aiton in North America. The grey dotted area indicates generalized distribution range of the species (modified from the E. J. Jäger map in Weber & Jakobs, 2005; restricted to east of the Rocky Mountains, Semple & Cook, 2006). Generalized distribution areas for each ploidy level are marked with a line and are labelled (diploid 2x, tetraploid 4x, hexaploid 6x). Possible contact zones between ploidy levels are designated. Insets A–C show the three contact zones between diploid and tetraploid populations. The coordinate system is the conic equal-area Albers projection for the contiguous USA.

Legend Ploidy levels (new data) tetraploid Ploidy levels (Literature) tetraploid

0 250 500 1’000Kilometers

Figure 2 Cytogeography of S. gigantea in Europe. The grey dotted area indicates generalized distribution range of the species (redrawn from the E. J. Jäger map in Weber & Jakobs, 2005). The coordinate system is the conic equal-area Albers projection for Europe.

33 Chapter 1 in southeastern Manitoba where we found one tetraploid population in a region otherwise occupied by hexaploids; however, the sampling was too sparse to determine how far this contact zone extends southwards into Minnesota. The three possible contact zones between diploid and tetraploid populations were located in: (i) southeastern Ontario and southern Québec (n = 23 diploid, 2 mixed-ploidy and 15 tetraploid locations), (ii) central northeastern USA/New England (n = 6 diploids, 1 mixed-ploidy and 11 tetraploids), and (iii) the southern Appalachian Mountains (n = 10 diploids, 1 mixed-ploidy and 10 tetraploids). Diploids and tetraploids were spatially segregated in central northeastern USA (Fisher’s exact test, p ≤ 0.050), whereas they were mixed in Ontario/Québec (p > 0.757) and in the southern Appalachian Mountains (p = 1.000). We found two mixed-ploidy populations (average number of plants measured per population = 5.5, minimum = 2, maximum = 14). In one of these—in the southern Appalachian Mountains, USA (population code SG277. SA015 in Appendix S1 in Supplementary Material)— we sampled two tetraploid plants and 11 diploids. In the second population—in Pennsylvania, USA, (US009.GJ033)—we found 11 tetraploids and two plants identified asS. cf. gigantea that were triploid (voucher Sch&J 1034) and pentaploid (voucher Sch&J 1035). We also know of two published reports of localities with mixed-ploidy levels in Québec: locality “Pointe-du-Lac” with two diploid and two tetraploid vouchers (Beaudry, 1970), and locality “Baldwin Mills” with two diploid and two tetraploid vouchers (Beaudry, 1970). At the population level, diploid and tetraploid plants were significantly segregated even in the contact zones in Ontario/Québec (2 mixed among 38 single-ploidy populations, expected frequency of mixed populations m = 26.6%, binomial distribution p < 0.001), in central northeastern USA (1 mixed among 17 single-ploidy populations, m = 25.9%, p < 0.033) and in the southern Appalachian Mountains (1 mixed among 20 single-ploidy populations, m = 27.4%, p < 0.011).

Table 1 Relative DNA content (given as the DNA index) of ploidy levels of Solidago gigantea Aiton from different areas.

DNA index Ploidy level Area Populations Plants Mean±SD Min Max Diploid North America 21 116 1.00±0.034 0.88 1.08 Triploid North America 1 1 1.42- - - Tetraploid North America 24 148 1.84±0.050 1.68 1.96 Tetraploid Europe 86 472 1.82±0.049 1.65 1.95 Tetraploid Japan & Russia 4 21 1.82±0.058 1.71 1.92 Pentaploid North America 1 1 2.23- - - Hexaploid North America 15 75 2.63±0.050 2.51 2.73 Populations = number of populations per ploidy level, mixed-ploidy populations are counted for each ploidy level; total number of populations = 149, total number of plants = 834. Note: Differences between means of the tetraploids were not accountable due to between date machine trends.

34 Ploidy level distribution

Table 2 Summary of distinct localities of S. gigantea with previously published ploidy levels for North America, Asia and Europe (see Supplementary Materials S2 for details).

Total Diploid Tetraploid Hexaploid Total 187 43 135 9

North America 156 43 104 9 Asia 2 0 2 0 Europe 29 0 29 0

Ploidy levels in the invasive range

Our samples from Europe, Russia and Japan contained only tetraploid plants (Tab. 1), and the published counts from Russia and Japan were also of tetraploids (Tab. 2). Tetraploids from North America and from Europe did not differ in DNA index values (t-test = 0.920, df = 28, p > 0.366). However, because some studies from Europe indicate that other ploidy levels occur—and in view of the absence of any diploids in our samples—we decided to re-examine these reports in more detail. Five papers from Europe report only tetraploid plants (Appendix S2 in Supplementary Material), but a further two refer to other ploidy levels. In one of these, Jurenitsch et al. (1988)—citing an undergraduate thesis from Maurer (1987)—reported eleven diploid populations from lower Austria. Because the herbarium vouchers for this work could no longer be found (R. Länger, Institute of Pharmacognosy, U of Vienna, Austria, personal communication), we obtained living material from three populations in the area, two collected close to populations 19–22 “Korneuburg” and one corresponding to population 27 “Hainburg” (Maurer, 1987). In all three populations, we found only tetraploid plants (populations SG214. AU001, SG215.AU002, SG216.AU003 in Appendix S1 in Supplementary Material). The second paper (Weber & Jakobs, 2005), citing the PhD thesis from Jakobs (2004), reported the presence of all three ploidy levels in Europe. For our study, 32 plants from 21 populations were measured again (the original plants from Jakobs’ PhD). 31 plants were found to be tetraploid. One plant turned out to be a misidentification and was actually a diploid Solidago canadensis (voucher taken from Jakobs’ plant: Sch&J 1029, EU004.GJ004.31). Additionally, E. Weber provided rhizomes from the same population that was reported as “100% diploid” (“EU7 - Sihlbrugg, Switzerland: 27 samples”, p. 66 and 91 in Jakobs, 2004). However, we measured them as tetraploid (population SG083.CH123 in Appendix S1 in Supplementary Material). Based on our re-examination of the putative non-tetraploid cytotypes in Europe, we omitted diploid and hexaploid records from the cytogeographic maps (Fig. 2).

35 Chapter 1

Di s c u s s i o n

In floras, statements about ploidy level of S. gigantea are usually based on morphological traits, but these cannot be relied upon to distinguish the ploidy levels. It is therefore important to determine the ploidy level of individual plant samples. The populations examined in this study refine the established cytogeography of S. gigantea in North America (Semple & Cook, 2006) and extend our knowledge of distribution on a local and population scale. The ploidy levels in North America occur in geographically almost distinct areas. Since polyploids can only originate within populations of their lower ploidy progenitors, the geographic separation of ploidy levels may be evidence that they have differing ecological requirements. There are many examples of such differentiation, for example between diploid and hexaploid Senecio carniolicus (Schönswetter et al., 2007), but also cases where no differences could be detected, e.g. between diploids and tetraploids of Ranunculus adoneus (Baack & Stanton, 2005). In attempting to explain geographic segregation, contact zones are especially interesting (Burton & Husband, 1999; Halverson et al., 2008). We identified four possible contact zones for S. gigantea in North America, but we focused especially on those between diploids and tetraploids, because the ranges of these ploidy levels include the most likely source area of the material introduced into Europe (Weber, 1997). Even within these contact zones, the ploidy levels tend to be segregated—at a smaller scale. This is the case, for example, in New England and in most of the contact zones of southeastern Ontario/southern Québec where diploid populations in southeastern Ontario are found in the Frontenac Axis region, whereas tetraploids occur mainly outside or along the border of this region (Melville & Morton, 1982; Semple et al., 1999). In contrast, only in the southern Appalachian Mountains do the ploidy levels co-occur with no obvious segregation. Mixed-ploidy populations can develop either when tetraploids are newly formed or when one ploidy level migrates into an area occupied by the other. In S. gigantea, we found only three mixed populations of diploids and tetraploids, suggesting that mixed-ploidy populations are rare despite geographic proximity in the contact zones; this was unexpected since Morton (1984) reported that these ploidy levels often occur together. In contrast, mixed-ploidy populations of the related species S. altissima appear to be rather common (Halverson et al., 2008), which has been attributed to a combination of recurrent polyploidization and the absence of ecological differences between ploidy levels. Neither triploid nor pentaploid plants of S. gigantea have been reported previously, and it is clear that they occur only rarely; indeed, this is the first report of a pentaploid in the genus Solidago. Naturally occurring triploids were found to be very rare in North American species of asters and goldenrods (Semple, 1992), with only eight triploid reports (0.12%) out of 6908 counts. As of September 2007, Semple’s chromosome data base on Solidago included data from 3598 individuals of which only two, neither of them S. gigantea, were 2n = 3x = 27. This very low level of naturally occurring triploids suggests either that triploids are rarely produced,

36 Ploidy level distribution even in mixed populations, or that they are unlikely to survive, or a combination of both. Melville & Morton (1982) report making an artificial cross between one diploid and one tetraploid plant, but this was not successful. We attempted two artificial crosses between diploids and tetraploids and one between a diploid and the triploid plant. One diploid-tetraploid cross was successful and produced three seeds that germinated; of these one seedling—a triploid— survived (unpublished data). This suggests that triploids are possible but do not form readily, and that gene flow across ploidy levels therefore occurs only rarely. The single triploid plant from a field population amounts to less than 0.2 % of all diploid and tetraploid individuals investigated in North America; this is a much smaller proportion than has been found in species such as Galax urceolata, for which 11 % of individuals were reported to be triploids (Burton & Husband, 1999). On the other hand, no triploids of Centaurea jacea have been found, even in a contact zone with mixed-ploidy populations (Hardy et al., 2000). In samples collected from Europe and East Asia, the two regions where S. gigantea has been introduced and become invasive, we found only tetraploid plants. Furthermore, only tetraploids have been reported from these regions, apart from two European studies reporting diploids (Jurenitsch et al., 1988; Weber & Jakobs, 2005). Our analyses of plant material from these or nearby populations did not confirm the presence of diploids, and we think it probable that the original studies were misdeterminations, perhaps because of technical difficulties. For example, Jakobs (2004) used frozen or refrigerated leaves to determine ploidy levels by flow cytometry, but we found that unless fresh material is used the results tend to be very inconsistent. Following these arguments, it seems reasonable to conclude that the tetraploid ploidy level is probably the only one that is widespread in Europe and Eastern Asia. Other invasive species show a similar pattern, e.g. Centaurea maculosa (U. Treier, personal communication, Müller- Schärer et al., 2004), Lythrum salicaria (Kubatova et al., 2008), Senecio inaequidens (Lafuma et al., 2003) and several other species (Verlaque et al., 2002; Pandit et al., 2006). In theory, there are three possibilities to explain why not all ploidy levels are detected in the area of introduction: (i) only certain ploidy levels were introduced, (ii) all ploidy levels were introduced, but some vanished, (iii) all ploidy levels are present in areas of introduction, but only some became widespread and the others are overlooked. For S. gigantea, hypotheses (i) and (ii) seem the most plausible, whereas hypothesis (iii) could only be proven by a positive finding, which despite intensive sampling we could not deliver. Arguments for a single-ploidy level introduction are twofold. Introduction to Europe may have occurred only a few times (Weber & Schmid, 1993), increasing the chances that the sources were the same or similar. Additionally, source areas were probably in the eastern part of North America (Weber, 1997), which would rule out hexaploids but not diploids. If both diploids and tetraploids were introduced, then the predominance of the tetraploids could be due either to ecological differentiation that favoured the tetraploids in the new range (i.e. wrong habitat template for diploids) and/or competitive exclusion of the diploids by tetraploids. Experience with transplanting S. gigantea has shown that tetraploids tend to be more successful than diploids (J.C. Semple, personal observation), suggesting that diploids are more sensitive to soil conditions and therefore less likely to spread

37 Chapter 1 into new areas, whether in Europe or elsewhere. We conclude that although the ploidy levels in S. gigantea cannot be consistently separated by morphology, they are likely to differ in their growth characteristics and ecology. Therefore, to investigate why this species—or any other species varying in ploidy—becomes invasive, it is essential to compare native and invasive populations of the same ploidy level.

Ac k n o w l e d g e m e n t s

We thank Marianne Wettstein-Bättig for help with and giving access to the Flow Cytometer, Lynda Hanson (Royal Botanical Gardens, Kew, United Kingdom) for organizing tomato seeds, Wilma Blaser for help with the artificial crosses and Markus Hofbauer for assistance during the fieldwork. We also thank Bruce Ford, Mark Gabel, Gary Larson, Garret E. Crow, Dwayne Estes, Joey Shaw, Larry Barden, Jim Matthews, and Larry Mellichamp, Nicolas Dassonville, Lars Leonardson and Lina Steinke, Beat Bäumler from the ZDSF, Jun Nishihiro and Toshikazu Mito for their help in localizing populations and giving us access to their herbaria. Further, we thank everyone who provided additional plant material. They are mentioned as collectors in Appendix S1 in Supplementary Material. The project is funded by the grant 0-20259-05 from the ETH Zurich, Switzerland.

Re f e r e n c e s

Anonymous (2004) Alien species recognized to be established in Japan or found in the Japanese wild (as of October 27, 2004) Ministry of the Environment Government of Japan. [pdf document, viewed 27 August 2007]. URL http://www.env.go.jp/en/nature/as.html

Baack, E.J. (2004) Cytotype segregation on regional and microgeographic scales in snow buttercups (Ranunculus adoneus, Ranunculaceae). American Journal of Botany, 91, 1783-1788

Baack, E.J. (2005) To succeed globally, disperse locally: effects of local pollen and seed dispersal on tetraploid establishment. Heredity, 94, 538-546

Baack, E.J. & Stanton, M.L. (2005) Ecological factors influencing tetraploid speciation in snow buttercups (Ranunculus adoneus): Niche differentiation and tetraploid establishment. Evolution, 59, 1936- 1944

38 Ploidy level distribution

Baltisberger, M. (1991) Chromosomenzahlen einiger Labiaten aus Albanien. Berichte des Geobotanischen Instituts der Eidgenössichen Technischen Hochschule, Stiftung Rübel, Zürich, 57, 165-181

Barrett, S.C.H. & Richardson, B.J. (1986) Genetic attributes of invading species. Ecology of biological invasions: an Australian perspective (ed. by R.H. Groves and J.J. Burdon), pp. 21-33. Australian Academy of Science, Canberra, Australia.

Beaudry, J.R. (1963) Studies on Solidago L. VI. Additional chromosome numbers of taxa of genus Solidago. Canadian Journal of Genetics and Cytology, 5, 150-174

Beaudry, J.R. (1970) Studies on Solidago L. X. Les Solidago gigantea Ait. dans l’est de l’Amerique du Nord. Naturaliste Canadien, 97, 35-42

Beaudry, J.R. (1974) Solidago shinnersii (Beaudry) stat. et comb. nov., une nouvelle espece du complexe du S. gigantea. Naturaliste Canadien, 101, 931-932

Beaudry, J.R. & Chabot, D.L. (1959) Studies on Solidago L. IV. The chromosome numbers of certain taxa of the genus Solidago. Canadian Journal of Botany - Revue Canadienne de Botanique, 37, 209-228

Bennett, M.D. & Leitch, I.J. (2004) Plant DNA C-values database [Internet database, viewed 12 June 2006]. URL http://www.rbgkew.org.uk/cval/database1.html

Bennett, M.D., Leitch, I.J. & Hanson, L. (1998) DNA amounts in two samples of angiosperm weeds. Annals of Botany, 82, 121-134

Bleeker, W. & Matthies, A. (2005) Hybrid zones between invasive Rorippa austriaca and native R. sylvestris (Brassicaceae) in Germany: ploidy levels and patterns of fitness in the field. Heredity, 94, 664-670

Brochmann, C., Brysting, A.K., Alsos, I.G., Borgen, L., Grundt, H.H., Scheen, A.-C. & Elven, R. (2004) Polyploidy in arctic plants. Biological Journal of the Linnean Society, 82, 521-536

Brown, A.H.D. & Marshall, D.S. (1981) Evolutionary changes accompanying colonization in plants. Evolution Today. Proceedings of 2nd international Congress of Systematics and Evolutionary Biology (ed. by G.G.E. Scudder and J.L. Reveal), pp. 351-363. Carnegie-Mellon University, Pittsburg.

Burton, T.L. & Husband, B.C. (1999) Population cytotype structure in the polyploid Galax urceolata (Diapensiaceae). Heredity, 82, 381-390

Dart, S., Kron, P. & Mable, B.K. (2004) Characterizing polyploidy in Arabidopsis lyrata using chromosome counts and flow cytometry. Canadian Journal of Botany - Revue Canadienne de Botanique, 82, 185-197

De Wet, J.M.J. (1980) Origins of polyploidy. Polyploidy - Biological relevance (ed. by W.H. Lewis), pp. 3-15. Plenum Press, New York, London.

Dolezel, J., Greilhuber, J. & Suda, J. (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nature Protocols, 2, 2233-2244

Dyer, A.F. (1963) The use of lacto-propionic orcein in rapid squash methods for chromosome preparations. Stain Technology, 38, 85-90

39 Chapter 1

Ehrendorfer, F. (1980) Polyploidy and distribution. Polyploidy - Biological relevance (ed. by W.H. Lewis), pp. 45-60. Plenum Press, New York, London.

Felber, F. (1991) Establishment of a tetraploid cytotype in a diploid population - effect of relative fitness of the cytotypes. Journal of Evolutionary Biology, 4, 195-207

Felber-Girard, M., Felber, F. & Buttler, A. (1996) Habitat differentiation in a narrow hybrid zone between diploid and tetraploid Anthoxanthum alpinum. New Phytologist, 133, 531-540

Fowler, N.L. & Levin, D.A. (1984) Ecological constraints on the establishment of a novel polyploid in competition with its diploid progenitor. American Naturalist, 124, 703-711

Fukuda, I. (1976) Distributions of Solidago altissima and S. gigantea introduced to the Japanese islands. Proceedings of the Japan Society of Plant Taxonomists, 3, 7-8

Gusev, Y.D. (1974) New data on adventive flora of Kaliningrad region.Botanicheskii zhurnal, 59, 1458- 1460

Halverson, K., Heard, S.B., Nason, J.D. & Stireman, J.O. (2008) Origins, distribution, and local co- occurrence of polyploid cytotypes in Solidago altissima (Asteraceae). American Journal of Botany, 95, 50-58

Hardy, O.J., Vanderhoeven, S., De Loose, M. & Meerts, P. (2000) Ecological, morphological and allozymic differentiation between diploid and tetraploid knapweeds (Centaurea jacea) from a contact zone in the Belgian Ardennes. New Phytologist, 146, 281-290

Husband, B.C. & Sabara, H.A. (2004) Reproductive isolation between autotetraploids and their diploid progenitors in fireweed, Chamerion angustifolium (Onagraceae). New Phytologist, 161, 703-713

Husband, B.C. & Schemske, D.W. (1998) Cytotype distribution at a diploid-tetraploid contact zone in Chamerion (Epilobium) angustifolium (Onagraceae). American Journal of Botany, 85, 1688- 1694

Jakobs, G. (2004) Evolution of increased competitive ability in the invasive perennial Solidago gigantea Aiton. Diss. 15536 ETH Zürich, Zürich.

Johnston, J.S., Bennett, M.D., Rayburn, A.L., Galbraith, D.W. & Price, H.J. (1999) Reference standards for determination of DNA content of plant nuclei. American Journal of Botany, 86, 609-613

Jurenitsch, J., Maurer, J., Rain, U. & Robien, W. (1988) Diterpenebutenolides in Solidago gigantea. Phytochemistry, 27, 626-627

Kao, R.H. (2008) Origins and widespread distribution of co-existing polyploids in Arnica cordifolia (Asteraceae). Annals of Botany, 101, 145-152

Keeler, K.H. (2004) Impact of intraspecific polyploidy in Andropogon gerardii (Poaceae) populations. American Midland Naturalist, 152, 63-74

Kubatova, B., Travnicek, P., Bastlova, D., Curn, V., Jarolimova, V. & Suda, J. (2008) DNA ploidy level variation in native and invasive populations of Lythrum salicaria at a large geographical scale. Journal of Biogeography, 35, 167-176

Lafuma, L., Balkwill, K., Imbert, E., Verlaque, R. & Maurice, S. (2003) Ploidy level and origin of the

40 Ploidy level distribution

European invasive weed Senecio inaequidens (Asteraceae). Plant Systematics and Evolution, 243, 59-72

Levin, D.A. (1975) Minority cytotype exclusion in local plant populations. Taxon, 24, 35-43

Li, B.H., Xu, X.M. & Ridout, M.S. (2004) Modelling the establishment and spread of autotetraploid plants in a spatially heterogeneous environment. Journal of Evolutionary Biology, 17, 562-573

Lumaret, R. (1988) Adaptive strategies and ploidy levels. Acta Oecologica-Oecologia Plantarum, 9, 83-93

Maceira, N.O., Jacquard, P. & Lumaret, R. (1993) Competition between diploid and derivative autotetraploid Dactylis glomerata L. from Galicia - implications for the establishment of novel polyploid populations. New Phytologist, 124, 321-328

Malysheva, V.G. (1980) On the appearance of new foreign plants in the Kalinin district. Botanicheskii zhurnal, 65, 1435-1439

Masterson, J. (1994) Stomatal size in fossil plants - evidence for polyploidy in majority of angiosperms. Science, 264, 421-424

Maurer, J. (1987) Über das Vorkommen von Diterpenbutenoliden und Flavoniden in cytologisch definierten Herkünften vonSolidago gigantea und Solidago canadensis. Diplomarbeit. Universität Wien, Wien, Österreich.

Melville, M.R. & Morton, J.K. (1982) A biosystematic study of the Solidago canadensis (Compositae) complex. 1. The Ontario populations. Canadian Journal of Botany - Revue Canadienne de Botanique, 60, 976-997

Morton, G.H. (1984) A practical treatment of the Solidago gigantea complex. Canadian Journal of Botany - Revue Canadienne de Botanique, 62, 1279-1282

Mosyakin, S.L. & Yavorska, O.G. (2002) The non-nnative flora of the Kiev (Kyiv) urban area, Ukraine: a checklist and brief analysis. Urban Habitats, 1

Müller, H. (1989) Growth pattern of diploid and tetraploid spotted knapweed, Centaurea maculosa Lam. (Compositae), and effects of the root mining moth Agapeta zoegana (L.) (Lep.: Cochylidae). Weed Research, 29, 103-111

Müller-Schärer, H., Schaffner, U. & Steinger, T. (2004) Evolution in invasive plants: implications for biological control. TRENDS in Ecology & Evolution, 19, 417-422

Nesom, G.L. (1993) Taxonomic infrastructure of Solidago and Oligoneuron (Asteraceae: Astereae) and observations on their phylogenetic position. Phytologia, 75, 1-44

Novak, S.J., Soltis, D.E. & Soltis, P.S. (1991) Ownbey’s Tragopogons: 40 years later. American Journal of Botany, 78, 1586-1600

Osborn, T.C., Pires, J.C., Birchler, J.A., Auger, D.L., Chen, Z.J., Lee, H.S., Comai, L., Madlung, A., Doerge, R.W., Colot, V. & Martienssen, R.A. (2003) Understanding mechanisms of novel gene expression in polyploids. Trends in Genetics, 19, 141-147

Otto, S.P. & Whitton, J. (2000) Polyploid incidence and evolution. Annual Review of Genetics, 34, 401- 437 41 Chapter 1

Pandit, M.K., Tan, H.T.W. & Bisht, M.S. (2006) Polyploidy in invasive plant species of Singapore. Botanical Journal of the Linnean Society, 151, 395-403

Rausch, J.H. & Morgan, M.T. (2005) The effect of self-fertilization, inbreeding depression, and population size on autopolyploid establishment. Evolution, 59, 1867-1875

Roy, J. (1990) In search of the characteristics of plant invaders. Biological invasions in Europe and the Mediterranean basin (ed. by F. Di Castri, A.J. Hansen and M. Debussche), pp. 335-352. Kluwer Academic Publishers, Dordrecht.

Rudyka, E.G. (1995) Chromosome numbers in vascular plants from the southern part of the Russian Far East. Botaniceskij Zurnal (Moscow & Leningrad), 80, 87-90

Schönswetter, P., Lachmayer, M., Lettner, C., Prehsler, D., Rechnitzer, S., Reich, D.S., Sonnleitner, M., Wagner, I., Huelber, K., Schneeweiss, G.M., Travnicek, P. & Suda, J. (2007) Sympatric diploid and hexaploid cytotypes of Senecio carniolicus (Asteraceae) in the Eastern Alps are separated along an altitudinal gradient. Journal of Plant Research, 120, 721-725

Semple, J.C. (1992) A geographic summary of chromosome number reports for North American Asters and Goldenrods (Asteraceae, Astereae). Annals of the Missouri Botanical Garden, 79, 95-109

Semple, J.C. & Cook, R.E. (2006) Solidago. Flora of North America North of Mexico (ed. by Flora of North America Editorial Committee), pp. 107-166. Oxford University Press, Oxford and New York.

Semple, J.C., Ringius, G.S., Leeder, C. & Morton, G. (1984) Chromosome numbers of Goldenrods, Euthamia and Solidago (Compositae, Astereae). II. Additional counts with comments on cytogeography. Brittonia, 36, 280-292

Semple, J.C., Ringius, G.S. & Zhang, J.J. (1999) The goldenrods of Ontario: Solidago L. and Euthamia Nutt. University of Waterloo Biology Series, 39, 1-90

Soltis, D.E., Soltis, P.S., Pires, J.C., Kovarik, A., Tate, J.A. & Mavrodiev, E. (2004) Recent and recurrent polyploidy in Tragopogon (Asteraceae): cytogenetic, genomic and genetic comparisons. Biological Journal of the Linnean Society, 82, 485-501

Soltis, D.E., Soltis, P.S. & Tate, J.A. (2003) Advances in the study of polyploidy since “Plant speciation”. New Phytologist, 161, 173-191

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 of the United States of America, 97, 7051-7057

Thompson, J.N., Nuismer, S.L. & Merg, K. (2004) Plant polyploidy and the evolutionary ecology of plant/animal interactions. Biological Journal of the Linnean Society, 82, 511-519

Verlaque, R., Aboucaya, A. & Fridlender, A. (2002) Invasive alien flora of France: ecology, life-forms and polyploidy. Botanica Helvetica, 112, 121-136

Voser-Huber, M.L. (1983) Studien an eingebürgerten Arten der Gattung Solidago L. Probleme mit S. gigantea, S. canadensis und S. graminifolia in Naturschutzgebieten des schweizerischen Mittellandes. Dissertationes Botanicae, 68, 97

42 Ploidy level distribution

Weber, E. (1997) Phenotypic variation of the introduced perennial Solidago gigantea in Europe. Nordic Journal of Botany, 17, 631-638

Weber, E. (2003) Invasive plant species of the world - a reference guide to environmental weeds. CABI Publishing, Zurich, Switzerland.

Weber, E. & Jakobs, G. (2005) Biological flora of central Europe: Solidago gigantea Aiton. Flora - Morphology, Distribution, Functional Ecology of Plants, 200, 109-118

Weber, E. & Schmid, B. (1993) Das Neophytenproblem. Dissertationes Botanicae, 196, 209-227

Zerbe, S., Choi, I.K. & Kowarik, I. (2004) Characteristics and habitats of non-native plant species in the city of Chonju, southern Korea. Ecological Research, 19, 91-98

43

Ploidy level distribution

Supplementary Material

Appendix S1 List of 149 new localities of Solidago gigantea Aiton with ploidy level determination. Appendix S2 List of distinct localities of Solidago gigantea Aiton with previously published ploidy level determination, including the associated literature references. Appendix S3 List of 187 localities of Solidago gigantea Aiton with previously published ploidy level determination with added geographic coordinates, including the associated references. References for Supplementary Material

Ap p e n d i x S1

List of 149 new localities of Solidago gigantea Aiton with ploidy level determination

New ploidy level determinations of S. gigantea from Asia, Europe and North America, sorted by continent, country, region, ploidy level, state or province. Population number given as SGxxx. LLxxx. Ploidy level (diploid = 2x, tetraploid = 4x, hexaploid = 6x) reported as pl = x, number of plants measured in parentheses. Locality description followed by coordinates in parentheses (WGS1984 latitude, longitude) negative values representing S and W. Seed collection: date (day, month, year) and collectors’ names which are abbreviated as follows: AM = Akos Malatinsky, AM = Aria Minder, CM = Carmen Rothenbühler, DRS = Daniel R. Schlaepfer, ER = Elvira Rudyka, EW = Ewald Weber, GJ = Gabi Jakobs, HB = Holger Buschmann, HD = Hansjörg Dietz, HM = Martin Hejda, HOV = Harry Olde Venterink, JB = Jill Bowling, JCS = John C. Semple, JK = Johannes Kollmann, JN = Jun Nishihiro, JP = James Partridge, MvK = Mark van Kleunen, MW = Marc Wälthi, NP = Nina Probatova, RB = Regula Billeter, RO = Regula Ott, RS = Rodolphe Schlaepfer, TB = Thomas Becker, TN = Toni Nikolic. V = voucher number(s) if available. Voucher specimens are deposited in herbarium Z+ZT (Zurich, Switzerland). Populations within the diploid-tetraploid contact zones are labelled (ON = southern Ontario/ Québec, NE = central Northeastern USA, SA = southern Appalachian Mountains) and the neighbouring ploidy level is indicated.

45 Chapter 1: Supplementary material

Asia: JAPAN: Hokkaido: SG284.JP001: pl(5) = 4x. ca. 4km E of Mombetsu, along a path in pasture, (42.491, 142.122); 22.10.2005 JN. V = Sch&N 1128. JAPAN: Hokkaido: SG285.JP002: pl(5) = 4x. ca. 8km NE of Mombetsu, on a road side, (42.531, 142.182); 23.10.2005 JN. JAPAN: Hokkaido: SG286.JP003: pl(5) = 4x. ca. 30km N of Mombetsu, between Mukawa and Hobetsu, along a streambank, (42.667, 142.053); 23.10.2005 JN. V = Sch&N 1129. RUSSLAND: Primorsky krai, Nadezhdinsky District Co.: SG320.RU001: pl(6) = 4x. in vicinity of the railway station Kiparissovo, near settlement Tayezhnoje, summer houses country area, along the road, (43.471, 131.925); 30.09.2006 ER&NP. V = Sch&R&P 1173.

Europe: AUSTRIA: Niederosterreich: SG214.AU001: pl(4) = 4x. Klosterneuburg, trail along river Donau, along bank of Donau, (48.315, 16.337); 25.07.2005 HD. V = Sch&D 1067. AUSTRIA: Niederosterreich: SG215.AU002: pl(11) = 4x, in proximity to populations 19-22 “Korneuburg” (Maurer, 1987). Klosterneuburg, camping site, along canal, (48.31, 16.333); 25.07.2005 HD. V = Sch&D 1143. AUSTRIA: Niederosterreich: SG216.AU003: pl(6) = 4x, corresponds to population 27 “Hainburg” (Maurer, 1987). Hainburg an der Donau, 200m N from the bridge over the Donau, slope on the E side of road, (48.15, 16.904); 25.07.2005 HD. V = Sch&D 1068. BELGIQUE: Région de Bruxelles-Capitale, Bruxelles Co.: SG287.BE001: pl(5) = 4x. S of Bruxelles, Forêt de Boitsfort, SE corner of Jct. between Avenue Van Kerm and the railway lines, (50.786, 4.417); 31.10.2005 DRS. BELGIQUE: Région de Bruxelles-Capitale, Bruxelles Co.: SG292.BE006: pl(10) = 4x. Schumanpark, S of Atticastraat and Avenue Marcel Thiry, (50.854, 4.426); 01.11.2005 DRS. V = Sch 1095. BELGIQUE: Vlaanderen, Vlaams-Brabant Co.: SG288.BE002: pl(5) = 4x. Kraainem, NE corner of Jct. between Rue Dezangrélaan (avenue dezangré) and RO (ring oost), (50.861, 4.476); 01.11.2005 DRS. V = Sch 1130. BELGIQUE: Vlaanderen, Oost-Vlaanderen Co.: SG291.BE005: pl(5) = 4x. Gent, park in the W corner of Jct. Nieuwewandeling and Jan Van Hembysebolwerk (along the canale Zuidkaai), land of construction project “De Groene Vallei”, (51.056, 3.707); 01.11.2005 DRS. V = Sch 1094. BELGIQUE: Vlaanderen, Vlaams-Brabant Co.: SG294.BE008: pl(5) = 4x. N of Zichem, poplar-silviculture in NW corner of Jct. between river Demer and canal “Perseiding”, ca. 150m NW of church, (51.002, 4.983); 02.11.2005 DRS. BELGIQUE: Wallonie, Namur Co.: SG289.BE003: pl(5) = 4x. 8km E of Namur, 2.5km E of Jct. with Hwy. E411, 460m W of Rue Petite Ruelle, between road Chaussée de Liège and the river Meuse, (50.478, 4.945); 01.11.2005 DRS. BELGIQUE: Wallonie, Hainaut Co.: SG290.BE004: pl(5) = 4x. ca. 0.6km NE of Saint-Ghislain, nature protecction area in the NW corner of Jct. between Hwy. E19 and railway lines, (50.454, 3.841); 01.11.2005 DRS. V = Sch 1131. CROATIA: Grad Zagreb: SG281.HR001: pl(5) = 4x. Zagreb, west part near river Sava, (45.807, 15.964); 14.10.2005 TN. CZECH REPUBLIC: Hlavni Mesto Praha: SG201.CR001: pl(5) = 4x. Prague, a semi-ruderal site in Prague, (50.093, 14.507); 25.10.2004 HM. V = Sch&H 1020. CZECH REPUBLIC: Hlavni Mesto Praha: SG205.CR002: pl(5) = 4x. Prague, road at the very suburb of

46 Ploidy level distribution

Prague, (50.127, 14.559); 07.12.2004 HM. V = Sch&H 1197. CZECH REPUBLIC: Hlavni Mesto Praha: SG206.CR003: pl(5) = 4x. Prague, construction site at the edge of Prague, (50.129, 14.503); 07.12.2004 HM. V = Sch&H 1025. DENMARK: Copenhagen: SG208.DK001: pl(5) = 4x. Copenhagen, Kongens Enghave, between road Spontinisvej and the railway lines, (55.648, 12.522); 26.02.2005 JK. V = Sch&K 1056. DENMARK: Copenhagen: SG209.DK002: pl(6) = 4x. Copenhagen, Hvidovre, between road-crossing of Strandvangen Magevej and highway E20, (55.617, 12.458); 26.02.2005 JK. V = Sch&K 1057. DENMARK: Copenhagen: SG210.DK003: pl(5) = 4x. Copenhagen, Hvidovre, N of road-crossing Avedore Havenvej and Hwy E20, along roads Magevej and Hvidovre Strandvej, (55.617, 12.477); 26.02.2005 JK. V = Sch&K 1058. GERMANY: Baden-Württemberg: SG204.GE003: pl(5) = 4x. railway station Waldshut, (47.622, 8.219); 31.10.2004 HB. V = Sch&B 1195. GERMANY: Brandenburg: SG202.GE001: pl(5) = 4x. railway station Pfirschheide in Potsdam, on both sides of the railroad at the end of platforms 7 and 8, and along an intersecting road, (52.372, 13.013); 21.10.2004 MvK. V = Sch&v 1199. GERMANY: Hessen: SG207.GE004: pl(6) = 4x. Kassel an der Fulda, (51.319, 9.496); 01.10.2003 TB&HB. V = Sch&B&B 1045. GERMANY: Niedersachsen: SG203.GE002: pl(5) = 4x. Schaumburger Land, in the quarry Liekwegen, (52.28, 9.181); 31.10.2004 HB. V = Sch&B 1194. HUNGARY: Pest Megye: SG280.HU001: pl(3) = 4x. fen meadows near Galgahév’iz village in the Central Hungarian Region, ca. 40km E of Budapest, (47.617, 19.567); 23.09.2005 AM. V = Sch&M 1125. ITALY: Emilia Romagna, Piacenza Co.: SG306.IT005: pl(5) = 4x. ca. 5km S of Piacenza, ca. 1km E of Vallera, roadside ditch along road SS28 to Gossolengo, 100-500m N of Via di Vallera, (45.019, 9.645); 14.11.2005 DRS. ITALY: Emilia Romagna, Piacenza Co.: SG307.IT006: pl(8) = 4x. E of Travo, along Fiume Trébbia, just N of bridge, (44.868, 9.551); 14.11.2005 DRS. V = Sch 1184. ITALY: Friuli-Venezia Gulia: SG213.IT003: pl(5) = 4x. north of Pinzano, in the riverine zone of river Tagliamento, (46.211, 12.985); 30.05.2005 RO. V = Sch&O 1065. ITALY: Friuli-Venezia Gulia: SG218.IT004: pl(6) = 4x. north of Pinzano, in the riverine zone of river Tagliamento, (46.211, 12.985); 20.08.2005 RO. V = Sch&O 1071. ITALY: Lombardia, Pavia Co.: SG308.IT007: pl(5) = 4x. ca. 1.5km SE of Stradella, old field along road “Via Emilia” SS10, 100m E of road “Regione San Zeno”, (45.072, 9.316); 14.11.2005 DRS. V = Sch 1098. ITALY: Lombardia, Milano Co.: SG309.IT008: pl(5) = 4x. ca. 4.5km E of Binasco, old field along road SP40, 2.9km E of Jct. with SS35, (45.332, 9.116); 15.11.2005 DRS. ITALY: Lombardia, Milano Co.: SG310.IT009: pl(5) = 4x. ca. 1km W of Melegnano, just E of railway lines and 640m E of Hwy E35, old field along road SP40, (45.358, 9.31); 15.11.2005 DRS. V = Sch 1135. ITALY: Lombardia, Lodi Co.: SG311.IT010: pl(8) = 4x. ca. 5km NW of Lodi, old field along road SP202, 250m N of Via Emilia, (45.323, 9.485); 15.11.2005 DRS. ITALY: Lombardia, Cremona Co.: SG312.IT011: pl(8) = 4x. ca. 3km SE of Castelleone, on road SS415, area in exit loop to Corte Madama, ca. 0.5km NE of Corte Madame, (45.273, 9.793); 15.11.2005 DRS. V = Sch 1136.

