EVOLUTIONARY DIFFERENTIATION in Sexualm U a MARIE O

EVOLUTIONARY DIFFERENTIATION IN SEXUALm

AND LIFE HISTORY IN POLYPLOID AGAMK

ANTENNARIA PARLM (ASTERACEAE)

UAMARIE O'CONNELL

A thesis submitted to the Department of Biology

in conformity with the requirements for

the degree of Master of Science.

Queen's University

Kingston, Ontario, Canada

September, 1997

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Antemarin parlinii is a hexaploid agamic complex found in deciduous forests and old fields throughout east-central North America. Northern and eastem populations of this dioecious plant conçist of females only ("female" populations) while southeastem populations contain both sexes ("mixed- sex"). Some individuals reproduce sexually, some through gametophytic apomixis (asexual seed production), while others may practice a mixture of sexual and apomictic seed production. 1investigated the pattern of variation in sexuality within and among populations and found that populations were highly differentiated in sexuality, and there was little or no variation in sexuality within populations. I also found no evidence that individuals commonly practiced partial apomixis. Natural seed set was measured in fifteen female and nine mixed-sex populations in Ohio, Ontario and Québec.

Female populations had a high autonomous seed set (average 75%), whereas in mixed-sex populations seed set was positively correlated with population size and the propotion of males, indicating a strong reliance on sexual reproduction. In a pollinator exdusion experiment conducted in the field in

Ohio, plants in two female populations set more than 70% of their seeds, while there was no evidence of autonomous seed set in two mixed-sex populations. Similar results were obtained in a pollination experiment in a common greenhouse environment. Overall these three experiments indicate that female populations are asexual and mixed-sex populations are sexual. I found that apomictic and sexual A. parlinii occupy different habitats where their ranges ovêrlap. Apornicts tend to occur in disturbed and ephemerd habitats while sexuals occur in more stable woodland habitats. I found that apomicts had a higher reproductive output than sexuals, producing more capitula per inflorescence, florets per capitulum and twice as many seeds per inflorescence. Diaspores from asexual plants had rnorphological traits, such as a larger pappus-toseed ratio and more barbs per pappus, which increased seed free-faU time, an estimate of dispersal potential.

The combination of asexuality with an inaeased reproductive output and seed dispersa1 potential cm lead to a superior colonizing ability of apomictic

A. parlinii and partly explain observed distributional differences between apomictic and sexual populations. ACKNOWLEDGMENTS

1 am indebted to numerous people who helped me throughout this

thesis. I thank my supervisor Chris Eckert for hiç help and encouragement

through regular "vibory jugs" at the grad club every step along the way. 1

also thank Bob Montgomerie and Heather Proctor for advice throughout this

study, and Katherine Mavraganis, Marcel Dorken, and Matthew Routley for

proofreading this manuscript. Thank you also to Joel Shore and Brian

Cumming for helpful comments to help me improve thiç thesis. My time spent in Ohio would not have been as enjoyable without the help and

Company of my field assistants MqlAllen and Russ Groves. 1 am particularly grateful to the Linehan family, John, Rita, Molly, Patick, Devin, and Christina for their hospitality by feeding me and providing a roof over my head on those cold, wet, spring nights in Ohio. 1 also thank the curators at the CAN, DAO, OS, and BH herbaria for access to Antennaria specimens. 1 am also grateful to the managers at the Kitty Todd Nature Conservancy

Preserve, Toledo metro parks (Oak Opening Nature Preserve), Irwin Prairie

Nature Preserve and Lake county metro parks (Indian Point) where 1 conducted parts of this study and collected plants.

This work was partly funded by a Natural Sciences and Engineering

Research Council postgraduate fellowship. TABLE OF CONTENTS

1 General introduction...... -1 Medianismsofapo~s...... 7 Partialapo- ...... 4 Selectionforapo- ...... 6 Polyploidy and hybridization...... -7 Geographical parthenogrnesis...... -8 Antennaria (Asteraceae) ...... 9 Herbanumswey ...... 12

2 The evolutionary significance of variation in sexuality in polyploid agamic Antemaria parlinii ...... 18

Materials and Methods ...... 21 Studypopulatiom...... 21 Natural seed set in female and mixed-sex populations ...... 23 Field pollinator-exclusion expriment ...... -25 Greenhouse pollination experiment ...... 26

Resdts ...... 27 Nahual seed set in female and mixed-sex populations ...... -27 Pohator-exdusion experiment ...... 32 Greenhouse pollination experiment ...... 32

Discussion ...... 36 Variation in sexuality among and within populations ...... 36 PartialapomWs ...... 38 Dismptive selection for sexuality ...... -42

3 Differences in distribution and colonizing ability between apomictic and sexual populations of Antennan'a parlinii ...... 43

Materials and Methods ...... 46 Habitatsurvey ...... 46 Popdationsurvey...... 46 Reproduaiveoutput...... 46 Diaspore measurements...... 48 Free-fautest ...... 50 Germination experirnent ...... 52

Results...... 53 Habitat differences between asexual and sexual populations ...... 53 Differences in reproductive output between asexual and sexual populationç ...... 53 Relation between diaspore traits and free-fall time ...... -57 Differences between asexual and sexual populations in diaspore traits ...... 57 Germination differences in asexual and sexual populations ...... 63

Discussion...... 63 Habitat differences ...... 63 Reproductiveoutput...... 65 Dispersal abiliq...... 66 Differences in cornpetitive ability ...... 69

4 General discussion and conclusions ...... 71 Mainhdings ...... 71 Partialapomixis ...... 71 Differences in reproductive output and dispersa1 potential ...... 72 Habitat differentiation ...... 73 Evolutionary origin of associations between sexuality. reproductive output. seed dispersal and habitat distribution ...... 74

APPENDICES ...... 86

VITA ...... 92 vii

LIST OF TABLES

2-1: Analysis of variation in natural seed set in female and mixed-sex

populations of An tmnaria parlinii...... -29

2-2: The effect of pollinator exclusion on seed set in female and rnixed-sex

populations of Antennaria parlinii ...... 34

3-1: Correlations arnong pappus traits in Antennaria parlinii...... 5 1

3-2: Anaiysiç of variation in reproductive output in Antennaria parlinii .56

3-3: Multiple regression of the effect of pappus traits on free-fall time in

Antemaria parlinii...... 59

3-4: Analysis of variation in diaçpore traits in Antennaria parlinii...... 61 LIST OF FIGURES

1-1. Range map of Antennaria parlinii and its three progenitor species . . . . .11

1-2. Distribution of herbarium collections of Antennaria parlinii...... 14

1-3. Distribution of herbarium collections of Antennaria parlinii in Ohio . . 15

1-4. Distribution of herbarium collections of Antennaria parlinii in Ontario

...... 16

Distribution of herbarium collections of Antennaria parlinii in New

York ...... 17

Location of study populations of Antennaria parlinii in Ohio ...... -22

Cornparisons in natural seed set in female and rnixed-sex populations of

Antennaria parlinii ...... 28

The effect of population size and the proportion of males in mixed-sex

populations of Antennaria parlinii ...... 30

Distance to the closest male inflorescence and seed set in mixed-sex

populations of Antemaria parlinii ...... 31

The effect of pollinator exclusion on seed set in female and muted-sex

populations of Antennaria parlinii ...... 33

Seed set in a greenhouse pollination experiment in plants from female

and mixed-sex populations of Antennaria parlinii ...... -35

Variation in sex-ratio of mixed-sex populations of Antennaria parlinii .41

Location of study populations of Anfennaria parlinii in Ohio ...... 47

Schematic diagram of an Antennaria parlinii diaspore ...... 49 3-3. Number of asexual and sexual populations ofAntennnria parlinii with or

wihoutatreecanopy...... 5-1

3-4. Cornparisons of reproductive output in asexual and sexual populations

of Antennaria parlinii ...... 55

3-5. Effect of diaspore traits on seed free-fall time in Antennaria parhii ....58