47 Chapter 1: Supplementary material

NETHERLANDS: Overijssel: SG283.NL002: pl(5) = 4x. Oldenzaal, near train station, (52.306, 6.934); 21.10.2005 HOV. V = Sch&O 1127. NETHERLANDS: Utrecht: SG282.NL001: pl(5) = 4x. abandoned garden in city centre of Utrecht, (52.091, 5.123); 21.10.2005 HOV. V = Sch&O 1126. POLAND: Wojewodztwo Malopolskie: SG217.PL001: pl(5) = 4x. Krakow; road next to Congress Venue Ill Campus of Jagiellonian University, Gronostajowa 7, 30-387 Krakow, (50.028, 19.901); 20.08.2005 AM&CR&MW. V = Sch&M&R&W 1146. SWEDEN: Skåne: SG298.SE004: pl(5) = 4x. W of Lomma, along road S. Västkuv., between crossing and Prästbergav., (55.68, 13.071); 08.11.2005 DRS. V = Sch&S 1132. SWEDEN: Skåne: SG300.SE011: pl(5) = 4x. ca. 1km E of Valdemarsrö, just S of road (Stafanstorpvägen) to Särslöv, vis-à-vis industial plant of ‘Akzo Novel’, (55.612, 13.088); 08.11.2005 DRS. V = Sch&S 1133. SWEDEN: Skåne: SG301.SE012: pl(5) = 4x. just S of road S of Revingeby, ca. 400m S of church, in old ‘quarry’ now military training area, (55.724, 13.459); 08.11.2005 DRS. V = Sch&S 1134. SWEDEN: Skåne: SG302.SE015: pl(5) = 4x. ca. 6km SE of Harlösa, 0-300m N of Svansjö along road through forest to Skartofta, (55.7, 13.597); 08.11.2005 DRS. SWEDEN: Skåne: SG304.SE018: pl(5) = 4x. NW of Slagtofta, 350m SE of Jct. with road to Hörby (at Hästäng), (55.875, 13.67); 09.11.2005 DRS. V = Sch&S 1097. SWEDEN: Skåne: SG305.SE019: pl(5) = 4x. ca. 2km SW of Tormestorp, ca. 350m SSW from Hovdala castle along the road, (56.103, 13.712); 09.11.2005 DRS. SWITZERLAND: Aargau: SG061.CH110: pl(5) = 4x. Wildegg, sawmill Wildegg, (47.413, 8.161); 13.10.2004 DRS. V = Sch 1007. SWITZERLAND: Aargau: SG062.CH111: pl(5) = 4x. Rothrist, behind train station, on road Bornweg, (47.307, 7.88); 13.10.2004 DRS. V = Sch 1014. SWITZERLAND: Aargau: SG086.CH023: pl(11) = 4x. Umiken bei Brugg, Schacheninsel, trail along river Aare, W of pedestrian bridge, (47.479, 8.191); 11.03.2005 DRS. V = Sch 1046. SWITZERLAND: Basel-Stadt: SG087.CH129: pl(6) = 4x. Basel, Kleinhüdingen, N side of embankment of stream Wiese, 100m S of bicicle bridge, (47.577, 7.596); 11.03.2005 DRS. V = Sch 1047. SWITZERLAND: Basel-Stadt: SG088.CH130: pl(11) = 4x. Riehen, along streambanks of Wiese, Lange Erle, (47.583, 7.632); 11.03.2005 DRS. V = Sch 1048. SWITZERLAND: Bern: SG090.CH133: pl(5) = 4x. near Niedermuhlern, farmhouse 200m SW from Oberblacken, (46.855, 7.451); 28.03.2005 DRS. V = Sch 1050. SWITZERLAND: Genève: SG065.CH114: pl(5) = 4x. Russin, along road chemin de Pirassay and river L’Allondon, (46.184, 6.011); 14.10.2004 DRS. V = Sch 1016. SWITZERLAND: Genève: SG066.CH115: pl(5) = 4x. Meyrin, Zone Industrielle 16, hedgerow at ABB Sécheron SA, (46.217, 6.057); 14.10.2004 DRS. V = Sch 1006. SWITZERLAND: GL: SG219.CH126: pl(6) = 4x. between train station Ziegelbrücke and Linth canal, (47.139, 9.054); 02.09.2005 DRS. V = Sch 1076. SWITZERLAND: Luzern: SG089.CH131: pl(6) = 4x. St. Kathrinen, at the highway A14 rest area direction Cham, 1.5km SW from exit Gisikon-Root, (47.119, 8.374); 11.03.2005 DRS. V = Sch 1049. SWITZERLAND: Schwyz: SG054.CH103: pl(5) = 4x. between Reichenburg and Buttikon, along slopes ca. 1.3km W of highway crossing A3 and A53, (47.184, 8.961); 12.10.2004 DRS. V = Sch 1002. SWITZERLAND: Schwyz: SG084.CH124: pl(5) = 4x. Schwyz, railway station, (47.024, 8.633); 03.09.2003 TB&HB. V = Sch&B&B 1021.

48 Ploidy level distribution

SWITZERLAND: Solothurn: SG063.CH112: pl(5) = 4x. Solothurn, behing Coop City West, at the end of road Fichtenweg, (47.207, 7.515); 13.10.2004 DRS. V = Sch 1018. SWITZERLAND: St. Gallen: SG055.CH104: pl(5) = 4x. ca. 1.2km W of Ragnatsch, between Flums and Mels, along banks of stream Seez, ca. 1.7km NW Hiltiweg in Plons, (47.067, 9.382); 12.10.2004 DRS. SWITZERLAND: St. Gallen: SG056.CH105: pl(5) = 4x. NW Bad Ragaz, along Hwy A13 and railway, just N of Mattenhoweg, (47.019, 9.489); 12.10.2004 DRS. V = Sch 1022. SWITZERLAND: St. Gallen: SG057.CH106: pl(5) = 4x. Buchs, industrial area in direction Grabs, (47.17, 9.485); 12.10.2004 DRS. V = Sch 1015. SWITZERLAND: St. Gallen: SG058.CH107: pl(5) = 4x. ca. 1.5km W of Diepoldsau, along the southern bridge “Oberrieterstrasse” over highway, (47.384, 9.64); 12.10.2004 DRS. V = Sch 1019. SWITZERLAND: St. Gallen: SG059.CH108: pl(5) = 4x. highway rest area S of Rorschach, (47.466, 9.481); 12.10.2004 DRS. V = Sch 1196. SWITZERLAND: Ticino: SG068.CH117: pl(5) = 4x. Contone,along road to Locarno, hedge behing Möbel Pfister, (46.152, 8.934); 15.10.2004 DRS. V = Sch 1192. SWITZERLAND: Ticino: SG069.CH118: pl(5) = 4x. Piano di Magadino, N Quartino, N end of bridge over Ticino in direction Locarno, (46.16, 8.888); 15.10.2004 DRS. V = Sch 1011. SWITZERLAND: Ticino: SG070.CH119: pl(5) = 4x. Biasca, E of Ticino, N of road to Iragna, area at “Construzioni Stradali ATAG”, (46.354, 8.956); 15.10.2004 DRS. V = Sch 1024. SWITZERLAND: Valais: SG020.CH014: pl(6) = 4x. Troistorrent, 200m W of railway station, (46.227, 6.914); 27.07.2004 DRS. V = Sch 1043. SWITZERLAND: Valais: SG021.CH022: pl(5) = 4x. Mollons, village centre, at the beginning of road rue de la Bourgoisie, (46.316, 7.521); 28.07.2004 DRS. V = Sch 1198. SWITZERLAND: Valais: SG022.CH035: pl(6) = 4x. Fiesch, parking area behing backery “Im Winkelried” along river, (46.402, 8.134); 30.07.2004 DRS. V = Sch 1053. SWITZERLAND: Valais: SG023.CH040: pl(5) = 4x. Sion, between road, river Rhône and canal, W of La Chandoline, (46.22, 7.359); 02.08.2004 DRS. V = Sch 1054. SWITZERLAND: Vaud: SG024.CH083: pl(6) = 4x. Aigle, Zone Industrielle 1, between quarry ponds, (46.301, 6.95); 06.08.2004 DRS. V = Sch 1055. SWITZERLAND: Vaud: SG064.CH113: pl(5) = 4x. Trélex, at the parking area of the Vita-Parcours in the forest, (46.425, 6.186); 14.10.2004 DRS. V = Sch 1023. SWITZERLAND: Vaud: SG067.CH116: pl(5) = 4x. Duiller, N of highway, below power supply line, 20m E of shooting range, (46.416, 6.238); 14.10.2004 DRS. V = Sch 1009. SWITZERLAND: Vaud: SG091.CH134: pl(3) = 4x. Ste-Croix, at second last house following footh path from church to Le Chasseron, (46.824, 6.504); 03.04.2005 DRS. V = Sch 1061. SWITZERLAND: Zug: SG001.CH003: pl(5) = 4x. Chastenrain, along road from Baar to caves ‘Höllgrotten’, (47.197, 8.553); 25.06.2004 DRS. SWITZERLAND: Zug: SG004.CH015: pl(5) = 4x. Gutschsagen, between Biberbrugg and Oberägeri, between road and sawmill ‘Besmer’, (47.151, 8.7); 25.06.2004 DRS. V = Sch 1059. SWITZERLAND: Zürich: SG002.CH004: pl(5) = 4x. Regensdorf, ruderal area S of main road to Adlikon, (47.446, 8.463); 22.06.2004 DRS. V = Sch 1042. SWITZERLAND: Zürich: SG003.CH007: pl(5) = 4x. small forest clearing right side on road from Sihlbrugg to Baar, (47.209, 8.549); 24.06.2004 DRS.

49 Chapter 1: Supplementary material

SWITZERLAND: Zürich: SG005.CH019: pl(8) = 4x. Zürich, NW side of industrial area Binz, (47.364, 8.512); 26.06.2004 DRS. V = Sch 1063. SWITZERLAND: Zürich: SG006.CH021: pl(4) = 4x. Riedikon, dumping area NW of gravel quarry ‘Eglli, Werk Riedikon’, (47.338, 8.705); 26.06.2004 DRS. V = Sch 1060. SWITZERLAND: Zürich: SG010.CH066: pl(14) = 4x. Kollbrunn, along road to Brünggen, left side at edge of the wood, (47.454, 8.771); 14.07.2004 DRS. V = Sch 1052. SWITZERLAND: Zürich: SG011.CH070: pl(5) = 4x. ca. 400m NNE of Billikon, along Kemptalstrasse between Billikon and Ettenhausen, just N of forest road Solenbachstrasse, (47.447, 8.73); 15.07.2004 DRS. SWITZERLAND: Zürich: SG050.CH100: pl(5) = 4x. between Adlikon and Dielsdorf, at the junction to Buchs, (47.459, 8.464); 11.10.2004 DRS. V = Sch 1012. SWITZERLAND: Zürich: SG051.CH101: pl(5) = 4x. between Embrach and Pfungen, between railway lines and road, (47.521, 8.61); 11.10.2004 DRS. V = Sch 1013. SWITZERLAND: Zürich: SG052.CH075: pl(5) = 4x. between Bauma and Steg along road 15, dumping area ca. 200m W from Lipperschwändi, (47.361, 8.916); 11.10.2004 DRS. V = Sch 1010. SWITZERLAND: Zürich: SG053.CH102: pl(5) = 4x. between Pfäffikon and Fehraltdorf, gravel quarry along main road, (47.377, 8.768); 11.10.2004 DRS. V = Sch 1004. SWITZERLAND: Zürich: SG060.CH109: pl(5) = 4x. highway rest area Kempthal, (47.451, 8.701); 12.10.2004 DRS. V = Sch 1005. SWITZERLAND: Zürich: SG083.CH123: pl(4) = 4x, corresponds to EU7 - Sihlbrugg, CH (p. 66 and 81, Jakobs, 2004). railway station Sihlbrugg, trail along right side of river Sihl, N of the two railway buildings, shortly before the tunnel, (47.243, 8.577); 17.12.2004 EW. UNITED KINGDOM: Lancashire: SG314.UK002: pl(2) = 4x. on allotment gardens near Withington, (53.431, -2.244); 26.01.2006 JP. V = Sch&P 1141. UNITED KINGDOM: Surrey: SG313.UK001: pl(4) = 4x. between Godalming and Guildford, west side along river Wey, 2/3 to Guildford, (51.215, -.578); 10.12.2005 RS&JB. UNITED KINGDOM: Warwickshire: SG315.UK003: pl(4) = 4x. railway bank along Princes’ Drive, (52.287, -1.551); 22.01.2006 JP. V = Sch&P 1174.

North America: CANADA: Manitoba, Winnipeg Capital Region Co.: SG236.MB001: pl(5) = 6x. Assiniboine Forest Park South, at Grand Rd. 105 and Calthcart Rd, along the Sagimay Trail in the aspen forest, (49.858, -97.25); 28.09.2005 DRS&RB. CANADA: Manitoba, Central Plains Region Co.: SG237.MB002: pl(5) = 6x. along TransCanada Hwy MB-1, Portage Campground, 0.5km E of Assiniboine River, along forest edge, (49.968, -98.093); 29.09.2005 DRS&RB. V = Sch&B 1191. CANADA: Manitoba, Central Plains Region Co.: SG238.MB003: pl(5) = 6x. along dirt road parallel to TransCanada Hwy MB-1, 5km E of Bagot (Rd. 242), between Rd. 48 West and Rd. 47 West, (49.976, -98.554); 29.09.2005 DRS&RB. V = Sch&B 1109. CANADA: Manitoba, Central Plains Region Co.: SG239.MB004: pl(5) = 6x. along TransCanada Hwy MB- 1, at exit for Rd. 34 to Austin, along old road remains, (49.957, -98.927); 29.09.2005 DRS&RB. CANADA: Manitoba, Westman Region Co.: SG240.MB005: pl(5) = 6x. NE corner of junction between Hwy 16 and Rd. 260, (50.226, -99.077); 29.09.2005 DRS&RB. CANADA: Manitoba, Parkland Region Co.: SG241.MB006: pl(5) = 6x. Riding Mountain National Park, ca. 5km NW of Lake Audy, Minnedosa River Camp, (50.798, -100.274); 30.09.2005 DRS&RB. 50 Ploidy level distribution

CANADA: Manitoba, Westman Region Co.: SG242.MB007: pl(5) = 6x. NW of Minnedosa, along the woods of the rest area at junction of Hwy 10 and Hwy 16, (50.271, -99.906); 01.10.2005 DRS&RB. CANADA: Manitoba, Westman Region Co.: SG243.MB008: pl(5) = 6x. Hwy 5 between Carberry and Glenboro, Spruce Woods Provincial Park, in the Marshs Lake Rest Area, (49.675, -99.268); 01.10.2005 DRS&RB. CANADA: Ontario, Lennox and Addington Co.: SG223.ON004: pl(5) = 2x. ca. 2km E of Kaladar, between Hwy 7 and Station Rd., (44.656, -77.104); 24.09.2005 JCS&DRS&RB. Contact zone ON (neigh=2x). CANADA: Ontario, Frontenac Co.: SG224.ON005: pl(5) = 2x. N of Mountain Grove, along Hwy 7, (44.754, -76.853); 24.09.2005 JCS&DRS&RB. Contact zone ON (neigh=2x). CANADA: Ontario, Lanark Co.: SG225.ON006: pl(5) = 2x. Hwy 7, W of Wemyss, 11.3km W of 5th Concession Rd. (= Cameronside Rd.), along Beaver Dam Creek, (44.837, -76.522); 24.09.2005 JCS&DRS&RB. Contact zone ON (neigh=2x). CANADA: Ontario, Stormont, Dundas and Glengarry Co.: SG226.ON007: pl(5) = 2x. 1.1km S of Hwy 43 on (Mountainship) Boundary Rd, vicinity of lot 632, on border to county Leeds and Greenville, (45.039, -75.556); 24.09.2005 JCS&DRS&RB. V = Sch&B&S 1078. Contact zone ON (neigh=2x). CANADA: Ontario, Leeds and Greenville Co.: SG228.ON009: pl(5) = 2x. NE of Westport, County Rd. 10, 0.5km NE of Parish Rd., (44.721, -76.4); 25.09.2005 JCS&DRS&RB. Contact zone ON (neigh=2x). CANADA: Ontario, Leeds and Greenville Co.: SG229.ON010: pl(5) = 2x. ca. 8km SW of Westport, County Rd. 10, 1.4km NE of Hutchings Rd., (44.633, -76.401); 25.09.2005 JCS&DRS&RB. Contact zone ON (neigh=2x). CANADA: Ontario, Waterloo Co.: SG220.ON001: pl(11) = 4x. University of Waterloo campus, along Laurel Creek, (43.47, -80.545); 23.09.2005 JCS&DRS&RB. V = Sch&B&S 1105. CANADA: Ontario, Lennox and Addington Co.: SG221.ON002: pl(5) = 4x. ca. 5km N of Selby, Hwy Rd. 41, S of Centreville Rd., along Mud Creek, (44.346, -77.006); 24.09.2005 JCS&DRS&RB. V = Sch&B&S 1193. Contact zone ON (neigh=4x). CANADA: Ontario, Lennox and Addington Co.: SG222.ON003: pl(7) = 4x. Roblin, just N of railway crossing, (44.371, -77.018); 24.09.2005 JCS&DRS&RB. V = Sch&B&S 1107. Contact zone ON (neigh=4x). CANADA: Ontario, Wellington Co.: SG230.ON011: pl(9) = 4x. between Damascus and Gordonville, Line 6, at bridge over Four Mile Creek, (43.906, -80.522); 26.09.2005 DRS&RB. V = Sch&B&S 1179. CANADA: Ontario, Dufferin Co.: SG232.ON013: pl(5) = 4x. N of Grand Valley, along Grand river at bridge of County Rd. 25, just N of Concession Rd. 4-5, (43.924, -80.321); 26.09.2005 DRS&RB. V = Sch&B&S 1083. CANADA: Ontario, Waterloo Co.: SG233.ON014: pl(3) = 4x. N of Conestogo, along Canagagigue Creek, at bridge of County Rd. 22, (43.579, -80.509); 27.09.2005 DRS&RB. V = Sch&B 1085. CANADA: Ontario, Waterloo Co.: SG234.ON015: pl(5) = 4x. S of Hwy 401, small creek at County Rd. 28, between the crossings of Dickie Settlement and with County Rd. 42, (43.384, -80.394); 27.09.2005 DRS&RB. V = Sch&B 1086. CANADA: Ontario, Waterloo Co.: SG235.ON016: pl(7) = 4x. along Grand River at the “Bean Ormston Park at Moyer’s Blair Landing”, County Rd. 28, (43.386, -80.385); 27.09.2005 DRS&RB. V = Sch&B 1087.

51 Chapter 1: Supplementary material

USA: Pennsylvania: US009.GJ033: pl(11) = 4x, pl(1) = 3x, pl(1) = 5x, mixed-ploidy population. Forest Hill, (40.97, -77.07); 01.09.2001 GJ. V = Sch&J 1034, Sch&J 1035, Sch&J 1181, Sch&J 1182. Contact zone NE (neigh=2x). USA: Massachusetts, Norfolk Co.: SG251.NE001: pl(5) = 2x. Dover, Centre Street, at town line of Dover and Medfield, opposite Hunt Rd., (42.215, -71.305); 09.10.2005 DRS. USA: Massachusetts, Worcester Co.: SG252.NE002: pl(5) = 2x. Mendon, vis-à-vis Nr. 88 at road that leads away from Providence St. and Hartford Ave. E just before the bridge, (42.091, -71.535); 09.10.2005 DRS. V = Sch&H 1139. USA: Massachusetts, Worcester Co.: SG253.NE003: pl(5) = 2x. Marlborough, I-495 exit 25B to Berlin, field margins between River Rd./South St. and Donald Lynch Blvd., (42.353, -71.622); 09.10.2005 DRS. Contact zone NE (neigh=2x). USA: Massachusetts, Worcester Co.: SG254.NE004: pl(5) = 2x. Leominster State Forest, gravel depot area on Rocky Pond Rd., ca. 200m E of Parking Area at Rd. 31, N of Paradise Pond, (42.512, - 71.855); 10.10.2005 DRS. Contact zone NE (neigh=2x). USA: Massachusetts, Middlesex Co.: SG255.NE005: pl(5) = 2x. NW of Groton, on Rd. 119, area opposite Gay Rd. and below a power line, (42.594, -71.542); 10.10.2005 DRS. Contact zone NE (neigh=2x). USA: Massachusetts, Essex Co.: SG256.NE006: pl(5) = 4x. Haverhill, along Merrimac River on Rt. 110, on property 266 River St. (on 10. Oct. 2005: “Paul Martin Antiques”), (42.765, -71.097); 10.10.2005 DRS. V = Sch&H 1114. Contact zone NE (neigh=2x). USA: New Hampshire, Grafton Co.: SG257.NE007: pl(5) = 4x. Swiftwater, on Rt. 112, parking area just N of woodbridge over Swiftwater River, (44.135, -71.951); 11.10.2005 DRS. V = Sch&H 1115. USA: Vermont, Washington Co.: SG258.NE008: pl(5) = 4x. between Montpellier and Barre, margin of roadside wetland between Berlin State Hwy. and Berlin Mall Rd., (44.22, -72.562); 12.10.2005 DRS. V = Sch&H 1137. USA: Vermont, Chittenden Co.: SG259.NE009: pl(5) = 4x. ca. 2km NW of I-89 exit 11 NW of Richmond, along Winooski River at PicNic area on Rt. 117, (44.442, -73.02); 12.10.2005 DRS. V = Sch&H 1116. USA: Vermont, Washington Co.: SG260.NE010: pl(9) = 4x. Colbyville near Waterbury, roadside slope on Guptil Rd., ca. 800m NE off Rt. 110, (44.358, -72.726); 12.10.2005 DRS. V = Sch&H 1091. USA: Vermont, Lamoille Co.: SG261.NE011: pl(5) = 4x. ca. 3.5km E of Morrisville, ca. 300m W of Jct. of 15A and 15, slope between Rt. 15 and Lamoille River, (44.567, -72.569); 12.10.2005 DRS. V = Sch&H 1117. USA: Vermont, Caledonia Co.: SG262.NE012: pl(8) = 4x. Joes Pond Dam in West Danville at Jct. 15 and 2, (44.409, -72.195); 12.10.2005 DRS. V = Sch&H 1140. USA: Georgia, Gilmer Co.: SG263.SA001: pl(6) = 2x. ca. 200m S of Cartecay, Rt. 52, behind fuel station “Crossroads” in Cartecay, at Jct. with Clear Creek Rd., (34.625, -84.383); 15.10.2005 DRS. USA: Georgia, Gilmer Co.: SG264.SA002: pl(6) = 2x. Ellijay, area between Post Office, railway tracks and Rt. 282, ca. 0.5km W of Jct. 5/52/76/282, (34.686, -84.482); 15.10.2005 DRS. V = Sch&H 1118. USA: Georgia, Murray Co.: SG265.SA003: pl(6) = 2x. old field next to “C. W. Bradley Bridge” on Rt. 52 over Holly Creek, (34.767, -84.76); 15.10.2005 DRS. USA: North Carolina, Transylvania Co.: SG279.SA017: pl(5) = 2x. silviculture between Rt. 276 and French Broad River, ca. 5.5km S of Jct. with Rt. 64 in Brevard, (35.192, -82.722); 19.10.2005 DRS.

52 Ploidy level distribution

USA: Tennessee, Hamilton Co.: SG266.SA004: pl(6) = 2x. N of Harrison, roadside on Rt. 58 at Jct. with Harrison Ooltewah Rd., ca. 4km S of Harrison State Park entrance at Harrison Bay Rd., 1.5km S of Jct. Hunter Rd., (35.126, -85.125); 16.10.2005 DRS. V = Sch&H 1119. Contact zone SA (neigh=2x). USA: Tennessee, Knox Co.: SG268.SA006: pl(5) = 2x. Knoxville, depression behind Sleep Inn, 214 Prosperity Dr., (35.923, -84.077); 17.10.2005 DRS. V = Sch&H 1178. Contact zone SA (neigh=2x). USA: Tennessee, Claiborne Co.: SG269.SA007: pl(5) = 2x. S of New Tazewell, creek along Rt. 33, at Jct. with Jesse Cir., (36.387, -83.709); 17.10.2005 DRS. Contact zone SA (neigh=4x). USA: Tennessee, Sullivan Co.: SG271.SA009: pl(5) = 2x. SE of Bristol, old field on Rt. 421, ca. 0.8km N of Jct. 394, (36.569, -82.164); 18.10.2005 DRS. Contact zone SA (neigh=4x). USA: Tennessee, Greene Co.: SG276.SA014: pl(6) = 2x. S of Greenville, along Paint Creek along Rt. 70, just N of lot 14050 Asheville Hwy., ca. 1.6km N of TN/NC state line, (35.989, -82.804); 19.10.2005 DRS. Contact zone SA (neigh=2x). USA: North Carolina, Madison Co.: SG277.SA015: pl(11) = 2x, pl(2) = 4x, mixed-ploidy population. 1.6km E of Jct. with 208, between Rt. 212 and Shelton Laurel Creek, ca. 10km N of Walnut, (35.934, -82.733); 19.10.2005 DRS. V = Sch&H 1176, Sch&H 1204. Contact zone SA (neigh=2x). USA: North Carolina, Watauga Co.: SG274.SA012: pl(5) = 4x. E of village of Beech Creek, 4.0km km E of TN/NC border, roadside along Rt. 321, 100m E of Creek Ridge Rd., (36.262, -81.895); 18.10.2005 DRS. V = Sch&H 1092. Contact zone SA (neigh=4x). USA: North Carolina, Henderson Co.: SG278.SA016: pl(6) = 4x. Mills River, just N at Davenport bridge on Rt. 280, 50m N of Jct. with S. Mills River Rd., (35.391, -82.569); 19.10.2005 DRS. V = Sch&H 1200. Contact zone SA (neigh=2x). USA: Tennessee, McMinn Co.: SG267.SA005: pl(8) = 4x. roadside ditch along Rt. 411, ca. 1.5km S of Etowah, ca. 3.5km S of Jct. Rt. 310E, (35.304, -84.536); 16.10.2005 DRS. V = Sch&H 1120. Contact zone SA (neigh=4x). USA: Tennessee, Claiborne Co.: SG270.SA008: pl(5) = 4x. Tazewell, just S of Jct. of Rt. 33 and 25E along creek, (36.455, -83.565); 17.10.2005 DRS. V = Sch&H 1121. Contact zone SA (neigh=2x). USA: Tennessee, Johnson Co.: SG272.SA010: pl(6) = 4x. ca. 4km N of Shady Valley, along Rt. 421, after 1st hairpin bend S of Jct. with Appalachian Trail, (36.534, -81.948); 18.10.2005 DRS. Contact zone SA (neigh=2x). USA: Tennessee, Johnson Co.: SG273.SA011: pl(5) = 4x. S of Mountain City, along Rt. 421 along Roan Creek (E of road), ca. 6.5km N of Jct. with Rt. 67E in Trade, (36.405, -81.761); 18.10.2005 DRS. Contact zone SA (neigh=4x). USA: Tennessee, Carter Co.: SG275.SA013: pl(6) = 4x. roadside along Rt. 321/67, 200m W of Watauga Point Recreation Area of Cherokee National Forest, ca. 9.5km E of Hampton Jct., (36.32, -82.083); 18.10.2005 DRS. V = Sch&H 1123. Contact zone SA (neigh=4x). USA: South Dakota, Lawrence Co.: SG244.SD001: pl(5) = 6x. Spearfish, along Spearfish Creek, from “Frank R. Brady” park to Lions Club Park, (44.488, -103.865); 03.10.2005 DRS. USA: South Dakota, Butte Co.: SG245.SD002: pl(5) = 6x. Belle Fourche, along Belle Fourche River, between bridge to soccer field at “Roundup Ground Ball Park” and railway bridge to the W, (44.673, -103.863); 04.10.2005 DRS. V = Sch&H 1110. USA: South Dakota, Lawrence Co.: SG246.SD003: pl(5) = 6x. Spearfish Canyon, ca. 8km S of Spearfish on Hwy 14A, along Spearfish Creek at Botany Bay Picnic Ground, just N of Bridal Veil Falls, (44.42, -103.881); 04.10.2005 DRS. V = Sch&H 1111. 53 Chapter 1: Supplementary material

USA: South Dakota, Lawrence Co.: SG247.SD004: pl(5) = 6x. Spearfish Canyon, ca. 4km S of Savoy at Hwy 14A, along Spearfish Creek, (44.332, -103.901); 04.10.2005 DRS. USA: South Dakota, Pennington Co.: SG248.SD005: pl(5) = 6x. Rapid City, Halley Park along Rapid Creek W of West Blvd., N of Omaha St. & E of Deadwood Ave., (44.085, -103.25); 05.10.2005 DRS. USA: South Dakota, Hughes Co.: SG249.SD006: pl(5) = 6x. Pierre, LaFramboise Island, along NE+NW shore of Missouri River, (44.356, -100.363); 06.10.2005 DRS. V = Sch&H 1112. USA: South Dakota, Hughes Co.: SG250.SD007: pl(5) = 6x. along mouth of Medicine Creek to Missouri River, ca. 15km SE of Pierre on Hwy. 34 just before Medicine Knoll Rest Area, (44.32, -100.072); 06.10.2005 DRS. V = Sch&H 1177.

54 Ploidy level distribution

Ap p e n d i x S2

List of distinct localities of Solidago gigantea Aiton with previously published ploidy level determination, including the associated literature references.

Table S2 Number of distinct localities of Solidago gigantea Aiton cited in the literature, reported for the first time, sorted by continent, ploidy level and literature source1 including remarks on the literature review. Number of total listed vouchers per literature source is given in parentheses. Numbers in brackets are miss-identified specimens. Counts in curly brackets for Europe could not been confirmed and were not included in sums.

Total Diploid Tetraploid Hexaploid Total 187 43 135 9

North America 156 43 104 9 Beaudry & Chabot (1959) 2 2 (10) 3 (4) 0 Beaudry (1963) 2 1 (1) 0 2 (9) Federov et al. (1969) 0 0 0 (1) 3 Beaudry (1970a) 4 5 (11+9) 8 (37+3) 0 Beaudry (1970b) 5 0 (0+1) 0 (0+1) 0 Semple et al. (1981) 2 (3) 10 (10) 0 Löve (1982) 0 1 (1) 0 Melville & Morton (1982) 12 (25) 54 (74) 0 Morton (1984) 8 (14) 4 (8) 0 [5] 6 Semple et al. (1984) 6 (6) 18 (21) 6 (7) [3] 6 Semple (1985) 0 0 0 [1] 6 Semple & Chmielewski (1987) 0 0 0 [1] 6 Semple et al. (1989) 1 (1) 0 0 Semple et al. (1993) 5 (5) 4 (4) 1 (1) Zhao (1996) 7 0 0 [1] 0 Semple et al. (2001) 0 0 0 [4] 6 Semple & Cook (2004) 1 (1) 2 (3) 0

Asia 2 0 2 0 Huziwara (1962) 0 1 (1) 0 Rudyka (1995) 0 1 (1) 0

Europe 29 0 29 0 Beaudry (1970a) 0 1 (1+1) 0 Májovsky (1974) 0 1 (1) 0 Májovsky & Murín (1987) 0 0 (1) 0 Skalinská et al. (1976) 0 3 (3) 0 Weber (1997) 0 20 (20) 0 Maurer (1987) cited in Jurenitsch et al. (1988) 0 {11} 4 (4) 0 Jakobs (2004) cited in Weber & Jakobs (2005) 0 {13} 0 {10} 0 {3}

1. Information from some papers is also mentioned in IPCN and IOPB reports (Goldblatt, 1981; Löve, 1982; Goldblatt, 1985, 1988; Goldblatt & Johnson, 1990; Goldblatt & Johnson, 1991, 1996, 1998; Goldblatt, 2000; and the IPCN database on the Internet included to Missouri Botanical Garden w3TROPICOS: Goldblatt & Johnson, 2006) and in the book from Federov et al. (1969). 2. Beaudry (1974) corrected earlier determinations. To our understanding, this involved the following changes: The specimen S. gigantea var. Shinnersii (voucher 58-286-1; Fort Saskatchewan, Alberta, Canada) earlier labelled with 2n = 18 should be 2n = 54 (Beaudry, 1963). The specimen S. gigantea var.

55 Chapter 1: Supplementary material leiophylla (voucher 55-126; Montréal, Québec, Canada) earlier labelled with 2n = 18 should be 2n = 36 (Beaudry & Chabot, 1959). 3. Federov et al. (p.137 1969) listed among other data “S. gigantea Ait: [2n = ] 54: Matbeeba [, T.C.], Tekhonovah [, A. D.]: neopubl.“. This entry misses a reference to a voucher specimen and its locality. Therefore, we omitted it from our analysis. 4. Beaudry (1970a) listed two localities with each diploid and tetraploid vouchers (counted once) and 12 vouchers that are already mentioned in Beaudry (1963) 5. Beaudry (1970b) listed two vouchers that are already mentioned in Beaudry (1970a) 6. Semple & Cook (2006) described the specimens that were reported inside and west of the Rocky Mountains as S. lepida due to glands in the capitulescence. J. C. Semple re-examined the vouchers. Therefore, literature localities that are inside or west of the Rocky Mountains were not included in Fig. 1 but were listed in Appendix S3 in the Supplementary Material and marked. 7. Zhao (1996) reported a tetraploid chromosome count for S. gigantea from Mexico; however, J. C. Semple re-examined the voucher “Nesom, Mayfield & Hinton 7739“ and determined it to be S. lepida var. salebrosa.

Remarks on the literature review

We could not confirm that S.“ gigantea has become naturalised in …, Hawaii, New Zealand (Hill, personal communication), Australia (Newfield, personal communication)…” (Weber & Jakobs, 2005). For New Zealand, a single population was reported for 1953 (Given, 1984; Webb, 1987; Webb et al., 1988-2004). M. Newfield stated in the “Aliens-l” discussion group that among other species S. gigantea is not reported as weed in New Zealand (M. Newfield, New Zealand, personal communication, Newfield, 2002). For Australia, there appear to be no records indicating that the species is naturalised (Anonymous, 1982 onwards; Thorp & Wilson, 1998 onwards; Randall & Kessal, 2006), though it is on the quarantine weed list for Western Australia (Randall, 2007). For Hawai’i, there are no records of the species in the “Flora of Hawaiian Islands” (Wagner & Herbst, 2003; Wagner et al., 2005-).

56 Ploidy level distribution

Ap p e n d i x S3

List of 187 localities of Solidago gigantea Aiton with previously published ploidy level determination with added geographic coordinates, including the associated literature references.

Voucher reports from the literature for S. gigantea localities with previously published ploidy level determination, sorted by continent, author of publication, ploidy level, country and state. Voucher number is listed as “V =” if available. † indicates localities inside or west of the Rocky Mountains for counts published as S. gigantea but which are for individuals of S. lepida (after Semple & Cook, 2006). # indicates localities in Europe that were reported to be diploid, but we could not confirm this ploidy level. * indicates voucher specimen for which the ploidy level was changed (Beaudry, 1974). We added coordinates to the locality descriptions: (latitude, longitude) negative values representing S and W (WGS1984). Precision (pr) of locality description is categorised as exact (0), close (1) or regional (2). Locations within the diploid-tetraploid contact zones are labelled (ON = southern Ontario/Québec, NE = central Northeastern USA, SA = southern Appalachian Mountains) and the neighbouring ploidy level is indicated.

East Asia: Huziwara (1962): tetraploid: Japan: Hokaido: (43.5, 142.8, pr=2), V=n.a. Rudyka (1995): tetraploid: Russia: Primorsky krai: (45.69, 136.24, pr=1), V=6909

Europe: Beaudry (1970a): tetraploid: France: (48.6, 7.7, pr=2), V=59-255-2; V=59-255-3 Májovsky (1974): tetraploid: Slovakia: Podunajská nízina: (48.15, 17.13, pr=1), V=n.a. Májovsky & Murín (1987): tetraploid: Slovakia: Podunajská nízina: V=n.a. Maurer (1987): diploid: Austria: Lower Austria: (48.34, 16.33, pr=1), pop=19#, V=n.a.; (48.34, 16.33, pr=1), pop=20#, V=n.a.; (48.34, 16.33, pr=1), pop=21#, V=n.a.; (48.34, 16.33, pr=1), pop=22#, V=n.a.; (46.72, 15.61, pr=1), pop=23#, V=n.a.; (48.211, 16.085, pr=0), pop=24#, V=n.a.; (48.223, 16.045, pr=0), pop=26#, V=n.a.; (48.146, 16.908, pr=0), pop=27#, V=n.a.; (48.17, 16.04, pr=1), pop=28#, V=n.a.; (48.438, 15.818, pr=0), pop=29#, V=n.a.; (48.55, 14.87, pr=1), pop=30#, V=n. a.; tetraploid: (48.602, 16.932, pr=0), pop=16, V=n.a.; (48.575, 16.914, pr=0), pop=17, V=n.a.; (48.47, 16.85, pr=1), pop=18, V=n.a.; (48.202, 16.109, pr=0), pop=25, V=n.a. Skalinská et al. (1976): tetraploid: Poland: (50.08, 19.92, pr=1), V=n.a.; (49.88, 19.48, pr=1), V=n.a.; (52.22, 17.57, pr=1), V=n.a. Weber (1997): tetraploid: Danemark: (55.33, 8.78, pr=1), V=n.a.; (55.4, 11.38, pr=1), V=n.a.; (55.47, 12.2, pr=1), V=n.a.; (55.7, 11.1, pr=1), V=n.a.; (55.72, 12.57, pr=1), V=n.a.; Finland: (60.5, 27.67, pr=1), V=n.a.; (61.72, 23.83, pr=1), V=n.a.; France: (44, 4.87, pr=1), V=n.a.; (47.65, 7.55, pr=1), V=n.a.; Germany: (51.9, 10.18, pr=1), V=n.a.; (52.62, 10.08, pr=1), V=n.a.; (54.08, 9.98, pr=1), V=n.a.; (54.32, 8.95, pr=1), V=n.a.; (54.48, 9.07, pr=1), V=n.a.; Italy: (44.65, 10.92, pr=1), V=n.a.; (44.65, 10.93, pr=1), V=n.a.; (45.05, 9.68, pr=1), V=n.a.; Switzerland: (46.15, 8.87, pr=1), V=n.a.; (46.15, 8.95, pr=1), V=n.a.; (47.6, 8.6, pr=1), V=n.a.

57 Chapter 1: Supplementary material

North America: Beaudry & Chabot (1959): diploid: Canada: Québec: (45.28, -72.98, pr=1, ON(4x)), V=56-299-1; V=56- 299-2; V=56-299-3; V=56-303-1; V=56-303-2; USA: Massachusetts: (42.1, -71.55, pr=1, NE(2x)), V=57-21-1; V=57-21-2; V=57-21-3; tetraploid: Canada: Québec: (45.5, -73.41, pr=1, ON(4x)), V=56-278-1; V=56-278-2; V=55-83; V=55-126*; (45.5, -73.6, pr=2, ON(4x)), V=55-212; (45.46, -74.09, pr=1, ON(2x)), V=55-226 Beaudry (1963): diploid: USA: Mississippi: (30.45, -89.09, pr=1), V=57-591; hexaploid: Canada: Alberta: (53.71, -113.21, pr=1), V=55-277-2; V=55-277-3; V=55-277-4; V=58-286-1*; V=58-299-1; V=58-300-2; V=58-301-1; Manitoba: (50.66, -96.98, pr=1), V=56-85; V=56-86 Beaudry (1970a): diploid: Canada: Ontario: (44.71, -75.52, pr=1, ON(4x)), V=62-204; V=62-206; V=62-208; (44.45, -75.86, pr=1, ON(2x)), V=62-228; Québec: (45.66, -74.34, pr=1, ON(4x)), V=55-219; V=56-299-1; V=56-299-2; V=56-299-3; V=56-303-1; V=56-303-2; (46.29, -72.69, pr=1, ON(4x)), V=AA 62-31, mixed-ploidy locality; V=AA 62-36, mixed-ploidy locality; (45.04, -71.91, pr=1, ON(4x)), V=63-111, mixed-ploidy locality; V=63-112, mixed-ploidy locality; (45.2, -71.5, pr=1), V=63-134; V=63-135; USA: Massachusetts: V=57-21-1; V=57-21-2; V=57-21-3; Mississippi: V=57-591; tetraploid: Canada: Ontario: (44.43, -78.14, pr=1), V=61-299; (45.02, -74.73, pr=1, ON(2x)), V=62-152; V=62-153; V=62-155; Québec: V=55-313; V=55-226; V=56-278-1; V=56-278- 2; V=AA 62-2; V=AA 62-3; V=AA 62-4; V=AA 62-6; V=AA 62-8; V=AA 62-9; V=AA 62-11; V=AA 62-12; V=AA 62-13; V=AA 62-15; (46.74, -71.46, pr=1), V=AA 62-16; V=AA 62-17; V=AA 62-18; V=AA 62-19; V=AA 62-20; (46.57, -72.2, pr=1), V=AA 62-21; V=AA 62-22; V=AA 62-23; V=AA 62-25; V=AA 62-27; (46.29, -72.69, pr=1, ON(4x)), V=AA 62-34, mixed-ploidy locality; V=AA 62- 35, mixed-ploidy locality; (45.03, -73.92, pr=1, ON(4x)), V=63-56; V=63-59; (45.12, -72.18, pr=1, ON(2.8x)), V=63-109; V=63-110; (45.04, -71.91, pr=1, ON(4x)), V=63-113, mixed-ploidy locality; V=63-114, mixed-ploidy locality; V=63-57; V=63-60; USA: Indiana: (39.94, -86.27, pr=1), V=61- 334; V=61-334-1 Beaudry (1970b): diploid: Canada: Québec: V=63-111, mixed-ploidy locality; tetraploid: Ontario: V=62-152 Löve (1982): tetraploid: Canada: Manitoba: (49.5, -97.05, pr=1), V=L&L 7327 Melville & Morton (1982): diploid: Canada: Ontario: (45.33, -75.25, pr=1, ON(4x)), V=MM 1276; V=MM 1277; V=MM 1278; V=MM 1281; V=MM 1285; V=MM 1286; V=MM 1294; V=MM 1295; (44.87, -76.38, pr=1, ON(2x)), V=MM1308; V=MM1310; V=MM1311; V=MM1316; V=MM1318; V=MM1321; V=MM1322; (44.38, -77.02, pr=1, ON(4x)), V=MM1736B; (44.74, -76.85, pr=1, ON(2x)), V=MM1743; (44.62, -75.76, pr=1, ON(2x)), V=MM1763; (45.06, -75.52, pr=1, ON(2x)), V=MM1771; (45.1, -75.23, pr=1, ON(2x)), V=MM1774; (45.12, -75.01, pr=1, ON(2x)), V=MM1777; (45.29, -74.86, pr=1, ON(4x)), V=MM1783; (45.23, -76.19, pr=1, ON(2x)), V=MM1807; (45.02, - 76.83, pr=1, ON(2x)), V=MM1813; (44.82, -77.18, pr=1, ON(4x)), V=MM1819; tetraploid: (43.96, -81.52, pr=1), V=MM52; V=MM53; V=MM58; (44.08, -79.68, pr=1), V=MM136; (46.26, -83.56, pr=1), V=MM672; (46.99, -83.18, pr=1), V=MM682b; V=MM683; V=MM684; (46.21, -82.08, pr=1), V=MM869; V=MM870; (49.78, -86.53, pr=1), V=MM974; (49.73, -86.95, pr=1), V=MM984; V=MM985; (48.76, -91.63, pr=1), V=MM1035; (48.73, -92.1, pr=1), V=MM1054; (48.61, -93.4, pr=1), V=MM1079; V=MM1080; V=MM1086; (49.78, -92.84, pr=1), V=MM1140; (43.46, -80.52, pr=1), V=MM1392; V=MM1395; V=MM1441; V=MM1442; (42.98, -82.41, pr=1), V=MM1481; V=MM1482; V=MM1483; V=MM1485; (42.32, -83.04, pr=1), V=MM1503; V=MM1509;

58 Ploidy level distribution

V=MM1512; V=MM1513; (46.78, -81.63, pr=1), V=MM1608; (49.22, -81.28, pr=1), V=MM1631; (49.27, -81.62, pr=1), V=MM1634; (51.28, -80.64, pr=1), V=MM1666; V=MM1671; (44.19, -79.03, pr=1), V=MM1702; (44.24, -78.4, pr=1), V=MM1707; (44.04, -78.22, pr=1), V=MM1711; (44.47, -77.31, pr=1), V=MM1725; (44.17, -77.38, pr=1), V=MM1731; (44.54, -76.68, pr=1, ON(2x)), V=MM1747; (44.42, -76.32, pr=1, ON(4x)), V=MM1753; (44.54, -76.2, pr=1, ON(4x)), V=MM1756; (44.84, -75.55, pr=1, ON(2x)), V=MM1765; (45.31, -74.63, pr=1, ON(2x)), V=MM1785; (45.27, - 75.28, pr=1, ON(2x)), V=MM1794; (44.84, -77.12, pr=1, ON(2x)), V=MM1815; (45.47, -76.68, pr=1), V=MM1824; (45.51, -77.56, pr=1), V=MM1831; (45.05, -78.51, pr=1), V=MM1843; (44.97, -78.27, pr=1), V=MM1846; (45.23, -77.94, pr=1), V=MM1847; (44.75, -78.09, pr=1), V=MM1852; V=MM1855; (44.69, -77.6, pr=1), V=MM1858; (42.99, -79.24, pr=1), V=MM1866; (42.95, - 79.86, pr=1), V=MM1870; V=MM1872; (43.04, -80.8, pr=1), V=MM1878; (42.69, -81.39, pr=1), V=MM1885; (42.57, -81.56, pr=1), V=MM1889; (42.33, -82, pr=1), V=MM1893; (42.13, -82.74, pr=1), V=MM1898; (42.4, -82.19, pr=1), V=MM1903; (42.98, -82.12, pr=1), V=MM1909; (43, -81.4, pr=1), V=MM1917; (43.09, -80.83, pr=1), V=MM1921; (43.19, -80.38, pr=1), V=MM1926; (43.51, -79.81, pr=1), V=MM1950; (45.1, -81.44, pr=1), V=NA10833; (45.98, -81.93, pr=1), V=NA10856; (45.9, -81.92, pr=1), V=NA10866; (46.93, -84.6, pr=1), V=S 2867 Morton (1984): diploid: USA: Alabama: (34.761, -85.675, pr=0), V=M4460; North Carolina: (35.944, - 82.73, pr=0, SA(2.92307692307692x)), V=M3882; V=M3883; (34.982, -80.463, pr=0), V=M3743; Tennessee: (35.997, -84.219, pr=0, SA(2x)), V=M2998; V=M4445; V=M3408; (35.967, -87.751, pr=0), V=M3912; V=M3913; (35.561, -85.471, pr=0, SA(4x)), V=M3852; (35.1, -85.17, pr=0, SA(2x)), V=M4443; Texas: (32.478, -95.292, pr=0), V=M3966; V=M3967; V=M3969; tetraploid: Kansas: (39.104, -94.497, pr=0), V=M5100; New Jersey: (40.85, -74.83, pr=1, NE(4x)), V=M5874; V=M5888; Tennessee: (36.455, -83.568, pr=0), V=M2920; V=M2921; V=M2923; (36.21, -82.47, pr=1, SA(4x)), V=M3879; V=M3880; hexaploid: Colorado: (39.71, -105.294, pr=0), V=M&A4993†; Oregon: (44.921, -117.946, pr=0), V=M&A5084†; (44.403, -117.234, pr=0), V=M&A5086†; (45.244, -121.157, pr=0), V=M&A5050†; Washington: (47.21, -117.361, pr=0), V=M&A5075† Semple et al. (1981): diploid: Canada: Québec: V=S&Bt 3387; (45.57, -72.52, pr=1, ON(2x)), V=S&Bt 3415; USA: Massachusetts: (42.25, -71.28, pr=1, NE(2x)), V=S&Bt 3538; tetraploid: Canada: Ontario: (46.88, -84.48, pr=1), V=S&Bt 2867; (44.07, -81.75, pr=1), V=S&Bt 2807; (44.04, -77.74, pr=1), V=S&Bt 2952; (44.8, -80.07, pr=1), V=S 2936; USA: Connecticut: (41.78, -72.61, pr=1, NE(2x)), V=S&Bt 3616; New Hampshire: (44.17, -71.97, pr=1), V=S&Bt 3453; New York: (43.1, -75.23, pr=1), V=S 3377; Vermont: (43.99, -72.45, pr=1), V=S&Bt 3506; (44.74, -72.47, pr=1), V=S&Bt 3441; (44.2, -72.5, pr=1), V=S&Bt 3449 Semple et al. (1984): diploid: Canada: Nova Scotia: (44.87, -65.22, pr=1), V=S & K 4883; (45.26, -63.01, pr=1), V=S & K 4721; USA: Georgia: (34.12, -83.43, pr=1), V=M 8591; North Carolina: (34.99, - 80.45, pr=1), V=M 3742; Pennsylvania: (40.91, -77.81, pr=1), V=M 6495; Vermont: (42.87, -72.79, pr=1, NE(4x)), V=S & K 4960; tetraploid: Canada: New Brunswick: (46.47, -67.58, pr=1), V=S & K 4668; Ontario: (43.31, -81.76, pr=1), V=R 1506; (43.22, -81.89, pr=1), V=R 1505; USA: Illinois: (39.82, -91.34, pr=1), V=S & Bt 7373; Michigan: (43.43, -85.39, pr=1), V=M 6677; New Jersey: (40.86, -74.43, pr=1, NE(4x)), V=M 6219; (40.94, -74.25, pr=1, NE(4x)), V=M 6302; (40.65, -74.64, pr=1, NE(4x)), V=M 5186; V=M 6303; (40.89, -75.07, pr=1, NE(4x)), V=M 6204; (40.86, -75.05, pr=1, NE(4x)), V=M 6169; (40.76, -74.98, pr=1, NE(4x)), V=M 6234; V=M 6235; New York: (42.1, -75.92, pr=1), V=S 6808; (44.39, -73.82, pr=1), V=R 1541; (41.58, -74.49, pr=1, NE(4x)), V=S, Ch &

59 Chapter 1: Supplementary material

R 6495; Tennessee: (35.05, -85.31, pr=1, SA(2x)), V=M 8500; (35.29, -84.95, pr=1, SA(2x)), V=M 8537; V=M 8538; Vermont: (43.19, -72.49, pr=1), V=S 6888; Wisconsin: (42.8, -88.6, pr=1), V=S & Bt 6931; hexaploid: Canada: Ontario: (49.8, -95.12, pr=1), V=S & S 6717; V=S & S 6718; USA: Colorado: (38.07, -103.22, pr=1), V=S & Bt 7292; (40.23, -105.27, pr=1), V=S et al. 5817†; Idaho: (44.51, -114.23, pr=1), V=S & Bt 7038†; (44.73, -116.08, pr=1), V=S & Bt 7066†; Minnesota: (45.94, -95.49, pr=1), V=S & Bt 6948; (44.77, -93.79, pr=1), V=5135; North Dakota: (47.98, -98.99, pr=1), V=S & S 6686; (46.27, -100.23, pr=1), V=S & S 6670 Semple (1985): hexaploid: USA: Colorado: (38.86, -104.92, pr=1), V=S&Bt 7269† Semple & Chmielewski (1987): hexaploid: USA: New Mexico: (33.4, -105.696, pr=0), V=S&H 8130† Semple et al. (1989): diploid: USA: Tennessee: (35.58, -86.69, pr=1), V=S&Ch 9118 Semple et al. (1993): diploid: USA: Kentucky: (37.09, -83.396, pr=0, SA(4x)), V=S&Su 9620; Mississippi: (33.506, -88.432, pr=0), V=S&Su 10165; North Carolina: (36.092, -78.123, pr=0), V=S&Su 9732; (35.934, -76.508, pr=0), V=S&Su 9748; South Carolina: (33.928, -82.32, pr=0), V=S&Su 9819; tetraploid: Illinois: (41.207, -88.007, pr=0), V=S&Su 9871; (37.966, -88.329, pr=0), V=S&Su 9424; Missouri: (36.811, -92.146, pr=0), V=S&Su 9928; (37.946, -93.401, pr=0), V=S&Su 9945; hexaploid: Nebraska: (42.043, -101.043, pr=0), V=S&Su&Ah 9200 Semple et al. (2001): hexaploid: USA: California: (41.614, -120.367, pr=0), V=S,Su&Ah 9294†; Montana: (46.347, -111.198, pr=0), V=S&Xg 10329†; (46.119, -111.064, pr=0), V=S&Xg 10243†; (46.941, -112.938, pr=0), V=S&Xg 10312† Semple & Cook (2004): diploid: USA: North Carolina: (35.164, -82.732, pr=0), V=S 10842; tetraploid: Canada: Ontario: V=C&S 8; (43.47, -80.545, pr=0), V=C&S 4; USA: North Carolina: (36.262, - 81.895, pr=0), V=S 10798

60 Ploidy level distribution

Re f e r e n c e s f o r Supplementary Ma t e r i a l

Anonymous (1982 onwards) Flora of Australia online - Australian biological resources study Australian Biological Resources Study. [Internet database, viewed 27 August 2007]. URL http://www. environment.gov.au/biodiversity/abrs/online-resources/flora/main/index.html Beaudry, J.R. (1963) Studies on Solidago L. VI. Additional chromosome numbers of taxa of genus Solidago. Canadian Journal of Genetics and Cytology, 5, 150-174 Beaudry, J.R. (1970a) Studies on Solidago L. X. Les Solidago gigantea Ait. dans l’est de l’Amerique du Nord. Naturaliste Canadien, 97, 35-42 Beaudry, J.R. (1970b) Studies on Solidago L. XI. Caryotypes additionnels de taxons du genre Solidago L. Naturaliste Canadien, 97, 431-445 Beaudry, J.R. (1974) Solidago shinnersii (Beaudry) stat. et comb. nov., une nouvelle espece du complexe du S. gigantea. Naturaliste Canadien, 101, 931-932 Beaudry, J.R. & Chabot, D.L. (1959) Studies on Solidago L. IV. The chromosome numbers of certain taxa of the genus Solidago. Canadian Journal of Botany - Revue Canadienne de Botanique, 37, 209-228 Federov, A., Bolkhovskikh, Z., Grif, V., Matvejeva, T. & Zakharyeva, O. (1969) Chromosome numbers of flowering plants - Chromosomnye cisla cvetkovych rastenij. Akademija Nauk SSSR; Botaniceskij Institut im. V.L. Komarova, Leningrad. Given, D.R. (1984) Checklist of dicotyledons naturalised in New Zealand 16. Compositae - tribes Vernonieae, Eupatorieae, Astereae, Inuleae, Heliantheae, Tageteae, Calenduleae, and Arctoteae. New Zealand Journal of Botany, 22, 183-190 Goldblatt, P. (1981) Index to plant chromosome numbers for 1975-1978. Missouri Botanical Garden, St. Louis, MO, USA. Goldblatt, P. (1985) Index to plant chromosome numbers for 1982-1983. Missouri Botanical Garden, St. Louis, MO, USA. Goldblatt, P. (1988) Index to plant chromosome numbers for 1984-1985. Missouri Botanical Garden, St. Louis, MO, USA. Goldblatt, P. (2000) Index to plant chromosome numbers for 1996-1997. Missouri Botanical Garden, St. Louis, MO, USA. Goldblatt, P. & Johnson, D. (1991) Index to plant chromosome numbers for 1988-1989. Monographs in Systematic Botany, 40 Goldblatt, P. & Johnson, D. (1996) Index to plant chromosome numbers for 1992-1993. Monographs in Systematic Botany, 58 Goldblatt, P. & Johnson, D. (1998) Index to plant chromosome numbers for 1994-1995. Monographs in Systematic Botany, 69, 1-208 Goldblatt, P. & Johnson, D. (2006) Index to plant chromosome numbers (IPCN) Missouri Botanical Garden. [Internet database, viewed 27 August 2007]. URL http://mobot.mobot.org/W3T/Search/ ipcn.html Goldblatt, P. & Johnson, D.E. (1990) Index to plant chromosome numbers for 1986-1987. Missouri Botanical Garden, St. Louis, MO, USA. Huziwara, Y. (1962) Karyotype analysis in some genera of Compositae VII. The chromosomes of Japanese Solidago species. Acta Phytotaxonomica et Geobotanica, 20, 176-179

61 Chapter 1: Supplementary material

Jakobs, G. (2004) Evolution of increased competitive ability in the invasive perennial Solidago gigantea Aiton. Diss. 15536 ETH Zürich, Zürich. Jurenitsch, J., Maurer, J., Rain, U. & Robien, W. (1988) Diterpenebutenolides in Solidago gigantea. Phytochemistry, 27, 626-627 Löve, A. (1982) IOPB chromosome number reports. LXXV. Taxon, 31, 342-368 Májovsky, J. (1974) Index of chromosome numbers of Slovakian flora (Part 3). Acta Facultatis Rerum Naturalium Universitatis Comenianae Botanica, 22, 1-20 Májovsky, J. & Murín, A. (1987) Karyotaxonomicky prehl’ad flóry Slovenska. Vydavatel’stvo Slovenskej Akadâemie Vied, Bratislava. Maurer, J. (1987) Über das Vorkommen von Diterpenbutenoliden und Flavoniden in cytologisch definierten Herkünften vonSolidago gigantea und Solidago canadensis. Diplomarbeit. Universität Wien, Wien, Österreich. Melville, M.R. & Morton, J.K. (1982) A biosystematic study of the Solidago canadensis (Compositae) complex. 1. The Ontario populations. Canadian Journal of Botany - Revue Canadienne de Botanique, 60, 976-997 Morton, G.H. (1984) A practical treatment of the Solidago gigantea complex. Canadian Journal of Botany - Revue Canadienne de Botanique, 62, 1279-1282 Newfield, M. (2002) Criteria: invasive elsewhere ISSG-IUCN, listserver aliens-l. [ISSG-IUCN, listserver aliens-l]. URL http://indaba.iucn.org/archives/aliens-l/2002-05/00002120.htm Randall, R. (2007) Permitted and Quarantine Species Lists Department of Agriculture and Food, Western Australia. [Excel file, viewed 27 August 2007]. URLhttp://www.agric.wa.gov.au/content/ PW/WEED/PERMITTED_PROHIBITED.HTM Randall, R.P. & Kessal, O. (2006) National List of Naturalised Invasive and Potentially Invasive Garden Plants in Australia, version 1.2 edn. WWF Australia, Sydney. Rudyka, E.G. (1995) Chromosome numbers in vascular plants from the southern part of the Russian Far East. Botaniceskij Zurnal (Moscow & Leningrad), 80, 87-90 Semple, J.C. (1985) Chromosome number determinations in fam. Compositae, tribe Astereae. Rhodora, 87, 517-527 Semple, J.C., Brammall, R.A. & Chmielewski, J. (1981) Chromosome numbers of Goldenrods, Euthamia and Solidago (Compositae-Astereae). Canadian Journal of Botany - Revue Canadienne de Botanique, 59, 1167-1173 Semple, J.C. & Chmielewski, J.G. (1987) Chromosome number determinations in fam. Compositae, tribe Astereae. II. Additional counts. Rhodora, 89, 319-325 Semple, J.C., Chmielewski, J.G. & Lane, M.E. (1989) Chromosome number determinations in fam. Compositae, tribe Astereae. III. Additional counts and comments on generic limits and ancestral base numbers. Rhodora, 91, 296-314 Semple, J.C. & Cook, R.E. (2004) Chromosome number determinations in fam. Compositae, tribe Astereae. VII. Mostly eastern North American and some Eurasian taxa. Rhodora, 106, 253-272 Semple, J.C. & Cook, R.E. (2006) Solidago. Flora of North America North of Mexico (ed. by Flora of North America Editorial Committee), pp. 107-166. Oxford University Press, Oxford and New York. Semple, J.C., Ringius, G.S., Leeder, C. & Morton, G. (1984) Chromosome numbers of Goldenrods, Euthamia and Solidago (Compositae, Astereae). II. Additional counts with comments on cytogeography. Brittonia, 36, 280-292 62 Ploidy level distribution

Semple, J.C., Xiang, C.S., Zhang, J., Horshburgh, M. & Cook, R.E. (2001) Chromosome number determinations in fam. Compositae, tribe Astereae. VI. Western North American taxa and comments on generic treatments of North American asters. Rhodora, 103, 202-218 Semple, J.C., Zhang, J. & Xiang, C.S. (1993) Chromosome number determinations in fam. Compositae, tribe Astereae. V. Eastern North American Taxa. Rhodora, 95, 234-253 Skalinská, M., Jankun, A. & Wcislo, H. (1976) Further studies in chromosome numbers of Polish angiosperms. XI contribution. Acta biologica Cracoviensia Polska Series botanica, 19, 107-148 Thorp, J.R. & Wilson, M. (1998 onwards) Weeds Australia - An Australian Weeds Committee National Initiative Australian Weeds Committee. [Internet database, viewed 27 August 2007]. URL http:// www.weeds.org.au/ Wagner, W.L. & Herbst, D.R. (2003) Flora of the Hawaiian Islands - electronic supplement to the manual of the flowering plants of Hawai’i [Internet database, viewed 27 August 2007]. URLhttp://ravenel. si.edu/botany/pacificislandbiodiversity/hawaiianflora/supplement.htm Wagner, W.L., Herbst, D.R. & Lorence, D.H. (2005-) Flora of the Hawaiian Islands website [Internet database]. URL http://ravenel.si.edu/botany/pacificislandbiodiversity/hawaiianflora/index.htm Webb, C.J. (1987) Checklist of dicotyledons naturalised in New Zealand 18. Asteraceae (Compositae) subfamily Asteroideae. New Zealand Journal of Botany, 25, 489-501 Webb, C.J., Sykes, W.R. & Garnock-Jones, P.J. (1988-2004) Flora of New Zealand. Volume IV. Naturalised Pteridophytes, Gymnosperms, Dicotyledons Botany Division, Department of Scientific and Industrial Research. [Internet database, viewed 27 August 2007]. URL http://FloraSeries. LandcareResearch.co.nz Weber, E. (1997) Phenotypic variation of the introduced perennial Solidago gigantea in Europe. Nordic Journal of Botany, 17, 631-638 Weber, E. & Jakobs, G. (2005) Biological flora of central Europe: Solidago gigantea Aiton. Flora - Morphology, Distribution, Functional Ecology of Plants, 200, 109-118 Zhao, Z. (1996) Documented chromosome numbers 1996: 2. Miscellaneous U. S. A. and Mexican species, mostly Asteraceae. SIDA - Contributions to Botany, 17, 259-263

63

Habitat differentiation

Ch a p t e r 2

Habitat differentiation between native ploidy levels and invasive tetraploids of Solidago gigantea (Asteraceae)

Daniel R. Schlaepfer, Peter J. Edwards, Regula Billeter

Institute of Integrative Biology, Plant Ecology, ETH Zurich, Zürich, Switzerland

65 Chapter 2

Ab s t r a c t

Many invasive plants are polyploids of species that also include diploids. To understand why particular ploidy levels become invasive it is important to determine whether ploidy levels in the native range differ ecologically. We investigated habitat differentiation between diploids, tetraploids and hexaploids of Solidago gigantea Aiton in the native range in North America. Because the species only occurs as a tetraploid in Europe (first introduced in 18th century) we also asked whether diploids have a different habitat template from tetraploids that might have excluded them from Europe, and whether there has been any niche shift in invasive populations compared with North American tetraploids. To do this, we described the habitat conditions of 78 populations in the native and invasive ranges and constructed environmental envelopes based upon the locations of 334 populations of known ploidy level. The geographic separation of ploidy levels in North America correlated partly with climatic, soil and habitat differences. At a more local scale, the most consistent difference between diploids and tetraploids was that diploids were calcifuge whereas tetraploids were indifferent to soil calcium. We conclude that the geographic separation between ploidy levels in North America cannot be explained by habitat differentiation alone. Similarly, the habitat template is unlikely to be the main reason why diploids do not occur in the invasive range, although we did find differences between populations from the invasive and the native range in total soil phosphorus, proximity to water and range of soil C/N values. More relevant is the fact that tetraploids of S. gigantea were successful colonizers in North America, extending their range well beyond that occupied by their diploid progenitors; the same traits that made them successful in North America, e.g. higher colonisation ability and wider habitat tolerance than diploids, may have predisposed tetraploids to becoming successful invaders.

Keywords: Calcium, ecological differentiation, field survey, habitat template, invasive alien species, niche breadth.

66 Habitat differentiation

In t r o d u c t i o n

Polyploidy is one of the important forces in plant evolution (Stebbins, 1971). This is because, after formation, a polyploid is reproductively isolated from its diploid progenitor(s), though this isolation may not be total (Husband & Sabara, 2004). Furthermore, a polyploid plant can differ profoundly from its diploid progenitor(s) in patterns of gene expression (Osborn et al., 2003; Soltis et al., 2004) and in its physiological (Soltis et al., 2003) and ecological behaviour (Lumaret, 1988; Thompson et al., 2004). Although polyploids may offer new ecological and evolutionary opportunities, the initial dominance of the diploid may restrict the chances of a newly formed polyploid becoming established. This is because new polyploids are likely to receive mostly diploid pollen, which greatly reduces their reproductive efficiency, a phenomenon referred to as minority cytotype disadvantage (Levin, 1975). However, if there is some form of ecological differentiation between the ploidy levels then the chances of polyploid establishment are likely to be greater (e.g. Thompson & Lumaret, 1992; Rodriguez, 1996). Such differentiation in habitat has been described between diploid and tetraploid Anthoxanthum alpinum (Felber-Girard et al., 1996) and between diploid and tetraploid Galax urcoleata (Johnson et al., 2003). Competitive and fitness advantages may also promote establishment of polyploids, as has been shown for tetraploid Dactylis glomerata (Maceira et al., 1993), or polyploids may be better colonizers than diploids (De Wet, 1980; Soltis & Soltis, 2000). For example, polyploids of the arctic specialist flora occur more frequently in previously heavily glaciated areas, whereas unglaciated areas tend to be occupied by diploids (Brochmann et al., 2004). However, non-adaptive explanations (Van Dijk & Bakx-Schotman, 1997; Baack & Stanton, 2005) have also been proposed to explain geographic separation of ploidy levels, including spatial structures of populations (Li et al., 2004; Baack, 2005) and stochastic effects (Rausch & Morgan, 2005). To help distinguish between these different types of explanation, zones in which ploidy levels overlap are of special interest; this is because interactions between ploidy levels can directly be observed in these areas (Mandakova & Münzbergova, 2006) and the strongest differentiation can be expected (Burton & Husband, 1999). Polyploids are more frequent among invasive alien plants than among angiosperms in general (Brown & Marshall, 1981; Bennett et al., 1998; Verlaque et al., 2002; Pandit et al., 2006), and many alien species are represented by a single higher ploidy level in the introduced range (U. Treier, personal communication, Verlaque et al., 2002; Lafuma et al., 2003; Müller- Schärer et al., 2004; Pandit et al., 2006). The simplest explanation for this phenomenon is that only one ploidy level of a species was introduced, as was apparently the case for Fallopia japonica in Europe (Hollingsworth & Bailey, 2000). However, in many other cases two or more ploidy levels were probably introduced but only one was successful. This could occur if the ploidy levels have differing ecological requirements, and the alien range only has habitat conditions suitable for one of them. It is also possible that the invasion is associated with a

67 Chapter 2 niche shift relative to that of the native populations (Broennimann et al. 2007), but that this shift is dependent on ploidy level. Because of the possibility of niche shifts, it is important to compare the habitat templates of species in both the alien and native ranges (Hierro et al., 2005), with these comparisons being made at the same ploidy level (chapter 1). Solidago gigantea Aiton (Asteraceae) is a good model species to study aspects of both biological invasion and polyploid establishment (chapter 1). In its native range in North America, Solidago gigantea occurs as diploids (NA.2x), tetraploids (NA.4x), and hexaploids (NA.6x). Populations consist mostly of plants of a single ploidy level. Diploids are restricted to eastern North America, occurring east of the Appalachian Mountains and in southern Ontario and Québec, while tetraploids are found in eastern North America as far west as Missouri. At least three overlap zones exist between diploids and tetraploids. Hexaploids occur in mid- western North America, westwards from Manitoba to the Rocky Mountains (chapter 1, Semple & Cook, 2006). The species is invasive in Europe, where it was introduced in 1758 (Weber & Schmid, 1993) and where only tetraploids have been detected (EU.4x, chapter 1). In an earlier paper, we recorded the geographic distributions of the different ploidy levels of S. gigantea (chapter 1). Here, we investigate how the ploidy levels differ in their habitat associations. We test the hypotheses that (i) ecological differentiation explains the geographic separation between ploidy levels in North America, (ii) ecological preferences (habitat templates) explain why no diploids have been found in Europe, and (iii) an ecological (niche) shift has occurred between North American and European tetraploids. For this purpose, we investigated habitat differences among ploidy levels in the native range, and between North American and European populations (NA.2x vs. EU.4x and NA.4x vs. EU.4x). We also compared the environmental envelopes of the various provenance types.

Me t h o d s

Field survey populations

We sampled a total of 78 S. gigantea populations in North America (NA) and Europe (EU) in autumn 2005, a population being defined as a stand of the species at a particular location (Fig. 1, Tab. 1, populations SG220–279 and SG287–312 from chapter 1). The populations selected all contained at least 12 individual patches of S. gigantea. Ploidy levels of plants were not known at the time of sampling, but subsequent determinations confirmed that we had sampled four cytotypes: NA.2x, NA.4x, NA.6x and EU.4x. The areas sampled in North America included zones of overlap with both NA.2x and NA.4x present. The mixed diploid-tetraploid

68 Habitat differentiation

Canada 55°N °N Sweden 55

Manitoba

Ontario Belgium

45°N °N 45 Italy Europe South Dakota New England

Southern Appalachian Mts.

35°N °N 35 USA

0500 1’000 2’000 Kilometers

Figure 1 Situation of field survey areas and location of populations ofSolidago gigantea Aiton used for the environmental envelopes. The cytogeographic maps from North America and Europe show the 334 locations from chapter 1 of diploid (grey circle), tetraploid (triangle) and hexaploid (square) S. gigantea (projection: Mollweide).

Table 1 Datasets of populations of Solidago gigantea Aiton from the field survey and for the environmental envelope

Field survey Environmental envelope Cytotype # locations, Occupied Cytotype Latitude # pop.s area (# cells) (# weighted) area European 4x 20 526 114 (44) 8% Northern Italy 45° 7 Belgium 50° 7 Southern Sweden 55° 6 North American 2x 21 110 64 (31) 28% S. Appalachian Mts. 35° 10 New England 40° 5 Ontario 45° 6 North American 4x 23 227 129 (66) 29% S. Appalachian Mts. 35° 8 New England 40° 7 Ontario 45° 8 North American 6x 15 175 24 (18) 10% South Dakota 45° 7 Southern Manitoba 50° 8 Latitude of region and number of populations (in total 78) that were sampled in the field. Cytotype area, the number of cells covered by the cytotype distribution (chapter 1) on a 1°*1° grid, number of cytotype locations (in total 334), number of grid-weighted locations (see text for details), and percentage of cells that were occupied with at least one location were retrieved with a geographic information system.

69 Chapter 2 location (SG277) was counted twice, once as NA.2x and once as NA.4x. The methods used for determining ploidy levels and preparing herbarium vouchers are described in chapter 1. In every population, we measured: locality, geographic location, three habitat variables and six soil variables (Tab. 2). The habitat was described in the field by the variables habitat type (two categories: water-adjacent, terrestrial), type of surrounding area (natural, agricultural, urban) and surrounding light conditions (closed, edge, open canopy). Three soil cores (5 cm Ø, 1–6 cm deep) were taken from below S. gigantea plants, mixed and air-dried (corrected for residual water-content). Soil pH was measured in a 0.01 M calcium chloride solution. Carbonate content was estimated in terms of effervescence classes after applying drops of 1 M hydrogen chloride (Anonymous, 2004). Dry soil samples were ground, 2 mm-sieved and analysed. For total phosphorus concentrations, sub-samples were digested in concentrated sulphuric acid (1 h refluxing at 420 °C with a potassium sulphate – copper sulphate Kjeltab; 2040 Digestor, Foss Tecator, Höganäs, Sweden) and analysed colorimetrically on a flow injection analyser (FIAstar 5000 Analyser, Foss Tecator, Höganäs, Sweden). Total carbon and total nitrogen concentrations were measured in 250 mg sub-samples with an elemental analyser (Leco CNS-2000, Leco Instrumente GmbH, Krefeld, Germany). Total calcium concentrations were measured in ammonium acetate extracts (Anonymous, 1996) with an AAS (SpectrAA 240 FS, Varian AG, Zug, Switzerland).

Environmental envelope

Environmental envelopes for each cytotype of S. gigantea were constructed with a dataset of 334 populations of known location and ploidy level (Fig. 1, Tab. 1, see chapter 1). Three mixed diploid-tetraploid locations were used both as NA.2x and as NA.4x. For each location we retrieved three soil variables and eight climatic variables from a geographic information system containing global data sets (Tab. 2; ArcGIS 9.1, ESRI, Redlands, USA). The soil types (106 categories, 17 occurring in dataset) were retrieved from a modified Zobler map (Post & Zobler, 2000). We retained for further analysis the six soil types that occurred in at least 10% of the locations of a cytotype (Acrisol, Cambisol, Chernozem, Kastanozem, Luvisol, Podzol). Acrisols are poor in many nutrients including calcium and occur mostly on acid rocks in Southeast USA (Driessen et al., 2001). Soil pH value of Cambisols is neutral to weakly acid (Driessen et al., 2001). Chernozems and Kastanozems, which are both rich in organic matter and usually in carbonates, are typical of mid-latitude steppes and prairies (Driessen et al., 2001). The surface soils of Luvisols are de-calcified, whereas subsurface soils may contain calcium carbonate (Driessen et al., 2001). Podzols are leached, calcium-poor, acidic soils (Driessen et al., 2001). Information was obtained from published sources on the phosphorus status (percentage of soils highly deficient in phosphorus: >60%, 40–60%, 20– 40%, 0–20%; Fairhurst et al. (1999) and carbonate content of soils (≤ 1%, > 1%; Batjes (2005).

70 Habitat differentiation 0.81 1 0.1 0.01 1.9 2 253 550 52 3.9 6.3 2.7 . 3± 7± 69± edge 4.6± <1% snow 927± 479± NA.6x 1.16± 0.03± 13.9± 21.1± <20% 1912± -19.1± water adj. water agricultural Chernozem S. gigantea 0.58 1 1.0 0.01 2.4 6.4 145 558 144 3.4 5.6 2.6 1± 58± edge 6.2± 6.8± <1% snow 809± 958± NA.4x 0.48± 0.03± 14.6± 20.4± >60% Podzol 1995± -12.8± water adj. water agricultural 0.26 0 1.2 0.02 2 4.7 225 1380 260 5.6 7.1 3.7 0± 54± edge 5.7± <1% -5.1± NA.2x 0.22± 0.03± 14.7± 23.4± 13.0± >60% Acrisol 1101± 3189± 1208± terrestrial agricultural warm temperate warm 0.68 1 0.3 0.03 4.4 8.4 93 315 2.1 1.2 223 1.6 1± 27± 6.8± 8.9± open <1% 657± 798± -2.6± EU.4x 0.68± 0.06± 14.5± 17.2± 1808± Luvisol terrestrial agricultural >60%, 20-40% warm temperate warm 1 1 1 1 1 2 2 2 2 1 1 1 1 1 2 2 1 1 2 Soil pH Habitat type Habitat type Soil C/N ratio Soil C/N ratio Carbonate level Carbonate level Light conditions Soil calcium (%) Zoebler soil units Zoebler Surrounding habitat Effervescence (1–4) Effervescence Koeppen climate types climate types Koeppen Continentality (Conrad) (Conrad) Continentality Total soil phosphorus (%) Total Annual precipitation (mm) Phosphorus deficiency level Phosphorus deficiency level Annual mean temperature (°C) Annual mean temperature Variables measured in field populations or obtained from global data sets for the environmental envelopes of cytotypes (EU.4x = European = (EU.4x cytotypes of envelopes environmental the for sets data global from obtained or populations field in measured Variables Growing degree days (base 5°C) Growing degree days Minimum annual temperature (°C) Minimum annual temperature Maximum annual temperature (°C) Maximum annual temperature Annual reference evaporation (mm) Annual reference evaporation Variables (unit) Variables Field populations (n = 78) Environmental envelopes (n = 334) Table 2 Table tetraploids; NA.2x = North American diploids; NA.4x NA.6x hexaploids) of 1, median±1.4826*mad; 2, mode

71 Chapter 2

Climate type (arid, warm temperate, snow) was retrieved from the updated Köppen-Geiger climate classification (Kotteket al., 2006). Climatic data for each location were retrieved from the CRU CL 2.0 global data set at 10’ * 10’ (New et al., 2002). Five variables representing average values for 1961–1990 were investigated: mean annual temperature, mean maximum temperature, mean minimum temperature and annual precipitation. In addition, using the same data, growing degree days (base 5°C) and a continentality index (Conrad, 1946) were calculated and annual reference evapotranspiration values (Allen et al., 1998) were retrieved from FAO-AGL (2005). Climatic ranges were comparable to other studies of S. gigantea (Weber, 2001).

Statistical analyses

Differences in mean habitat conditions among cytotypes were compared with Kruskal-Wallis tests. In the case of nominal variables the frequency distributions among cytotypes were compared with c2-tests. To test for differences in niche breadth (Debussche & Thompson, 2003), two-tailed F-tests on the variance ratios were calculated (Sokal & Rohlf, 1995). For nominal variables, the number of categories was used as a measure of niche breadth. Differences between NA.2x and NA.4x at a regional scale within the three zones of overlap were also tested with Kruskal-Wallis and F-tests. Associations between habitat type and both climate variables and continent were tested with Spearman rank correlations. We used the rectilinear approach of extremes for each variable to construct the environmental envelopes (Carpenter et al., 1993). However, at least four shortcomings appear in the presence-only dataset. First, the locations do not cover the whole geographic range (Tab. 1), though they do include the extremes of the ranges. Secondly, the locations are geographically clustered, which affects the resulting environment envelope (Araujo & Guisan, 2006). Thirdly, the sampling was biased towards roadside sites. Fourthly, cytotype datasets did not meet the recommended number of observations (50–75) for maximal model performance (Kadmon et al., 2003). To decluster the data, we calculated grid cell occupancy (number of locations within each 1°*1° grid cell) for each cytotype (Tab. 1) and weighted each location with the inverse of its grid cell occupancy (pp. 241 in Isaaks & Srivastava, 1989). Because latitude is correlated with the independent variable cytotype (i.e. locations in Europe occur at higher latitudes than in North America with only a small range of overlap), we could not include latitude as a covariate in our analyses. Instead, the geographic cline was estimated by Pearson correlation coefficients between latitude and the climatic variables. Sequential Bonferroni tests applicable to multiple comparisons (Rice, 1989) were applied to construct cytotype-wide significance levels of 5%. Statistics were calculated with SPSS 13.0.0 (SPPS Inc., Chicago, USA) and R 2.5.0 (R Development Core Team, 2007).

72 Habitat differentiation

Re s u l t s

Field populations

We found evidence for habitat differentiation between NA.2x and NA.4x at a continental scale, mainly in terms of soil calcium and carbonate contents (Tab. 3). NA.2x was restricted to soils with effervescence between categories 0 and 2, whereas NA.4x occurred across a wider range of values (up to 4) and the mean effervescence was higher. NA.4x had a non-significantly higher median of soil calcium content, but a significantly larger niche breadth. At a regional scale, differentiation between NA.2x and NA.4x was only found in Ontario, where there were significant differences in soil calcium (KW=8.1, p<0.005), effervescence (KW=10.7, p<0.001) and soil pH (KW=9.3, p<0.002). In all three regions, though, maximum values for both soil calcium and effervescence were higher for NA.4x than for NA.2x. Niche breadth of NA.4x for soil calcium was significantly larger in New England (v-ratio=0.03, p<0.004) and in Southern Appalachian Mts. (v-ratio=0.01, p<0.001; Fig. 2(a)). Niche breadth of NA.4x for soil pH was significantly wider in Ontario than for NA.2x (v-ratio=27.5, p<0.001). There was also habitat differentiation between NA.4x and NA.6x (Tab. 3), with NA.4x growing in soils with a lower median pH and a wider pH range than NA.6x. Habitats also differed between NA.2x and EU.4x (Tab. 3). Whereas NA.2x occurred frequently on sites beside water, EU.4x grew mainly on terrestrial sites (Fig. 3). Compared with the soils on which EU.4x was found, NA.2x soils differed as follows: (i) lower median calcium content and a narrower range of values (Fig. 2(a)); (ii) lower effervescence values and a narrower range; (iii) significantly lower pH; (iv) much lower mean phosphorus content (Fig. 2(b)); (v) significantly narrower range of C/N values. Finally, habitat differentiation was also found between NA.4x and EU.4x (Tab. 3), with NA.4x often growing close to water (Fig. 3) while EU.4x occurred mainly on terrestrial sites. Soil chemical conditions also differed significantly in two main respects: (i) the mean phosphorus concentration at EU.4x sites was twice as high as at NA.4x sites, and the range of values was also higher (Tab. 3, Fig. 2(b)), and (ii) EU.4x soils had a significantly larger range of C/N values. The habitat type of all sites correlated significantly with continent (r=0.42, p<0.001), and also with continentality (r=0.30, p<0.008), evapotranspiration (r=0.30, p<0.008) and minimum temperature (r=-0.33, p<0.003).