3-6. Comparisons of diaspore traits in asexual and semai populations of

Antennaria parlinii ...... 60

3-7. Cornparisons in estimate of diaspore free fall the in asexual and sexual

populations of Antanaria parlinii ...... 62

3-8. Comparisons of germination in asexual and sexual populations of

Antennaria parlinii ...... 64 CHAPTER 1 : General Introduction

Two major trends in the evoiution of reproductive systems in flowering plants are the transition from outcrossing to se&fertilization, and the transition from sexual to asexual reproduction (Frpcell, 1957). The evolution of selfing has received considerable attention while the transition from sexuality to apomixis, the production of asexual seeds, has been largely neglected. There has been much theoretical work identïfymg the likely selective factors involved in the evolution of self-fertilization (Lloyd, 1980; Uyenoyama et al., 1993; Holsinger, 1996; Harder and Barrett, 1996) and estimates of the selfing rate have been obtained for many species (129 species in Barrett and Eckert, 1990; > 200 species, Barrett et al., 1996). In contrast, there is little theory dealing with the evolution of apomixis in plants (Marshall and Weir, 1979; Charlesworth, 1980; Marshall and Brown, 1981; Harper, 1982) and few studies have examined the pattern of variation in apomictic reproduction within (Bayer et al., 1990; Schmidt and Antlfinger, 1992) or among populations (Soreng, 1986; Gadella, 1987). Apomictic specieç have been viewed as evolutionary dead-ends incapable of tracking environmental changes or producing new species (Darlington, 1939; Stebbinç, 1941, 1950). In contrast to this view, recent work has revealed considerable genetic variation in asexual plant populations (Ellstrand and Roose, 1987; Hamrick and Godt, 1990). Many workers have hypothesized that partial sexuality is responsible for maintaining genetic variation in asexual lineages and thereby enhancing the capacity for evolutionary change (Gustafsson, 1946-1947; Asker, 1979, 1980; Marshall and Weir, 1979; Asker and Jerling, 1992). In many çpecies, it is suspected that individuals regularly produce both sexual and apomictic seeds and some potential for sexuality may remain in al1 apomicts (Nogler, 1984; Asker and Jerling, 1992). More recentiy, some have suggested that apomWs should not be viewed as partial or obligate but rather as a continuum from complete sexuality to complete apo-s (Nogler 1984, Bayer et al., 1990). However, there has been relatively Little effort to quantify the relative sexual to apomictic seed production in apomictic speues (Bayer et al., 1990; Schmidt and Antlfinger, 1992). In the first part of this study, 1examine the pattern of variation in sexuality among and within populations of apomictic Antennaria parlinii. 1 also attempt to assess the importance of partial apomixk in these populations. To provide some background for this work, 1 first give an overview of the different mechanisms of apomictic reproduction and their ecological consequences. 1 then discuss the selection of apombcis. Like self-fertilization, apomixis is strongly associated with partidar ecological and lifehistory traits. In the second part of this study 1address the differences in ecological and geographical distribution between sexual and apomictic Antennaria parlinii and show that correlations between apomixis and colonizing traits may help explain these distributional differences. To set the stage, 1 first review the comrnon distributional differences between sexual and asexual species and the hypotheses advanced to explain these differences. 1 then provide an overview of the genus Anfennaria in North America and describe the distribution, ecology and sexuality of taxa most closely related to A. parlinii.

Mechanisrns of apomixis The taxonomie distribution of apomixis has been the subject of several comprehewive reviews (Stebbins, 1941; Gustafsson, 1946-1947; Nygren, 1954, 1967; Nogler, 1984; Asker and Jerling, 1992). To date, it has been confirmed in 46 families of angiosperms including 134 genera, and is especially common in the Asteraceae, Poaceae and Rosaceae (Asker and Jerling, 1992). Two main categories of apomixis are recognized: adventitious embryony and gametophytic apomixis (Koltunow, 1993). In adventitious embryony, the embryo arises diredy from a somatic cell outside the megagametophyte, usually a nucellar ce11 of the ovule. The sporophyte gives rise to a new sporophytic generation through mitosis and the female gametophyte (embryo sac) is only involved to provide resources for the maturation of the adventitious embryo (Gustafsson, 1946-1947). This mode of apomixis is well known in many species of Citnrs and occurs in the Liliaceae and Orchidaceae (Grant, 1971). In gametophytic apomïxis, which cm be Merdivided into diplospory and apospory, the egg cell arises from inside the female garnetophyte but the egg nucleus remains unreduced. In apospory, somatic cellç of the ovule (usually the nucellus) give rise to the female gametophyte through mitosis. Apospory occurs in many Rosaceae, particularly Alchemilln, Malus, Pofentilla and Rubus (Asker and Jerling, 1992). In diplospory the embryo-sac mother cell (EMC) gives rise to the female gametophyte but again the egg nucleus remains unreduced (Gustafsson, 1946- 1947; Grant, 1971). Diplospory can be further divided into meiotic diplospory (Taraxacum type) where there is a restitution of the somatic chromosome complernent in the egg cell after a first meiotic division, and rnitotic diplospory (Anfennaria type) where the egg cell arises from mitotic divisions of the EMC (Asker and Jerling, 1992). Meiotic diplospory provides some oppomuiity for recombination, whereas mitotic diplospory does not (Richards, 1986). Diplospory is common in the Asteraceae, particularly in species of Antennaria, Hieracium, Taraxacum and Chondrilla (Nogler, 1984). Like self-fertilization, apomixis is a form of uniparental reproduction and provides reproductive assurance in environments where polhator service is inadequate and/or potential mates are scarce (Lloyd, 1980). However, some forms of apomixk, especiauy adventitious embryony and apospory require that the polar nudei be fertilized to initiate endosperm development and thus pollen production is maintained in these species possessing these forms of apomixis (Nygren, 1967; Asker and Jerling, 1992). These types of apomixis are often lumped together as "pseudogamy", meaning "false mamage". Even in pseudogamous species where fertilization of the egg ce11 rarely occurs, the presence of pollen rnay ensure some amount of sexuality. In contrast, most forms of diplospory involve spontaneous endosperm development, thus apomicts can reproduce in the complete absence of pollen. The main consequence of pseudogamy is that plants still need pollen to produce seeds and, therefore, apomixis does not provide much reproductive assurance.

Partial apomixis - There is a lot of opportunity for partial apomws in species with apospory and adventitious embryony, where the developrnent of an asexual embryo does not exclude the presence of a sexud embryo within the same embryo sac (adventitious embryony) or ovule (apospory; Richards, 1986). In diplospory, there may be less scope for partial apornUas because each ovule produces either a reduced or an unreduced embryo sac (Richards, 1986). Nevertheless, partial apomixis has been reported in diplosporous species of Antennnria (Bayer and Stebbins, 1983; Bayer et aL, 1990). It has been suggested that the potential for partial apomixis rernains in al1 species (Nogler, 1984; Asker and Jerling, 1992) and embryological evidence of remauiing sexuality has been reported in taxa which were thought of as obligately asexual (e.g. [Hieracizim], Skalinska, 1971; [Taraxaciim], Malecka, 1973).