Environmental envelope

There were many differences between the climatic conditions in the ranges of the various ploidy levels: (i) NA.2x and NA.4x differed significantly in the five climatic variables recorded, 73 Chapter 2

*** *** n.a. n.a. n.a. *** n.a. n.a. - . 1.1 0.8 0.8 6.5 5.2 1.0 1.9 1.0 1.0 1.3 0.4 0.7 1.9 0.4 0.2 1.0 1.0 0.8 breadth (v1/v2, p) test value) 2 *** *** *** *** *** *** *** *** *** *** S. gigantea 0.1 0.1 1.4 0.0 4.5 0.0 0.7 3.6 1.6 EU.4x vs. NA.4x EU.4x location 17.3 19.1 65.1 74.5 38.4 24.4 11.3 60.3 25.8 37.0 (test, p) *** *** *** n.a. n.a. n.a. *** *** *** *** n.a. n.a. - 8.9 8.8 0.5 2.3 5.7 1.0 1.3 1.0 1.0 2.6 0.2 0.3 1.4 0.2 0.2 0.7 2.3 1.7 breadth (v1/v2, p) test value) and differences in niche 2 ** *** ** ** *** *** *** *** *** ** *** *** *** EU.4x vs. NA.2x EU.4x 5.0 9.0 9.8 0.0 7.3 1.1 0.0 8.0 5.6 1.5 location (test, p) 11.9 13.7 48.2 36.5 15.3 35.9 31.2 42.8 19.5 *** n.a. n.a. n.a. n.a. n.a. - 0.4 2.2 9.7 1.8 0.5 1.0 0.4 1.0 1.0 1.1 0.5 1.8 3.4 1.4 1.5 0.9 1.3 2.7 breadth test (likelihood ratio c 2 (v1/v2, p) *** *** *** *** *** *** *** 2.7 1.5 0.3 0.3 0.5 0.2 0.0 9.7 3.2 0.3 3.7 0.2 NA.4x vs. NA.6x 15.8 22.2 38.8 11.8 74.4 26.2 16.5 location (test, p) *** *** n.a. n.a. n.a. n.a. n.a. - 0.1 0.1 1.5 2.8 0.9 1.0 1.4 1.0 1.0 0.5 1.6 2.7 1.3 1.6 1.1 1.5 0.4 0.4 breadth (v1/v2, p) ** *** *** *** *** *** *** *** *** *** 4.0 9.1 3.5 0.0 0.4 3.3 1.5 0.0 0.0 NA.2x vs. NA.4x location (test, p) 13.9 12.0 13.1 15.1 37.3 18.6 20.0 11.5 15.9 12.0 1 1 1 1 1 2 1 2 2 1 1 1 1 1 1 1 2 1 1 Soil pH Habitat type Habitat type Soil C/N ratio Soil C/N ratio Effervescence Effervescence Carbonate level Carbonate level Light conditions Soil calcium (%) Zoebler soil units Zoebler Surrounding habitat Koeppen climate types climate types Koeppen Continentality (Conrad) (Conrad) Continentality Total soil phosphorus (%) Total Annual precipitation (mm) Phosphorus deficiency level Phosphorus deficiency level Comparison of niche location and breadth of field populations and environmental envelopes between cytotypes (EU.4x = European Annual mean temperature (°C) Annual mean temperature Growing degree days (base 5°C) Growing degree days Minimum annual temperature (°C) Minimum annual temperature Maximum annual temperature (°C) Maximum annual temperature Annual reference evaporation (mm) Annual reference evaporation Variables Field populations Environmental envelope Table 3 tetraploids; NA.2x = North American diploids; NA.4x NA.6x hexaploids) of Significance levels1 are Differences and in per cytotype differences 2 niche Differences sequential-Bonferroniin niche location in (frequencies) between cytotypes were location tested adjustedwith c breadth were estimated as ratio of number categories. niche (median breadth values) were (***, between tested p cytotypes < 0.001; with were F-test tested (variance with **, of Kruskal-Wallis p < first test 0.01; (c cytotype n.a., not applicable). divided by variance of second).

74 Habitat differentiation

7 (a) 6

5

4

3

Soil calcium (%) 2

1

0 0.16 (b) 0.14

0.12

0.10

0.08

0.06

0.04 Soil phosphorus (%)

0.02

0 35 40 45 50 55 Latitude (° N) Figure 2 Soil calcium (a) and soil phosphorus (b) niche locations (median) and breadths (minimum– maximum) of S. gigantea for the cytotypes North American diploids (black bars)/tetraploids (densely dashed)/hexaploids (grey) and European tetraploids (widely dashed) at different latitudes (corresponding to regions per continent).

but they occurred within the same climatic ranges (Tab. 3); (ii) NA.4x and NA.6x differed in the median values of continentality, annual precipitation and minimum temperature, but also shared the same climatic ranges; (iii) EU.4x and NA.2x differed significantly in four of the five climatic variables (the exception was minimum annual temperature), with ranges of these variables being larger for NA.2x; (iv) EU.4x and NA.4x differed significantly in all variables except in growing degree days, but ranges were only different for minimum temperature. Most climatic variables were significantly and strongly correlated with latitude; exceptions to this were minimum temperature for EU.4x (due to the overall oceanic climate of Europe) and continentality and annual precipitation for NA.6x (data not shown).

75 Chapter 2

10 0 Number of water adjacent sites

8 2

6 4

4 6

2 8 Number of terrestrial sites

0 10 35 40 45 50 55 Latitude (° N) Figure 3 Distribution of habitat types of populations of S. gigantea between terrestrial sites (left axis, bottom bars) and sites next to water (right axis, top bars) for the cytotypes North American diploids (NA.2x, black)/tetraploids (NA.4x, densely dashed)/hexaploids (NA.6x, grey) and European tetraploids (EU.4x, widely dashed) at different latitudes (corresponding to regions per continent).

NA.2x and NA.4x did not differ in soil types. EU.4x shared three soil types with NA.2x and NA.4x, i.e. Podzols, Luvisols and Cambisols. NA.2x and NA.4x also occurred on Acrisols, a soil type that does not exist in Europe. In Eastern Europe, some sites were on Chernozems that do not exist in areas of NA.2x and NA.4x. The soils on which NA.6x grew —Chernozems and Kastanozems—were different from those of NA.4x. The general level of carbonate content in soils did not differ between cytotypes, except that NA.6x occurred on more carbonate- rich soils than NA.4x (Tab. 3). However, the phosphorus status of soils did vary significantly among cytotypes (Tab. 3), with NA.2x being associated with the most, and NA.6x with the least phosphorus-deficient soils. The soils where NA.4x grew were of intermediate phosphorus status and did not differ significantly from those of EU.4x.

Di s c u s s i o n

Habitat differentiation between ploidy levels in North America

The main soil variable influencing the occurrence of NA.2x was soil calcium content, which was consistently lower at NA.2x than NA.4x sites. However, there was no significant difference in soil carbonate, even though concentrations of carbonate levels are usually closely correlated

76 Habitat differentiation with those of calcium. In Ontario, NA.2x occurred on more acidic soils than NA.4x sites, which supports previous observations made in the region by J.C. Semple (personal communication). We also found a similar but non-significant association of NA.2x with acidic soils in New England, but not in Southern Appalachian Mts. These results suggest that NA.2x is a calcifuge sensu stricto, being excluded primarily from calcium rich soils. However, strength of habitat differentiation varies regionally, being strong in Ontario, less so in New England and very weak in the Southern Appalachians. Several mechanisms have been proposed to explain calcifuge behaviour. In addition to excessive calcium being toxic for calcifuge plants, they must be able to tolerate high concentrations of aluminium, manganese and iron (reviewed by Lee, 1999). In contrast, calcicole species are less vulnerable to calcium toxicity but must be able to cope with very low concentrations of available iron. And because phosphorus is bound in the form of apatite or apatite-like calcium compounds (Sumner, 2000), its availability is also much lower in calcareous soils than in silicate soils (Tyler, 1996). Calcicole plants can solubilise these compounds from calcareous soils by excreting high amounts of organic acids from their roots (Tyler & Strom, 1995); however, calcifuges do not excrete these compounds to the same extent and may therefore experience phosphorus deficiency in base-rich soils (Tyler, 1994, 1996). For example, ina study with five calcifuge herbs, plants grown in calcareous soil were found to take up excessive amounts of calcium but had low concentrations of phosphorus and a lower proportion of the total phosphorus was present as inorganic phosphate (Zohlen & Tyler 2004). Through an increase in number of alleles, polyploids may show increased variation in dosage-regulated gene expression, altered regulatory interactions and rapid genetic and epigenetic changes (Osborn et al., 2003; Soltis et al., 2003). In one of the few studies to relate calcium nutrition to ploidy level, expression of the calcium-dependent protein kinase gene IiCPK2 was greater in tetraploids of Isatis indigotica than in diploids; this gene is active in various pathways including responses to cold, high salinity and certain hormones (Lu et al., 2006). If there are similar changes in gene expression in S. gigantea, they could make tetraploids less sensitive to direct toxic effects of calcium or better able to acquire other nutrients such as phosphorus from calcium rich soils. Various lines of evidence suggest that NA.4x plants are more tolerant of soil conditions than NA.2x plants. For example, in the field NA.2x only occurred on five soil types whereas NA.4x occurred on nine. And in an experimental study, adding calcium decreased the growth of NA.2x while NA.4x and EU.4x were unaffected (unpublished data). This greater sensitivity of diploids than tetraploids may also apply to other species; for example, when NA.2x and lower ploidy levels of some aster species were transplanted to alien soils, they were less successful than when higher ploidy levels were transplanted (J.C. Semple, personal communication). The habitat differentiation in soil calcium and partly in pH value may explain the observed separation of diploid and tetraploid populations at small scales, especially within the overlap zones. However, this effect does not explain large-scale distribution patterns, since the ranges

77 Chapter 2 of both ploidy levels include soils with a wide range of calcium concentrations. Therefore, other mechanisms must be responsible for the separation between ploidy levels at a geographic scale. In the case of the separation between tetraploid and hexaploid populations we found significant differences in both climate and soil variables. Although this result could partially be due to sample bias—because we had no samples on the western side of the NA.4x range— NA.6x occur mainly in the prairies where continentality is high and precipitation and minimum temperatures are lower than in NA.4x area. Compared with the soils on which NA.4x was found, soil types of NA.6x tended to be richer in carbonate level, less phosphorus deficient and have a higher pH.

Habitat templates as barrier for introduction

Among the barriers faced by a newly introduced plant, poor adaptation to local abiotic conditions is thought to be particularly important (Richardson & Pysek, 2006; Theoharides & Dukes, 2007). If diploid S. gigantea differed greatly in their habitat requirements from tetraploids, then a habitat barrier could explain why no diploids are observed in Europe while tetraploids are very successful. We found that the habitat differences between NA.2x and EU.4x populations were broadly similar to those between NA.2x and NA.4x populations, e.g. soil calcium, effervescence and soil pH value, though there were continent-specific differences in soil phosphorus, habitat type and the range of C/N values. However, the observed habitat differences hardly seem adequate to explain absence of diploids from Europe, especially since large areas of Europe have calcium-poor soils. Possible alternative explanations could be that diploids were never introduced to Europe, or that life history traits of diploids make them poor colonisers. It is also possible that the tetraploids were introduced first and preempted suitable sites, so that diploids had no chance to establish in Europe. Other invasive species show similar patterns of distribution in regard to ploidy level. Centaurea maculosa is diploid and tetraploid in the native range in Europe, but only tetraploids occur in North America (U. Treier, personal communication, Müller-Schärer et al., 2004), and Senecio inaequidens is diploid and tetraploid in South Africa, but only tetraploids have been found in the introduced range in Europe (Lafuma et al., 2003). Also, five invasive species in Singapore are represented in only one of their ploidy level (Pandit et al., 2006) and similar examples have be identified in the alien flora of France (Verlaque et al. (2002). Although biological invasions cannot be explained by polyploidy alone (Ehrendorfer, 1980; Barrett & Richardson, 1986; Roy, 1990), polyploids do appear to be more frequent among invasive alien plants than among angiosperms in general. This has been shown for the ‘world’s worst weeds’ (Brown & Marshall, 1981), for British garden weeds (Bennett et al., 1998), for the flora of France (Verlaque et al., 2002) and for the flora of Singapore (Pandit et al., 2006).

78 Habitat differentiation

Niches shifts in introduced species

No evidence was found that European populations of S. gigantea have a different climatic range from that of NA.4x populations and a homoclime analysis indicates that large parts of Europe are climatically suitable (Weber, 2001). Jakobs et al. (2004) also found that climatic differences explained only a small part of variation in distribution of S. gigantea between the continents. However, for other species such as Centaurea maculosa, a climatic niche shift has been detected in alien populations (Broennimann et al., 2007). If such a shift occurs, the potential distribution of an invasive species cannot be reliably predicted from models based on climatic matching with the native range (Peterson & Vieglais, 2001; Broennimann et al., 2007). In North America, S. gigantea tends to grow in relatively moist habitats. For example, in a comparative study of five Solidago species in Pennsylvania, USA, S. gigantea was the species most strongly associated with permanently moist soils (Abrahamson et al., 2005). In our study, S. gigantea in North America occurred more consistently close to water than it did in Europe, confirming a similar trend found by Jakobs et al. (2004). This difference could be due a niche shift to a broader ecological range in Europe (i.e. an evolutionary change), but it could also reflect the less continental climate in Europe that allowed the species to grow in a wider range of habitats (i.e. an ecological response). Indeed, because the habitat difference was the same between EU.4x and both NA.2x and NA.4x, a climatic explanation seems more likely than a niche shift. In general, the populations of S. gigantea in North America occurred on soils with less total phosphorus than those in Europe. Like habitat type, this difference was the same between EU.4x and both NA.2x and NA.4x, and probably reflects differences in the frequency of phosphorus- rich sites in the two ranges. This in turn could be due both to differing soil types (with a higher proportion of more weathered soils such as Acrisols and Podzols in North America) and to a greater impact of agriculture on soil phosphorus concentrations in Europe. It may also be that S. gigantea enhances phosphorus turnover rates in invaded ecosystems; for example, sites in Belgium with S. gigantea had higher concentrations of extractable phosphorus than sites without S. gigantea (Chapuis-Lardy et al., 2006; Vanderhoeven et al., 2006). However, Güsewell et al. (2005) found no such differences between invaded and uninvaded sites in nutrient poor wetlands in Switzerland. Furthermore, phosphorus concentrations in the biomass were the same as in co-occuring native vegetation (Güsewell et al., 2005; Chapuis-Lardy et al., 2006) and as in North American S. gigantea grown in a common garden (Güsewell et al., 2006). Therefore, an explanation involving differences between continents, such as different soil types, again seems more likely than a niche shift.

79 Chapter 2

Co n c l u s i o n s

In North America, diploids, tetraploids and hexaploids of S. gigantea are geographically separated, with little overlap in the ranges of the three ploidy levels. Although this geographic separation is broadly associated with differences in climate, soil and habitat, these continental scale distributions are unlikely to have arisen simply through ecological differentiation, and other factors—such as differences in the ability of the various ploidy levels to colonise after the last glaciation—were more important. Similarly, it seems unlikely that habitat factors can explain the absence of diploids in the invasive range. Although we found differences in total soil phosphorus concentration and proximity to water between populations in Europe and North America, these may simply reflect differences in climatic and soil conditions on the two continents. Finally, there is no need to assume that any adaptive evolution was needed to make tetraploid S. gigantea invasive in Europe. NA.4x had already been a successful plant in North America. It has colonised a large new range not occupied by the diploid progenitor and is considered a weed in bottomland forest reforestations (Kruse & Groninger, 2003). This success of tetraploid S. gigantea, which can be partly related to good colonisation ability and to a wider habitat tolerance (e.g. soil calcium) compared to diploids, could be one reason for it becoming a successful invader in Europe.

Ac k n o w l e d g m e n t s

We thank John C. Semple (U of Waterloo, Canada) for help in the field and information on Solidago, Rose Trachsler and Marilyn Gaschen for soil nutrient analyses, Sabine Güsewell for statistical advice and Markus Hofbauer for assistance during the field survey. The project is funded by the grant 0-20259-05 from ETH Zurich, Switzerland.

Re f e r e n c e s

Abrahamson, W.G., Dobley, K.B., Houseknecht, H.R. & Pecone, C.A. (2005) Ecological divergence among five co-occurring species of old-field goldenrods. Plant Ecology, 177, 43-56

Allen, R.G., Pereira, L.S., Raes, D. & Smith, M. (1998) Crop evapotranspiration - Guidelines for computing crop water requirements. FAO Irrigation and drainage paper 56. FAO - Food and Agriculture Organizations of the United Nations, Rome

80 Habitat differentiation

Anonymous (1996) Schweizereische Referenzmethoden der Eidgenössischen landwirtschaftlichen Forschungsanstalten, Band 1 “Boden- und Substratuntersuchungen zur Düngeberatung”. Eidgenössische Forschungsanstalten FAL, RAC, FAW

Anonymous (2004) Assessing carbonates in the field with a dilute hydrochloric acid (HCl) solution. United States Department of Agriculture (USDA) - Natural Resources Conservation Service (NRCS).

Araujo, M.B. & Guisan, A. (2006) Five (or so) challenges for species distribution modelling. Journal of Biogeography, 33, 1677-1688

Baack, E.J. (2005) To succeed globally, disperse locally: effects of local pollen and seed dispersal on tetraploid establishment. Heredity, 94, 538-546

Baack, E.J. & Stanton, M.L. (2005) Ecological factors influencing tetraploid speciation in snow buttercups (Ranunculus adoneus): Niche differentiation and tetraploid establishment. Evolution, 59, 1936- 1944

Barrett, S.C.H. & Richardson, B.J. (1986) Genetic attributes of invading species. Ecology of biological invasions: an Australian perspective (ed. by R.H. Groves and J.J. Burdon), pp. 21-33. Australian Academy of Science, Canberra, Australia.

Batjes, N.H. (2005) ISRIC-WISE global data set of derived soil properties on a 0.5 by 0.5 degree grid (Version 3.0). Report 2005/08. ISRIC - World Soil Information, Wagenhingen, the Netherlands (with data set)

Bennett, M.D., Leitch, I.J. & Hanson, L. (1998) DNA amounts in two samples of angiosperm weeds. Annals of Botany, 82, 121-134

Brochmann, C., Brysting, A.K., Alsos, I.G., Borgen, L., Grundt, H.H., Scheen, A.-C. & Elven, R. (2004) Polyploidy in arctic plants. Biological Journal of the Linnean Society, 82, 521-536

Broennimann, O., Treier, U.A., Muller-Scharer, H., Thuiller, W., Peterson, A.T. & Guisan, A. (2007) Evidence of climatic niche shift during biological invasion. Ecology Letters, 10, 701-709

Brown, A.H.D. & Marshall, D.S. (1981) Evolutionary changes accompanying colonization in plants. Evolution Today. Proceedings of 2nd international Congress of Systematics and Evolutionary Biology (ed. by G.G.E. Scudder and J.L. Reveal), pp. 351-363. Carnegie-Mellon University, Pittsburg.

Burton, T.L. & Husband, B.C. (1999) Population cytotype structure in the polyploid Galax urceolata (Diapensiaceae). Heredity, 82, 381-390

Carpenter, G., Gillison, A.N. & Winter, J. (1993) Domain - a flexible modeling procedure for mapping potential distributions of plants and animals. Biodiversity and Conservation, 2, 667-680

Chapuis-Lardy, L., Vanderhoeven, S., Dassonville, N., Koutika, L.S. & Meerts, P. (2006) Effect of the exotic invasive plant Solidago gigantea on soil phosphorus status. Biology and Fertility of Soils, 42, 481-489

Conrad, V. (1946) Usual formulas of continentality and their limits of validity. Transactions of the American Geophysical Union, 27, 663-664

81 Chapter 2

De Wet, J.M.J. (1980) Origins of polyploidy. Polyploidy - Biological relevance (ed. by W.H. Lewis), pp. 3-15. Plenum Press, New York, London.

Debussche, M. & Thompson, J.D. (2003) Habitat differentiation between two closely related Mediterranean plant species, the endemic Cyclamen balearicum and the widespread C. repandum. Acta Oecologica-International Journal of Ecology, 24, 35-45

Driessen, P., Deckers, J. & Nachtergaele, F. (2001) Lecture notes on the major soils of the world. FAO, Rome.

Ehrendorfer, F. (1980) Polyploidy and distribution. Polyploidy - Biological relevance (ed. by W.H. Lewis), pp. 45-60. Plenum Press, New York, London.

Fairhurst, T., Lefroy, R., Mutert, E. & Batjes, N.H. (1999) The importance, distribution and causes of phosphorus deficiency as a constraint to crop production in the tropics. Agroforestry Forum, 9, 2-8

Fao-Agl (2005) Aquastat - Climate Information Tool FAO’s Information System on Water and Agriculture. [Internet database, viewed 26 June 2006]. URL http://www.fao.org/ag/AGL/AGLW/ aquastat/gis/index3.stm

Felber-Girard, M., Felber, F. & Buttler, A. (1996) Habitat differentiation in a narrow hybrid zone between diploid and tetraploid Anthoxanthum alpinum. New Phytologist, 133, 531-540

Güsewell, S., Jakobs, G. & Weber, E. (2006) Native and introduced populations of Solidago gigantea differ in shoot production but not in leaf traits or litter decomposition. Functional Ecology, 20, 575-584

Güsewell, S., Zuberbühler, N. & Clerc, C. (2005) Distribution and functional traits of Solidago gigantea in a Swiss lakeshore wetland. Botanica Helvetica, 115, 63-75

Hierro, J.L., Maron, J.L. & Callaway, R.M. (2005) A biogeographical approach to plant invasions: the importance of studying exotics in their introduced and native range. Journal of Ecology, 93, 5-15

Hollingsworth, M.L. & Bailey, J.P. (2000) Evidence for massive clonal growth in the invasive weed Fallopia japonica (Japanese Knotweed). Botanical Journal of the Linnean Society, 133, 463-472

Husband, B.C. & Sabara, H.A. (2004) Reproductive isolation between autotetraploids and their diploid progenitors in fireweed, Chamerion angustifolium (Onagraceae). New Phytologist, 161, 703-713

Isaaks, E.H. & Srivastava, R.M. (1989) Applied geostatistics. Oxford University Press, New York.

Jakobs, G., Weber, E. & Edwards, P.J. (2004) Introduced plants of the invasive Solidago gigantea (Asteraceae) are larger and grow denser than conspecifics in the native range. Diversity and Distributions, 10, 11-19

Johnson, M.T.J., Husband, B.C. & Burton, T.L. (2003) Habitat differentiation between diploid and tetraploid Galax urceolata (Diapensiaceae). International Journal of Plant Sciences, 164, 703- 710

Kadmon, R., Farber, O. & Danin, A. (2003) A systematic analysis of factors affecting the performance of climatic envelope models. Ecological Applications, 13, 853-867

82 Habitat differentiation

Kottek, M., Grieser, C., Beck, B., Rudolf, B. & Rubel, F. (2006) World map of the Köppen-Geiger climate classification updated. Meteorologische Zeitschrift, 15, 259-263

Kruse, B.S. & Groninger, J.W. (2003) Vegetative characteristics of recently reforested bottomlands in the lower Cache River Watershed, Illinois, USA. Restoration Ecology, 11, 273-280

Lafuma, L., Balkwill, K., Imbert, E., Verlaque, R. & Maurice, S. (2003) Ploidy level and origin of the European invasive weed Senecio inaequidens (Asteraceae). Plant Systematics and Evolution, 243, 59-72

Lee, J.A. (1999) The calcicole-calcifuge problem revisited. Advances in Botanical Research Incorporating Advances in Plant Pathology, Vol 29, 29, 1-30

Levin, D.A. (1975) Minority cytotype exclusion in local plant populations. Taxon, 24, 35-43

Li, B.H., Xu, X.M. & Ridout, M.S. (2004) Modelling the establishment and spread of autotetraploid plants in a spatially heterogeneous environment. Journal of Evolutionary Biology, 17, 562-573

Lu, B.B., Ding, R.X., Zhang, L., Yu, X.J., Huang, B.B. & Chen, W.S. (2006) Molecular cloning and characterization of a novel calcium-dependent protein kinase gene IiCPK2 responsive to polyploidy from tetraploid Isatis indigotica. Journal of Biochemistry and Molecular Biology, 39, 607-617

Lumaret, R. (1988) Adaptive strategies and ploidy levels. Acta Oecologica-Oecologia Plantarum, 9, 83-93

Maceira, N.O., Jacquard, P. & Lumaret, R. (1993) Competition between diploid and derivative autotetraploid Dactylis glomerata L. from Galicia - implications for the establishment of novel polyploid populations. New Phytologist, 124, 321-328

Mandakova, T. & Münzbergova, Z. (2006) Distribution and ecology of cytotypes of the Aster amellus aggregates in the Czech Republic. Annals of Botany, 98, 845-856

Müller-Schärer, H., Schaffner, U. & Steinger, T. (2004) Evolution in invasive plants: implications for biological control. TRENDS in Ecology & Evolution, 19, 417-422

New, M., Lister, D., Hulme, M. & Makin, I. (2002) A high-resolution data set of surface climate over global land areas. Climate Research, 21, 1-25

Osborn, T.C., Pires, J.C., Birchler, J.A., Auger, D.L., Chen, Z.J., Lee, H.S., Comai, L., Madlung, A., Doerge, R.W., Colot, V. & Martienssen, R.A. (2003) Understanding mechanisms of novel gene expression in polyploids. Trends in Genetics, 19, 141-147

Pandit, M.K., Tan, H.T.W. & Bisht, M.S. (2006) Polyploidy in invasive plant species of Singapore. Botanical Journal of the Linnean Society, 151, 395-403

Peterson, A.T. & Vieglais, D.A. (2001) Predicting species invasions using ecological niche modeling: New approaches from bioinformatics attack a pressing problem. Bioscience, 51, 363-371

Post, W.M. & Zobler, L. (2000) Global soil types, 0.5-degree grid (modified Zobler) data set. Available on- line [http://www.daac.ornl.gov] from Oak Ridge National Laboratory Distributed Active Archive Center, Oak Ridge, Tennessee, U.S.A. [Internet database, viewed 5 January 2007]. URL http:// www.daac.ornl.gov/SOILS/guides/ZoblerSoilDerived1.html

83 Chapter 2

R Development Core Team (2007) R: A language and environment for statistical computing. ISBN 3-900051-07-0. R Foundation for Statistical Computing, Vienna, Austria

Rausch, J.H. & Morgan, M.T. (2005) The effect of self-fertilization, inbreeding depression, and population size on autopolyploid establishment. Evolution, 59, 1867-1875

Rice, W.R. (1989) Analyzing tables of statistical tests. Evolution, 43, 223-225

Richardson, D.M. & Pysek, P. (2006) Plant invasions: merging the concepts of species invasiveness and community invasibility. Progress in Physical Geography, 30, 409-431

Rodriguez, D.J. (1996) A model for the establishment of polyploidy in plants. American Naturalist, 147, 33-46

Roy, J. (1990) In search of the characteristics of plant invaders. Biological invasions in Europe and the Mediterranean basin (ed. by F. Di Castri, A.J. Hansen and M. Debussche), pp. 335-352. Kluwer Academic Publishers, Dordrecht.

Semple, J.C. & Cook, R.E. (2006) Solidago. Flora of North America North of Mexico (ed. by Flora of North America Editorial Committee), pp. 107-166. Oxford University Press, Oxford and New York.

Sokal, R.R. & Rohlf, F.J. (1995) Biometry the principles and practice of statistics in biological research, 3rd edn. Freeman, New York.

Soltis, D.E., Soltis, P.S., Pires, J.C., Kovarik, A., Tate, J.A. & Mavrodiev, E. (2004) Recent and recurrent polyploidy in Tragopogon (Asteraceae): cytogenetic, genomic and genetic comparisons. Biological Journal of the Linnean Society, 82, 485-501

Soltis, D.E., Soltis, P.S. & Tate, J.A. (2003) Advances in the study of polyploidy since “Plant speciation”. New Phytologist, 161, 173-191

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 of the United States of America, 97, 7051-7057

Stebbins, G.L. (1971) Chromosomal Evolution in Higher Plants. Edward Arnold, London.

Sumner, M.E. (2000) Handbook of soil science. CRC Press, Boca Raton, Fla.

Theoharides, K.A. & Dukes, J.S. (2007) Plant invasion across space and time: factors affecting nonindigenous species success during four stages of invasion. New Phytologist, 176, 256-273

Thompson, J.D. & Lumaret, R. (1992) The evolutionary dynamics of polyploid plants - origins, establishment and persistence. Trends in Ecology & Evolution, 7, 302-307

Thompson, J.N., Nuismer, S.L. & Merg, K. (2004) Plant polyploidy and the evolutionary ecology of plant/animal interactions. Biological Journal of the Linnean Society, 82, 511-519

Tyler, G. (1994) A new approach to understanding the calcifuge habit of plants. Annals of Botany, 73, 327-330

Tyler, G. (1996) Mineral nutrient limitations of calcifuge plants in phosphate sufficient limestone soil. Annals of Botany, 77, 649-656

84 Habitat differentiation

Tyler, G. & Strom, L. (1995) Differing organic acid exudation pattern explains calcifuge and acidifuge behavior of plants. Annals of Botany, 75, 75-78

Van Dijk, P. & Bakx-Schotman, T. (1997) Chloroplast DNA phylogeography and cytotype geography in autopolyploid Plantago media. Molecular Ecology, 6, 345-352

Vanderhoeven, S., Dassonville, N., Chapuis-Lardy, L., Hayez, M. & Meerts, P. (2006) Impact of the invasive alien plant Solidago gigantea on primary productivity, plant nutrient content and soil mineral nutrient concentrations. Plant and Soil, 286, 259-268

Verlaque, R., Aboucaya, A. & Fridlender, A. (2002) Invasive alien flora of France: ecology, life-forms and polyploidy. Botanica Helvetica, 112, 121-136

Weber, E. (2001) Current and potential ranges of three exotic goldenrods (Solidago) in Europe. Conservation Biology, 15, 122-128

Weber, E. & Schmid, B. (1993) Das Neophytenproblem. Dissertationes Botanicae, 196, 209-227

85

Common garden experiment

Ch a p t e r 3

Ploidy-level precise common garden with native and invasive Solidago gigantea (Asteraceae): differences in clonal growth and nutrient responses indicate colonization success of tetraploids

Daniel R. Schlaepfer, Peter J. Edwards, Regula Billeter

Institute of Integrative Biology, Plant Ecology, ETH Zurich, Zürich, Switzerland

87 Chapter 3

Su m m a r y

1. Many studies have compared the growth of plants from native and invasive populations, but few have considered the role of ploidy. In its native range in North America, Solidago gigantea Aiton (Asteraceae) occurs as diploid, tetraploid and hexaploid, with considerable habitat differentiation and geographic separation amongst these ploidy levels. In the introduced range in Europe, however, only tetraploid populations are known. 2. We investigated the growth performance and life history characteristics of plants from 12 European and 24 North American (12 diploid, 12 tetraploid) populations in a common garden experiment involving two nutrient and two calcium treatments. Twelve plants per population were grown in pots for two seasons. We measured leaf nutrient concentrations, plant size, biomass production, phenology and sexual reproduction, and counted numbers of shoots and rhizomes. 3. Native diploid plants had a higher specific leaf area and higher leaf nutrient concentrations than native tetraploids, but tetraploids produced many more shoots and rhizomes. Diploids grown with additional calcium produced less biomass whereas tetraploids were not affected. European plants were less likely to flower and produced smaller capitulescences than North American tetraploids, but biomass production, shoot and rhizome number did not differ. 4. We conclude that a knowledge of ploidy level is essential in comparative studies of invasive and native populations. While clonal growth is important for invasion success of S. gigantea, its potential was not acquired by adaptation after introduction but by evolutionary processes in the native range.

Key-words: Calcium, latitudinal cline, plant introductions, polyploidy, rhizome system.

88 Common garden experiment

In t r o d u c t i o n

One approach to understanding why some introduced plants become invasive is to compare the growth of native and invasive species (e.g. Daehler, 2003). This approach has been used to investigate many of the proposed attributes of successful invaders—high specific leaf area (SLA), high nutrient concentrations (Daehler, 2003), rapid nutrient cycling (Ehrenfeld, 2003)—as well as specific mechanisms such as the enemy release (Colautti et al., 2004) and the novel weapons hypotheses (Callaway & Ridenour, 2004). Another approach is to compare the growth of populations of the same species in the native range and introduced ranges (e.g. Hierro et al., 2005). In some cases, plants from invasive populations have been found to grow larger than plants from the native range (Bossdorf et al., 2005), especially in the absence of competition (Blumenthal & Hufbauer, 2007) or enemies (Colautti et al., 2004); in other cases, however, no such difference has been found (e.g. Clidemia hirta, Dewalt et al., 2004; Lepidium draba, McKenney et al., 2007). Recently there has been considerable interest in the idea that these differences may have arisen through evolutionary changes in introduced populations. Such evolutionary processes may include niche shifts to more suitable habitats in the invasive range (Broennimann et al., 2007), hybrid vigour due to mixing of previously isolated populations (Ellstrand & Schierenbeck, 2000) and the evolution of increased competitive ability due to resource transfer from defence to plant growth because of enemy release (EICA, Blossey & Notzold, 1995). However, despite recent research efforts, there is no general understanding about where and when such processes may be important (Dietz & Steinlein, 2004; Theoharides & Dukes, 2007). Solidago gigantea Aiton (Asteraceae) was introduced in 1758 to Europe (Weber & Schmid 1993) and is now invasive in a variety of habitats including grassland, wetland and forest on both nutrient-poor and more fertile sites (Weber & Jakobs, 2005). It is a perennial herb 0.5– 2.5 m tall, with annual, partially sexual shoots and persistent, clonal rhizomes. The breeding system involves obligate outcrossing (Melville & Morton, 1982). In its native range in North America, S. gigantea occurs as a diploid, tetraploid and hexaploid. Populations on the eastern side of the Appalachian Mountains and also in southern Ontario and Québec are diploid (NA.2x). Tetraploids are found in eastern North America as far west as Missouri (NA.4x), while hexaploids occur in mid-western North America, westwards from Manitoba to the Rocky Mountains (chapter 1, Semple & Cook, 2006). In Europe, only tetraploids have been detected (EU.4x, chapter 1). Various processes may account for the success of S. gigantea as an invasive species in Europe. First, S. gigantea has been shown to increase concentrations of nitrogen (Vanderhoeven et al., 2005) and labile phosphorus in the soil, probably because of higher rates of mineralization (Chapuis-Lardy et al., 2006) and organic matter turnover (Koutika et al., 2007). However, in a mesotrophic wetland lakeshore site, invasive S. gigantea had no impact on total soil nitrogen and phosphorus (Güsewell et al., 2005). Furthermore, with the exception of its effects on soil

89 Chapter 3 phosphorus, which appear to be site specific (Vanderhoeven et al., 2006), the impact of S. gigantea on soil conditions is rather small. Secondly, in the closely related species, Solidago canadensis, allelopathic effects have been detected, e.g. arbuscular mycorrhizal colonization of native plant species are inhibited (Zhang et al., 2007) and germination rate and seedling growth of native species are decreased (Yang et al., 2007); it seems plausible that similar affects may also apply in S. gigantea. Thirdly, introduced plants may suffer less damage from natural enemies than plants in the native range (i.e. enemy release), and there is some evidence for associated evolutionary changes. For instance, in one study invasive populations of S. gigantea had lowered levels of some secondary defences compared to native populations (Johnson et al., 2007), but not in another (Hull-Sanders et al., 2007). While larvae of a generalist lepidopteran grew better on plants from invasive than native populations, no effect was detected for three specialist herbivores (Hull-Sanders et al., 2007). Also, invasive populations were more susceptible to some leaf pathogens than native populations (Meyer et al., 2005), though both compensated for insect herbivory to the same degree, albeit with different strategies (Meyer & Hull-Sanders, 2007). Such differences in enemy defence and susceptibility could be explained by evolutionary changes after introduction in response to enemy release (Meyer & Hull-Sanders, 2007) or, non-exclusively, by founder effects coupled with clonal spread (Johnson et al., 2007). Since only a few tetraploid haplotypes of S. gigantea appear to have been introduced (chapter 4), and because particular genotypes were probably selected as ornamentals and nectar plants for bees (Weber & Jakobs, 2005), founder effects may well have played a significant role. While enemy release may have contributed to the success of S. gigantea in Europe, the evidence for EICA is far from conclusive (Güsewell et al., 2006; Meyer & Hull-Sanders, 2007). However, none of the studies on S. gigantea has taken account of ploidy level, although this factor has considerable implications for both growth performance (Maceira et al., 1993; Bretagnolle & Thompson, 1996; Petit & Thompson, 1997), life history (Müller, 1989) and plant- enemy interactions (Thompson et al., 2004; Münzbergova, 2006; Halverson et al., 2008). For S. gigantea, habitat differentiation in regard to soil calcium tolerance has been detected in the native range, with diploids being calcifuge and tetraploids insensitive to calcium concentration (chapter 2). Similarly, geographic separation between ploidy levels in North America suggests different colonization abilities (chapter 2). The aims of this study were: (i) to investigate whether native diploids and tetraploids differ in growth performance, life history and responses to nutrient and soil calcium treatments, (ii) to relate any such differences to the habitat requirements and geographic distribution of these ploidy levels, and (iii) to compare the same characteristics of native and invasive tetraploids and evaluate whether evolutionary change after introduction was more important in enabling introduced populations to become invasive than being polyploid.

90 Common garden experiment

Me t h o d s

Experimental setup

In autumn 2005, we measured population performance and sampled seeds of S. gigantea in North America (six NA.2x and six NA.4x populations per region: Southern Appalachian Mts. (SA, ~35°N), and Southern Ontario (ON, ~45°N)), and in Europe (six EU.4x populations per region: Northern Italy (IT, ~45°N), and Belgium (BE, ~50°N)). Population locations, vouchers and ploidy level determination are described in chapter 1. Subsequently, seeds from 16 randomly chosen individual shoots per population were germinated in climate chambers; one plant per seed-family was re-potted and transferred to a cold green house. To minimize possible maternal effects, experimental plants were pre-cultivated for four months after germination and all but one shoot was eliminated at the start of the experiment, so that initial biomass corresponded to 4% of first season biomass production. At the end of April 2006, 12 plants per population (432 plants in total) were chosen for their similar size. These were randomly assigned to a treatment combination (three replicates) and re-potted to 7.5 L pots in rhododendron soil (Klasmann-Deilmann GmbH, Geeste, Germany). The pots were transferred to the experimental garden in Zürich (Switzerland) at 570 m a.s.l. To prevent edge effects, all pots were fully re-randomised once every month until July and once at the beginning of the second season. For the nutrient treatment, a calcium-free, slow-release fertilizer was used (Osmocote exact standard 8–9 months, Scotts, Heerlen, The Netherlands). In the second season, the plants under the high nutrient level were fertilized with a commercial complete fertilizer (Wuxal, Maag Agro, Dielsdorf, Switzerland). In total, plants at the low nutrient level were supplied with 1.0 g N and 0.4 g P and at the high level with 2.5 g N and 0.9 g P. The calcium treatment was administered as calcium carbonate (Sigma-Aldrich, Seelze, Germany) to the plants under the high treatment level. These plants received in total 39.8 g Ca. The plants under the low level received no extra calcium. The rhododendron soil itself contained ≈ 5.5 g Ca per pot. At the end of the experiment, the soil pH was 3.8 ± 0.06 (n = 12) in the low calcium pots and 5.3 ± 0.13 (n = 12) in the high calcium pots. There was a significant difference between calcium treatment levels (ANOVA, F = 131.1, p < 0.001), but neither among continents, ploidy levels nor nutrient treatment levels. An additional experiment with 30 plants on a soil pH gradient (4–7), adjusted with sodium hydroxide, showed no effect of soil pH on plant biomass (ANOVA, F = 0.2, p ≥ 0.942). Aphid attack was treated with Paraderil (active ingredient: rotenon; Maag Agro). Fungal attack, mostly mildew, was treated with Funginex (triforine; Maag Agro). Attack rates were low and differed not between cytotypes (data not shown). The high nutrient level increased pathogen attack significantly.

91 Chapter 3

In 2006, May and August were exceptionally wet, whereas July and September to November were exceptionally hot. In 2007, April was exceptionally hot and dry, whereas May was exceptionally hot and wet. Therefore, in many months of the growing season the plants were watered daily with (calcium-free) osmosis water.

Measurements

To control for possible differences in initial size, the experimental plants were measured at the start of the experiment (shoot number, diameter and height and leaf length and width) and pruned to one shoot of similar size. An additional 70 plants were measured and harvested. Initial biomass of experimental plant was estimated based on a linear multiple regression 2 (r adj = 0.65, p < 0.001). During 2006, various parameters (height of tallest shoot, number of shoots and portion of shoot having senescent leaves) were recorded in May, June, July, September and at the harvest in November. Any dead leaves were also collected and dried. Differential growth during the season was quantified as age of the plant when reaching 50% of maximum shoot height (in days after 1 May). Leaf senescence was assessed by age of the plant when 50% of shoot height was covered with dead leaves (in days after 1 May). Allocation to vegetative reproduction was quantified as percentage of non-flowering shoots. At the harvest in November 2006, shoot diameter and number of latent buds visible on the soil surface were recorded. (S. gigantea is a hemicryptophytic species, and the number of buds provides an indication of potential vegetative reproduction). Above-ground biomass was harvested and dried at 72 °C for two days before weighing (together with dead leaves). Three healthy leaves per plant were harvested in mid August, dried and pooled per population and treatment combination. For nitrogen and total phosphorus concentrations, samples were digested in concentrated sulphuric acid (1 h refluxing at 420 °C with a potassium sulphate - copper sulphate Kjeltab; 2040 Digestor, Foss Tecator, Höganäs, Sweden) and analysed colorimetrically on a flow injection analyser (FIAstar 5000 Analyser, Foss Tecator, Höganäs, Sweden). Total calcium concentrations were measured in hydrochloric acid extracts (Hunt, 1982) with an AAS (SpectrAA 240 FS, Varian AG, Zug, Switzerland). Additionally, one healthy mature mid-shoot leaf per plant was collected (Garnier et al., 2001). Leaf fresh and dry mass and area (LI-3100 Area Meter, Licor Inc., Lincoln, USA) were determined. Specific leaf area (SLA, area per dry mass) and leaf dry matter content (LDMC, dry per fresh mass) were calculated. To assess sexual reproduction and phenology in 2006, plants were checked every 2nd–4th day during the growing season. Pollination was guaranteed by a large number of pollinators. Pollinator flights within ploidy level were as frequent as between ploidy levels (Binomial test, p ≥ 0.383; n = 21 pollinators visits). First appearance of flower buds, onset of flowering (first flower head open) and end of flowering (last flower head closed) were recorded for every shoot

92 Common garden experiment

(capitulescence). Percentage of flowering plants, number of flowering shoots, duration of flower per shoot and per plant, plant age at start of flowering, at peak flower (median date of most shoots in flower) and at end of flowering were calculated (in days after 1 May). At the end of flowering, capitulescences were measured, clipped, dried and weighted. Capitulescence biomass and total seed production are highly correlated in S. altissima (Meyer et al., 2005). Allocation to sexual reproduction was calculated as ratio of capitulescence biomass to total biomass. In 2007, plants were harvested when the first flower buds appeared (beginning of June). Height of the longest shoot and number of shoots were measured and above-ground biomass was harvested. The rhizomes were counted along two meridians on the outside of root balls to provide an index of total rhizome length. Rhizome diameter and number of visible buds per 10 cm of rhizome were measured on three rhizome pieces from one plant per population and treatment combination.

Data analysis

We considered the treatments as nested in population and aggregated the data into the unit population*treatments (i.e. mean of the three replicates). Therefore, interactions were tested against populations and not against plants. Threefold and higher interactions were considered significant at the 0.01 level, whereas other significance levels were at 0.05. To test whether measured variables differed between NA.2x and NA.4x (2x-4x comparison), linear mixed models (LMM) with a crossed design were used and included ploidy level, region, calcium treatment and nutrient treatment as fixed factors, population (nested within region) as a random factor and initial biomass as a covariate. Variables were transformed if needed to meet the assumptions of the model. To test whether measured variables differed between NA.4x and EU.4x (NA-EU comparison), LMMs with a nested design were used and included continent, region (nested within continent), calcium treatment and nutrient treatment as fixed factors, population (nested within region) as random factor and initial biomass as covariate. Interactions only with the factor continent were included (region interactions are redundant). Variables were transformed if needed to meet the assumptions of the model. For significant interactions between treatments and ploidy level or continent, responses were calculated as reaction norms, i.e. difference between means for high and low treatment level divided by the overall mean (Güsewell, 2005). We calculated coefficients of variation for each cytotype and tested for differences among cytotypes with c2 tests (Zar, 1999). We also calculated Pearson correlation coefficients between variables measured in the common garden and at the original field sites. Differences in numbers of significant correlations between cytotypes were tested with two-sample tests on

93 Chapter 3

Poisson counts (Zar, 1999). Significance levels were adjusted for both tables with the sequential Bonferroni method (Rice, 1989). All analyses were calculated with SPSS 13.0.0 (SPPS Inc., Chicago, USA).

Re s u l t s

General remarks about plant performance

All 432 plants survived the experiment and 291 flowered (Tab. 1). The phenology of the experimental plants was similar to that of wild populations growing nearby. In the 2x-4x comparison, diploid plants grew faster than tetraploids in the first season but not in the second (Tab. 2). However, in the NA-EU comparison, similar trends were obtained in both years (Tab. 3). Both treatments affected tissue nutrient concentrations, indicating that the treatment levels were adequate to influence plant performance. The nutrient treatment significantly increased the concentration of leaf nitrogen in plants in the 2x-4x comparison (low nutrients, mean±SD = 1.8±0.5% N; high nutrients, mean = 2.0±0.6% N; Tab. 2), though there was a similar but non-significant trend in the NA-EU comparison (low mean = 1.6±0.5% N; high mean = 1.8±0.5% N; Tab. 3). Leaf phosphorus was not significantly increased in either comparison (mean = 0.14±0.03% P). The leaf N/P ratios varied between 7 and 19 suggesting that growth was limited primarily by N (mean N/P ratio = 12.5 < 14 (Güsewell, 2004), t-test = - 7.6, p < 0.001). The calcium treatment significantly increased leaf calcium in both comparisons (low mean = 0.84±0.18% Ca; high mean = 0.97±0.17% Ca; Tabs 2 and 3).

Variation among populations and regions

The variation in plant performance among populations within regions was mainly small, and any differences in mean parameter values were only weakly significant (Tabs 1–3). However, there was considerable variation between the two North American regions: thus, biomass

Table 1 Parameters of plant growth, vegetative and sexual reproduction and phenology measured on plants from 12 diploid, 12 North American and 12 European tetraploid populations of Solidago gigantea Aiton grown in a common garden. Coefficient of variation (CV) for each cytotype and c2 test for differences among cytotypes. 1The largest CV is indicated in bold if the corresponding c2 test is significant.2 Differences among cytotypes were tested per variable with c2 tests (Zar, 1999). Significance levels were sequential Bonferroni adjusted (***, p < 0.001, **, p < 0.01).

94 Common garden experiment 2 *** *** *** *** ** *** *** *** *** *** *** *** 1 test value 2 2 0.34 1.65 1.30 2.52 1.43 1.01 4.91 4.71 6.62 0.00 1.61 1.28 5.42 0.83 2.89 2.85 9.16 0.99 6.54 8.35 9.28 c c 17.54 13.98 35.78 24.08 13.53 50.87 10.99 74.14 29.21 26.01 20.75 28.18 28.34 5% 6% 6% 4x EU 31% 49% 45% 40% 12% 24% 17% 14% 22% 28% 19% 24% 28% 10% 29% 31% 47% 61% 18% 46% 36% 86% 85% 88% 29% 21% 17% 14% 12% 67% 127% 8% 8% 7% 4x CV 38% 51% 49% 35% 17% 31% 20% 21% 21% 32% 17% 22% 24% 18% 29% 37% 56% 57% 20% 53% 24% 62% 61% 69% 24% 14% 14% 82% 21% 17% 100% NA Coefficient of variation Coefficient of 5% 9% 7% 9% 49% 44% 20% 17% 26% 17% 30% 40% 11% 30% 31% 56% 25% 53% 54% 49% 66% 67% 46% 36% 20% 2x 61% 89% 29% 51% 24% 14% 30% 104% 168% ±2.1 ±0.34 ±3.9 ±0.19 ±0.1 ±3 ±2 ±1 ±1 ±0.03 ±0.1 ±0 ±1.1 ±11 ±1.7 ±0 ±0.01 ±0 ±1 ±1 ±1 ±0.1 ±0 ±0.05 ±0.1 ±0.01 ±0.7 ±0.63 ±4 ±2 ±1 ±3 ±3 ±3 4x EU 5 69 62 31 12 15 12 87 19 21 36 33 7.6 1.7 4.2 1.2 5.2 145 118 154 263 134 45.5 4.79 59.8 3.37 0.95 0.14 0.33 0.94 0.49 0.08 4.58 121.9 ±2.8 ±0.45 ±4.2 ±0.19 ±0.2 ±5 ±2 ±2 ±2 ±0.03 ±0.1 ±0 ±0.8 ±8 ±1.5 ±0 ±0.02 ±0 ±1 ±1 ±1 ±0.1 ±0 ±0.03 ±0.3 ±0.01 ±0.7 ±0.5 ±7 ±2 ±1 ±3 ±3 ±3 4x NA 6 NA-EU comparison NA-EU 76 57 11 10 16 17 50 50 39 4.2 7.7 8.1 1.7 2.2 144 112 164 111 218 137 51.3 6.18 59.6 3.74 0.88 0.14 26.6 0.31 0.79 0.85 0.15 5.02 126.3 ±2.3 ±0.54 ±5.5 ±0.23 ±3 ±3 ±2 ±0.04 ±1.1 ±1.8 ±0 ±0.02 ±1 ±1 ±2 ±0.1 ±0 ±0.03 ±0.01 ±0.8 ±0.65 ±3 ±2 ±0.2 ±3 ±0.1 ±0.01 ±10 ±2 ±0.2 ±5 ±2 ±1 ±3 4x 5 70 52 10 10 20 93 89 19 41 48 36 7.5 0.9 1.9 3.8 1.6 145 191 117 144 41.9 5.17 52.2 3.06 24.2 0.31 0.82 0.88 0.23 10.3 7.44 0.14 130.9 Ontario Mean ± SE ±1.6 ±0.16 ±4.8 ±0.3 ±2 ±1 ±2 ±0.03 ±0.6 ±2.3 ±0 ±0.01 ±0 ±3 ±0 ±0.2 ±1 ±0.06 ±0.02 ±0.7 ±0.53 ±2 ±2 ±0.2 ±4 ±0.1 ±0 ±4 ±1 ±0.1 ±4 ±2 ±1 ±2 2x 0 6 74 77 12 47 57 12 20 29 26 0.3 4.1 4.7 6.8 2.4 1.3 151 122 151 123 134 26.8 2.43 46.6 3.86 0.85 0.17 19.3 0.95 0.39 0.09 3.84 159.9 ±4.4 ±0.67 ±6.2 ±0.23 ±3 ±2 ±2 ±0.03 ±1.1 ±2 ±0.01 ±0.03 ±1 ±1 ±1 ±0.2 ±1 ±0.05 ±0.01 ±0.8 ±0.27 ±2 ±2 ±0.3 ±7 ±0.1 ±0 ±9 ±1 ±0.5 ±13 ±3 ±1 ±2 4x 7 2x-4x Comparison 2x-4x 83 63 11 11 12 16 58 51 42 8.8 1.5 0.3 4.6 2.7 5.1 2.6 134 142 245 132 156 183 60.7 7.18 66.9 4.43 0.85 29.1 0.75 0.82 0.07 0.14 121.8 ±4.5 ±1.32 ±5.6 ±0.45 ±0.3 ±4 ±3 ±2 ±3 ±0.03 ±0.1 ±0 ±1.2 ±12 ±2.8 ±0 ±0.04 ±0 ±0 ±1 ±1 ±0.2 ±0 ±0.02 ±0.2 ±0.01 ±1.1 ±0.4 ±30 ±2 ±1 ±2 ±2 ±2 2x 8 3 5 5 98 51 10 18 50 35 S. Appalachian Mts. S. 1.7 0.3 2.6 161 121 234 248 119 144 170 87.5 58.2 5.77 0.95 31.2 0.58 0.97 0.12 11.2 5.11 11.2 0.14 15.77 138.3 Leaf mass (mg) Leaf area (cm2) Sexual mass ratio Number of rhizomes Vegetative shoot ratio Vegetative Number of shoots 2006 Rhizome diameter (mm) Rhizome Flowering plants (% of all) Rhizome buds (per 10 cm) Rhizome Age at end of flowering (d) Biomass per plant 2006 (g) Biomass per plant 2007 (g) Specific leaf area (cm2 g-1) Biomass per shoot 2006 (g) Biomass per shoot 2007 (g) Number of latent buds 2006 Age at 50% shoot height (d) Age at onset of flowering (d) Age with 50% dead leaves (d) Age with 50% dead leaves (June) shoot length 2007 (cm) Leaf calcium concentration (%) Leaf calcium concentration Flowering duration per plant (d) Flowering duration Leaf nitrogen concentration (%) Leaf nitrogen concentration Flowering duration per shoot (d) Flowering duration Maximum shoot length 2006 (cm) Number of shoots 2007 (≥ 15 cm) Leaf phosphorus concentration (%) Leaf phosphorus concentration Age at peak of flowering shoots (d) Flowering shoots per flowering plant Volume of main capitulescence (cm3) Volume Main shoot diameter (at harvest, mm) Main shoot diameter (at harvest, Leaf dry matter content (dry/fresh biomass) Capitulescence biomass per flowering plant (g) Capitulescence biomass per flowering shoot (g) Size: Phenology: Leaf traits: Vegetative reproduction Shoots: Rhizomes: Sexual reproduction Biomass production: Size: Phenology: Plant growth: Biomass production: Variable

95 Chapter 3 production, plant size (Fig. 1) and timing of flowering were all lower in the more northerly ON populations than in SA, while leaf senescence, SLA and vegetative shoot ratio were higher (Tab. 2). The nutrient treatment had a stronger effect on the growth of the ON plants—significant for biomass production and shoot number—than it did on SA plants. The latitudinal trends in EU.4x were less pronounced than those for NA4x, but were broadly congruent with those in NA (Tab. 3): biomass production, shoot length (both second season) and timing of flowering were negatively correlated with latitude while some reproductive traits were positively correlated.

Effects of cytotypes and cytotype-region interactions

Most parameters varied significantly with ploidy level, and there were also several significant interactions between ploidy and region (Tab. 2). NA.4x plants differed from NA.2x plants in producing heavier leaves with a lower SLA and less nitrogen than NA.2x (Fig. 2), more shoots (Fig. 3(b)) and more biomass (Fig. 3(c), both second season); they also had a longer flowering period than NA.2x plants. However, by far the greatest difference between the ploidy levels was in the number of rhizomes, with a mean count of 2 rhizomes per pot for NA.2x and 16 for NA.4x (Fig. 4). The significant cytotype-region interactions arose because the values of several variables were higher for SA.2x than for SA.4x, but lower for ON.2x than for ON.4x (Fig. 1), or in the case of phenological variables the reverse of this pattern. Thus, NA.4x plants tended to vary less between regions than did NA.2x plants.

180 c 160 d 140 120 a 100 a a 80 b 60 40 Shoot height 2006 (cm) 20

Belgium Italy Ontario S App Figure 1 Variation of shoot height of Solidago gigantea Aiton in the first season of the common garden experiment between regions of origin (S App, Southern Appalachian Mts.) and cytotypes (EU.4x, black bars; NA.2x; widely hashed; NA.4x, densely hashed). Error bars indicate ± 1 SE of the means. Homogeneous subsets are based on Dunnett T3 post hoc tests.

96 Common garden experiment

180 160 c 140 b 120 a 100 aba 80 60 EU.4x 40 NA.2x 20 NA.4x Specific leaf area (cm2 g-1) 0 0 0.5 1 1.5 2 2.5 3 Leaf N concentration (%) Figure 2 Leaf nitrogen concentration and specific leaf area differ between ploidy levels ofS. gigantea. Mean and one standard deviation are shown for each cytotype (EU.4x, NA.2x, NA.4x). Homogeneous subsets are based on LMM main effects (Tabs 2 and 3).

There were no significant differences between NA and EU tetraploids in the number of rhizomes (Fig. 3(a)) and shoots (Fig. 3(b)), nor in biomass production (Fig. 3(c), Tab. 3). However, EU.4x plants produced more latent buds and larger leaves with a lower SLA, while NA.4x plants (Fig. 2) were taller, and produced larger capitulescences that persisted for a longer period.

Responses to nutrient treatment

Plant growth was stimulated by the addition of nutrients, with most variables responding positively to this treatment. The only parameters showing a consistently negative response were vegetative shoot ratio in the 2x-4x comparison (nutrients increased number of flowering shoots) and leaf dry matter content in the NA-EU comparison (Tabs 2 and 3). NA.2x and NA.4x plants differed in their responses to nutrients. NA.4x responded more strongly than NA.2x in numbers of rhizomes (Fig. 3(d)), latent buds and shoots (Fig. 3(e)), but more weakly in biomass production (Fig. 3(f)) and rhizome diameter. Comparing the effect of the nutrient treatment between continents, NA.4x plants responded more strongly than EU.4x plants in both shoot number (Fig. 3(e), second season) and duration of flowering.

97 Ploidy Nutrients Ploidy Region Calcium Ploidy Region N Pop Variable Ploidy Region * region = N * N * N = Ca * Ca * Ca * Ca (region) Plant growth: Biomass production: Biomass per plant 2006 (g) -5.3* -62.0*** -23.9*** +228.1*** +0.2 +19.7*** -4.8* ±3.1 -0.0 0.4 2.7** Biomass per shoot 2006 (g) 1 -6.1* -57.9*** -54.8*** +22.2*** +3.5 +9.2** -2.7 -1.8 ±0.1 1.6 1.2 Biomass per plant 2007 (g) +7.0* -16.7*** -1.4 +1874.2*** +6.4* +15.6*** -3.2 ±11.6** -0.3 0.7 2.1* Biomass per shoot 2007 (g) -26.9*** -18.6*** -0.4 +151.8*** +18.5*** +7.2** +0.4 ±7.4** +0.4 0.4 1.2 Size: Main shoot diameter (at harvest, mm) -10.5** -38.2*** -17.1*** +27.2*** +0.6 +2.4 -5.6* -0.5 -0.6 0.0 2.1* Maximum shoot length 2006 (cm) +0.3 -77.9*** -18.1*** +2.6 +0.2 ±1.6 -3.1 ±2.6 -0.0 0.3 1.8 (June) shoot length 2007 (cm) -28.1*** -34.0*** -4.4* +149.1*** +0.4 +0.4 +0.2 ±8.4** ±0.1 0.7 1.2 Phenology: Age at 50% shoot height (d) -2.1 +0.2 ±44.2*** +0.4 ±0.0 +0.7 -1.1 -0.3 -0.0 0.0 1.8 Age with 50% dead leaves (d) +14.8*** +10.5** +20.6*** +3.3 +0.0 +1.1 -1.9 ±1.0 -0.0 1.6 1.1 Leaf traits: Leaf calcium concentration (%) -0.2 -1.5 ±6.2* -3.4 -0.1 -0.2 +27.0*** +0.5 +0.9 2.0 1.0 Leaf nitrogen concentration (%) -13.5** +21.4*** +1.7 +5.8* +0.7 +0.0 -0.3 ±0.7 ±0.7 0.9 0.6 Leaf phosphorus concentration (%) -9.1** +8.2** ±4.4* +3.9 ±0.8 ±0.0 +0.4 ±0.3 ±0.8 3.5 0.6 Leaf area (cm2) +0.3 -26.5*** -8.6** +9.5** +2.4 +1.2 +0.0 ±0.0 ±0.0 1.1 Leaf mass (mg) +11.3** -30.5*** -6.2* +6.3* +1.1 +1.4 -3.8 -0.0 -0.9 2.3 0.0 Specific leaf area (cm2 g-1) -93.3*** +17.4*** +5.5* +0.9 +0.0 +0.6 +19.4*** +0.0 +1.6 2.1 0.2 Leaf dry matter content (dry/fresh biomass) +4.0 +0.0 ±2.7 -2.8 -0.0 -1.3 -24.0*** -0.6 -12.8*** 0.0 0.5 Vegetative reproduction Vegetative shoot ratio 2 +0.0 +21.9*** +16.1*** -4.4* -2.0 -0.8 +0.9 ±1.2 +0.2 2.2 2.2* Shoots: Number of shoots 2006 +2.1 +3.5 ±9.4** +1.5 ±1.3 ±3.8 +0.5 ±0.6 ±0.3 2.0 2.1* Number of shoots 2007 (≥ 15 cm) +35.4*** +1.6 +0.3 +143.7*** +21.2*** +7.0* -6.4* -1.7 -0.0 0.0 2.1* Number of latent buds 2006 -132.4*** +30.9*** ±63.5*** +25.8*** +6.2* +8.8** -11.9** -2.1 -2.5 1.6 1.5 Rhizomes: Number of rhizomes 3 +175.9*** +0.3 ±19.8*** +3.3 ±7.9** +0.2 -7.2** -0.3 -1.2 0.5 1.9 Rhizome diameter (mm) -5.6* -13.0*** -0.0 +18.0*** +5.7* +1.0 -0.1 ±3.0 ±1.0 1.3 Rhizome buds (per 10 cm) 4 +1.6 +1.2 ±3.8 +9.4** +0.0 +0.0 -2.3 ±1.0 -0.1 0.2 Sexual reproduction Flowering plants (% of all) +8.9** -10.6** ±49.9*** +3.5 +0.6 ±3.2 -2.3 ±1.7 -0.1 3.9 0.0 Flowering shoots per flowering plant5 +0.0 -8.5** -1.5 +11.0** +0.1 +1.6 +0.0 +0.0 ±0.4 1.8 2.1* Sexual mass ratio +5.7* +29.8*** ±36.7*** +1.6 ±0.5 +0.3 +0.4 -0.1 ±2.2 0.2 1.0 Biomass production: Capitulescence biomass per flowering plant (g) -0.9 -1.1 ±25.1*** +36.2*** +0.2 +2.1 +0.0 ±0.7 ±0.3 0.0 2.0* Capitulescence biomass per flowering shoot (g) +0.0 +26.1*** ±29.4*** +8.6** +0.6 +0.3 +0.0 ±1.2 +0.0 0.0 0.8 Size: Volume of main capitulescence (cm3) 6 -7.2* -12.5** -32.8*** +6.3* +0.8 +0.2 +3.1 +0.1 +0.4 0.5 1.1 Phenology: Flowering duration per plant (d) +11.2** -7.3* -9.8** +17.2*** +0.2 +2.9 +1.9 +0.0 +1.1 0.1 1.3 Flowering duration per shoot (d) +53.8*** -27.3*** -1.7 +1.7 +0.9 ±0.2 +2.4 +0.8 +0.1 0.3 0.7 Age at onset of flowering (d) -3.4 -46.0*** ±40.7*** +0.2 ±0.1 ±3.1 -0.9 ±1.3 -0.0 0.5 1.4 Age at peak of flowering shoots (d) -0.1 -72.0*** -23.1*** +4.1* +0.0 +0.4 +0.1 ±0.6 ±0.5 0.5 1.4 Age at end of flowering (d) +1.9 -75.7*** -11.6** +9.0** +0.1 +0.1 +0.0 +0.5 ±0.7 0.2 0.6 Table 2 data. 3 effect. interactions are negative/positive if there is in both ploidy levels resp. in both regions a negative/positive positive if in both ploidy levels there is a negative/positive latitudinal cline. F ratios for ploidy and region calcium treatments are negative for negative effects. F ratios for and region ploidy interactions nutrient are negative/ for ratios F gradient. latitudinal negative a is there if negative are effect region F for tetraploids. ratios for than higher is diploids for value variable if negative are effect level F ploidy for < 0.05). ratios p *, < 0.01; p **, < 0.001; p (***, given are levels significance and ratios F covariate. fixed as biomass intial and factor random as region in nested population factors, fixed as treatment nutrient and treatment Calcium region, level, ploidy with models mixed linear with tested were tetraploids and diploids American North between Differences buds). and diameter rhizome concentrations, (nutrient pooled plants three from or combination treatment and population per plants three of means the from For each variable, means and SE of the North American diploid and tetraploid populations were calculated grown inacommongarden. of populations tetraploid American North 12 and diploid American North 12 Table 2 Calculated from inverse-hyperbolic-sine-transformed data. inverse-hyperbolic-sine-transformed from Calculated Ploidy Ploidy Ploidy Region Ploidy Initial Pop Variable * region * region * N * N * region

biomass (region) 5

* N * Ca * Ca * Ca * N * Ca Calculated frominverse-square-root-transformeddata. 1 acltd rm nes-rnfre data. inverse-transformed from Calculated

Plant growth: Biomass production:

Comparison of plant growth, vegetative and sexual reproduction and phenology of plants from Biomass per plant 2006 (g) 5.9* 0.2 0.1 1.5 0.1 22.1*** 2.7** (contd.) Biomass per shoot 2006 (g) 1 3.5 2.6 0.0 4.6* 0.1 9.3** 1.2 Biomass per plant 2007 (g) 1.1 0.0 1.6 4.3* 0.6 7.3** 2.1* Biomass per shoot 2007 (g) 2.7 0.6 0.9 0.3 0.1 6.5* 1.2 Size: Main shoot diameter (at harvest, mm) 4.0 0.6 0.2 5.6* 0.4 8.3** 2.1* Maximum shoot length 2006 (cm) 0.7 0.0 0.0 5.9* 1.3 9.0** 1.8 (June) shoot length 2007 (cm) 0.0 0.2 0.5 0.2 0.1 7.4** 1.2 Phenology: Age at 50% shoot height (d) 1.0 1.6 0.0 0.3 2.1 21.2*** 1.8 Age with 50% dead leaves (d) 10.0** 0.5 0.9 0.4 0.0 11.1** 1.1 Leaf traits: Leaf calcium concentration (%) 0.4 2.8 0.0 2.1 0.4 1.3 1.0 Leaf nitrogen concentration (%) 1.0 1.1 1.1 0.1 0.3 0.1 0.6 Leaf phosphorus concentration (%) 0.8 0.0 1.1 0.3 0.8 1.2 0.6 Leaf area (cm2) 0.6 0.4 5.7* 0.4 0.7 7.6** Leaf mass (mg) 1.8 0.7 5.2* 0.3 0.0 10.3** 0.0 Specific leaf area (cm2 g-1) 4.6* 0.9 0.0 0.2 2.1 6.9* 0.2 Leaf dry matter content (dry/fresh biomass) 0.7 0.1 1.5 1.0 0.6 0.1 0.5 Vegetative reproduction

2 2

Vegetative shoot ratio 0.8 0.1 0.1 4.4* 5.6* 1.2 2.2* data. power-2.5-transformed from Calculated Shoots: Number of shoots 2006 0.4 0.0 0.0 3.0 0.0 0.0 2.1* 4

Number of shoots 2007 (≥ 15 cm) 0.2 0.8 0.3 4.3* 0.6 1.2 2.1* square-root-transformed from Calculated 6 Number of latent buds 2006 5.8* 11.6** 2.3 4.0* 0.4 3.5 1.5 Calculated fromln-transformeddata. Rhizomes: Number of rhizomes 3 0.2 0.7 0.3 3.5 0.2 1.6 1.9 Rhizome diameter (mm) 0.2 0.9 2.1 0.5 0.2 0.4 Rhizome buds (per 10 cm) 4 0.2 0.0 2.0 1.9 0.1 1.2 Sexual reproduction Flowering plants (% of all) 1.2 1.2 0.0 7.6** 0.4 8.0** 0.0 Flowering shoots per flowering plant5 0.3 0.0 0.1 0.1 2.7 0.0 2.1* gigantea Solidago Sexual mass ratio 0.2 0.0 0.1 0.2 0.5 8.8** 1.0 Biomass production: Capitulescence biomass per flowering plant (g) 3.3 2.3 0.0 0.0 0.4 11.4** 2.0* Capitulescence biomass per flowering shoot (g) 0.3 0.6 0.0 1.2 2.8 14.3*** 0.8 Size: Volume of main capitulescence (cm3) 6 0.1 3.3 0.0 0.6 1.9 6.2* 1.1 Phenology: Flowering duration per plant (d) 0.1 2.1 0.0 0.0 0.1 4.0 1.3 Flowering duration per shoot (d) 0.1 0.2 0.7 0.5 4.0 0.0 0.7 Aiton Age at onset of flowering (d) 1.8 2.0 1.1 0.0 0.1 8.2** 1.4 Age at peak of flowering shoots (d) 1.6 0.5 0.7 0.1 2.1 2.3 1.4 Age at end of flowering (d) 1.2 0.1 0.4 0.0 0.2 0.0 0.6 Chapter 3 (region) Population 2.7** 2.2* 2.0* 1.9 2.2* 1.8 0.9 1.5 0.5 0.9 0.5 0.7 1.6 1.8 0.1 2.0* 2.4* 2.6* 2.2* 2.2* 0.3 0.9 2.2* 0.6 1.2 1.2 0.9 0.1 0.5 Initial 7.5** 4.8* 3.6 4.4* 9.5** 1.1 2.9 0.2 1.9 0.2 1.2 1.1 1.5 0.8 0.1 0.2 1.6 2.7 0.2 5.6* 4.0* 4.3* 2.2 2.1 3.0 2.4 1.3 0.1 biomass 12.2*** 10.4** 10.4** 11.7*** 16.7*** 16.4*** Cont *Ca*N 3.4 0.0 1.5 0.7 0.0 0.4 0.1 0.0 1.7 0.7 0.1 1.8 1.9 3.6 2.2 0.4 0.2 0.3 3.0 2.2 0.1 0.4 1.1 1.8 1.0 0.1 0.4 0.0 1.1 2.7 3.8 0.1 0.1 0.8 Ca * N 1.4 1.2 0.1 6.7* 0.0 0.0 0.1 0.0 0.4 0.1 0.3 0.3 4.2* 3.3 0.1 0.1 0.0 0.1 4.2* 3.0 0.9 0.1 0.0 0.0 0.8 0.1 0.2 0.1 0.1 1.8 4.1* 0.9 1.7 3.1 * Ca Cont -1.7 -1.7 -0.2 -1.9 -1.3 -0.1 -6.5* -2.8 -2.7 ±1.9 +0.3 +1.3 ±0.6 +1.9 ±2.6 ±5.6* +0.5 ±0.0 ±0.5 ±0.6 +0.2 ±2.6 +0.0 ±2.9 +0.1 ±0.1 ±2.4 +0.3 ±2.3 +0.2 +0.1 ±4.5* +4.8* +4.7* * N Cont -0.5 -0.5 +0.5 +1.9 +0.8 +1.1 +1.2 +0.7 ±0.4 ±1.4 ±0.6 +0.0 ±0.5 +2.8 +1.3 +0.9 +0.3 +5.2* +0.1 +1.2 +0.8 +1.2 +1.7 +0.2 ±1.0 +0.0 +0.3 +0.4 ±4.3* ±2.5 ±0.1 ±0.9 ±2.8 +0.3 = Ca -2.8 -3.2 -0.8 -0.2 -0.6 -5.3* -4.3* -2.8 -6.1* -0.8 -2.8 -8.7** -4.2* -5.2* -2.8 +0.6 +7.9** +2.7 +1.3 +0.0 +0.3 +1.6 +1.6 +4.5* +0.2 +0.0 +0.7 +0.5 +4.9* +6.6* +7.4** Calcium -11.7** +13.7*** +19.6*** = N -1.2 -4.7* -2.8 -1.1 +4.4* +0.0 +1.3 +1.9 +0.1 +7.9** +2.5 +7.8** +7.9** +9.1** +4.4* +4.7* +8.3** +2.2 +7.2** +8.0** +1.8 +0.2 +0.2 +0.1 +16.2*** +11.3** +99.0*** +11.3** +32.9*** +16.0*** Nutrients +244.3*** +111.7*** +137.7*** +1249.9*** -5.8** -4.7* -3.3 -4.3* -8.6** -1.2 -0.7 -0.1 -0.5 -6.1** (Cont) Region ±5.3* ±3.2 ±0.3 ±0.0 ±4.7* ±1.7 +5.4* ±2.5 +0.8 ±5.2* ±3.1* +6.6** ±1.9 ±0.3 ±3.3 ±5.5** -38.2*** -35.5*** -32.1*** ±11.9*** ±14.6*** +30.4*** +10.5*** ±18.6*** -2.2 -0.5 -1.4 -5.3* -0.4 -2.8 -0.0 -2.1 -7.8* -6.6* -0.6 -0.6 -5.5* -0.9 = Cont +0.4 +0.7 +0.1 +1.1 +0.0 +0.9 +1.1 +5.4* +2.5 -10.1** -15.1*** -27.4*** -25.3*** -10.7** -23.9*** -19.3*** +13.9** +15.6*** +22.7*** +27.4*** Continent 1 2 1 3 4 5 5 5 1 6 Leaf mass (mg) Leaf area (cm2) Sexual mass ratio Number of rhizomes Vegetative shoot ratio shoot ratio Vegetative Number of shoots 2006 Rhizome diameter (mm) Rhizome Flowering plants (% of all) Age at end of flowering (d) Biomass per plant 2007 (g) Biomass per plant 2006 (g) Biomass per shoot 2007 (g) Rhizome buds (per 10 cm) Rhizome Age at 50% shoot height (d) Age at onset of flowering (d) Specific leaf area (cm2 g-1) Biomass per shoot 2006 (g) Number of latent buds 2006 Age with 50% dead leaves (d) Age with 50% dead leaves (June) shoot length 2007 (cm) Flowering duration per plant (d) Flowering duration Leaf nitrogen concentration (%) Leaf nitrogen concentration Flowering duration per shoot (d) Flowering duration Leaf calcium concentration (%) Leaf calcium concentration Maximum shoot length 2006 (cm) Number of shoots 2007 (≥ 15 cm) Leaf phosphorus concentration (%) Leaf phosphorus concentration Age at peak of flowering shoots (d) Flowering shoots per flowering plant Main shoot diameter (at harvest, mm) Main shoot diameter (at harvest, Volume of main capitulescence (cm3) Volume Leaf dry matter content (dry/fresh biomass) Capitulescence biomass per flowering plant (g) Capitulescence biomass per flowering shoot (g) Plant growth: Biomass production: Size: Phenology: Leaf traits: Vegetative reproduction Shoots: Rhizomes: Sexual reproduction Biomass production: Size: Phenology: Variable

100 Common garden experiment

Table 3 Comparison of plant growth, vegetative and sexual reproduction and phenology of plants from 12 North American tetraploid and 12 European tetraploid populations of S. gigantea grown in a common garden. For each variable, means and SE of the North American and European populations were calculated from the means of three plants per population and treatment combination or from three plants pooled (nutrient concentrations, rhizome diameter and buds). Differences between North American and European tetraploids were tested with linear mixed models with continent, region nested in continent, Calcium treatment and nutrient treatment as fixed factors, population nested in region as random factor and intial biomass as fixed covariate. F ratios and significance levels are given (***, p < 0.001; **, p < 0.01; *, p < 0.05). F ratios for continent effect are negative if variable value for European tetraploids is higher than for North American. F ratios for region effect are negative/positive if there is a negative/ positive latitudinal gradient in both continents. F ratios for nutrient and calcium treatments are negative for negative effects. F ratios for continent nutrient or calcium treatment interactions are negative/ positive if the effect is negative/positive in both continents. 1Calculated from inverse-square-root- transformed data. 2Calculated from inverse-transformed data. 3Calculated from power-5-transformed data. 4Calculated from ln-transformed data. 5Calculated from square-root-transformed data. 6Calculated from arsine-transformed data.

Responses to calcium treatment

For both ploidy levels, adding calcium tended to reduce plant performance, though the effects were mainly small; thus, there were reductions in the numbers of rhizomes (Fig. 3(g)), latent buds and shoots (Fig. 3(h), second season), and in shoot diameter and LDMC (this effect being stronger for SA than for ON). Only for SLA were the effects of added calcium significantly positive (Tab. 2). Similar effects were found in the between-continent comparisons (Tab. 3), with numbers of rhizomes (Fig. 3(g)) and shoots (Fig. 3(h), second season), capitulescence biomass, leaf mass and LDMC being significantly reduced by calcium, whereas SLA, rhizome diameter and shoot biomass (second season) increased. There were also differences between ploidy levels: while NA.2x responded negatively to calcium in the second season for biomass production (Fig. 3(i); plant and shoot) and shoot height, NA.4x responded indifferently or positively. Between continents, calcium significantly decreased sexual mass ratio of EU.4x plants and postponed flowering time and leaf senescence, whereas NA.4x plants were indifferent.

Coefficients of variation and correlations among variables differ between cytotypes

Diploids were the cytotype that showed most often the largest coefficient of variation of those that differed significantly among cytotypes (Tab. 1). This was especially so for parameters of plant growth and rhizome number. There were more significant correlations between the parameters recorded in the experiment and those recorded in the field for diploids (n = 16 of 44) than for either North American (n = 3; Z = 9.8, p < 0.001) or European tetraploids (n = 1; Z = 16.5, p < 0.001). This

101 Chapter 3 x a b b EU.4 a b ab NA.4x a a a high calcium a NA.2x x 0.04 b 0.01 b - - a b b EU.4 a b b NA.4x responses NA.2x = -0.12 NA.4x = EU.4x = response = -0.20** response = -0.13* a a a low calcium NA.2x (g ) (h ) (i ) U.4x c bc xE c cc c NA.4 c b a igh nutrients b a b NA.2x a b a b a b x 0.50 0.50 b - - b b s s EU.4 s b ab NA.4x EU.4x = response NA.2x = 0.36 NA.4x = 0.51 EU.4x = 0.32 response NA.2x = -0.67 NA.4x = response NA.2x = 0.92 NA.4x = 0.90 EU.4x = 0.84 a a a low nutrient sh NA.2x (d) (e) (f) b bb U.4x xE bb b b NA.4 cytotypes a a a NA.2x (a) (b) (c) 5 5

25 20 15 10 25 20 15 10 90 80 70 60 50 40 30 20 10

100

Rhizome number Rhizome ) (2007 number Shoot ) (g 2007 Biomass

102 Common garden experiment

Figure 3 Differences between cytotypes of S. gigantea (a-c), in response to nutrients (d-f) and to calcium (g-i) in the second season for rhizome number, shoot number and above-ground biomass. Error bars indicate ± 1 SE of the means. Homogeneous subsets are based on LMMs and on Dunnett T3 post hoc tests (Tabs 2 and 3).