Although it is clear that apomixis has a genetic basis, the exact genetic mechanism has been the subject of ongoing debate (e.g., Stebbins, 1941; Asker, 1979; Mogie, 1988). Environmental factors such as photoperiod can also influence the level of apomictic reproduction, as was found in aposporous Dichanthium aristaturn (Knox and Heslop-Harrison, 1963; Knox 1967). Although many have pointed out that partial apomixis is important in introducing genetic variation in predominantly apomictic lineages, few studies have attempted to estimate the relative amount of sexual vs. apomictic seed production within individuals (Marshall and Brown, 1981). To estimate the level of apomixis in individual plants, most studies have counted the proportion of unreduced (asexual) vs. reduced (sexual) embryo sacs, or the occurrence of multiple embryos within individual ovules (Saran and de Wei, 1970; Kellogg, 1987; Schmidt and Antlfinger, 1992). However, the proportion of unreduced embryo sacs is not necessarily a reliable indicator of the level of apomixis and probably overestimates the actual amount of sexual seeds produced (Nogler, 1984). Insufficient pollen or degeneration of embryo sacs can change the ratio of asexual to sexual seeds. A better way of measuring the level of apomkis in partial apomicts is to use genetic markers to estimate the fraction of seeds produced through apomixis for a larger number of individuals and populations (Marshall and Brown, 1974). To date, only one study has used genetic markers to estimate the relative amount of sexual vs. apomictic reproduction in natural populations (Bayer et al., 1990). Selection for apornixis When should apo& or partial apom3cis be favoued by natural selection? Unlike self fertilization, where inbreeding depression imposes a major cost to uniparental reproduction, the disadvantages of asexual reproduction are less obvious (Marshall and Brown, 1981). In fa&, the prevalence of sex over the various modes of asexual reproduction remains one of the central problems in evolutionary biology (reviewed in Williams, 1975; Maynard Smith, 1978; Bell, 1982; Steams, 1987). Apomkis has an automatic advantage over sexual reproduction by avoiding the cost of meiosis: an apornictic mother passes on its fd genetic complement to each offspring while only half of the genes from a sexual mother are passed on to each offspring (an individual level benefit). Apomicts also avoid the cost of producing males: sexual females can only produce half as many daughters as asemial females (a population level benefit; Maynard Srnith, 1971; Williams, 1975; Maynard Smith, 1978; Marshall and Brown, 1981). Apomicts also have the advantage of reproductive assurance provided by asexuality (Bierzychudek, 198%). Most of the hypotheses for the maintenance of sex propose that there is a direct benefit of producing genetically diverse offpring. The short term benefits of sex have been examined in two types of models: an individual produces diverse offspring to (1) increase the likelihood of offspring success in a spatially or temporally heterogeneous environment ("lottery" or "tangled bank models", Williams, 1975; Bell, 1982) or (2) decrease cornpetition with siblings or parents ("sib-cornpetition model" Williams, 1975; Maynard Smith 1976; "elbow-room model", Young, 1981). However, there is little experimental work testing these hypotheses and the available studies have produced mixed results. For example, a series of experiments using Anthoxanthurn odoratum were performed to test if genetically diverse offspring performed better than genetically identical offsp~g(Antonovics and Ellstrand, 1984; Schmitt and Antonovics, 1986; Kelley et al., 1988; Kelley, 1989a,b). Clondy-derived mers and seed-derived mers, were grown in monocultures or mixtures at different densities. Antonovics and Schmitt (19û4) fond that, when grown together, groups of unrelated plants sometimes enjoyed a two fold increase in fifness over groups of related plants, but the actual fitness difference was highly variable. Schmitt and Antonovics (1986) found no difference in insect infestation between monocultures and mixtures of plants. in the field, Kelley (1989a) and Kelley et al. (1988) found that sexual plants performed better than asexual plants but this advantage was not related to sib-competition. Furthemore, there was no difference in fitness between asexual and sexual tillers in a two year greenhouse experiment (Kelley, 1989b). Overall, studies of sib-cornpetition provide little evidence for its role in maintainhg sexual reproduction (Burt and Bell, 1992). The few theoretical modeh that explore the evolution of apomixis in plants predict that either apomixis or sexual reproduction should become fixed depending of the balance between the obvious benefits of asexuality and the fitness cos& of producing genetically-homogeneous offspruig (Charlesworth, 1980; Marshall and Brown, 1981; Harper, 1982; Kondrashov, 1985). It is not clear that partial apomh wiU be favoured, so it is often considered a transitional state between apomixis and sexuality (Harper, 1982).

Polyploidy and hybridization Because apomicts are usually of hybrid origin (Asker and Jerling, 1992) they often possess the genome of two different sexual progenitor species and thus novel gene combinations. There are several polyploid agamic complexes well-known for their taxonomie difficulty due to their hybrid origin. In these complexes, the diploid progenitor species usually reproduce sexually while the allopolyploid hybrids reproduce sexually or apomictically (Grant, 1971). Genera with well-known agamic complexes include Crepis (Asteraceae; Babcock and Stebbins, 1938), Taraxacum (Asteraceae; Richards, 1973; Mogie and Ford, 1988), Hieracium (Asteraceae; Ostenfeld, 1912), Pua (Poaceae; Hiesey and Nobs, 1952), Potentilla (Rosaceae; Muntzing, 1958; Asker 1970a, b, 1971), and Rubus (Rosaceae; Gustafsson 1946-1947). The association between polyploidy and apomixis is, however, far from perfect. Most polyploids reproduce sexually, and many apomicts are diploid, especially in genera with adventitious embryony su& as Citrus, Poncirus and Fortunelln (Gustafsson, 1946-1947; Stebbins, 1950).

Geogrnphical parthenogenesis. One common trend in both asexual plants and animalc is that they have a different geographical and ecological range than closely related sexual species (Bierzychudek, 1987a; Bell 1982; Lynch, 1984; Glesener and Tilman, 1978). Apomictic species usually occur at higher latitudes, higher altitudes, and in deglaciated areas compared to closely related sexual species. ApomWs may lead to greater colonizing ability due to reproductive assurance (Stebbins, 1950). A single apomiaic individual can fomd a new population, while an ourcrossing hermaphrodite will require the presence of at least two plants and a sexual dioecious species requires that the founders indude at least one male and one female. If asexuality facilitates the colonization of new sites, then there may be selection for a colonizing life history in apomictic plants. The association between asexuality and other colonizing traits has received little attention (Michaels and Bazzaz, 1986). The wider geographical and ecological range of apomicts compared to related sexual taxa may be due to differences in ploidy along with or instead of differences in reproductive mode between sexuals and apomicts (Bierzychudek, 1987a). Polyploidy per se can contribute to an increase in ecological tolerance in plants through a slower development, delayed reproduction, longer Me span, larger seeds, and greater defense against pest and pathogew (Levin, 1983), though many of the potential consequences of polyploidy have not been thoroughly investigated. Polyploid plants also have larger geographical ranges than related diploids, and their distribution often extends to higher latitudes and altitudes, deglaciated areas and occur in more disturbed habitats (Stebbhs, 1950; Lewis, 1979). Detecting the relative importance of reproductive mode vs. ploidy has been a major problem in interpreting distributional differences between sexual and apomictic taxa (Bierzychudek, 1987a).