Population area (m2) Population density

Capitulescence size

Shoot height European 4x -0.49

Population area (m2)

Population density -0.46 -0.49 -0.58

Capitulescence size in the common garden experiment and in and experiment garden common the in Shoot height North American 4x Field sites

Population area (m2) S. gigantea S. 0.58 -0.55 -0.55 -0.57

Population density 0.48 0.56 0.49 0.75 -0.72

Capitulescence size 0.69 0.47 0.66 0.70 -0.69

North American 2x Shoot height 0.46 0.45 Correlations between variables measured on plants of plants on measured variables between Correlations Common garden Shoot length 2006 (cm) Shoot length 2007 (cm) Capitulescence per plant (g) Capitulescence per shoot (g) Biomass per plant 2006 (g) Biomass per shoot 2006 (g) Biomass per plant 2007 (g) Biomass per shoot 2007 (g) Number of shoots 2006 Number of shoots 2007 number 2007 Rhizome Table 4 Table populations from the original field sites. Only Pearson correlation coefficients that areCommon significantgarden data are means of three plants per atpopulation. Shoot height and capitulescence the sequentialsize of the original Bonferroni populations adjustedat the field sites werelevel are shown. estimated as population averages in four categories at end ofdensity was estimated in Braun-Blanquet categories. season. Population

103 Chapter 3 pattern was similar to those of correlations amongst parameters recorded in the experiment (data not shown). For the diploid plants, most of the correlations between performance in the field and in the common garden experiment were obtained using data from thefirst experimental year, with biomass and number and length of shoots being significantly correlated with field parameters (Tab. 4). For NA.4x, common garden variables (capitulescence biomass and rhizome number) correlated only with field population density (negatively). For EU.4x, shoot number (first season) in the common garden was negatively correlated to shoot height in the field.

Di s c u s s i o n

The experimental plants, although grown in pots, reached a ‘normal’ size and expressed a phenology comparable to that of wild populations. We cannot exclude the possibility that ‘maternal’ effects influenced our results, especially in the first season (Roach & Wulff, 1987), although we attempted to minimise these by pre-cultivating the plants. In the case of NA.2x plants, germination time and first season biomass were correlated (r = -0.31, p < 0.034), which could reflect a maternal effect; furthermore, correlations between experimental plants and field sites were significant for the first season and SA.2x grew differently in the first than the second season. However, in a previous common garden experiment with the same species (Güsewell et al., 2006), correlations with field parameters were also obtained and these results were clearly due to genetic rather than maternal effects. Whenever possible we focus the discussion on growth variables available from the second season, since these are less likely to be affected by any maternal effects. There were clear differences in the performance of diploid and tetraploid plants of S. gigantea, and also between plants of the same ploidy level in different regions. These regional differences in time of flowering and plant size, which were found for both native and invasive populations, probably reflect clinal variation associated with latitude, and are comparable to results from an earlier study with invasive S. gigantea (Weber & Schmid, 1998). Similar latitudinal clines in both native and invasive populations have also been found in some other species, e.g. Hypericum perforatum (Maron et al., 2004). For S. gigantea, however, latitudinal clines were less congruent between native and invasive tetraploids than between diploid and tetraploid native populations. This suggests that the populations in the introduced range have not yet adapted fully to local conditions, either because there has not been sufficient time (although other studies have demonstrated rapid adaptation in introduced populations of this species, Weber & Schmid, 1998), or because of the limited genetic diversity in invasive populations, as was observed for S. gigantea (chapter 4). In addition to latitudinal variation, we detected strong interactions between region of origin and ploidy level in native populations. While the reasons for this are not fully clear, they reflect the fact that diploids are more genetically distinct between regions than tetraploids

104 Common garden experiment

Ontario S. Appalachian Mts. Belgium Italy diploid aploid tetr

10 cm

Figure 4 Variation of rhizome system of S. gigantea between ploidy levels and between regions of origin. Rhizomes from plants grown with a high nutrient and low calcium treatment combination are shown.

(chapter 4). The greater differentiation and genetic variation of diploids is also reflected in a higher coefficient of variation for many parameters than was found for tetraploids, and in the more numerous correlations amongst individual parameters.

Soil calcium as growth modifier ofS. gigantea

Adding calcium affected the growth of S. gigantea, and especially of diploid plants. Although calcium carbonate is often applied to reduce soil acidity (Grewal & Williams, 2003), we do not think that this effect was due to altered pH, since the small additional experiment showed S. gigantea plants to be relatively insensitive to pH. It is also unlikely to have been due to reduced P availability in the calcium treatment, since the N/P ratios suggest that N was the more limiting nutrient. We suppose, therefore, that the result was a direct effect of higher calcium concentrations. Besides being an essential component of cell walls and membranes (Hirschi, 2004), calcium is an important chemical messenger, involved in many physiological processes

105 Chapter 3 including responses to hormones and stress signals (Reddy & Reddy, 2004). Calcium is also potentially toxic, and to prevent negative effects (White & Broadley, 2003) and to facilitate effective signal transduction upon small concentration changes in the cytoplasm, free calcium is tightly controlled and kept at low levels (Plieth, 2005), despite being influenced by external calcium concentrations (White & Broadley, 2003). For instance, poplar trees increase calcium concentrations in phloem and cambium with increasing calcium supply, which translates to significant alteration of the structure and physiology of wood formation (Lautner et al., 2007). The calcium treatment affected plant development in all cytotypes, reducing the numbers of shoots and rhizomes and increasing SLA. However, there were also differences in the responses of the cytotypes to increased soil calcium, with NA.2x plants showing reduced growth in the second season. Similar physiological differentiation between ploidy levels has been found in other studies; for instance, tetraploid Isatis indigotica expresses higher levels than diploids of the calcium-dependent protein kinase gene IiCPK2 which is active in various pathways including responses to cold, high salinity and certain hormones (Lu et al., 2006). Our result links such physiological differentiation in calcium response to habitat differentiation observed in wild populations, with NA.2x showing calcifuge behaviour whereas NA.4x does not (chapter 2). We also found that soil calcium postponed flowering and leaf senescence in EU.4x, but not in NA.4x. While calcium is known to retard leaf senescence and abscission (Hepler, 2005), the more oceanic climate in Europe compared to a more continental climate in North America (chapter 2) could also promote a longer vegetation period and delayed flowering.

Pre-disposition of tetraploid S. gigantea for colonization

This experiment has revealed many differences between NA.2x and NA.4x of S. gigantea, mostly in life history traits and less so in plant performance. Other studies have shown varying effects of ploidy upon plant performance and life history; while no effects were found in some cases, e.g. in Aster amellus (Münzbergova, 2007) or Ranunculus adoneus (Baack & Stanton, 2005), in others there were considerable differences between ploidy levels, e.g. in Arrhenatherum elatius (Petit & Thompson, 1997) and Dactylis glomerata (Maceira et al., 1993; Bretagnolle & Thompson, 1996). Similarly, in Centaurea maculosa, diploids were monocarpic, producing only a single flowering shoot, whereas tetraploids from both native and invasive populations were polycarpic perennials and produced several flowering shoots in the second season (Müller, 1989). It has been argued that polycarpic perennials are fitter than monocarpic plants in the absence of specialist root-feeding herbivores (Müller, 1989), and that release from these enemies may explain the success of the C. maculosa tetraploid in the invasive range (Müller- Schärer et al., 2004). Compared with the tetraploids, diploid S. gigantea shows functional characteristics associated with more rapid growth and a shorter life span. Thus, the largest shoots obtained in

106 Common garden experiment our experiment were of NA.2x plants in the first season, with 163 g biomass, 2.2 m height and 2.0 cm shoot diameter. The NA.2x plants have generally high values for leaf N and SLA (Fig. 2), a combination that is characteristic of fast-growing species (Reich et al., 1999; Shipley et al., 2005). Similarly, NA.2x have a high allocation of biomass to shoots rather than to rhizomes. However, the diploids also showed a wider range of growth performance than NA.4x and greater differentiation among regions. On the other hand, NA.4x appears to be a longer-lived, clonal perennial with a much larger, denser rhizome system than the diploid (Fig. 4) and it responds more strongly to added nutrients. More rhizomes translate into more shoots, and the plant biomass of NA.4x overtook that of NA.2x in the second year. These results support our previous hypothesis that NA.4x plants were able to extend their range in North America because of their superior colonising ability, e.g. well developed rhizome system, and broader ecological niche, e.g. tolerance to calcium, (chapter 2).

Invasive tetraploid populations of S. gigantea

Several experiments comparing invasive with native populations of S. gigantea have been conducted (Jakobs, 2004; Meyer et al., 2005; Güsewell et al., 2006; Hull-Sanders et al., 2007; Johnson et al., 2007; Meyer & Hull-Sanders, 2007), but this is the first to take ploidy levels into account. Because invasive populations of S. gigantea are only tetraploid (chapter 1), they should be compared with native tetraploid populations. While we found many differences between NA.2x and NA.4x, differences between NA.4x and EU.4x were less obvious. Studies to investigate whether introduced populations of S. gigantea exhibit increased plant performance and competitive ability have produced conflicting results. A field study found supportive results (Jakobs et al., 2004), while experimental studies, including this, found no consistent increase in plant height or biomass in invasive populations compared to native populations (Meyer et al., 2005; Meyer & Hull-Sanders, 2007). However, invasive S. gigantea plants were found to produce more shoots through clonal growth (Güsewell et al., 2006) and allocated more resources to rhizomes than to flowers (Meyer & Hull-Sanders, 2007), but these plants were less likely than native plants to flower (Meyer & Hull-Sanders, 2007). Our results support the finding that invasive populations were less likely to flower and invested less biomass into flowers, but the differences for shoot and rhizome number were not significant. While the outcome of common garden experiments may depend on location (Maron et al., 2004), it is also possible that other studies mixed native diploid plants, having much smaller rhizome systems, with tetraploid plants. Such non-intentional mixing of ploidy levels could also explain the large population variation found in some other studies. The ability of S. gigantea to invade dense, established vegetation and nutrient-poor sites (Weber & Jakobs, 2005) has been attributed to its clonal growth (Güsewell et al., 2006). Furthermore, this successful ability seems not to be restricted to the invasive range but

107 Chapter 3 applies also to tetraploids in the native range (chapter 2). The much smaller colonising ability of diploids could be one reason why diploids are absent from Europe. Based on our ploidy- level precise comparison, we conclude that the invasion success of tetraploid S. gigantea is a consequence of its vigorous rhizome system, perhaps coupled with other factors such as enemy release, evolutionary changes, founder effects and allelopathy. Therefore, comparative studies should take care to compare like genotypes with like (Dlugosch & Parker, 2008), which implies thorough knowledge of a species’ native populations (chapter 1). Even though genetic change may be detected in invasive populations, the potential to become invasive is not necessarily acquired in the introduced range but may be a property of particular genotypes in the native range (Bastlova et al., 2004; Hooftman et al., 2006). The fact, that the vigorous rhizome growth of S. gigantea is not unique to invasive populations but rather to polyploids of this species, supports this idea.

Ac k n o w l e d g m e n t s

We thank Sabine Güsewell for statistical advice, Martin Fotsch for help with the experiment, Philipp Streckeisen for the green house (FAL, Reckenholz, Switzerland), Andreas Wolf for measuring leaf traits and Rose Trachsler for nutrient analyses. We also thank Jake Alexander, Daniela Eichenberger. Harry Eggenschwiler, Min Hahn, Edith Lang, Myriam Poll, Carmen Rothenbühler, Rodolphe Schlaepfer and Mila Trtiková for help during the harvest. The project is funded by the grant 0-20259-05 from the ETH Zurich, Switzerland.

Re f e r e n c e s

Baack, E.J. & Stanton, M.L. (2005) Ecological factors influencing tetraploid speciation in snow buttercups (Ranunculus adoneus): Niche differentiation and tetraploid establishment. Evolution, 59, 1936- 1944

Bastlova, D., Cizkova, H., Bastl, M. & Kvet, J. (2004) Growth of Lythrum salicaria and Phragmites australis plants originating from a wide geographical area: response to nutrient and water supply. Global Ecology and Biogeography, 13, 259-271

Blossey, B. & Notzold, R. (1995) Evolution of increased competitive ability in invasive nonindigenous plants - a hypothesis. Journal of Ecology, 83, 887-889

Blumenthal, D.M. & Hufbauer, R.A. (2007) Increased plant size in exotic populations: A common- garden test with 14 invasive species. Ecology, 88, 2758-2765 108 Common garden experiment

Bossdorf, O., Auge, H., Lafuma, L., Rogers, W.E., Siemann, E. & Prati, D. (2005) Phenotypic and genetic differentiation between native and introduced plant populations. Oecologia, 144, 1-11

Bretagnolle, F. & Thompson, J.D. (1996) An experimental study of ecological differences in winter growth between sympatric diploid and autotetraploid Dactylis glomerata. Journal of Ecology, 84, 343-351

Broennimann, O., Treier, U.A., Müller-Schärer, H., Thuiller, W., Peterson, A.T. & Guisan, A. (2007) Evidence of climatic niche shift during biological invasion. Ecology Letters, 10, 701-709

Callaway, R.M. & Ridenour, W.M. (2004) Novel weapons: invasive success and the evolution of increased competitive ability. Frontiers in Ecology and the Environment, 2, 436-443

Chapuis-Lardy, L., Vanderhoeven, S., Dassonville, N., Koutika, L.S. & Meerts, P. (2006) Effect of the exotic invasive plant Solidago gigantea on soil phosphorus status. Biology and Fertility of Soils, 42, 481-489

Colautti, R.I., Ricciardi, A., Grigorovich, I.A. & Macisaac, H.J. (2004) Is invasion success explained by the enemy release hypothesis? Ecology Letters, 7, 721-733

Daehler, C.C. (2003) Performance comparisons of co-occurring native and alien invasive plants: Implications for conservation and restoration. Annual Review of Ecology Evolution and Systematics, 34, 183-211

Dewalt, S.J., Denslow, J.S. & Hamrick, J.L. (2004) Biomass allocation, growth, and photosynthesis of genotypes from native and introduced ranges of the tropical shrub Clidemia hirta. Oecologia, 138, 521-531

Dietz, H. & Steinlein, T. (2004) Recent advances in understanding plant invasions. Progress in Botany, 65, 539-573

Dlugosch, K.M. & Parker, I.M. (2008) Founding events in species invasions: genetic variation, adaptive evolution, and the role of multiple introductions. Molecular Ecology, 17, 431-449

Ehrenfeld, J.G. (2003) Effects of exotic plant invasions on soil nutrient cycling processes. Ecosystems, 6, 503-523

Ellstrand, N.C. & Schierenbeck, K.A. (2000) Hybridization as a stimulus for the evolution of invasiveness in plants? Proceedings of the National Academy of Sciences of the United States of America, 97, 7043-7050

Garnier, E., Shipley, B., Roumet, C. & Laurent, G. (2001) A standardized protocol for the determination of specific leaf area and leaf dry matter content. Functional Ecology, 15, 688-695

Grewal, H.S. & Williams, R. (2003) Liming and cultivars affect root growth, nodulation, leaf to stem ratio, herbage yield, and elemental composition of alfalfa on an acid soil. Journal of Plant Nutrition, 26, 1683-1696

Güsewell, S. (2004) N : P ratios in terrestrial plants: variation and functional significance. New Phytologist, 164, 243-266

Güsewell, S. (2005) Responses of wetland graminoids to the relative supply of nitrogen and phosphorus. Plant Ecology, 176, 35-55

109 Chapter 3

Güsewell, S., Jakobs, G. & Weber, E. (2006) Native and introduced populations of Solidago gigantea differ in shoot production but not in leaf traits or litter decomposition. Functional Ecology, 20, 575-584

Güsewell, S., Zuberbühler, N. & Clerc, C. (2005) Distribution and functional traits of Solidago gigantea in a Swiss lakeshore wetland. Botanica Helvetica, 115, 63-75

Halverson, K., Heard, S.B., Nason, J.D. & Stireman, J.O. (2008) Differential attack on diploid, tetraploid, and hexaploid Solidago altissima L. by five insect gallmakers. Oecologia, 154, 755-761

Hepler, P.K. (2005) Calcium: A central regulator of plant growth and development. Plant Cell, 17, 2142- 2155

Hierro, J.L., Maron, J.L. & Callaway, R.M. (2005) A biogeographical approach to plant invasions: the importance of studying exotics in their introduced and native range. Journal of Ecology, 93, 5-15

Hirschi, K.D. (2004) The calcium conundrum. Both versatile nutrient and specific signal. Plant Physiology, 136, 2438-2442

Hooftman, D.A.P., Oostermeijer, J.G.B. & Den Nijs, J.C.M. (2006) Invasive behaviour of Lactuca serriola (Asteraceae) in the Netherlands: Spatial distribution and ecological amplitude. Basic and Applied Ecology, 7, 507-519

Hull-Sanders, H.M., Clare, R., Johnson, R.H. & Meyer, G.A. (2007) Evaluation of the evolution of increased competitive ability (EICA) hypothesis: Loss of defense against generalist but not specialist herbivores. Journal of Chemical Ecology, 33, 781-799

Hunt, J. (1982) Dilute hydrochloric acid extraction of plant material for routine cation analysis. Communications in Soil Science and Plant Analysis, 13, 49-55

Jakobs, G. (2004) Evolution of increased competitive ability in the invasive perennial Solidago gigantea Aiton. Diss. 15536 ETH Zürich, Zürich.

Jakobs, G., Weber, E. & Edwards, P.J. (2004) Introduced plants of the invasive Solidago gigantea (Asteraceae) are larger and grow denser than conspecifics in the native range. Diversity and Distributions, 10, 11-19

Johnson, R.H., Hull-Sanders, H.M. & Meyer, G.A. (2007) Comparison of foliar terpenes between native and invasive Solidago gigantea. Biochemical Systematics and Ecology, 35, 821-830

Koutika, L.S., Vanderhoeven, S., Chapuis-Lardy, L., Dassonville, N. & Meerts, P. (2007) Assessment of changes in soil organic matter after invasion by exotic plant species. Biology and Fertility of Soils, 44, 331-341

Lautner, S., Ehlting, B., Windeisen, E., Rennenberg, H., Matyssek, R. & Fromm, J. (2007) Calcium nutrition has a significant influence on wood formation in poplar. New Phytologist, 173, 743-752

Lu, B.B., Ding, R.X., Zhang, L., Yu, X.J., Huang, B.B. & Chen, W.S. (2006) Molecular cloning and characterization of a novel calcium-dependent protein kinase gene IiCPK2 responsive to polyploidy from tetraploid Isatis indigotica. Journal of Biochemistry and Molecular Biology, 39, 607-617

Maceira, N.O., Jacquard, P. & Lumaret, R. (1993) Competition between diploid and derivative

110 Common garden experiment

autotetraploid Dactylis glomerata L. from Galicia - implications for the establishment of novel polyploid populations. New Phytologist, 124, 321-328

Maron, J.L., Vila, M., Bommarco, R., Elmendorf, S. & Beardsley, P. (2004) Rapid evolution of an invasive plant. Ecological Monographs, 74, 261-280

McKenney, J.L., Cripps, M.G., Price, W.J., Hinz, H.L. & Schwarzlander, M. (2007) No difference in competitive ability between invasive North American and native European Lepidium draba populations. Plant Ecology, 193, 293-303

Melville, M.R. & Morton, J.K. (1982) A biosystematic study of the Solidago canadensis (Compositae) complex. 1. The Ontario populations. Canadian Journal of Botany - Revue Canadienne de Botanique, 60, 976-997

Meyer, G., Clare, R. & Weber, E. (2005) An experimental test of the evolution of increased competitive ability hypothesis in goldenrod, Solidago gigantea. Oecologia, 144, 299-307

Meyer, G.A. & Hull-Sanders, H.M. (2007) Altered patterns of growth, physiology and reproduction in invasive genotypes of Solidago gigantea (Asteraceae). Biological Invasions, in press. Available online 29 June 2007. doi:10.1007/s10530-007-9131-z

Müller, H. (1989) Growth pattern of diploid and tetraploid spotted knapweed, Centaurea maculosa Lam. (Compositae), and effects of the root mining moth Agapeta zoegana (L.) (Lep.: Cochylidae). Weed Research, 29, 103-111

Müller-Schärer, H., Schaffner, U. & Steinger, T. (2004) Evolution in invasive plants: implications for biological control. TRENDS in Ecology & Evolution, 19, 417-422

Münzbergova, Z. (2006) Ploidy level interacts with population size and habitat conditions to determine the degree of herbivory damage in plant populations. Oikos, 115, 443-452

Münzbergova, Z. (2007) No effect of ploidy level in plant response to competition in a common garden experiment. Biological Journal of the Linnean Society, 92, 211-219

Petit, C. & Thompson, J.D. (1997) Variation in phenotypic response to light availability between diploid and tetraploid populations of the perennial grass Arrhenatherum elatius from open and woodland sites. Journal of Ecology, 85, 657-667

Plieth, C. (2005) Calcium: Just another regulator in the machinery of life? Annals of Botany, 96, 1-8

Reddy, V.S. & Reddy, A.S.N. (2004) Proteomics of calcium signaling components in plants. Phytochemistry, 65, 1745-1776

Reich, P.B., Ellsworth, D.S., Walters, M.B., Vose, J.M., Gresham, C., Volin, J.C. & Bowman, W.D. (1999) Generality of leaf trait relationships: A test across six biomes. Ecology, 80, 1955-1969

Rice, W.R. (1989) Analyzing tables of statistical tests. Evolution, 43, 223-225

Roach, D.A. & Wulff, R.D. (1987) Maternal effects in plants. Annual Review of Ecology and Systematics, 18, 209-235

Semple, J.C. & Cook, R.E. (2006) Solidago. Flora of North America North of Mexico (ed. by Flora of North America Editorial Committee), pp. 107-166. Oxford University Press, Oxford and New York. 111 Chapter 3

Shipley, B., Vile, D., Garnier, E., Wright, I.J. & Poorter, H. (2005) Functional linkages between leaf traits and net photosynthetic rate: reconciling empirical and mechanistic models. Functional Ecology, 19, 602-615

Theoharides, K.A. & Dukes, J.S. (2007) Plant invasion across space and time: factors affecting nonindigenous species success during four stages of invasion. New Phytologist, 176, 256-273

Thompson, J.N., Nuismer, S.L. & Merg, K. (2004) Plant polyploidy and the evolutionary ecology of plant/animal interactions. Biological Journal of the Linnean Society, 82, 511-519

Vanderhoeven, S., Dassonville, N., Chapuis-Lardy, L., Hayez, M. & Meerts, P. (2006) Impact of the invasive alien plant Solidago gigantea on primary productivity, plant nutrient content and soil mineral nutrient concentrations. Plant and Soil, 286, 259-268

Vanderhoeven, S., Dassonville, N. & Meerts, P. (2005) Increased topsoil mineral nutrient concentrations under exotic invasive plants in Belgium. Plant and Soil, 275, 169-179

Weber, E. & Jakobs, G. (2005) Biological flora of central Europe: Solidago gigantea Aiton. Flora - Morphology, Distribution, Functional Ecology of Plants, 200, 109-118

Weber, E. & Schmid, B. (1998) Latitudinal population differentiation in two species of Solidago (Asteraceae) introduced into Europe. American Journal of Botany, 85, 1110-1121

White, P.J. & Broadley, M.R. (2003) Calcium in plants. Annals of Botany, 92, 487-511

Yang, R.Y., Mei, L.X., Tang, J.J. & Chen, X. (2007) Allelopathic effects of invasive Solidago canadensis L. on germination and growth of native Chinese plant species. Allelopathy Journal, 19, 241-247

Zar, J.H. (1999) Biostatistical analysis, 4th edn. Prentice Hall, Upper Saddle River, New Jersey.

Zhang, Q., Yao, L.J., Yang, R.Y., Yang, X.Y., Tang, J.J. & Chen, X. (2007) Potential allelopathic effects of an invasive species Solidago canadensis on the mycorrhizae of native plant species. Allelopathy Journal, 20, 71-77

112 Phylogeography

Ch a p t e r 4

Phylogeography of native ploidy levels and invasive tetraploids of Solidago gigantea (Asteraceae)

Daniel R. Schlaepfer1, Peter J. Edwards1, Alex Widmer2, Regula Billeter1

1Institute of Integrative Biology, Plant Ecology, ETH Zurich, Zürich, Switzerland 2Institute of Integrative Biology, Plant Ecological Genetics, ETH Zurich, Zürich, Switzerland

113 Chapter 4

Ab s t r a c t

Ploidy level is an important factor in the ecological and genetic makeup of a species and may be a factor affecting its invasiveness. We used a phylogeographic approach to elucidate history of polyploidization and colonization success of diploid and tetraploid Solidago gigantea Aiton (Asteraceae) in North America. We also compared spread in the invasive range in Europe with pattern of colonization in its native range and identified source material of the haplotype lineages invasive in Europe and Asia. To do this, we sequenced 1275 bp of chloroplast intergenic spacer DNA in 268 individuals from 57 populations. Inheritance of chloroplasts was confirmed to be maternal. The phylogeographic analysis showed a complex pattern of 20 haplotypes of diploid and tetraploid plants. In North America, we found significant differentiation among regions, private haplotypes, and isolation by distance. Ploidy levels were more differentiated in the northern regions than in the South. The haplotype network was shallow with one tetraploid- only star-shaped structure that was successful in colonization. Post-glacial migration of diploid S. gigantea occurred mainly northwards east of the Appalachian Mts., and to a lesser degree also southward. Our data suggest that tetraploids have formed several times in North America. Haplotype number and diversity were lower in European populations than in the native range, and we found evidence that four haplotypes were introduced to Europe from two source areas, New England and the Southern Appalachian Mts. We found no evidence for isolation by distance during the process of invasive spread.

Keywords: Multiple introduction, multiple polyploidization, invasive alien species, cpDNA, maternal chloroplast inheritance.

114 Phylogeography

In t r o d u c t i o n

The migration routes and colonization events of many taxa, for example after the last glacial maximum (Bettin et al., 2007), have been elucidated using classical phylogeographic techniques (Avise, 2000). In eastern North America, many species of both plants and animals show a discontinuous distribution on both sides of the Appalachian Mountain, indicating that there were glacial refugia on both sides of this range from which species subsequently migrated; however, distributional patterns of other species indicate a more complex phylogeographic structure, often reflecting several glacial refugia as well as barriers to migration (Soltis et al., 2006). In the genus Solidago of Asteracean forbs, mainly occurring in North America, rather little is known about post-glacial distribution and movement, although it seems clear that past glaciations have shaped their current distribution patterns. For instance, Semple & Cook (2006) argued that diploid populations of species from the S. canadensis complex were isolated at glacial times and that polyploid populations subsequently acquired overlapping ranges and shared morphological traits, either through hybridisation or convergence. And in the case of S. flexicaulis, Chmielewski & Semple (1985) concluded that the present-day distribution of ploidy levels in Ontario also reflects events during the last glaciation. Invasive alien species are a major and increasing concern for ecosystem management worldwide (Chapin et al., 2000; Colautti et al., 2006). Despite considerable recent research, no general mechanisms to explain why some species become invasive could be found (Dietz & Edwards, 2006; Mitchell et al., 2006). However, all invasions pass through a series of stages—transport, colonization, establishment and spread—and success at each of these stages is controlled by filters that are similar for all invasions (Theoharides & Dukes, 2007). Phylogeographic approaches can help in identifying these filters, e.g. source area(s) in the native range, genetic makeup of introduced populations and pattern of spread, and in studying the recent range expansion of invasive plants (Trewick et al., 2004). Furthermore, directly comparing invasive spread with past native range expansion can lead to a better understanding of the invasion process (Dlugosch & Parker, 2007). For example, it may demonstrate whether invasive spread is species-specific (with a similar pattern of spread both in native and invasive ranges of a species) or specific to biological invasions (with the same pattern of invasive spread for different species that differs from native range expansion). Solidago gigantea Aiton (Asteraceae) is a suitable species for studying aspects of both biological invasion in Europe and phylogeographic processes in the native range of North America. Solidago gigantea belongs to subsection Triplinerviae (Torrey & A. Gray) G. L. Nesom, together with S. canadensis, S. lepida and others (Semple & Cook, 2006). It is a 0.5–2.5 m tall herb with annual, partially sexual shoots and persistent, clonal rhizomes. The breeding system involves obligate outcrossing, and pollination is brought about by a wide range of insects (Melville & Morton, 1982). In its native range, S. gigantea occurs as diploids, tetraploids, and hexaploids. Populations consist mostly of plants of a single ploidy level (chapter 1). The range

115 Chapter 4 of diploids spans along the eastern side of the Appalachian Mountains and also in southern Ontario and Québec; tetraploids are found in eastern North America as far west as Missouri; hexaploids occur in mid-western North America, westwards from Manitoba to the Rocky Mountains (chapter 1, Semple & Cook, 2006). In Europe, where the species was introduced in 1758 (Weber & Schmid, 1993), only tetraploids have been detected (chapter 1). Solidago gigantea is also naturalised in northern Japan, Russia and various other parts of the world (chapter 1). The aim of this study was to investigate the significance of ploidy level as a factor affecting the spread of S. gigantea, both in its native range and as an alien species in Europe. We used a phylogeographic approach based on chloroplast DNA sequence data to determine haplotype diversity, history of polyploidization and colonization success of diploid and tetraploid S. gigantea in North America and to identify the source material of the haplotype lineages invasive in Europe and Asia.

Ma t e r i a l s a n d Me t h o d s

Sampling

Seeds of 268 individuals of S. gigantea were collected from 57 populations. We sampled diploids and tetraploids in the native area in three zones of overlap (Ontario, New England and Southern Appalachian Mts.) and tetraploids in two areas of introduction (Europe and two populations from Asia; Fig. 1, Supplementary Material Table S1). Seed families were collected in the field from individual shoots at least 2 m apart to reduce the risk of resampling the same clone. The methods used for ploidy level determination are described in chapter 1. Vouchers are deposited in herbarium Z+ZT. Leaf material from one seedling each from five seed families per population was lyophilised and stored at -80 °C. We included two populations of each Erigeron annuus (L.) Pers., S. virgaurea L. (subsection Solidago), S. lepida DeCandolle and S. canadensis L. (both subsection Triplinerviae, Semple & Cook, 2006) as possible outgroups.

Figure 1 Geographical locations of the North America diploid (A), tetraploid (B) and European tetraploid (C) populations of Solidago gigantea Aiton. Also shown are the proportions of the 20 detected haplotypes. Colour segments indicate shared haplotypes among populations (see legend in A) and white represent haplotypes private to one population. Migration rates of diploids among regions are visualized by arrows (migrants per generation, A).

116 Phylogeography

$1RUWK$PHULFD [  1HZ(QJODQG 2QWDULR

&2817 ( 1  ) 2 * 3  + 4  , 5 $ - 6 % . 7 & / ' 0 6RXWKHUQ$SSDODFKLDQ0WV    ¶.LORPHWHUV

%1RUWK$PHULFD [

2QWDULR

1HZ(QJODQG

6RXWKHUQ$SSDODFKLDQ0WV

&(XURSH [

117 Chapter 4

Analysis of chloroplast inheritance and artificial crosses

We conducted eight reciprocal crosses (four among tetraploids, one among diploids, two among diploids and tetraploids and one among a diploid and a triploid) to check success of mixed-ploidy mating. Although S. gigantea is reported to be self-incompatible, we re-checked self-incompatibility of one diploid and six tetraploid plants by selfing. From each mother, well- developed achenes of 12 flower heads were counted and germination rates were tested in Petri dishes. Ploidy level of mixed-ploidy crosses was determined with flow cytometry (chapter 1). In angiosperms, chloroplasts are maternally inherited, though paternal inheritance does also occur in some taxa. In a phylogenetic study on the genus Solidago, Zhang (1996) assumed a maternal inheritance. However, deviations from strict maternal inheritance appear to be relatively frequent (Mccauley et al., 2007). Therefore, we tested mode of chloroplast inheritance of S. gigantea with 91 offspring plants of five crosses (three among tetraploids, one among diploids, one plant of a diploid–tetraploid cross) with parent plants differing in haplotype. A power analysis with a binomial model estimates the maximal degree of paternal transmission to p = 1 - (1 - b)1/n, given a sample size n of only maternal inheritance instances and power b (Milligan, 1992).

DNA extraction, amplification and sequencing

DNA was extracted from lyophilised leaf tissue using either the CTAB method (Doyle, 1991), modified for 1.5 ml tubes, or, for most samples, a Silica based method (Elphinstone et al., 2003) in 96-well format. First, we screened cpDNA variation in intergenic spacers with 18 primer pairs and selected three: trnH(tRNA-His)–trnK(tRNA-Lys) (Demesure et al., 1995), trnK5’r–rps16-4547mod and trnV5f–atpE(S1022) (Kress et al., 2005). The PCR mixtures (50 µL) contained for trnH-trnK/(trnK5r-rps16mod and trnV-atpE) 28.1/30.6 µL deionized water, 2 U Taq polymerase (GoTaq Flexi DNA Polymerase, Promega, Madison, WI, USA), 10 µL 5*Taq-Buffer, 3 µL 25 mM MgCl2, 2.5/1 µL 2 mM dNTP mixture and 2/1.5 µL 10 mM of each primer. The amplifications were carried out using one cycle of 5/3 min at 94 °C, 25/30 cycles of 30 s at 94 °C, 30 s at 60/(48/55) °C, 1.5/1 min at 72 °C and one cycle of 10/7 min at 72 °C. The PCR products were purified using glass filter plates (MAFB N0B, Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. The PCR product was mixed with equal amount of binding buffer (350 mM guanidine-hydrochloride, 184 mM 2-(N-morpholino) ethanesulfonic acid buffer (MES) free acid and 20 mM MES sodium salt, adjusted to pH 5.6) and filtered using a centrifuge. The filters were washed twice with 80% ethanol, dried and eluted with TE buffer. The BigDye sequencing PCR mixture (10 µL) contained 0.8 µL BigDye Terminator v3.1 (Applied Biosystems, Foster City, CA, USA), 1.6 µL 5*sequencing-buffer, 1.6 µL 1 mM of one

118 Phylogeography primer (using trnH, trnK5r and trnV) and 6 µL of purified PCR product. The amplification was carried out using 60 cylces of 10 s at 94 °C, 5s at 50 °C, 3 min at 60 °C. The product was purified with Sephadex G-50 superfine (GE Healthcare, Little Chalfont, England) and sequenced on an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems).

Sequence analysis

Sequences were manually checked with Sequencher 4.7 (Gene Codes Corp., Ann Arbor, MI, USA). Sequence alignments were made using ClustalX 1.83.1 (Thompson et al., 1997) with default parameters and edited manually following recommendations for non-coding cpDNA (Kelchner, 2000; Borsch et al., 2003; Löhne & Borsch, 2005). In trnV-atpE, a 5 bp long inversion (site 222) of a stem-loop (15 bp stem) was reverse complemented and binary coded. In trnK- rps16, an 8–15 bp long mononucleotide-A string (site 405) was excluded. In trnH-psbA, a region of uncertain homology (hotspot sensu Borsch et al., 2003) mostly of TA repeats was excluded (sites 38–96) as well as a 6–8 bp long mononucleotide-A string (site 221). Variable sites are listed in Supplementary Material Table S2. We retained indels (insertion/deletion; mostly simple sequence repeats) for analyses because they provide much phylogenetic information (Egan & Crandall, 2008). We applied simple indel coding (sic, Simmons & Ochoterena, 2000) with SeqState (Müller, 2005) because it performs well (Ogden & Rosenberg, 2007) and is applicable downstream for MrBayes and TCS analysis.

Phylogenetic relationships

Parsimony analyses were performed with PAUP* 4.0beta10 (Swofford, 2002), using heuristic searches with 100 replicates of random additions, tree-bisection-reconnection branch swapping, collapsing branches if minimum length is zero and saving all most parsimonious trees. Partition homogeneity was tested with the incongruence-length difference (ILD) test (Farris et al., 1995) for 100 replicates under parsimony. Phylogenetic relationships were reconstructed with maximum parsimony (MP). A majority-rule consensus tree of all MP trees was reconstructed. Bootstrap analyses were performed for 500 replicates. Because maximum likelihood methods treat gaps only as missing data (Egan & Crandall, 2008), a Bayesian inference (BI) tree was constructed with mixed models using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003). The best model of sequence evolution was determined as F81 (Felsenstein, 1981) with Modeltest 3.7 (Posada & Crandall, 1998) by both criteria, the AIC and the hLRTs. Sic were treated as binary data under a variable model. Metropolis-coupled Markov Chain Monte Carlo (MC-MCMC) was run for 2’000’000

119 Chapter 4 generations with twice four incrementally heated chains, starting from random trees and sampling one out of every 100 generations. A burn-in of 2000 trees was determined through likelihood score stabilization with Tracer 1.4 (Rambaut & Drummond, 2007). The remaining 18’001 trees were summarized in a majority-rule consensus tree with posterior probabilities as nodal support. We tested whether a single or multiple origins of tetraploid S. gigantea were significantly more likely with a Swofford-Olsen-Waddell-Hilli (SOWH) parametric bootstrap test (Swofford et al., 1996; Goldman et al., 2000). This test determines the significance of the difference between the score of the maximum likelihood (ML) tree (i.e. multiple origin tree) with the score of the best tree under a topological constraint (i.e. monophyly). Because ML treats gaps as missing data, we excluded sic and inversion binary states and retained eight haplotypes, three of which were both diploid and tetraploid. The monophyly constraint tree was constructed in MacClade 4.08 (Maddison & Maddison, 2005). F81+I was determined as the best evolutionary model with Modeltest to estimate the best ML tree consistent with the topological constraint using PAUP*. Null distribution for the test was estimated from 500 replicate datasets simulated with Seq-Gen 1.3.2 (Rambaut & Grassly, 1997) accessed with SGRunner 1.5.3 (Wilcox, 2004).

Phylogeographic analysis

For use in subsequent analyses, the binary codes of indels and of the inversion were recoded to “A” and “T” (Watanabe et al., 2006). Completeness of haplotype sampling was estimated by calculating the probability that all haplotypes were sampled assuming equal frequency of haplotypes (Dixon, 2006). Additionally, the probability that all haplotypes were sampled that occur in at least 5% of all plants was calculated (Holt et al., 2007). Statistical parsimony was applied with TCS 1.21 (Clement et al., 2000) to calculate a 95% minimum spanning network of diploid haplotype genealogy. Because of multiple formations of tetraploids, we added into this backbone network those tetraploid-only haplotypes whose relationships were inferred from a network using all data. The network was verified with the one obtained from Arlequin 3.11 (Excoffier et al., 2005). We used Arlequin to calculate population genetic parameters such as haplotype and private haplotype number (confined to one region), nucleotide diversity (p), haplotype diversity (h) and number of segregating sites. We compared p and h with t-tests for unequal variances. We also calculated Tajima’s D and tested it with 1000 replicates with Arlequin. A phylogeographic signal in the data was inferred by testing Nst > Gst with 1000 replicates using Permut 2.0 (Pons

& Petit, 1996; Burban et al., 1999). A higher Nst than Gst indicates that closely related haplotypes occur more likely in the same population than less related haplotypes. Gst and Nst measure both genetic differentiation while Nst additionally accounts for similarities between haplotypes.

120 Phylogeography

Analyses of molecular variance (AMOVA) were used in Arlequin to partition haplotype composition hierarchically (i) between North American regions, populations and individuals and (ii) between diploids and tetraploids for each North American region. Calculations for both diploid and tetraploid S. gigantea were run with 1023 replicates. We tested for isolation by distance with the ISOLDE option in GenePop 4.0 (Rousset, 2008) between log transformed geographic distance and Slatkin-transformed genetic distance, Fst/(1-Fst) (Rousset, 1997). Significance was assessed with a Mantel test of 10’000 random matrix permutations. Migration patterns of diploids between regions in North America were estimated with migrate-n 2.3 (Beerli & Felsenstein, 2001) based on the coalescent theory and maximum likelihoods. We implemented a stepping-stone model and determined with initial tests the length of chains for convergence. After a first run to estimate start parameters, we re-ran the program with the following Markov chain settings: 10 short chains with 200’000 trees sampled, 4 long chains with 20’000’000 trees sampled, averaging over 4 replicates. We also analyzed our data with nested clade analysis (NCA, Templeton et al., 1987). Some authors have criticized its use (Petit & Grivet, 2002; Petit, 2008), because a simulation analysis (Panchal & Beaumont, 2007) found a high rate of false negatives and because NCA does not assess errors in inference between different scenarios (Knowles & Maddison, 2002). However, the NCA results in our case—showing that several clades of S. gigantea have a history of restricted gene flow with isolation by distance (Supplementary Material Tab. S3 and Fig. S4)— were consistent with those of the other analyses.

Re s u l t s

Artificial crosses and inheritance of chloroplasts

Crosses within a ploidy level of S. gigantea produced well-developed achenes (~40%) that germinated to 100%. In contrast, only one cross between ploidy levels produced viable achenes. This cross—between a tetraploid mother and a diploid father—produced two achenes which both germinated, though only one survived. This single cross-ploidy offspring was triploid. The triploid mother, fertilized by a diploid father, produced one developed achene. It did not survive after germination. Three out of the seven selfed plants, including the selfed diploid, produced in total seven offspring (~0.2%). In the 91 offspring investigated from five crosses, the chloroplast haplotypes indicated only maternal inheritance. The probability of missed paternal inheritance was p ≤ 0.049, given power b = 0.99.

121 Chapter 4

Chloroplast DNA sequence data

Within S. gigantea, our sequences comprised 432 bp of trnV-atpE (GenBank nos EU337706– EU337973), 404 bp of the trnK-rps16 (EU337393–EU337660) and 439 bp of the trnH-psbA (EU337125–EU337392) regions. The corresponding positions on the chloroplast of Lactuca sativa (GenBank AP007232) are 51’984–52’362, 4’471-4’793 and 83–663. One inversion and seven indels in the trnV-atpE, one indel in the trnK-rps16 and nine indels in the trnH-psbA region were found in our sample of 268 individuals. Among these individuals, 10 trnV-atpE, six trnK-rps16 and 11 trnH-psbA haplotypes were distinguished with nucleotide substitutions, indels and an inversion. Because the three partitions were not heterogeneous (ILD, p ≥ 0.34), the sequence data were combined to a dataset of 1275 bp. Twenty haplotypes were recognized from the combined sequences based on 27 variable sites, of which 11 were parsimony-informative (Supplementary Material Table S2). The geographical distribution and relative frequencies of the haplotypes are shown for North America and Europe in Figure 1. To infer phylogenetic relationships with haplotypes of the outgroup species (EU337661– EU337705), sequence data from all species were combined to a dataset of 1359 bp (ILD, p ≥ 0.27). Twenty-six haplotypes were recognized based on 62 variable sites, of which 44 were parsimony-informative. The phylogenetic structure within the set of Solidago species investigated was shallow (Fig. 2) and haplotypes did not group species monophyletically. Within subsection Triplinerviae, three haplotypes were shared among species. Erigeron annuus was a clear outgroup connected to the Solidago clade by 33 changes. Further analyses were, therefore, conducted only with unrooted S. gigantea haplotypes.

Multiple polyploidization in North America and multiple introductions to Europe of tetraploids

Multiple polyploidization events of tetraploids were significantly more likely than a single origin (SWOH-test, ln(L) = -1568.71 vs. -1583.71, p < 0.012). The ML tree used contained only three tetraploid haplotypes, of which two grouped together with the corresponding diploid haplotypes, suggesting that tetraploids were formed at least twice. However, seven origins can be inferred (assuming each haplotype evolved only once) from the diploid and tetraploid haplotypes that co-occur on branches (haplotype T in Fig. 3) or share the same haplotypes (A, C, E, G, Q, S). In the Southern Appalachian Mts., polyploidization events happened between two (S, T) to five (A, E, Q) times. In New England, tetraploids of haplotype C were formed and possibly haplotypes A, E and Q. In Ontario, polyploidization occurred either once (G) or perhaps twice (A). In Europe, we found three common haplotypes (C, D, F) and a fourth one (E) that occurred

122 Phylogeography ns ns ns ns ns ns ns ns ns ns Solidago 1.687 1.101 0.272 -0.924 -0.687 -0.963 -0.622 -0.679 -0.485 -0.809 Tajima‘s D Tajima‘s ns ns * ------test 0.584 0.508 0.407 (Nst>Gst) (SD) ±0.038 ±0.021 ±0.035 ±0.010 ±0.044 ±0.041 ±0.064 ±0.059 ±0.103 ±0.069 h 0.585 0.835 0.798 0.889 0.676 0.704 0.753 0.754 0.684 0.748 (SD) ±0.0013 ±0.0015 ±0.0011 ±0.0014 ±0.0013 ±0.0015 ±0.0015 ±0.0008 ±0.0012 ±0.0008 p 0.0021 0.0026 0.0018 0.0025 0.0021 0.0024 0.0025 0.0012 0.0019 0.0011 7 8 8 9 6 S 22 14 25 13 10 - 0 57 50 50 75 50 38 83 17 private private HT (%) 4 6 4 6 8 6 6 14 12 20 HT ) and Tajima’s neutrality test. Significance level (*, p < 0.05; ns, not significant). st 76 87 91 42 27 25 43 20 21 178 >G # of st Plants 8 6 5 8 4 4 18 17 18 34 # of Populations — (4x) — (4x) — (4x) Ontario (2x) S. App. Mts. (2x) App. S. Genetic diversity indices for North American diploid (2x) and tetraploid (4x) and European tetraploid populations of Aiton, grouped per cytotype and, for North America, ploidy level each region. New England (2x) Cytotype/Region Europe (4x) North America (2x) North America (4x) North America (2x+4x) Table 1 gigantea HT, number of haplotypes; private HT, haplotypes confined to one region; S, number of segregating sites; p, nucleotide diversity; h, haplotypic h, diversity; nucleotide p, sites; segregating of number S, region; one to confined haplotypes HT, private haplotypes; of number HT, (gene) diversity, phylogeographic signal test (N

123 Chapter 4 in two populations in Switzerland (Fig. 1C). None of them was unique to Europe, and we could infer the potential source areas from the distribution of haplotypes in North America. As a tetraploid the rare haplotype E occurred in the Southern Appalachian Mts., though it also occurred as a diploid in New England. The common introduced haplotype C occurred only in New England, while the other two haplotypes were either shared between New England and Ontario (D) or between all three regions (F). In the Asian populations, we found two haplotypes (D, F). Potential source areas for haplotype D were New England, Ontario and Europe, and for haplotype F also the Southern Appalachian Mts.

Phylogeographic structure

Completeness of haplotype sampling was estimated for diploids according to Dixon to a probability of 0.978 and according to Holt to 0.789. The equivalent probabilities for North American tetraploids were 0.995 and 0.807, respectively, and for European tetraploids, 1.000 and 0.653. In North America, diploid and tetraploid populations harboured a comparable number of haplotypes (mean = 2.3±0.2 se, t-test = -0.85, p ≥ 0.403). European tetraploid populations also commonly contained more than one haplotype per population, though the mean number was less than in North American populations (1.7±0.2, t = -2.47, p < 0.017). For North American diploids and European tetraploids there was no tendency for haplotypes to be more closely related within populations than among populations, as indicated by a non-significant phylogeographic signal (Tab. 1). However, there was a significant phylogeographic signal in the case of North American tetraploids. Haplotypic diversity was lower among North American tetraploids than among diploids, and there were differences between ploidy levels in the spatial distribution of diversity. For tetraploids, the highest number of haplotypes was found in New England and the percentage of private haplotypes declined with increased latitude. For diploids, the total number of haplotypes was the same in every region, but percentage of private haplotypes was highest in Ontario. Haplotypic diversity peaked for both ploidy levels in New England (Tab. 1), but not significantly. None of the Tajima’s D neutrality tests was significant. The haplotype diversity of European tetraploids was significantly lower than that of tetraploids in the native range (p < 0.002; Table 1). The minimum spanning network connected all haplotypes of our sample (Fig. 3) with a maximum of 14 mutational steps at 95% confidence. Haplotype A had the highest outgroup probability based on coalescent theory (Castelloe & Templeton, 1994). As a diploid, this haplotype A was found in all three regions and was at the centre of the entire network. Only two other diploid haplotypes (E, M) occurred across regions, namely the Southern Appalachian

124 Phylogeography

Erigeron annuus (1*) Erigeron annuus (2*)

S. virgaurea (3*) 0.76/- S. lepida (1*) R (2x) S. canadensis (1*) 0.71/- M (2x) 0.96/100 T (4x) F (4x, EU, AS) + S. lepida (1*) A (2x+4x) + S. canadensis (5*) B (4x) + S. canadensis (2*) C (2x+4x, EU) 0.53/- D (4x, EU, AS) S (2x+4x) E (2x+4x, EU) 0.99/100 G (2x+4x) N (2x) 0.98/57 O (2x) 1.00/100 K (2x) L (4x) P (4x) 0.87/85 H (2x) J (2x) I (2x) 0.001 substitutions/site Q (2x+4x) Figure 2 Chloroplast phylogram of maternal inherited haplotypes from diploid (2x) and tetraploid (4x) Solidago gigantea (A–T, see Fig. 1) and outgroups (number of plants in parentheses) based on DNA sequence variation of 1359 bp from the trnV-atpE, trnK-rps16 and trnH-psbA regions. Haplotypes introduced to Europe (EU) and Asia (AS) are labelled. The majority-rule consensus tree of 18’001 trees obtained with Bayesian inference (BI) is shown. The majority-rule consensus tree of 5778 maximum parsimony (MP) trees with 500 bootstrap replicates (length = 84 steps, consistency index = 0.738, retention index = 0.645, rescaled consistency index = 0.476) is overlaid. Numbers give support values for the respective nodes (posterior probability (BI)/%-bootstrap (MP)). A hyphen indicates values below 50%.

Mts. and New England. Most other diploid haplotypes were rare and private. Haplotype F formed the centre of a tetraploid-only star-shaped structure. It was linked by one mutational step to haplotype A; this was not only the most widespread diploid haplotype but was also found in tetraploid plants in New England and Ontario. While haplotype F occurred in all three regions, three of its four satellites (B, D, L) were still found in two regions (New England and Ontario) and only its satellite P was private to New England. All other tetraploid haplotypes (C, E, G, Q, S, T) were private to a region. Geographic structuring of haplotypes in North America, as indicated by private haplotypes (Fig. 1), was corroborated by AMOVA. Variation among regions was significant both for diploid and tetraploid populations (Tab. 2), suggesting moderate structuring at a larger geographic scale. In both ploidy levels, populations were significantly differentiated within groups as well as individuals within populations. The highest portion of the variance was found within populations, indicating that most of the genetic diversity existed over small spatial scales. Variation among ploidy levels was significant for each region (Tab. 2). Whereas in Ontario the largest percentage of variation was found between ploidy levels, genetic differentiation

125 Chapter 4

Table 2 Results of analyses of molecular variance (AMOVA) of chloroplast DNA sequence data from diploid and tetraploid populations of Solidago gigantea from North America. Tests among regions (ON, Ontario; NE, New England; SA, Southern Appalachian Mts.) and among ploidy levels (2x, diploids; 4x, tetraploids). Significance levels are based on 1023 permutations (***p < 0.001, **p < 0.01, *p < 0.05; ns, not significant).

Fixation Percentage of Grouping Source of variation d.f. indices variation Among regions

2x of (ON, NE, SA) Among groups 2 Fct =0.16 16.36*

Among pop.s within groups 13 Fsc =0.45 37.59***

Among individuals within pop.s 70 Fst =0.54 46.05***

4x of (ON, NE, SA) Among groups 2 Fct =0.15 15.37**

Among pop.s within groups 15 Fsc =0.39 33.07***

Among individuals within pop.s 73 Fst =0.48 51.56*** Among ploidy levels

ON of (2x, 4x) Among groups 1 Fct =0.48 48.26*

Among pop.s within groups 6 Fsc =0.10 5.19ns

Among individuals within pop.s 33 Fst =0.53 46.55***

NE of (2x, 4x) Among groups 1 Fct =0.33 32.72***

Among pop.s within groups 11 Fsc =0.34 22.82***

Among individuals within pop.s 55 Fst =0.56 44.46***

SA of (2x, 4x) Among groups 1 Fct =0.15 15.18*

Among pop.s within groups 11 Fsc =0.60 51.03***

Among individuals within pop.s 55 Fst =0.66 33.79***

between ploidy levels (Fct) was lower in New England and even more so in the Southern Appalachian Mts.; in these regions, the largest percentage of variation was found within or between populations. Mantel tests for both diploid and tetraploid North American populations showed a weak but significant isolation by distance (diploids: b = 0.049, p < 0.004; tetraploids: b = 0.042, p < 0.019). There was no such trend for European populations (b = 0.021, p ≥ 0.167). A migration analysis of diploids between regions in North America indicated that the main direction of movement of migrants was northwards from Southern Appalachian Mts. to New England and from there to Ontario (Fig. 1, Tab. 3). The results suggest that Ontario was not a source of migrants, while New England not only received migrants from Southern Appalachian Mts. but to a lesser degree was also a source.

Table 3 Pairwise likelihood estimates of migration rates (migrants per generation) of diploid S. gigantea between regions in North America (95% confidence intervals in parentheses). Receiving populations are shown on the left and source populations are given along the top (S. App. Mts., Southern Appalachian Mts.). S. App. Mts. New England Ontario S. App. Mts. - 1.04(0.78–1.34) - New England 3.36(0.6–13.53) - 0(0–0) Ontario - 2.65(0.76–20.58) -

126 Phylogeography

Di s c u s s i o n

History of diploid S. gigantea in North America

The genus Solidago, with its many similar and highly variable species, is known to be taxonomically difficult (Semple & Cook, 2006). In a chloroplast RFLP study on thegenus Solidago, Zhang (1996), could distinguish S. gigantea from S. canadensis but two subspecies of S. canadensis did not group together as sisters, underlining the taxonomic difficulty of this group. Similarly, we were unable to resolve relationships among species using chloroplast DNA sequence data, and found that some haplotypes were shared by more than one species within the subsection Triplinerviae. While it is possible that our resolution was too weak to find species-specific markers, haplotypes shared among species are not uncommon, e.g.Hieracium (Trewick et al., 2004), Pinus (Syring et al., 2007). In the light of the taxonomic difficulties in the genus Solidago, two other explanations seem equally plausible: first, incomplete lineage sorting of haplotypes between species could have led to a sampling from an ancestral gene pool (Avise, 2000) and, second, formerly monophyletic species may have hybridized or captured chloroplasts from other species following secondary contact. Both processes are consistent with recent speciation (Trewick et al., 2004), and differentiating between them is difficult (but see Buckley et al., 2006). The distribution of diploid S. gigantea (east of the Appalachian Mts.) conspicuously follows the “Appalachian Mountain discontinuity”—a phylogeographic break that is reported for both plant and animal species (Soltis et al., 2006). This pattern could be explained by glacial survival in different refugial areas and separate migration routes on opposite sites of the Appalachians; a hypothetical refugium for the eastern side is on the Florida peninsula (Soltis et al., 2006). While the Southern Appalachian Mts. remained ice free, New England and Ontario were glaciated during the last glacial maximum (Dyke et al., 2002). Vegetation maps of the last glacial maximum indicate that temperate woodlands, the present habitat of S. gigantea, occurred only in Florida (Ray & Adams, 2001). We therefore expected to find evidence for a northward colonization by diploids. Furthermore, although the Southern Appalachian Mts. was not a glacial refugium, we expected a northward decline of genetic diversity because of founder effects along the migration route (Austerlitz et al., 1997). In general, regions that have been occupied for a long period tend to contain higher levels of genetic variation than those more recently occupied (Avise, 2000), though depending on historical processes, gene diversity is not necessarily elevated in glacial refugia (Widmer & Lexer, 2001). For diploid S. gigantea, the evidence for a predominantly northward movement of migrants supports our hypothesis of a sequential post-glacial colonization. However, the data also indicate more complex patterns of migration within the main diploid range along the eastern side of the Appalachian Mts. As a result, populations harbour multiple haplotypes that

127 Chapter 4

R G

E* H S K N Q J O A M I T

L C* F*†

B P D*†

Figure 3 Haplotype network of S. gigantea based on chloroplast DNA sequence variation of 1275 bp from the trnV-atpE, trnK-rps16 and trnH-psbA regions. Haplotype names correspond to Fig. 1. Size of circles is proportional to haplotype frequency; region is coded with colours (yellow, Southern Appalachian Mts.; grey, New England; blue, Ontario). Chequerboard patterns indicate tetraploid plants. Haplotypes found in Europe (*) and Asia (†) are indicated. Each line between haplotypes corresponds to one mutational change. Criteria of Crandall & Templeton (1993) resolved dashed lines. Small empty circles indicate hypothetical haplotypes not found in the data set of 268 plants. are not necessarily closely related, and even widely separated regions share half of their diploid haplotypes. Only at the range margin, was this not the case; thus Ontario, although close to New England, shared only one common haplotype. This finding of extensive migration contrasts with the genetic differentiation among regions and the presence of many private haplotypes; we attribute these patterns to a rather complex history of migration, with occasional long-distance dispersal and also local extinction. These results suggest that phylogeographic studies of past migration following deglaciation may need to account for more complex dispersal patterns, sometimes over large distances, than is usually assumed.

128 Phylogeography

Formation and colonization of tetraploid S. gigantea in North America

We found evidence for up to seven independent events of tetraploid formation in North America. These events were not distributed equally among regions, but declined towards the north. Multiple formations of higher ploidy levels seem to be the norm (Soltis, 2005). Polyploidization is one of the important forces in plant evolution (Stebbins, 1971) also because a polyploid is reproductively isolated from its diploid progenitor(s), though this isolation may not be total (Husband & Sabara, 2004). Isolation between ploidy levels of S. gigantea seems to be very strong (Melville & Morton, 1982) but not complete (here, one triploid offspring from mixed-ploidy crosses). Each polyploidization event creates novel adaptive and genomic changes (Adams & Wendel, 2005) and leads to an enrichment of the species’ ecological potential (Comai, 2005). Ecological differentiation between ploidy levels is one of the factors promoting establishment of newly formed polyploids (Thompson & Lumaret, 1992) and is reported for many species, e.g. Anthoxanthum alpinum (Felber-Girard et al., 1996), Galax urcoleata (Johnson et al., 2003) and also S. gigantea (chapter 2). Furthermore, polyploidization provides new potential for the development of invasive types. Maybe as a result, a larger proportion of invasive plants is polyploid than is the case among angiosperms in general (Brown & Marshall, 1981; Bennett et al., 1998; Verlaque et al., 2002; Pandit et al., 2006). In cases of multiple formations of polyploids, i.e. polyphly, there is no common ancestor. Therefore, tetraploid-only phylogenetic trees, networks or coalescent approaches may yield a distorted result of true relationships. Nevertheless, we found regional differences between tetraploid populations, with isolation by distance that was similar to that found in diploid populations. The distribution of tetraploid S. gigantea (west of the Appalachian Mts.) contrasts the diploids’ range (east of the Appalachian Mts.). Their distribution generally follows the “Appalachian Mountain discontinuity” (Soltis et al., 2006). Many studies support the hypothesis that polyploids were better colonizers after the last glaciation than diploids (Ehrendorfer, 1980; Soltis, 1984; Brochmann et al., 2004; Joly & Bruneau, 2004). Coalescent theory predicts that older lineages are more likely to be widespread (Donnelly & Tavare, 1986; Hudson, 1990). Because all tetraploid lineages must be younger than their diploid progenitors, a tetraploid lineage is more successful if it is more widespread than its diploid progenitor (i.e. five widespread tetraploid vs. one widespread diploid haplotype). Both diploids and tetraploids of S. gigantea expanded their ranges, but tetraploids seem to have been far more successful in this respect. The various tetraploid haplotypes differed in their distribution range and therefore presumably in their colonizing ability. In addition to the many haplotypes that did not spread far, there was one notably successful lineage that gave rise to the widely distributed ‘star- shaped cluster’. Such a cluster can indicate recent population expansion (Avise, 2000). It was mainly due to this successful lineage that we found a significant phylogeographic signal, even

129 Chapter 4 though many populations harboured plants originating from independent polyploidization events. Furthermore, all plants from two hexaploid populations of S. gigantea additionally included for sequencing (Manitoba, South Dakota; Supplementary Material Tab. S1) as well as the only triploid and pentaploid plants (population US009, Tab. S1) were of this lineage. Thus, this successful lineage colonized not only our three study regions but expanded also to the west of the tetraploid range.

Introductions of invasive S. gigantea

This expanding tetraploid lineage was not only successful in colonizing North America, but was also successful in invading other regions of the world. Two of the three haplotypes that contribute to the invasion success of S. gigantea in Europe and both of the two haplotypes detected in Asian populations are from this lineage. However, the third European haplotype (C), although successful in Europe, was less widespread in North America. The limited range of this haplotype in North America may be explained by its recent formation; we estimate the time of deglaciation as upper age limit for those polyploidization events that were only found in previously glaciated areas (haplotypes C and G). Our data indicate that tetraploid S. gigantea must have been introduced to Europe as at least four mothers from at least two source regions in North America, namely New England and Southern Appalachian Mts. Obviously, there is a small possibility that we missed the fourth haplotype among tetraploids in New England, which may reduce the source regions to one. Multiple introductions are commonly found in invasive plants species (Bossdorf et al., 2005), e.g. Hypericum perforatum (Maron et al., 2004), Alliaria petiolata (Durka et al., 2005) and may even be an important factor in their success as invaders. Through introduction of genetic variation, extent of founder effects and inbreeding after bottlenecks can be mitigated (Durka et al., 2005) and new evolutionary potential created when previously isolated populations are mixed (Ellstrand & Schierenbeck, 2000). Our results support the conclusion of Weber & Schmid (1993), based upon historical data, that S. gigantea was only introduced to Europe on a few occasions. The eastern part of North America, New England, was identified as potential source area (Weber, 1997). Solidago was first introduced to botanical gardens in London (S. gigantea in 1758) and Paris (Weber & Schmid, 1993). From these gardens, plant material was further distributed all over Europe so that S. gigantea did not expand its range gradually but appeared at several places almost simultaneously (Weber & Schmid, 1993). Our result from the isolation by distance analysis, which found no correlation, is in concordance with the proposed non-uniform pattern of expansion. In principle, the absence of isolation by distance could be due to range expansion (Slatkin, 1993), or to multiple introductions of the same haplotypes into different areas, or to introductions of unrelated haplotypes to the same area. In our case, a combination of all

130 Phylogeography three explanations is plausible. A tentative comparison between invasive spread and native colonization indicates that in both ranges the successful lineage spread at least partly through range expansion. In S. gigantea, haplotype number and haplotypic diversity are reduced in the invasive range and we found no evidence that novel haplotypes have been formed through mutation after introduction. Despite reduced diversity, S. gigantea is invasive in Europe, maybe because polyploidization created this successful star-shaped lineage that was pre-adapted to successful spread. This supports the hypothesis that tetraploid S. gigantea is successful because of strong colonization ability and a wider ecological niche than diploids (chapter 2). In an experimental study, it was shown that the tetraploid S. gigantea, unlike the diploid, produces an extensive rhizome system and benefits more from available nutrients (chapter 3). It seems plausible that these differences could allow tetraploids to expand successfully both in their native and invasive ranges.

Ac k n o w l e d g m e n t s

We thank Claudia Michel for assistance in the lab, Carmen Rothenbühler, Alex Kocyan, Patrick Brunner and Rolf Holderegger for advice with genetic analyses, Wilma Blaser for germination tests, John C. Semple and Markus Hofbauer for help in the field. We also express our thanks to all of the many collectors of seeds. The project is funded by the grant 0-20259-05 from ETH Zurich, Switzerland.

Re f e r e n c e s

Adams, K.L. & Wendel, J.F. (2005) Polyploidy and genome evolution in plants. Current Opinion in Plant Biology, 8, 135-141

Austerlitz, F., Jungmuller, B., Godelle, B. & Gouyon, P.H. (1997) Evolution of coalescence times, genetic diversity and structure during colonization. Theoretical Population Biology, 51, 148-164

Avise, J.C. (2000) Phylogeography: the history and formation of species. Harvard University Press, Cambridge, Mass.

Beerli, P. & Felsenstein, J. (2001) Maximum likelihood estimation of a migration matrix and effective population sizes in n subpopulations by using a coalescent approach. Proceedings of the National Academy of Sciences of the United States of America, 98, 4563-4568

Bennett, M.D., Leitch, I.J. & Hanson, L. (1998) DNA amounts in two samples of angiosperm weeds. Annals of Botany, 82, 121-134 131 Chapter 4

Bettin, O., Cornejo, C., Edwards, P.J. & Holderegger, R. (2007) Phylogeography of the high alpine plant Senecio halleri (Asteraceae) in the European Alps: in situ glacial survival with postglacial stepwise dispersal into peripheral areas. Molecular Ecology, 16, 2517-2524

Borsch, T., Hilu, K.W., Quandt, D., Wilde, V., Neinhuis, C. & Barthlott, W. (2003) Noncoding plastid trnT-trnF sequences reveal a well resolved phylogeny of basal angiosperms. Journal of Evolutionary Biology, 16, 558-576

Bossdorf, O., Auge, H., Lafuma, L., Rogers, W.E., Siemann, E. & Prati, D. (2005) Phenotypic and genetic differentiation between native and introduced plant populations. Oecologia, 144, 1-11

Brochmann, C., Brysting, A.K., Alsos, I.G., Borgen, L., Grundt, H.H., Scheen, A.-C. & Elven, R. (2004) Polyploidy in arctic plants. Biological Journal of the Linnean Society, 82, 521-536

Brown, A.H.D. & Marshall, D.S. (1981) Evolutionary changes accompanying colonization in plants. Evolution Today. Proceedings of 2nd international Congress of Systematics and Evolutionary Biology (ed. by G.G.E. Scudder and J.L. Reveal), pp. 351-363. Carnegie-Mellon University, Pittsburg.

Buckley, T.R., Cordeiro, M., Marshall, D.C. & Simon, C. (2006) Differentiating between hypotheses of lineage sorting and introgression in New Zealand alpine cicadas (Maoricicada dugdale). Systematic Biology, 55, 411-425

Burban, C., Petit, R.J., Carcreff, E. & Jactel, H. (1999) Rangewide variation of the maritime pine bast scale Matsucoccus feytaudi Duc. (Homoptera: Matsucoccidae) in relation to the genetic structure of its host. Molecular Ecology, 8, 1593-1602

Castelloe, J. & Templeton, A.R. (1994) Root probabilities for intraspecific gene trees under neutral coalescent theory. Molecular Phylogenetics and Evolution, 3, 102-113

Chapin, F.S., Zavaleta, E.S., Eviner, V.T., Naylor, R.L., Vitousek, P.M., Reynolds, H.L., Hooper, D.U., Lavorel, S., Sala, O.E., Hobbie, S.E., Mack, M.C. & Diaz, S. (2000) Consequences of changing biodiversity. Nature, 405, 234-242

Chmielewski, J.G. & Semple, J.C. (1985) The cytogeography and post-glacial migration of Solidago flexicaulis (Compositae) into southern Ontario (Canada). Naturaliste Canadien (Quebec), 112, 307-312

Clement, M., Posada, D. & Crandall, K.A. (2000) TCS: a computer program to estimate gene genealogies. Molecular Ecology, 9, 1657-1659

Colautti, R.I., Bailey, S.A., Van Overdijk, C.D.A., Amundsen, K. & Macisaac, H.J. (2006) Characterised and projected costs of nonindigenous species in Canada. Biological Invasions, 8, 45-59

Comai, L. (2005) The advantages and disadvantages of being polyploid. Nature Reviews Genetics, 6, 836-846

Crandall, K.A. & Templeton, A.R. (1993) Empirical tests of some predictions from coalescent theory with applications to intraspecific phylogeny reconstruction. Genetics, 134, 959-969

Demesure, B., Sozi, N. & Petit, R.J. (1995) A set of universal primers for amplification of polymorphic noncoding regions of mitochondrial and chloroplast DNA in plants. Molecular Ecology, 4, 129- 131 132 Phylogeography

Dietz, H. & Edwards, P.J. (2006) Recognition that causal processes change during plant invasion helps explain conflicts in evidence. Ecology, 87, 1359-1367

Dixon, C.J. (2006) A means of estimating the completeness of haplotype sampling using the Stirling probability distribution. Molecular Ecology Notes, 6, 650-652

Dlugosch, K.M. & Parker, I.M. (2007) Molecular and quantitative trait variation across the native range of the invasive species Hypericum canariense: evidence for ancient patterns of colonization via pre-adaptation? Molecular Ecology, 16, 4269-4283

Donnelly, P. & Tavare, S. (1986) The ages of alleles and a coalescent. Advances in Applied Probability, 18, 1-19

Doyle, J.J. (1991) DNA protocols for plants. Molecular Techniques in Taxonomy (ed. by G.M. Hewitt, J.P.W. Young and A.W.B. Johnston), pp. 283-293. NATO ASI Series H, Berlin etc.

Durka, W., Bossdorf, O., Prati, D. & Auge, H. (2005) Molecular evidence for multiple introductions of garlic mustard (Alliaria petiolata, Brassicaceae) to North America. Molecular Ecology, 14, 1697- 1706

Dyke, A.S., Andrews, J.T., Clark, P.U., England, J.H., Miller, G.H., Shaw, J. & Veillette, J.J. (2002) The Laurentide and Innuitian ice sheets during the Last Glacial Maximum. Quaternary Science Reviews, 21, 9-31

Egan, A.N. & Crandall, K.A. (2008) Incorporating gaps as phylogenetic characters across eight DNA regions: ramifications for North American Psoraleeae (Leguminosae). Molecular Phylogenetics and Evolution, in press. Available online 17 October 2007. doi:10.1016/j.ympev.2007.10.006

Ehrendorfer, F. (1980) Polyploidy and distribution. Polyploidy - Biological relevance (ed. by W.H. Lewis), pp. 45-60. Plenum Press, New York, London.

Ellstrand, N.C. & Schierenbeck, K.A. (2000) Hybridization as a stimulus for the evolution of invasiveness in plants? Proceedings of the National Academy of Sciences of the United States of America, 97, 7043-7050

Elphinstone, M.S., Hinten, G.N., Anderson, M.J. & Nock, C.J. (2003) An inexpensive and high- throughput procedure to extract and purify total genomic DNA for population studies. Molecular Ecology Notes, 3, 317-320

Excoffier, L., Laval, G. & Schneider, S. (2005) Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online, 1, 47-50

Farris, J.S., Kallersjo, M., Kluge, A.G. & Bult, C. (1995) Constructing a significance test for incongruence. Systematic Biology, 44, 570-572

Felber-Girard, M., Felber, F. & Buttler, A. (1996) Habitat differentiation in a narrow hybrid zone between diploid and tetraploid Anthoxanthum alpinum. New Phytologist, 133, 531-540

Felsenstein, J. (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of Molecular Evolution, 17, 368-376

Goldman, N., Anderson, J.P. & Rodrigo, A.G. (2000) Likelihood-based tests of topologies in phylogenetics. Systematic Biology, 49, 652-670

133 Chapter 4

Holt, J.A., Holt, S.D.S. & Bures, P. (2007) Experimental design in intraspecific organelle DNA sequence studies III: statistical measures of sampling success. Taxon, 56, 847-856

Hudson, R.R. (1990) Gene genealogies and the coalescent process. Oxford surveys in evolutionary biology (ed. by D. Futuyama and J. Antonovics), pp. 1-44. Oxford University Press, Oxford.

Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17, 754-755

Husband, B.C. & Sabara, H.A. (2004) Reproductive isolation between autotetraploids and their diploid progenitors in fireweed, Chamerion angustifolium (Onagraceae). New Phytologist, 161, 703-713

Johnson, M.T.J., Husband, B.C. & Burton, T.L. (2003) Habitat differentiation between diploid and tetraploid Galax urceolata (Diapensiaceae). International Journal of Plant Sciences, 164, 703- 710

Joly, S. & Bruneau, A. (2004) Evolution of triploidy in Apios americana (Leguminosae) revealed by genealogical analysis of the histone H3-D gene. Evolution, 58, 284-295

Kelchner, S.A. (2000) The evolution of non-coding chloroplast DNA and its application in plant systematics. Annals of the Missouri Botanical Garden, 87, 482-498

Knowles, L.L. & Maddison, W.P. (2002) Statistical phylogeography. Molecular Ecology, 11, 2623-2635

Kress, W.J., Wurdack, K.J., Zimmer, E.A., Weigt, L.A. & Janzen, D.H. (2005) Use of DNA barcodes to identify flowering plants.Proceedings of the National Academy of Sciences of the United States of America, 102, 8369-8374

Löhne, C. & Borsch, T. (2005) Molecular evolution and phylogenetic utility of the petD group II intron: A case study in basal angiosperms. Molecular Biology and Evolution, 22, 317-332

Maddison, D.R. & Maddison, W.P. (2005) MacClade 4: Analysis of phylogeny and character evolution. Version 4.08. Sinauer Associates, Sunderland, MA, USA

Maron, J.L., Vila, M., Bommarco, R., Elmendorf, S. & Beardsley, P. (2004) Rapid evolution of an invasive plant. Ecological Monographs, 74, 261-280

Mccauley, D.E., Sundby, A.K., Bailey, M.F. & Welch, M.E. (2007) Inheritance of chloroplast DNA is not strictly maternal in Silene vulgaris (Caryophyllaceae): evidence from experimental crosses and natural populations. American Journal of Botany, 94, 1333-1337

Melville, M.R. & Morton, J.K. (1982) A biosystematic study of the Solidago canadensis (Compositae) complex. 1. The Ontario populations. Canadian Journal of Botany - Revue Canadienne de Botanique, 60, 976-997

Milligan, B.G. (1992) Is organelle DNA strictly maternally inherited? Power analysis of a binomial distribution. American Journal of Botany, 79, 1325-1328

Mitchell, C.E., Agrawal, A.A., Bever, J.D., Gilbert, G.S., Hufbauer, R.A., Klironomos, J.N., Maron, J.L., Morris, W.F., Parker, I.M., Power, A.G., Seabloom, E.W., Torchin, M.E. & Vazquez, D.P. (2006) Biotic interactions and plant invasions. Ecology Letters, 9, 726-740

Müller, K. (2005) SeqState: Primer design and sequence statistics for phylogenetic DNA datasets. Applied Bionformatics, 4, 65-69 134 Phylogeography

Ogden, T.H. & Rosenberg, M.S. (2007) How should gaps be treated in parsimony? A comparison of approaches using simulation. Molecular Phylogenetics and Evolution, 42, 817-826

Panchal, M. & Beaumont, M.A. (2007) The automation and evaluation of nested clade phylogeographic analysis. Evolution, 61, 1466-1480

Pandit, M.K., Tan, H.T.W. & Bisht, M.S. (2006) Polyploidy in invasive plant species of Singapore. Botanical Journal of the Linnean Society, 151, 395-403

Petit, R.J. (2008) The coup de grâce for the nested clade phylogeographic analysis? Molecular Ecology, 17, 516-518

Petit, R.J. & Grivet, D. (2002) Optimal randomization strategies when testing the existence of a phylogeographic structure. Genetics, 161, 469-471

Pons, O. & Petit, R.J. (1996) Measuring and testing genetic differentiation with ordered versus unordered alleles. Genetics, 144, 1237-1245

Posada, D. & Crandall, K.A. (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics, 14, 817-818

Rambaut, A. & Drummond, A. (2007) Tracer v1.4, Available from http://beast.bio.ed.ac.uk/Tracer [Last visited 16 January 2008].

Rambaut, A. & Grassly, N.C. (1997) Seq-Gen: An application for the Monte Carlo simulation of DNA sequence evolution along phylogenetic frees. Computer Applications in the Biosciences, 13, 235- 238

Ray, N. & Adams, J.M. (2001) A GIS-based vegetation map of the world at the Last Glacial Maximum (25,000-15,000 BP). Internet Archaeology, 11, http://intarch.ac.uk/journal/issue11/rayadams_ index.html

Ronquist, F. & Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19, 1572-1574

Rousset, F. (1997) Genetic differentiation and estimation of gene flow from F-statistics under isolation by distance. Genetics, 145, 1219-1228

Rousset, F. (2008) Genepop’007: a complete re-implementation of the genepop software for Windows and Linux. Molecular Ecology Resources, 8, 103-106

Semple, J.C. & Cook, R.E. (2006) Solidago. Flora of North America North of Mexico (ed. by Flora of North America Editorial Committee), pp. 107-166. Oxford University Press, Oxford and New York.

Simmons, M.P. & Ochoterena, H. (2000) Gaps as characters in sequence-based phylogenetic analyses. Systematic Biology, 49, 369-381

Slatkin, M. (1993) Isolation by distance in equilibrium and non-equilibrium populations. Evolution, 47, 264-279

Soltis, D.E. (1984) Autopolyploidy in Tolmiea menziesii (Saxifragaceae). American Journal of Botany, 71, 1171-1174

135 Chapter 4

Soltis, D.E., Morris, A.B., McLachlan, J.S., Manos, P.S. & Soltis, P.S. (2006) Comparative phylogeography of unglaciated eastern North America. Molecular Ecology, 15, 4261-4293

Soltis, P.S. (2005) Ancient and recent polyploidy in angiosperms. New Phytologist, 166, 5-8

Stebbins, G.L. (1971) Chromosomal Evolution in Higher Plants. Edward Arnold, London.

Swofford, D.L. (2002) PAUP*: Phylogenetic analysis using parsimony (and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts, USA

Swofford, D.L., Olsen, G.J., Waddell, P.J. & Hillis, D.M. (1996) Phylogenetic inference. Molecular systematics (ed. by D.M. Hillis, C. Mortiz and B.K. Mable), pp. 407–514. Sinauer Associates, Sunderland, MA, USA.

Syring, J., Farrell, K., Businsky, R., Cronn, R. & Liston, A. (2007) Widespread genealogical nonmonophyly in species of Pinus subgenus Strobus. Systematic Biology, 56, 163-181

Templeton, A.R., Boerwinkle, E. & Sing, C.F. (1987) A cladistic analysis of phenotypic associations with haplotypes Inferred from restriction endonuclease mapping .1. Basic theory and an analysis of alcohol dehydrogenase activity in Drosophila. Genetics, 117, 343-351

Theoharides, K.A. & Dukes, J.S. (2007) Plant invasion across space and time: factors affecting nonindigenous species success during four stages of invasion. New Phytologist, 176, 256-273

Thompson, J., Gibson, T., Plewniak, F., Jeanmougin, F. & Higgins, D. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Research, 25, 4876-4882

Thompson, J.D. & Lumaret, R. (1992) The evolutionary dynamics of polyploid plants - origins, establishment and persistence. Trends in Ecology & Evolution, 7, 302-307

Trewick, S.A., Morgan-Richards, M. & Chapman, H.M. (2004) Chloroplast DNA diversity of Hieracium pilosella (Asteraceae) introduced to New Zealand: Reticulation, hybridization, and invasion. American Journal of Botany, 91, 73-85

Verlaque, R., Aboucaya, A. & Fridlender, A. (2002) Invasive alien flora of France: ecology, life-forms and polyploidy. Botanica Helvetica, 112, 121-136

Watanabe, K., Kajita, T. & Murata, J. (2006) Chloroplast DNA variation and geographical structure of the Aristolochia kaempferi group (Aristolochiaceae). American Journal of Botany, 93, 442-453

Weber, E. (1997) Phenotypic variation of the introduced perennial Solidago gigantea in Europe. Nordic Journal of Botany, 17, 631-638

Weber, E. & Schmid, B. (1993) Das Neophytenproblem. Dissertationes Botanicae, 196, 209-227

Widmer, A. & Lexer, C. (2001) Glacial refugia: sanctuaries for allelic richness, but not for gene diversity. Trends in Ecology & Evolution, 16, 267-269

Wilcox, T.P. (2004) SG Runner: A Mac OS X shell-wrapper for Seq-Gen. Published by the author, Long Key, FL, USA

Zhang, J. (1996) A molecular biosystematic study of North American Solidago and related genera (Asteraceae: Astereae) based on chloroplast DNA RFLP Analysis. PhD thesis. University of Waterloo, Canada. 136 Phylogeography

Supplementary Material

Appendix Table S1 Populations of Solidago gigantea Aiton with sampling location, ploidy level, number of plants investigated and haplotype composition. Appendix Table S2 Haplotypes identified in our sample of 268S. gigantea chloroplast DNA sequences: 432 bp from the trnV-atpE, 404 bp from the trnK-rps16 and 439 bp from the trnH- psbA regions. Appendix Table S3 Nested clade analysis for 17 diploid and 18 tetraploid populations of Solidago gigantea from North America. Appendix Figure S4 Nested cladogram obtained from the minimum spanning network of S. gigantea haplotypes (Fig. 3). References for Supplementary Material

Ap p e n d i x S1

Table S1 Populations of Solidago gigantea Aiton with sampling location, ploidy level, number of plants investigated and haplotype composition (sampling details, see chapter 1). Vouchers are deposited in herbarium Z+ZT.