An tennaria (Asteraceae) Antennaria is a genus of srnall perennials occurring in temperate and arctic regions of the northem hemisphere (Bayer and Stebbins, 1987a). Bayer and Stebbins (1993) recognized 35 species of Antennaria in North America with several subspecies within those species. Diploid species of Antennaria are always sexual while polyploid species are either sexual, apomictic or both (Bayer and Stebbins, 1981; 1987a). Agamic complexes in Antennaria arose through hybridization arnong diploid sexual species (Bayer and Crawford, 1986). Bayer and Stebbins (1987a), recognized five agamic complexes: A. alpina, A. neodioica ( = howelli, Bayer and Stebbins, 1993), A. parlinii, A. pnraifolia and A. rosea. Asexual reproduction in Antennaria is through autonomous, mitotic diplosporous apomws and offspring are genetically identical to the2 mother (Stebbins, 1932). The genus has offered some of the best systems for examinuig the costs

and benefits of sex (Bayer and Stebbins, 1983; Michaels and Bazzaz, 1986,1989; Bierzychudek 1987b, 1989, 1990; Bayer et al., 1990). Antennaria is dioeaous (separate male and female plants) and, in species with apomixk, some populations are composed entirely of females while others contain both males and females. Three agamic complexes A. parlinii, A. media, A. paroifolia audesexual and apomictic individuals with the same ploidy level, which allows us to directly assess the role of apomixis in causing distributional differences between sexual and asexual populations (Bayer, 1984; Bayer and Stebbins, 1987). ln eastem North America, Bayer (1982,1985a) described two polyploid agamic complexes, A. parlinii and A. neod ioica (howelli) . Three diploid progenitor species are believed to be involved in the origin of the A. parlinii species complex: A. plantaginifolia, A. solitaria and A. racemosa (Bayer, 1985a). Two additional diploid species gave rise to the A. neodioica (howelli) complex: A. neglecta and A. uirginica (Bayer and Crawford, 1986; Bayer 198%). Only female plants are found in the A. neodioica complex while both male and female plants are found in A. parlinii. Antennaria parlinii is distributed throughout eastem-central North America, and its range overlaps and extends furthe1 north than two of its progenitor species (Fig. 1-1; Bayer and Stebbins, 1982, 1987). The third species, A. racemosa, only occurs further west and is geographically isolated from A. parlinii and the other two sexual taxa. Bayer (1985a) hypothesized that A. racemosa may have been sympahic in the past with A. plantaginifolia or Fig. 1-1: Distribution of Antennaria parlinii and its three sexual, diploid progenitor species, A. plantaginifolia, A. racernosa and A. solitaria (After Bayer and Stebbins, 1987). A. solitaria and gave rise to A. parhii during that period of sympatry. In Ohio, Antennaria purlinii is predominantly hexaploid (20 populations; 2x1 = M). Only a single location has been identified as octoploid (2x1 = 112). Throughout the rest of its the range, more than 50 of 58 populations surveyed were hexaploid and only seven were tetraploid (2x1 = 56) and two were pentaploid (2n = 70; Bayer, 1980,1984; Bayer and Stebbins, 1981). 1 also obtained chromosome counts for three Ohio and one Ontario population which were dl hexaploid. All three progenitor species of A. parlinii are found in wooded environrnents. Antennaria plantaginifolia occurs on dry ridge tops and slopes, in forest openings, A. solitaria in moist forest slopes, and A. racemosa in dry coniferous forest (Bayer and Crawford, 1986; Bayer and Stebbins, 1993). Bayer and Stebbinç (1993) described the habitat of A. parlinii as forest openings in deciduous forests its habitat resembling that of A. plantaginifolia. Michaels and Bazzaz (1986) observed, however, that there was a habitat difference between sexual and apomictic A. parlinii in IUinois. They found that asexual populations occur in disturbed habitats such as old fields, while sexual plants were found in more stable wooded habitats as weU as old fields. The range of Antennaria parlinii overlaps with other species of Antennaria involved in the neodioica complex. Antennaria oirginica occurs on shale barrens, while A. neglecta occurs in prairies and pastures (Bayer and Crawford, 1986). 1 obsemed that A. neodioica was common in grassy roadside ditches in southem Ontario and southem Québec and occured occasionally in Ohio.

Herbarium survey Because Antennaria is dieocious, herbarium specimens can be used to roughly infer the geographical distribution of sexual and apomictic individuals. The absence of males in an area can indicate the presence of female-ody populations and thus apomixis. However the presence of males does not necessarily indicate that apomicts are absent from an area. Based on herbarium specimens, seed set in the greenhouse, and field surveys of sex ratios, Bayer (1980) concluded that females predominate in the northem and eastem part of the range and thus populations in these regions were asexual, while males were common in the southwest and thus populations in this region are primarily sexual. 1 fomd a similar distribution of males and females in herbarium specirnens (n = 898; Fig. 1-2). In all regions represented by more than 10 records, there was a disproportionate number of female specimens. This bias increased towards the northeast part of the range, and al1 collection in the far northeast were female. Males were also absent from the southeast part of the range (Virginia, North Carolina and Georgia) however the number of specimens representing these areas was small (n = 9). Large numbers of specirnens were examined for Ohio (n = 453), Ontario (n = 108) and New York (n = 167). In Ohio, where most of my work was conducted, males make up 36 % of herbarium specimens and were collected throughout the state, though somewhat less frequently in the northwest (Fig. 1-3). In Ontario, males were rare (8%) and oc~edonly in the south of the province (Fig. 1-4). In New York, males were also rare (9.5%) and were absent in most of the state (Fig. 1-5). This herbariurn survey suggests that, in the same way that sexual and apomictic species pairs differ in distribution, apomictic individuals of A. parlinii are more widely distributed and occur at higher latitudes than sexual individuals. '3 Number of herbarium specimens

Fig. 1-2: Regional sex ratios in Antennaria parlinii based on herbarium specimens. The dark part of the circle is the proportion of female plants collected and the light part is the proportion of male plants. Specimens were from the CAN, DAO, QK, BH and OS herbaria. Fig. 1-3: Location of collection sites for herbarium specimens of Antennaria parlinii in Ohio. The empty circles are females and filled cirdes are males. The circles do not indicate the exact location specimens were collected but are within the same county. Fig. 1-4: Location of collection sites for herbarium specimens of Antennaria parlinii in Ontario. The empty cirdes are females and filled circles are males. Fig. 1-5: Location of collection sites for herbarium specimens of Antennaria parlinii in New York. The empty circles are females and filled circles are males. The cirdes do not indicate the exact location specimens were collected but are within the same county. CHAPTER 2: The evolutionary significance of variation in sexuality in polyploid agamic Antennan'a parlinii

Flowering plants are well-known for their diversity of breeding systems. Although most plants are predominantly sexual and outcrossing many taxa practice uniparental reproduction through either self-fertilization or apomkis. Stebbins (1958) presented the two systerns as alternative methods of reproductive assurance under conditions where pollinators or potential mates are scarce such as in temporary habitats. Many studies have estimated the level of self-fertilization in plant populations and determined the extent to which the selfing rate varies within and between species (129 species in Barrett and Edcert, 1990; s 200 species in Barrett et al., 1996). Like self-fertilization, the relative importance of apomixis varies among species and is widely expected to vqwithin species (Bayer et al., 1990). However, few studies have estimated the relative amount of sexual and apomictic seed production in natural populations (Bayer et al., 1990; Sdimidt and Antlfinger, 1992) and fewer still have examined the extent to which the frequency of apomixis varies among populations within species (Soreng, 1986; Gadella, 2987). In dioeuous species, among-population variation in sexuality is sometimes suggested by wide variation in sex ratio. For instance, Antennaria parlinii is a hexaploid agamic complex which originated from hybridization among three sexual, diploid species (Bayer and Stebbins, 1981; Bayer, 1984, 1985; Bayer and Crawford, 1986). It possesses a dioecious sexual system and is pollinated by small flies, bees and wasps (pers. obs.). Asexual reproduction occurs through mitotic diplosporous apomkis (Stebbins, 1932). It also spreads vegetaüvely through stolons (pers. obs). Populations occur throughout eastern North Amerka, and in the southeastem part of the range populations

are composed 1of both male and female plants (hereafter "mixed-sex" populations) while in the northem and eastern regions, males are rare and most populations are entirely female ("female" populations; Bayer, 1980; Chapter 1, Fig. 1-2). The absence of males from a population indicates that females have at least some capacity for apomictic seed production. In a survey of 63 populations in Ohio, Bayer and Stebbins (1983) found wide variation Vi population sex ratios, ranging from &73% males, which suggests wide variation in the relative importance of sexual and apomictic seed production among populations. Mu& of the effort to quanbfy the pattern of variation in self- fertilization within and among natural populations has been stimulated by theoretical models that make clear predictionç as to what sorts of mating systems we should observe in nature. In contrast, there are relatively few models for the evolution of apombcis, despite the fact that similar selection pressures may operate in the evolution of both forms of uniparental reproduction (Charlesworth, 1980; Marshall and Brown, 1981). Both self- fertilization and apomixis increase an individual's genetic contribution to the next generation (Maynard Smith, 1971, 1978; Williams, 1975), and can provide reproductive assurance when pollinators or potential mates are scarce (Bierzychudek, 198%). However, the selection of self-fertilization is strongly opposed by inbreeding depression, whereas genetic load is not expected to cause a short-tenn decrease in the fitness of asexually-produced offspring (Marshall and Brown, 1981). Accordingly, existing models predict that, under most conditions, either apomixis or sexual reproduction will become fked in a population (Charlesworth, 1980; Marshall and Brown, 1981; Harper, 1982). Species exhibiting wide variation in sexuality, such as A. parlinii, can provide excellent opportunities for testing these models. It is generally expected that, in moçt apomictic species, sexual and apomictic seeds can be produced by the sarne individual (Gustafsson, 1946- 1947). Partial apomixis has been found in several plants species and it has been suggested that some capauty for sexual reproduction rernains in all apomicts (Asker, 1979,1980; Nogler, 1984; Asker and Jerhg, 1992). Although partial apomixis has been reported in a variety of speues, little is known about how often it occurs and how important it is for introducing genetic variation into predominantly asexual Lineages. Again the few theoretical models exploring the selection of partial apodssuggest that it is unlikely to evolve (Marshall and Brown, 1981; Harper, 1982). Of course, many apomictic species are pseudogarnous and require pollen for the production of endosperm. Because pollination is required, some sexuality may be maintained (Nygren, 1967; Asker and Jerling, 1992). In contrast, apomixis in many taxa, including Antennaria, is autonomous and can occur in the complete absence of males. Thus we would expect that partial apomkis is unlikely to be selected. However, in Antennaria parlinii, Bayer and Stebbins (1983) found that, under greenhouse conditions, there was a wide variation in autonomous çeed set (0-100%) among plants sampled from several Ohio populations. The addition of pollen to a separate inflorescence on plants with low levels of autonomous seed set signihcantly increased seed production suggesting that, in contrast to theoretical predictions, partial apomixis may be common in this species. The potentially widespread occurrence of partial apomixis complicates the interpretation of sex ratios in terms of among-population differentiation in sexuality. For instance, if sexuality in Antennaria parlinii is a continuous trait, all populations may have some potential for both sexual and apornictic seed production but the realized sexual reproduction would be limited by the distribution of males. Female populations could, therefore, be composed of partially apomictic plants which can found new populations in the absence of males yet çtill retain the ability to produce seeds sexually if males were present. Similarly, mixed-sex populations could contain a mixture of sexuals, apomicts and partial apomicts. III this study, 1examine patterns of natural seed set, and conduct field and greenhouse pollinator-exclusion experiments to address two specific questions concerning variation in sexuality in Antennaria parlinii: 1) What is the pattern of variation in sexuality within and among populations? and 2) how common is partial apomixis?

MATERlALS AND METHODS Study populations This study involved 21 populations in Ohio (Fig 24), two in Frontenac (KL) and Leeds & Grenville (QU) counties, eastem Ontario, and another population in Gatineau (FG) county, western Québec. Ohio populations were distributed throughout a region where both entirely female and mixed-sex populations occur. The Ontario and Québec populations were located in a region where only females are found (see Figs. 1-2 and 1-4). Most populations had clear boundaries and were at separated by a least 2 km. Because clones of Antennaria parlinii often overlap, it is sometimes difficult to distinguish individual genets. Therefore, the number of inflorescences was used as an estimate of population size and the proportion of male inflorescences was used to estimate population sex ratio. Michaels and Bazzaz (1986) found that in sexual A. parlinii, male genets were larger Fig. 2-1. Location of 15 fernale and nine mixed-sex populations of Antennaria parlinii in Ohio. The shaded part of the circle is the proportion of females in the population and the light part the proportion of males. The dark line represents the southem limit of the Wisconsin glaciation. and could therefore produce more inflorescences than female genets. In the mixed-sex populatiow 1 studied, it also appeared that males produced more inflorescences than females (pers. obs.). As a result, caIculating sex ratio using inflorescence numbers probably overestimates the ratio of male to female genets in a population, and populations with even genotypic sex ratios rnay appear male-biased. Voucher specimens from each population are in the Fowler Herbarium (QK) at Queen's University in Kingston, Ontario, Canada.