137 Chapter 4: Supplementary material

Population Region Latitude Longitude Ploidy Voucher no. Count Asia (2 Pop.s) 10 SG284 Japan 42.49 142.12 4 Sch&N 1128 5 SG320 Russia 43.47 131.93 4 Sch&R&P 1173 5 Europe (18 Pop.s) 76 SG005 Switzerland 47.36 8.51 4 Sch 1063 4 SG219 Switzerland 47.14 9.05 4 Sch 1076 2 SG201 Czech Republic 50.09 14.51 4 Sch&H 1020 5 SG203 Germany 52.28 9.18 4 Sch&B 1194 5 SG210 Denmark 55.62 12.48 4 Sch&K 1058 5 SG215 Austria 48.31 16.33 4 Sch&D 1143 5 SG217 Poland 50.03 19.90 4 Sch&M&R&W 1146 5 SG218 Italy 46.21 12.99 4 Sch&O 1071 1 SG307 Italy 44.87 9.55 4 Sch 1184 5 SG309 Italy 45.33 9.12 4 5 SG280 Hungary 47.62 19.57 4 Sch&M 1125 3 SG281 Croatia 45.81 15.96 4 5 SG282 Netherlands 52.09 5.12 4 Sch&O 1126 5 SG289 Belgium 50.48 4.94 4 5 SG305 Sweden 56.10 13.71 4 5 SG313 United Kingdom 51.22 -0.58 4 4 SG314 United Kingdom 53.43 -2.24 4 Sch&P 1141 4 SG315 United Kingdom 52.29 -1.55 4 Sch&P 1174 3 North America (2x; 17 Pop.s) 87 SG251 New England 42.22 -71.31 2 5 SG252 New England 42.09 -71.54 2 Sch&H 1139 5 SG253 New England 42.35 -71.62 2 5 SG254 New England 42.51 -71.86 2 5 SG255 New England 42.59 -71.54 2 5 SG223 Ontario 44.66 -77.10 2 5 SG224 Ontario 44.75 -76.85 2 Sch&B&S 1077 5 SG225 Ontario 44.84 -76.52 2 5 SG229 Ontario 44.63 -76.40 2 5 SG263 Southern Appalachian Mts. 34.63 -84.38 2 5 SG264 Southern Appalachian Mts. 34.69 -84.48 2 Sch&H 1118 1 SG266 Southern Appalachian Mts. 35.13 -85.13 2 Sch&H 1175 5 SG268 Southern Appalachian Mts. 35.92 -84.08 2 Sch&H 1178 5 SG269 Southern Appalachian Mts. 36.39 -83.71 2 5 SG276 Southern Appalachian Mts. 35.99 -82.80 2 5 SG277 Southern Appalachian Mts. 35.93 -82.73 2 SG277.10 11 SG279 Southern Appalachian Mts. 35.19 -82.72 2 5 North America (4x; 18 Pop.s) 91 SG256 New England 42.76 -71.10 4 Sch&H 1114 5 SG257 New England 44.13 -71.95 4 Sch&H 1115 5 SG258 New England 44.22 -72.56 4 Sch&H 1137 5 SG259 New England 44.44 -73.02 4 Sch&H 1116 5 SG260 New England 44.36 -72.73 4 Sch&H 1091 5 SG261 New England 44.57 -72.57 4 Sch&H 1117 5 SG262 New England 44.41 -72.20 4 Sch&H 1140 5 US009 New England 40.97 -77.07 4 8 SG220 Ontario 43.47 -80.54 4 Sch&B&S 1105 6 SG222 Ontario 44.37 -77.02 4 Sch&B&S 1107 5 SG230 Ontario 43.91 -80.52 4 Sch&B&S 1179 5 SG235 Ontario 43.39 -80.38 4 Sch&B 1087 5 SG267 Southern Appalachian Mts. 35.30 -84.54 4 Sch&H 1120 5 SG272 Southern Appalachian Mts. 36.53 -81.95 4 5 SG273 Southern Appalachian Mts. 36.41 -81.76 4 5 SG275 Southern Appalachian Mts. 36.32 -82.08 4 Sch&H 1123 5 SG277 Southern Appalachian Mts. 35.93 -82.73 4 2 SG278 Southern Appalachian Mts. 35.39 -82.57 4 Sch&H 1200 5 North America (others) – US009 New England 40.97 -77.07 3 Sch&J 1034 1 US009 New England 40.97 -77.07 5 Sch&J 1035 1 SG236 Manitoba 49.86 -97.25 6 2 SG245 South Dakota 44.67 -103.86 6 Sch&H 1110 2 Outgroup species – Erigeron annuus Europe, Switzerland (mes)* - 1 Erigeron annuus Europe, Switzerland (sch)* - 2 Solidago virgaurea Europe, Switzerland (SV001) 8.58 47.35 - Sch 1207 3 Solidago canadensis Europe, Switzerland (SC002) 47.38 8.57 - Sch 1206 2 Solidago canadensis USA, Ontario (SC038) 43.45 -80.54 - Sch&B 1100 5 Solidago lepida USA, Oregon 45.30 -117.81 - Sch&A&D&P 1040 1 Solidago lepida USA, Oregon (SL012)† - 1 *Provided by Miluse Trtiková; †Provided by Jake Alexander

138 Phylogeography

Haplotyptes Population A B C D E F G H I J K L M N O P Q R S T EA1 EA2 SV SC1 SL1 2 8 SG284 2 3 SG320 5 25 42 2 7 SG005 1 2 1 SG219 1 1 SG201 5 SG203 3 1 1 SG210 4 1 SG215 5 SG217 5 SG218 1 SG307 1 1 3 SG309 5 SG280 3 SG281 1 4 SG282 3 2 SG289 1 3 1 SG305 5 SG313 4 SG314 4 SG315 3 24 5 13 11 2 2 1 1 20 1 1 1 4 1 SG251 5 SG252 1 1 3 SG253 2 1 1 1 SG254 1 4 SG255 1 4 SG223 1 4 SG224 4 1 SG225 2 1 2 SG229 2 1 1 1 SG263 1 4 SG264 1 SG266 4 1 SG268 5 SG269 2 3 SG276 4 1 SG277 2 9 SG279 4 1 5 6 3 9 11 37 1 7 2 2 7 1 SG256 5 SG257 1 3 1 SG258 1 1 1 2 SG259 1 4 SG260 1 1 1 2 SG261 1 1 2 1 SG262 2 3 US009 2 1 5 SG220 1 2 3 SG222 1 3 1 SG230 1 4 SG235 1 3 1 SG267 3 2 SG272 5 SG273 5 SG275 2 3 SG277 1 1 SG278 4 1

US009 1 US009 1 SG236 2 SG245 2

Erigeron annuus 1 Erigeron annuus 1 1 Solidago virgaurea 3 Solidago canadensis 1 1 Solidago canadensis 4 1 Solidago lepida 1 Solidago lepida 1

139 Chapter 4: Supplementary material

Ap p e n d i x S2

Table S2 Haplotypes identified in our sample of 268 S. gigantea chloroplast DNA sequences: 432 bp from the trnV-atpE, 404 bp from the trnK-rps16 and 439 bp from the trnH-psbA regions. Only variable sites are shown. Site numbers refer to the aligned position in our concatenated 1275 bp dataset after simple indel coding (sic). Site 1144 is the code for the inversion in the trnV-atpE region. Sic are at positions 1145–1161; question marks ‘?’ indicate undeterminable shorter indels within longer indels.

trnV-atpE trnK-rps16 trnH-psbA inversion_222_226 indel1_300_300 indel2_303_310 indel3_325_330 indel4_325_337 indel5_331_337 indel6_344_349 indel7_421_426 indel8_497_514 indel9_861_870 indel10_883_888 indel11_900_914 indel12_905_939 indel13_950_955 indel14_950_960 indel15_956_960 indel16_969_981 indel17_971_975 37

Haplotype 273 491 527 567 759 794 934 1144 1145 1146 1147 1148 1149 1150 1151 1152 1106 1153 1154 1155 1156 1157 1158 1159 1160 1161 A C 0 A 0 1 0 0 0 0 1 C 0 T G T C T T 0 1 0 1 ? 1 ? 0 1 B C 0 A 0 1 0 0 0 0 1 C 0 T A T C T T 1 1 0 1 ? 1 ? 0 1 C C 1 A 1 1 0 0 1 0 1 C 0 T G T C T T 0 1 0 1 ? 1 ? 0 1 D C 0 A 0 1 0 0 0 0 1 C 0 T G T C T T 1 1 0 1 0 0 1 0 1 E T 0 A 0 0 0 0 0 0 1 C 0 T G T C T T 0 1 0 1 ? 1 ? 0 1 F C 0 A 0 1 0 0 0 0 1 C 0 T G T C T T 1 1 0 1 ? 1 ? 0 1 G T 0 A 0 0 0 0 1 0 1 C 0 T G T C T T 0 1 0 1 ? 1 ? 0 1 H C 0 T 0 1 ? 1 ? 0 1 C 0 T G T C T T 0 1 0 0 ? 1 ? 0 1 I C 0 A 0 1 0 0 1 0 1 C 0 T G T C T T 0 0 0 1 ? 1 ? 0 1 J C 0 A 0 1 ? 1 ? 0 1 C 1 T G T C T T 0 1 0 1 ? 1 ? 0 1 K C 0 A 0 1 0 0 1 1 1 C 0 T G T C G T 0 1 0 1 ? 1 ? 0 1 L C 0 A 0 1 0 0 1 0 1 C 0 T G T C T T 1 1 0 1 ? 1 ? 0 1 M C 0 A 0 1 0 0 0 0 1 T 0 T G A C T T 0 1 0 1 ? 1 ? 0 1 N C 0 A 0 1 0 0 1 0 1 C 0 T G T C G A 0 1 0 1 ? 1 ? 0 1 O C 0 A 0 1 0 0 1 0 1 C 0 T G T C G T 0 1 0 1 1 0 0 0 1 P C 0 A 0 1 0 0 0 0 1 C 0 T G T C T T 1 1 0 1 ? 1 ? 1 ? Q C 0 A 0 1 1 0 0 0 1 C 0 T G T C T T 0 1 0 1 ? 1 ? 0 1 R C 0 A 0 1 0 0 1 1 1 C 0 G G T G T T 0 1 1 0 ? 1 ? 0 1 S C 0 A 0 1 0 0 1 0 0 C 0 T G T C T T 0 1 0 1 ? 1 ? 0 0 T C 0 A 0 1 0 0 0 0 1 T 0 T G A C T T 1 1 0 1 ? 1 ? 0 1

140 Phylogeography

Ap p e n d i x S3

Table S3 Nested clade analysis for 17 diploid and 18 tetraploid populations of S. gigantea from North America. NCA was performed on the 95% parsimonious haplotype network (Fig. 3) with the automation of ANeCA 1.0 (Panchal, 2007) incorporating GeoDis (Posada et al., 2000) and the latest inference key (11 November 2005). Nesting of clades according to Figure S4. ***p<0.001; *p<0.05; ns, not significant. Significance levels are based on 1000 permutations.

Clade c2 statistic Significance Chain of inference Demographic event 1-1 36.00 0.000 *** 1-19-20-2-3-4 Restricted gene flow with isolation by NO distance (restricted dispersal by distance in non-sexual species) 1-6 86.91 0.023 * 1-2-3-4 NO Restricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species) 1-10 21.00 0.090 ns 1-2-3-4 NO Restricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species) 2-1 312.84 0.000 *** 1-2-3-5-6*-7-8 Restricted gene flow/dispersal but with YES some long-distance dispersal over intermediate areas not occupied by the species; or past gene flow followed by extinction of intermediate populations. Too few Clades: Insufficient genetic resolution to discriminate between range expansion/colonization and restricted dispersal/gene flow. 2-2 36.00 0.000 *** 1-19-20-2-3-5- Geographic sampling(s) inadequate to 15-16-18 NO discriminate between fragmentation, range expansion and isolation by distance 3-1 3.00 1.000 ns 1-2-3-4 NO Restricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species) 3-2 3.00 0.329 ns 1-2-3-5-6-7 YES Restricted gene flow/dispersal but with some long-distance dispersal

141 Chapter 4: Supplementary material

Ap p e n d i x S4

Total Cladogram 3-1 2-3 1-8 1-5 JH

2-1 1-1 1-10 G E M T

1-7 1-6 A F P

Q B DL

3-2 2-2 1-2 1-4 1-3 S C I

2-5 1-9 1-13 N K O

2-4 1-11 1-12 R

Figure S4 Nested cladogram obtained from the minimum spanning network of S. gigantea haplotypes (Fig. 3). We applied criteria form Crandall & Templeton (1993) to solve loops in the TCS network. Clade numbers correspond to clades in Table S3.

142 Phylogeography

Re f e r e n c e s f o r Supplementary Ma t e r i a l

Crandall, K.A. & Templeton, A.R. (1993) Empirical tests of some predictions from coalescent theory with applications to intraspecific phylogeny reconstruction. Genetics, 134, 959-969 Panchal, M. (2007) The automation of nested clade phylogeographic analysis. Bioinformatics, 23, 509- 510 Posada, D., Crandall, K.A. & Templeton, A.R. (2000) GeoDis: a program for the cladistic nested analysis of the geographical distribution of genetic haplotypes. Molecular Ecology, 9, 487-488

143

General conclusions

Ge n e r a l Co n c l u s i o n s

This project was the first to study ecology, genetics, plant performance and life-history of different ploidy levels of S. gigantea and to relate invasion potential to ploidy. The perspective has been a biogeographic one, and the combination of different approaches used, field surveys, common garden experiments and genetic investigations, have clearly shed light on the ecological differentiation of ploidy levels in the native range in North America, and helped explain why tetraploids are invasive. Altogether, this project has furnished evidence that polyploidy in S. gigantea does matter ecologically and plays a significant role in biological invasion.

What do differences between the ploidy levels of S. gigantea teach us?

The ploidy levels of S. gigantea—diploids, tetraploids and hexaploids—are now treated as one taxon. This is because morphological characters do not consistently separate them, and therefore no reliable infraspecific categories can be made (Melville & Morton, 1982; Morton, 1984; Semple et al., 1984). However, in the past, three species (sometimes also referred to as subspecies or as varieties) differing in ploidy level, S. gigantea Aiton, S. serotina Aiton, S. shinnersii (Beaudry) Beaudry, and one form, S. serotina forma huntingdonensis Beaudry have been named. These taxa were thought to be distinguishable by hairiness and size of leaves (Beaudry & Chabot, 1959; Beaudry, 1963, 1970, 1974). Most plant material Beaudry studied, however, originated from the northeastern part of North America. The discrepancy between taxonomic treatments may thus be explained by incomplete range sampling. Recent work (carried out as part of this project) emphasizes the importance of considering the complete range and suggests that any differences in morphology between diploid and tetraploid S. gigantea could vary with region, with these differences being partially apparent in Ontario but not in the Southern Appalachian Mts. (Wolf, 2007). Genetic isolation between ploidy levels of S. gigantea seems to be strong (no successful crosses, Melville & Morton, 1982) but not complete (in this study, one triploid offspring from mixed-ploidy crosses was produced). Because of such low levels of gene flow between ploidy levels, the open question of species delineation should perhaps be re-addressed (Soltis et al., 2003). However, multiple formations of tetraploid S. gigantea have been detected (up to seven

145 General conclusions origins detected), though there is no information on how recent or recurrent the events have been. Furthermore, no study has addressed whether the higher ploidy levels of S. gigantea arose through polyploidization between plants of this species (auto-polyploidy) or involving plants of a different species (allo-polyploidy). Although hybridization between higher ploidy levels of the closely related S. canadensis complex is hypothesized to explain this taxonomically difficult species assemblages (Semple & Cook, 2006), auto-polyploidy in S. gigantea cannot be excluded, especially given the absence of morphological differentiation between its ploidy levels. A striking difference between ploidy levels of S. gigantea, on the other hand, is the spatial separation—on large scale, but also at the population level. Because the higher ploidy levels need to be formed in the vicinity of their progenitors (be they of allo- or auto-polyploid origins), later separation calls for mechanisms influencing establishment and spread. However, the field survey revealed no habitat differentiation that is strong enough to act at a continental scale. Nevertheless, calcifuge behaviour was detected in diploids whereas tetraploids and hexaploids are apparently indifferent to soil calcium. Altogether, diploids tend to show the narrowest niche width and the smallest range size among the three ploidy levels. Linked to the history of spread and range size is the fact that the distribution of genetic variation is different between diploid and tetraploid S. gigantea and overall genetic variation is smaller in tetraploids. In particular, the difference in genetic variation between diploids and tetraploids depends on the region, with Ontario harbouring the largest contrast (Tab. 1). The region-dependent pattern of differentiation between ploidy levels for genetic variation

Table 1 Degree of differentiation between diploid and tetraploid S. gigantea in three region of overlap along a North-South gradient in the native range in North America

Ontario New England S. Appalachian (45° N) (40° N) Mts. (35° N) Tests, p Tests, p Tests, p 1 Genetic variation Fct 0.5* 0.3*** 0.2* Habitat differentiation2 Soil calcium (%) 8.1** 2.9ns 0.2ns Soil pH 9.3** 6.3ns 0.6ns Plant performance3 Biomass (g) 29.0*** nd 18.3*** Plant height (cm) 33.1*** nd 12.5** Rhizome number 108.3*** nd 35.5*** Flowering plants (#) 47.0*** nd 8.4** Morphology4 Hairy main leaf veins5 107.47*** nd 73.10*** Glabrous leaves5 76.04*** nd 30.54*** Leaf area (log(cm2))6 21.09*** nd 2.05ns

1 2 2 AMOVA (Fct, genetic variation between diploids and tetraploids; ch. 4); Kruskal-Wallis tests (χ value; ch. 2); 3Linear mixed models (ch. 3); 4Data obtained from plants from ch. 3 by Wolf (2007), 5Likelihood ratio χ2 test for crosstabs, Beaudry (1970) distinguished by presence/absence of hairy main veins between diploids and tetraploids, 6ANOVA (F value). nd, no data available. Significance levels (ns, not significant; *, p<0.05, **, p<0.01; ***, p<0.001).

146 General conclusions is reflected in morphological characteristics and also for habitat requirements and plant performance (Tab. 1). This pattern of regional differentiation gives further support to the hypothesis—inferred from the pattern of haplotypes—that more polyploidization events occurred in the South than in the North of North America. Although tetraploids have been formed many times, one tetraploid lineage proved to be especially effective in colonising a large range. Size of the range a lineage occupied can be indicative for the age and, in the case of polyploids compared to their diploid progenitors, of success in spread. The common garden experiment showed that the extent of the rhizome system differs enormously, with diploid plants producing a few short rhizomes and tetraploids having many long rhizomes. As rhizomes serve for over-wintering, nutrient storage and clonal reproduction, a more extensive rhizome system of tetraploids translates into a higher potential for spread—colonising new areas and invading sites with established vegetation. Future research should address whether different lineages of tetraploids vary in the extent of their rhizome system and whether such variation could be related to their distribution and hence to colonization ability. A more physiological research question, aimed at understanding the habitat differentiation of diploid and tetraploid S. gigantea, would be to identify what mechanisms cause the switch from calcifuge to calcium-indifferent behaviour.

Why has diploid S. gigantea not become invasive in Europe?

Since no diploid has been recorded in Europe, either in this study or in the literature, no definitive answer can be given to this question. However, the basic prerequisites for an introduction of diploid S. gigantea seem to be given: the plants are morphologically identical/similar to introduced tetraploids and occur in geographic vicinity to source areas of tetraploid plants. This project has provided some arguments supporting the hypothesis that diploid S. gigantea has never been introduced to Europe. In particular, only a small number of haplotypes are found in Europe. This increases the chance of missing diploid source material for introductions. Similarly, this project has found arguments supporting the notion that diploids may have been introduced but failed to spread. Based on the habitat differentiation between diploid and tetraploid S. gigantea in North America, it seems unlikely that the diploid was excluded because no suitable habitat was available. However, it is possible that both diploid and tetraploid were originally introduced onto calcium-rich soil. Because of the calcifuge behaviour of diploids, these plants would have grown less well and gardeners may have unknowingly selected tetraploids for further propagation. However, a more substantial argument is the difference between ploidy levels in the rhizome system. As already discussed, tetraploids were successful in North America in spreading probably also due to their vigorous rhizome system. This indicates that the potential for invasiveness of tetraploid S. gigantea is conferred by polyploidy and thus is lacking in diploids.

147 General conclusions

Future research could address mechanisms that lead to novel traits (rhizome system, calcium indifference, ability to attain large range size) in tetraploid S. gigantea compared to their diploid progenitors; this could include investigating the mode of polyploidy (e.g. were these novel traits conferred through hybridization in form of allo-polyploidy?) and possible differences among diploid lineages, relating any differences to the traits of the corresponding tetraploids. A particular relevant question is whether polyploidization creates these traits anew or merely realises the potential already present in the original diploids.

What contributes to the invasion success of tetraploid S. gigantea?

Successful biological invasions are recognized as resulting from an interaction of properties of invading species, invaded communities and transport vectors (Facon et al., 2006; Richardson & Pysek, 2006). Whereas this study focused on how genetic change (in particular, polyploidy) influences invasiveness of S. gigantea, other research has investigated additional factors to determine contribution to the success of S. gigantea (see General Introduction). This project revealed that compared to corresponding native tetraploid populations, invasive populations of S. gigantea exhibit reduced haplotypic diversity, as well as minor differences in habitat and plant growth. The major differences were not found between native and invasive populations but rather between diploid and tetraploid populations. Because the characters affected—such as a stronger rhizome system, larger niche width and stronger nutrient responses—seem to be important for colonization ability and the potential for invasiveness, we conclude that polyploidy, rather than post-introduction genetic changes, plays a fundamental role in the invasion success of S. gigantea. This shows that knowledge of the native range is indispensable for a comparative biogeographic approach to studying any invasive alien species (Dlugosch & Parker, 2008). For instance, by neglecting ploidy level, a comparative approach could yield the wrong conclusion that the strong rhizome system is absent in the native range of S. gigantea (by sampling in the diploids’ range, for example) and was therefore acquired in the invasive range. Polyploidy is an important factor, too, in other invasive species. For instance, diploid and tetraploid Centaurea maculosa differ in having a monocarpic and polycarpic life- history, respectively; this in turn can be related to susceptibility to enemy attack, suggesting that tetraploids have an advantage as invaders under a particular set of conditions (Müller, 1989; Müller-Schärer et al., 2004). While this project aimed to improve our knowledge of biological invasions, it was not designed to give new insights into the management of invasive populations of S. gigantea. However, the results highlight the importance of clonal reproduction via the rhizome system for spread of S. gigantea, and thus corroborate the need not only to control seed dispersal but also to weaken the rhizome system and—as recommended in current management practices—

148 General conclusions to restrict transport of rhizome pieces in contaminated soil (Anonymous, 2000, 2006). The main finding of this study, i.e. that the tetraploid S. gigantea in North America was predisposed to become a successful coloniser, does not preclude other factors explaining its invasion success. The advantage of tetraploids corresponds rather to the fundamental potential that together with several other circumstances contributes to invasion success. For S. gigantea, it has been shown that post-introduction genetic change has occurred, e.g. geographic cline for plant growth (Weber & Schmid, 1998), enemy defence and susceptibility (Meyer & Hull-Sanders, 2007), and even in patterns of clonal growth (Güsewell et al., 2006). A further aspect is enemy release, which is occurring at least partially in invasive populations of S. gigantea (Bopp, 1997; Hahn, 2005). It is also possible that horticulture selected for specific traits to increase the ornamental value of S. gigantea (Weber, 1994). Another factor increasing invasion success could be propagule pressure which is important in the case of S. gigantea, as garden trade and bee keepers were involved in large-scale distribution and release (Meier, 1958; Voser-Huber, 1983). And given that allelopathy has been detected in the closely related species, S. canadensis (Yang et al., 2007; Zhang et al., 2007), it seems quite probable that this mechanism also plays a role in S. gigantea. This project showed that polyploidy is ecologically significant for invasive S. gigantea. Nevertheless, future research should investigate how commonly polyploidy confers the potential to become an invader, and elucidate which relevant plant characteristics are altered through polyploidy. Furthermore, future research should attempt to evaluate the relative importance of the different factors recognized as contributing to the invasion success of polyploid taxa.

Li t e r a t u r e

Anonymous (2000) Spätblühende und Kanadische Goldruten. Problempflanzen. Amt für Landschaft und Natur des Kantons Zürich, Fachstelle Naturschutz

Anonymous (2006) Späte Goldrute. Exotische Problempflanzen: Bedrohung für Natur, Gesundheit und Wirtschaft - Art der Schwarzen Liste der Schweizerischen Kommission für die Erhaltung von Wildpflanzen. www.cps-skew.ch. CPS-SKEW

Beaudry, J.R. (1963) Studies on Solidago L. VI. Additional chromosome numbers of taxa of genus Solidago. Canadian Journal of Genetics and Cytology, 5, 150-174

Beaudry, J.R. (1970) Studies on Solidago L. X. Les Solidago gigantea Ait. dans l’est de l’Amerique du Nord. Naturaliste Canadien, 97, 35-42

Beaudry, J.R. (1974) Solidago shinnersii (Beaudry) stat. et comb. nov., une nouvelle espece du complexe du S. gigantea. Naturaliste Canadien, 101, 931-932

Beaudry, J.R. & Chabot, D.L. (1959) Studies on Solidago L. IV. The chromosome numbers of certain taxa of the genus Solidago. Canadian Journal of Botany - Revue Canadienne de Botanique, 37, 209-228 149 General conclusions

Bopp (1997) Die amerikanische Goldrute Solidago canadensis L. und S. gigantea Ait. als Neophyten in Mitteleuropa: Besiedlung durch Insekten. Göttinger naturkundliche Schriften, 4, 181-206

Dlugosch, K.M. & Parker, I.M. (2008) Founding events in species invasions: genetic variation, adaptive evolution, and the role of multiple introductions. Molecular Ecology, 17, 431-449

Facon, B., Genton, B.J., Shykoff, J., Jarne, P., Estoup, A. & David, P. (2006) A general eco-evolutionary framework for understanding bioinvasions. Trends in Ecology & Evolution, 21, 130-135

Güsewell, S., Jakobs, G. & Weber, E. (2006) Native and introduced populations of Solidago gigantea differ in shoot production but not in leaf traits or litter decomposition. Functional Ecology, 20, 575-584

Hahn, M. (2005) Pathogen infection and herbivory on the invasive plant species Solidago gigantea. Master thesis. ETH Zurich, Zurich.

Meier, K. (1958) Goldruten (Solidago)-Arten für die Verbesserung der Spätsommer- und Herbstpollentracht. Schweizerische Bienen-Zeitung, 81, 452-456

Melville, M.R. & Morton, J.K. (1982) A biosystematic study of the Solidago canadensis (Compositae) complex. 1. The Ontario populations. Canadian Journal of Botany - Revue Canadienne de Botanique, 60, 976-997

Meyer, G.A. & Hull-Sanders, H.M. (2007) Altered patterns of growth, physiology and reproduction in invasive genotypes of Solidago gigantea (Asteraceae). Biological Invasions, in press. Available online 29 June 2007. doi:10.1007/s10530-007-9131-z

Morton, G.H. (1984) A practical treatment of the Solidago gigantea complex. Canadian Journal of Botany - Revue Canadienne de Botanique, 62, 1279-1282

Müller, H. (1989) Growth pattern of diploid and tetraploid spotted knapweed, Centaurea maculosa Lam. (Compositae), and effects of the root mining moth Agapeta zoegana (L.) (Lep.: Cochylidae). Weed Research, 29, 103-111

Müller-Schärer, H., Schaffner, U. & Steinger, T. (2004) Evolution in invasive plants: implications for biological control. TRENDS in Ecology & Evolution, 19, 417-422

Richardson, D.M. & Pysek, P. (2006) Plant invasions: merging the concepts of species invasiveness and community invasibility. Progress in Physical Geography, 30, 409-431

Semple, J.C. & Cook, R.E. (2006) Solidago. Flora of North America North of Mexico (ed. by Flora of North America Editorial Committee), pp. 107-166. Oxford University Press, Oxford and New York.

Semple, J.C., Ringius, G.S., Leeder, C. & Morton, G. (1984) Chromosome numbers of Goldenrods, Euthamia and Solidago (Compositae, Astereae). II. Additional counts with comments on cytogeography. Brittonia, 36, 280-292

Soltis, D.E., Soltis, P.S. & Tate, J.A. (2003) Advances in the study of polyploidy since “Plant speciation”. New Phytologist, 161, 173-191

Voser-Huber, M.L. (1983) Studien an eingebürgerten Arten der Gattung Solidago L. Probleme mit

150 General conclusions

S. gigantea, S. canadensis und S. graminifolia in Naturschutzgebieten des schweizerischen Mittellandes. Dissertationes Botanicae, 68, 97

Weber, E. (1994) Evolutionary trends in european neophytes: a case study of two Solidago species. Ph.D. dissertation. University of Basel, Basel, Switzerland.

Weber, E. & Schmid, B. (1998) Latitudinal population differentiation in two species of Solidago (Asteraceae) introduced into Europe. American Journal of Botany, 85, 1110-1121

Wolf, A.P. (2007) Blattmorphologie von Solidago gigantea in Abhängigkeit von Ploidiestufe und Calciumdüngung. Term paper. ETH Zurich, Zürich.

Yang, R.Y., Mei, L.X., Tang, J.J. & Chen, X. (2007) Allelopathic effects of invasive Solidago canadensis L. on germination and growth of native Chinese plant species. Allelopathy Journal, 19, 241-247

Zhang, Q., Yao, L.J., Yang, R.Y., Yang, X.Y., Tang, J.J. & Chen, X. (2007) Potential allelopathic effects of an invasive species Solidago canadensis on the mycorrhizae of native plant species. Allelopathy Journal, 20, 71-77

151

Ac k n o w l e d g e m e n t s

First, I express my gratitude to my wife Sandra Joachim for her wonderful love, support and encouragement during the past years and for being very generous with whims a PhD student can generate at odd times.

I am very thankful to Peter Edwards and Regula Billeter for supervising my thesis, and to Heinz Müller-Schärer for being co-examiner.

Regula and Peter offered me the opportunity and freedom to develop together the outline of this project—a process from which I could profit enormously—and to take this project to a finish. It is amazing to realize in retrospect how many opportunities, people and places I could meet and how many new things I could learn, all of which is ultimately due to them, their invaluable critical comments and moral support.

Min Hahn has carried out under challenging circumstance but with success his master thesis on pathogen infection and herbivory on invasive populations of Solidago gigantea. Andreas Wolf has completed endless hours of leaf measurements and has written a term paper on leaf morphology of native diploid and tetraploid S. gigantea. Wilma Blaser has conducted a short term work, though not short in hours of counting seeds, on artificial crosses and germination ability in diploid and tetraploid S. gigantea. Many thanks to all of you!

Many thanks to all members of both the plant ecology and the plant ecological genetics groups at the IBZ (former Geobot). In particular, I thank my peers with whom I could spend many funny hours, share sorrows and also have stimulating discussions, and who helped me on several occasions either setting up or harvesting my experimental plants, Jake Alexander, Holger Buschmann, Patrick Cech, Hans Göransson, Stephanie Halsdorf, Albert Kölbener, Christoph Küffer, Steffi Lorenz, Aria Minder, Myriam Poll, Joelle Rahmé, Carmen Rothenbühler, Charlotte Salzmann, Deborah Scharfy, Eva Schuhmacher, Fabrizio Steinebrunner, Miluse Trtikova, Georg von Arx, Marc Wälti. I further extend my thanks to Sabine Güsewell for much appreciated statistical guidance, Markus Hofbauer who accompanied me through many hours of snow and rain storms in the field in North America, Karsten Rohweder and Hans- Heini Vogel for always providing smoothly running computer equipment, Tino Fotsch who

153 watered endlessly my plants, Dieter Ramseier for organizing my experimental sites, Rose Trachsler and Marylin Gaschen for assistance with chemical analyses, René Graf for all kinds of technical support and introduction to a new world of tastes, Matthias Baltisberger for advice on chromosome counts and herbarium vouchers, Erika Aeschbach and Kathrin Rentsch for solutions to my administrative issues and Harry Olde Venterink who dropped often by my office for good chats. I am especially thankful to Alex Widmer for his discussions, support and encouragement to carry out my genetic analyses in his lab. A lab I loved to work which is impeccably led by Claudia Michel who showed me all the neat methods and tricks and who made possible together with Alex Widmer my thesis chapter about phylogeography. I am very grateful to Carmen Rothenbühler, Jake Alexander and Alex Kocyan for their help in the lab and with the analyses on many occasions.

John C. Semple gave me a warmhearted and in-depth welcome and introduction to the world of Solidago at the beginning of my project. I owe him many thanks for taking his time to show me around in the field in Ontario from which this project and I could profit a lot. Without the flow cytometer from the lab of Peter Stamp and with the help of Marianne Wettstein-Bättig, I could measure no ploidy level—a pivotal element for this project. Many thanks to them and to Philipp Streckeisen who offered me space in a green house at the FAL in Reckenholz! Many thanks to all people who supported me generously with information and help during my field surveys in North America and Europe, Beat Bäumler from the ZDSF in Genève, Garret E. Crow at the University of New Hampshire, Nicolas Dassonville at the Université Libre de Bruxelles, Bruce Ford at the University of Manitoba, Mark Gabel at the Black Hills State University in Spearfish and Lars Leonardson and Lina Steinke at the Lund Universitet. Also many thanks to all those who went outdoors, collected seeds and rhizomes from my study species and mailed it to me, Akos Malatinsky, Elvira Rudyka, Ewald Weber, Gabi Jakobs, Holger Buschmann, Hansjörg Dietz, Martin Hejda, Harry Olde Venterink, Jill Bowling, Johannes Kollmann, Jun Nishihiro, James Partridge, Mark van Kleunen, Marc Wälthi, Nina Probatova, Regula Ott, Rodolphe Schlaepfer, Sandra Joachim, Thomas Becker, Toni Nikolic, Toshikazu Mito.

An ETH Zurich research grant and the Stiftung Rübel supported this project financially.

Last but least, special thanks to my friends, both old and new, and my families who put up with not being able to escape from hearing my stories of invasive plants, goldenrods and tigers in the forest.

154 Cu r r ic u l u m Vi t a e

Name Daniel Rodolphe Schlaepfer Date of birth 17th January 1977 Citizen Montreux (VD) and Rehetobel (AR), Switzerland

Ed u c a t i o n a n d Qu a l i f ic a t i o n s 1998–2003 Study of Environmental Sciences, ETH Zurich: Degree: Dipl. Umwelt-natw. ETH Thesis title: “Rapid Expansion-Contraction Dynamics and Ecosystem- Processes in a Large Intermittent Floodplain River (Tagliamento, NE-Italy)”. Projects: • “Water quality modelling with SWAT in the catchment of the river Thur (NE-Switzerland)” • “Project soil protection ‑ Phosphates in the soil of the field- laboratory Hönggerberg: content, distribution and influence on plant growth“ • “Investigation on sand lizards (Lacerta agilis) in highway- embankments (Fricktal, N-Switzerland)” • “Investigation on smooth snakes (Coronella austriaca) in highway- embankments (Fricktal, N-Switzerland)” 1997–1998 Study of Physics and Mathematics, ETH Zurich 1991–1997 Matura (high school graduation), Kantonsschule Oerlikon KSOe

Wo r k Ex p e r i e n c e Mai 2004–­ Scientific assistant/PhD student, Institute of Integrative Biology, ETH Zurich Oct–Nov 2003 Scientific assistant, Department of Limnology, Swiss Federal Institute for Environmental Science and Technology (EAWAG), Dübendorf Jul–Dec 2001 Research assistant, Centre for Swiss Plant Databases (ZDSF/CRSF), Geneva: leaflets (Merkblätter Artenschutz) on conservation issues of four rare and endangered plant species March 2000 Assistant, Forest Ecosystems and Ecological Risks at the Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Birmensdorf Aug 1998–Aug 1999 IT supporter for PADMA AG, Schwerzenbach: developed and managed company databases

155 Pu b l ic a t i o n s Schlaepfer, D.R., Edwards, P.J., Semple, J.C. & Billeter, R. (in print) Cytogeography of Solidago gigantea (Asteraceae) and its invasive ploidy level. Journal of Biogeography Schlaepfer, D.R., Edwards, P.J. & Billeter, R. (submitted) Habitat differentiation between native ploidy levels and invasive tetraploids of Solidago gigantea (Asteraceae). Diversity and Distributions Schlaepfer, D.R., Edwards, P.J., Widmer, A. & Billeter, R. (submitted) Phylogeography of native ploidy levels and invasive tetraploids of Solidago gigantea (Asteraceae). Molecular Ecology Doering, M., Uehlinger, U., Rotach, A., Schlaepfer, D.R. & Tockner, K. (2007) Ecosystem expansion and contraction dynamics along a large Alpine alluvial corridor (Tagliamento River, Northeast Italy). Earth Surface Processes and Landforms, 32, 1693-1704

In v i t e d Pr e s e n t a t i o n s Schlaepfer, D.R. (2007): Influence of polyploidy on Solidago gigantea. „Community ecology“ seminar series of Zoological Institute at the University of Bern, Bern, Switzerland. 8 Oct 2007. Invited by Prof. Dr. Wolfgang Nentwig.

Co n f e r e n c e Co n t r i b u t i o n s Schlaepfer, D.R. & R. Billeter (2007): Solidago gigantea: Plant performance in a ploidy-level precise common garden. 37th Annual Conference Gesellschaft für Ökologie (GfÖ). 10– 14 Sept 2007. Marburg, Germany. (oral) Schlaepfer, D.R. (2007): Solidago gigantea: A ploidy-level precise alien - native range comparison. Workshop: Colonization versus invasion: do the same traits matter? 25 Feb – 2 March 2007. Monte Verità, Ascona, Switzeland. (oral) Schlaepfer, D.R. & R. Billeter (2006): Not all ploidy levels of Solidago gigantea Ait. are invasive! Symposium: An evolutionary perspective of biological invasions. 2 – 3 Oct 2006, Fribourg, Switzerland. (poster) Schlaepfer, D.R. & R. Billeter (2006): Not all ploidy levels of Solidago gigantea Ait. are invasive! 4th European Conference on Biological Invasions NEOBIOTA - From Ecology to Conservation. 27 – 29 Sept 2006, Vienna, Austria. (poster) Schlaepfer, D.R. & R. Billeter (2004): The Role of Polyploidy in Invasive Plants – A Case Study on Solidago gigantea Ait. 3rd International Conference on Biological Invasions NEOBIOTA - From Ecology to Control. 30 Sept – 1 Oct 2004. Bern, Switzerland. (poster)

156