Natural seed set in female and mixed-sex populations The extent to which populations are differentiated for sexuality might be reflected by patterns of natural seed set If female populations are composed predominantly of partial apornicts, then seed set will be reduced by the absence of males relative to mixed-sex populations. Female populations consisting of obligate apomiaç, on the other hand, will exhibit high seed set. To compare the level of open-pollinated seed set between female and mixed- sex populations, 1 collected inflorescences from 20 plants in each of the 24 populations just before seed dispersa1 in late May 1996. For small populations (less than 20 individuals), 1 attempted to collect an inflorescence from every plwt. When genets were difficult to distinguish, inflorescences were collected at least 2 m apart to minimize sampling from the same genet more than once (see Bayer, 1980). Each inflorescence included an average of 6 capitula per inflorescence with 150 florets per capitulum. Seed set was determined for the central capitulum of each inflorescence. Viable seeds were dark and plump while undeveloped seeds were small, white and empty. A nested ANOVA model was used to test for differences in seed set between female and mixed-sex populations. Population type was a fixed effect and population, nested within population type, was a random effect. The sample variances between population types and among populations within types were significantly heterogeneous, so seed set was arcsine ( Y = ar arcs in fi]) transformed. However, the variance among populations remained heterogencouç. Nested ANOVA with arcsine-trançformed and rank- transformed data gave the same result as the raw data indicating that the original analysis was probably robust to violations of ANOVA assumptions. Accordingly, nested ANOVA on the untransforrned data is presented. This mode1 as well as other statistical analyses reported below were performed using JMP (version 3.1.6, SAS Institute, 1989). Because sample size varied among populations the F-test for population type used a synthetic denominator calculated using the Satterthwaite method (Sokal and Rohlf, 1995, pp. 292-300). If mixed-sex populations are predominantly sexual, seed set may be influenced by the relative frequency of males and the level of pollinator activity (Bierzydiudek, 1987b). The latter may, in tum, increase with population size. If, on the other hand, populations with female-biased sex ratios include either partial or obligate apornicts, seed set may show little or no correlation with sex ratio or population size. To see whether pollen availabiüty limited seed set in mixed-sex populations, 1 tested whether mean population seed set increased with population size and sex ratio. To test if seed set within a population was correlated to pollen availability, the distance from each female inflorescence to the closest male inflorescence was measured in two populations with a sex ratio near 0.5 (CC and JL) and two populations with a single male clone (CCand GR). I then tested whether seed set was negatively correlated with the distance to the closest male inflorescence. Field pollinafor-exclusion experiment To more directly compare the capacity of plants for apomixis between female and mixed-çex populations, 1conducted a pollinator exclusion experiment during May and June 1996, in two mixed-sex (CC and JL)and two female (IP and KT) populations in northern Ohio. These populations were chosen because they were all located in the recently glauated region of Ohio, where we might expect to find partially or fully apomictic plants in mixed-sex populations (Bayer and Stebbins, 1983). For each plant, one inflorescence was bagged before any stigmas were presented with two layers of fine bridal veil to exclude pollinators, and another was left unbagged to serve as an open- pollinated control. The bag was left on throughout seed maturation. Inflorescences were collected in Iate May and seed set was determined for the central capitulum in each. 1 used a patially-nested mixed-mode1 ANOVA to test whether female and mixed-sex populations differed in the capacity for apomictic seed production. Only plants receiving both treatrnents were included in the analysis. Population type (PT), treatment and interaction between those effects were fixed effects while population (P; nested within PT), and plant (nested within P and PT) were random effects. In mixed-sex populations, 31 of 60 bagged inflorescences set some seeds. 1 waç able to sarnple stigmas from 17 of the 31 inflorescences which set seed and examined thern for pollen contamination. Contaminant pollen was found on stigmas from 16 of these 17 inflorescences, and therefore these 16 inflorescences were excluded from the analysis. Flower stalks continued growing after bagging and some stigmas pushed through the bridal veil bags and were exposed to pollinators. Low seed set could indicate partial apomixis, however, in this case contaminant pollen could account for the low seed set # found in the remaining 14 bagged inflorescences which set seed. Greenhouse pollination experiment The capacity for apomixk for a larger sample of rnixed-sex and female populations was cornpared in a common environment using a controlled pollination experiment in a pohator-free greenhoue. In August 1996, plants hm20 Ohio and two Ontario popuiations were collected and transferred to an outdoor garden in Kingston, Ontario. Twenty plants were sampled for each female population, and 30 plants were sampled for each mixed-sex population (because males and females could not be differentiated in late summer). In October, plants were moved to a dark, coldroom (5'C) to overwinter. Late the following March, after flower buds had formed and flower stalks had begun to elongate, plants were moved to the greenhouse. Inflorescences were produced by û4 plants from 13 female populations and 59 plants from nine mixed-sex populations. Because half of the flowering plants from mixed-sex populations were male, only 30 flowering females from mixed-sex populations were available. Sorne populations only had one or two flowering individuals, therefore, plants were pooled for each population type and the variation arnong population was not analyzed. To test for apomixis, inflorescences were left unpollinated. On plants with more than one inflorescence, an additional inflorescence was hand-pollinated 1-3 times using a small paintbmsh to test whether pollination increased seed set. Inflorescences were collected just before seed dispersal, and seed set was determined for the entire inflorescence. There were, on average, 5 capitula and 500 florets per inflorescence. Paired t-tests were used to test for an increase in seed set after hand-pollination. RESULTS Natriru1 seed set in fernale and rnixed-sex popdations Nested ANOVA did not detect a significant difference in seed set between the 15 female (Fig. 2-2, TABLE 2-1; mean rt SE = 0.73 t 0.04, range = 0.33-0.94, CV = 19.4%) and nine mixed-çex populations (0.69 f 0.04, range = 0.48-0.83, CV = 15.4%). Within each population type, seed set varied significantly arnong individual populations (TABLE 2-1). The seed set (S) of nine miwed-sex populations was positively correlated with both population size (N; Fig. 2-3a; r5~= +0.68,1-tailed P =

0.022) and the proportion of males (M; Fig. 2-3b; YSM = +0.80,1-tailed P = 0.005). Since population size and the proportion of males were not significantly correlated (rNM= +0.35, 2-tailed P = 0.35), partial correlation coefficients between seed set and the proportion of males, and seed set and population size remained signihcant when the other variable was held constant (rSN.J,f= +0.71, 1-tailed P c 0.025 and rs~.~= +0.82,1-tailed P < 0.005 ). In contrast, seed set in female populations was not sigruhcantly correlated with population size (rSN= +0.37, n = 15,l-tailed P = 0.09); however, the correlation coefficients for seed set and population size in ded-sex and female populations were not signihcantly different (tS= 0.88, P = 0.19; Sokal and Rohlf, 1995, p. 582). In two mixed-sex populations with even sex ratios, seed set was negatively correlated with the distance to the closest male inflorescence (Fig. 2-4, population JL, n = 16, r = -0.52,l-tailed P = 0.019; population CC, n = 31, r = -0.39, 1-tailed P = 0.013). In contrast, seed set in the two populations with a single male clone did not decrease with increased distance from the maie. In one population (GC) seed set exceeded 80% in all individuals regardless of the distance to the male (n = 14, r = iO.14,l-tailed P = 0.31). When part of this male clone was brought into the greenhouse it produced starninate flowers 0.0 ' Female Mixed-sex Population type

Fig. 2-2. Natural seed set in 15 female and nine mixed-sex populations of Antennaria pnrlinii. Box plots show the distribution of population means. The top-edge, middle line and bottom edge of the box are the upper quartile, median, and lower quartile, respectively. The broken line is the mean. The whiskers extend to include al1 the points, or 1.5 times the interquartile range from the upper and lower quartile, whichever is least. Analysis of these data is in TABLE 2-1. TABLE 2-1. Analysis of variation in natural seed set in 15 female and nine mixed-sex populations of Antennaria parlinii. Population type (PT) was a fixed effect and population (nested within PT) a random effect. The F-test for PT used (0.89MSpopdaoon + O.llMSResidual)as denominater and the one for Population, MSResidual.For the whole model, r2 = 0.25. Means are in Fig. 2-2.

-- - Source of variation df SS F P

Population Type 1 0.2 0.9 0.34 Population [PT] 22 5.5 5.9 <0.0001 Residual 402 17.0 O 500 1000 1500 2000 2500 3000 3500 Population size

Proportion of males

Fig. 2-3. The effect of population size (a), and the proportion of males @) on seed set in nine mixed-sex populations of Anfennaria parlinii. (a) JL,n = 16, r= -0.27 @) CC,n=31,r=-0.40

Distance from male (m)

Fig. 2-4. Relation between distance to the closest male inflorescence and seed set in four mixed-sex populations of Antennaria parhii. Two populations (a, b) had approximately even sex ratios (JL = 0.54, CC = 0.58) and two had a very female-biased sex ratio (cf d; GC = 0.04; GR = 0.008). but the anthers produced no pollen. In the second population (GR), the correlation between seed set and distance to male was in the opposite direction of what was expected (n = 14, r = +0.55,2-tailed P = 0.04). Because seed set in the two singlemale populations was high and not negatively correlated with the distance to the male, these populations and another with only two males (HU, sex ratio = 0.012) were considered all-female populations in other analyses.

Pollinator-exclusion experiment There was no difference in seed set between bagged (0.72 I: 0.024, range

= 0.28-0.97) and open-pobated (0.71 & 0.027, range = 0.06-0.97) inflorescences in female populations (Fig. 2-5; paired t = 0.44, df = 45, P = 0.66). In contrast, there was a large difference in seed set between bagged (0.01 + 0.004, range = 0- 0.09) and open-polhated (0.68 f 0.04,0.11-1.00) inflorescences in mixed-sex populations (Fig. 2-5; paired t = 15.18, df = 30, P < 0.0001). As a result of the strong effect of poLlinator exclusion in mixed-sex but not in female populations, ANOVA showed a strong significant interaction between the effects of population type and treatrnent (TABLE2-2).

Greenhouse pollination experimen t Results from the greenhouse experiment generally reflected those from the field pollinator-exdusion experiment (Fig. 2-6). Eighty-four unpollinated inflorescences (1 inflorescence/plant) from 13 female populations had a high autonomous seed set (0.66 t 0.03; range û-û.97). In contrast, 29 of 30 unpollinated inflorescences from nine mixed-sex populatiow produced no seeds. One inflorescence produced a single seed out of 424 florets. In plants receiving both treatments, hand-pollination of 14 inflorescences from mixed- Female Mixed-Sex ~*OTT T T T T

n = 19 27 19 12 Population

Fig. 2-5. The effect of pollinator exclusion on seed set in four Ohio populations of Antennaria parlinii. n is the nurnber of plants that received both pollination treatments (pollinators-excluded bagged] and open- pollinated). Andysis of these data is in TABLE 2-2. See Fig. 2-2 for details on boxplots. 34

TABLE 2-2: The effect of pollinator-exclusion on seed set in two female and two mixed-sex populations of Antemaria parlinii. Population type (PT; female or mixed-sex) and treatment (pollinators-excluded or open-pollinated) were hed effects. Population (P), nested within PT, and plant were random effects. The F-test for population type used (O-96MSPopulation - 0.57MSplant[p, PT] + 0.61MSResidual)as a denominator, the one for population used 1.22MSPlant[P,PT1- O.ZWResidual and au others used MSResidual*For the whole model r2 = 0.88. Means are in Fig. 2-5.

-. Source of variation df SS F P

Population Type

Population [PT]

Plant (P[PT])

Trea tment

PT x T

PxTImI

Residuaf Population type

Fig. 26. Seed set of plants from female and mixed-sex populations of Antennaria parlinii under greenhouse conditions. n is the number of plants that received both pohation treatments (unpollinated and hand- pollinated). See Fig. 2-2 for details on boxplots. sex populations increased mean seed set from O to 0.42 t 0.061 (paired t = 6.78, df = 13, P < 0.0001). In female populations, pollinated (0.64 + 0.038) inflorescences did not set more seedç than unpohated (0.68 f 0.039) inflorescences (paired t = 1.38, df = 41, P = 0.18).

DISCUSSION Variation in sexuality among and within populations This study shows that populations of Antennaria parlinii in Ohio are highly differentiated in sexuality, and that there is little or no variation in sexuality within populations. 1 found no evidence that sexual and asexual individuals occurred in the sarne population, or that individuals commonly pradiced partial apomixis. High seed set was observed in plants from female populations under natural conditions, in pohator-exclusion bags and in an insect-hee greenhouse. Three predominantly female populations included ody one or two male clones, however, the scarcity of males was not associated with reduced seed set, suggesting predorninant apomixis. Ln mixed-sex populations, seed set increased with both population size and the proportion of males suggesting that these populations are predominantly sexual. Low Ievels of seed set was observed in the pollinator exclusion experiment, however, it is likely that al1 of the seed set by plants from mixed- sex populations in thiç experiment could be attributed to accidental pollen contamination (see Methods). This is supported by the complete lack of autonomous seed set under greenhouse conditions in 30 plants representing nine mixed-sex populations. The strong differentiation in sexuality among populations and the apparent lack of variation in sexuality within populations is somewhat at odds with the general view of continuous sexual variation in apomictic species (Nogler, 1984, Bayer et al., 1990). Bayer and Stebbins (1983) found fully- sexual, fully-apomictic and partially-apomictic plants in two Ohio populations of A. parlinii. Michaels and Bazzaz (1986,1989) ako found both sexual and apomicts in an Illinois population of A. parlinii. Sexuals, apomicts and partial apomicts also CO-occurwithin' populations of Antennaria media (Bayer et al., 1990) and A. paroifolia (Bierzydiudek, 1989). However, all these studies involved a small number of populations so it is not clear whether the extensive variation in sexuality observed is representative of these species in general. The few largescale surveys of species with both sexual and asexual individuals have found only one mode of reproduction in most populations (Soreng, 1986; Gadella, 1987; Herbert et al., 1988,1993; Ward et al., 1994). Gadella (1987) examirted 157 populations of Hieracium pilosella on three Dutch islands and found that sexuals and apomicts always occurred in separate populations. Soreng (1986) tested for differences in seed set between 11 mixed-sex and four all-fernale populations in dioecious Poa fendleriana in New Mexico. Among the mixed-sex populations, seed set in those with fernale-biased sex ratios was pollen limited; thus the populations appeared to be predominantly sexual. Only three mixed-sex populations with strongly female-biased sex ratios (>75%) may have contained a mixture of both reproductive -es. Based on patterns of variance in seed set, he estimated that in the geographical area of overlap between all-female and mixed-sex populations, there were 16 fully-apomictic populations, 26 fully-sexual and only four with a mixture of both sexuals and apomicts. In animals, large scale studies of the distribution of asexual and sexual reproduction have been conducted in Daphnia pulex (Hebert et al., 1988, 1993; Ward et al., 1994). The general trend is that southem populations are sexual and northern populations asexual. In the transition zone from sexual to asexual reproduction, few populations with both reproductive types were found. The differentiation in sexuality 1 observed among populations of A. parlinii could possibly be due to environmental variation among habitats. In Dichantium aristatum, a partial apomict, the proporfion of apornictic seeds is intluenced by photopenod (Knox and Heslop-Harrison, 1963; Knox, 1967). In Antennaria parlinii female and mixed-sex populations were found in different habitat types: female populations occurred in open habitats and mixed-sex populations occurred under a tree canopy (see Chapter 3; Fig 3-3). However it is unlikely that this environmental variation made a significant contribution to the obsenred differentiation in sexuality. Plants from 22 populations which were grown in the greenhouse and subjected to the same temperature and light regime had autonomous seed set values very sirnilar to plants from the same population under field conditions. The level of apomictic seed set was not influenced by the range of environmental conditions involved in the transition hmfield to greenhouse.

Partial apomixis Partial apomixis has been found in many plant species and its possible role in introducing variation in asexual lineages has been discussed by several papers (Marshall and Brown, 1974; Marshall and Weir, 1979; Harper, 1982; Nogler, 1984; Kondrashov, 1985). Based on data from Antennaria media, Bayer et al. (1990) suggested that rather than categorizing apomicts as obligate or facultative (partial), apornixis should be viewed as a continuum ranging from complete sexuality through facultative apomixis to nearly obligate apomixis. Bayer and Stebbiw (1983) also observed a wide range of apornictic seed production among plants in two Ohio populations of A. parlinii. In contrast, this study suggests that partial apomicts are probably rare in Ohio populations of A. parlinii. In mixed-sex populations, partial apomixis is easily detected by isolating plants from pollinators and looking for autonomous seed set. None of the plants from mixed-sex populations in this study set seed autonomously under greenhouse conditions. Detecting partial apoxnixis in female populations is more difficult since departures from 100% seed set when isolated from pollinators is not necessarily indicative of incomplete apomixis. Less than 100% seed set could be due to resource limitation (Willson, 1983), genetic load (Charlesworth, 1989) and/or malformed ovules (Wiens, 1984) in addition to or instead of the occurrence of reduced embryo sacs (partial apomkis). For example, in apomictic Antennaria paraifolin Bierzychudek (1989) obsenred that autonomous seed set is limited by water stress rather than incomplete apomixis. In this study, the seed set of plants from female populations isolated in the greenhouse could not be increased by hand-pollination suggesting little or no capacity for sexual reproduction in female populations. Nevertheless, one female population exhibited particularly low natural seed set (see Fig. 2-2), setting less than 40% of its seeds in 11 of 12 inflorescences, suggesting incomplete apomixis. Unfortunately, no plants from this population were included in the greenhouse experiment. Detecting partial apomkis in individual plants with low autonomous seed set is difficult since an increase in seed set after pollination is not necessarily due to the production of sexual seeds. Different inflorescences within the same clone cm vary greatly in autonomous seed set. In plants from the two female populations involved in the pollinator-exclusion experiment (IP and KT) seed set of the same plant measured in the field and greenhouse was negatively correlated (n = 24; r = -0.42; P = 0.04). Because autonomous seed set is so variable within a clone, a combination of controlled pollination experiments and genetic markers should be used to detect partial apomkis (see Bayer et al., 1990). From an adaptationist point of view, partial apomixis might be advantageous in pollen limited environrnents such as female-biased populations. However, biases in sex ratio could also be a result of partial apomix6 instead of a selective pressure for it. Bayer and Stebbins' (1983) results suggest that in A. parlinii the presence of apomixis in mixed-sex populations gives rise to female-biased sex ratios (see Bayer 1980). My results did not support this hypothesis. 1 found low natural seed set in female-biased populations indicating that these populations were predominantly sexual. Furthemore, sex ratios in dioecious plants often Vary greatly (Willson, 1983). Such variance is even more likely in perennials like A. parlinii with a well- developed capacity for clona1 propagation (Eckert and Barrett, 1992, 1995). The sex ratios for 40 mixed-sex populations in Bayer and Stebbins' (1983) study have a mean and median of 0.50 and there are as many female-biased as male-biased sex ratios indicating that female biases were more likely due to random drift than variation in sexuality among mixed-sex populations (Fig. 2-7). In the nine mixed-sex populations 1 studied, sex ratios also varied greatly and ranged from 29 to 73 % males even though al1 these populations were sexual. I found that populations with fernale-biased sex ratios were usually small (sometirnes less than 20 plants), while the large populations had balanced sex ratios. This is consistent with the role of random drift in biasing sex ratios. Only populations with highly skewed sex ratios (~5%males), with only one or two males were apomictic. Population

Fig. 2-7. Sex ratio of 40 mixed-sex populations of Antennnria parlinii populations in Ohio (data from Bayer and Stebbins 1983). The lines are the 95% confidence interval. Sex-ratios with confidence intervals which do not cross the broken line are sigruficantly different from 0.5. Disru ptive selection for sexuality The strong differentiation in sexuality arnong populations of Antennaria parlinii and the absence or rarity of partial apomicts is in accord with theoretical models for the evolution of apomixis in plants (Charlesworth, 1980; Marshall and Brown, 1981). These models predict that there should usually be strong dismptive selection for sexuality, depending on the relative fitness of sexuals and apomicts. Populations with partial apomixis or a polymorphism for sexuality are stable only under restrictive conditions (Marshall and Brown, 1981). Partial apomWs or the CO-occurence of sexuals and apomicts may, therefore, be a transitional stage towards complete apomWs or complete sexuality (Harper, 1982). Because both mixed- sex and female populations of A. parlinii in Ohio occur nearby but in different habitats types (Chapter 3), 1 would expect that the shift between these two evolutionary trajectories depends on differences in fitness between sexual and apomict individuals in contrasting habitats. CHAPTER 3: Differences in distribution and colonizing ability between apomictic and senial populations of Antennaria parlinii

A common pattern in both plants and animals is that asexual species have a different ecological and geographical distribution than dosely related semial species (Glesener and Tilman, 1978; Bell, 1982; Lynch, 1984; Bierzychudek, 1987a). Compared to semial species, asenial species usually have larger geographical ranges and occur at higher altitudes and latitudes, in harsher environments, and in more epherneral and disturbed habitats. Stebbins (1950) attributed the greater range of asexual plants to çuperior colonizing ability due to the reproductive assurance afforded by their uniparental reproductive mode; a single individual can colonize a new habitat and quickly build up a new population. Stebbins (1958) further claimed that genera in which apoWis common also have special dispersa1 structures (eg. Hieracium, Youngia, Ixeris, Taruxucum, ChondriIla, and Crepis) which would also promote colonization of ephemeral habitats. However, Bierzychudek (1987a) questioned the association between habitat disturbance and apomkis. In the studies she reviewed, some apomicts occurred in more disturbed and ephemeral habitats than their sexual relatives but there was no difference in habitat type between rnost other sexual- apomictic pairs. In addition, a correlation between asexuality and polyploidy confounds the correlation between asexuality and geographic distribution (Bierzychudek, 1987a). Asexual species usually have a higher ploidy level than closely related sexual species, and polyploidy alone cm enhance ecological tolerance through slower development, delayed reproduction, longer life span, larger seeds, and greater chernical defense against pests and pathogens (Levin, 1983). In some species, both asexual and sexual individuals have the same ploidy level, allowing a more direct assessrnent of the relation between asexuality and distribution. Antennaria parlinii is a polyploid agamic complex in which both asexuai and sexual plants are almost always hexaploid (2n = 86; Bayer, 1981). Asexud reproduction is through autonomous, mitotic diplosporous apodxis (Stebbiw, 1932). It also reproduces vegetatively through stolons (pers. obs.). The species possesses a dioecious sexual system and there is a wide variation in sexuality among populations which is reflected by the population sex ratio. Some populations are composed entirely of females and are obligately asexual while others have both males and females and are obligately sexual (Chapter 2). The species is distributed throughout eastem North Amenca (Bayer, 1982), and the range of apomictic plants extends further north and east than sexual plants (Bayer, 1980; Chapter 1, Fig. 1-2). In Ohio, there is a transition from sexual populations in the south to asexual populations in the north, a pattern which is associated with the glacial terminal moraine of the Wisconsin glaciation (Bayer and Stebbinç, 1983). Michaelç and Bazzaz (1986) also observed that, in Illinois, asexual A. pnrlinii occurred in disturbed habitats such as old fields, while sexual plants were found in more stable woodland habitats as well as old fields. The differences in ecological and geographical distribution of sexual and apomictic A. parlinii mirror the general pattern found in interspecific comparisonç. These distributional differences may, in part, anse due to greater colonizing ability of asexual plants afforded by apomixk. Because Antennaria parlinii is dioecious, sexual plants have a much more limited colonization potential since both a male and a female are required before a new population cm become established. In addition, sexual females suffer reduced seed set in small populations with few males (Chapter 2). If uniparental reproduction facilitates colonization, other colonizing traits such as the production of many small seeds with high dispersa1 ability may become iinked to apomixis through selection for increased colonizing ability. Greater reproductive output increases the diance of some seeds encountering suitable sites (Harper, 1965; Willson, 1983) and allows founders to quickly increase population size (Stebbins, 1958). In three Illinois populations, Michaels and Bazzaz (1986) found that asexual A. pnrlinii plants produced more numerous but smaller seeds than sexual plants suggesting that asexual plants were r-selected (see also Midiaels and Bazzaz, 1989). In addition to elevated reproductive output, traits that increase seed dispersal might ako be çelected because in ephemeral habitats the chance of a seed making it to a newly disturbed site is greater than its chance of sumiving in its present location (Harper, 1965; Howe and Smallwood, 1982). The wind- dispersed seeds of A. parlinii are equipped with a plume-like pappus which keeps the seed aloft and enhances dispersal. Michaels and Bazzaz (1986) report a casual observation that, in A. parlinii, seeds from apomictic plants had a longer free-fall time than sexual seeds. Morphological traits of diaspores which increase seed free-fall time can be measured to compare differences in dispersa1 potential between reproductive types (Sheldon and Burrows, 1973). In this study, 1 begin by showing that in regions where sexual and apomictic A. parlinii co-occur, apornicts occur in more disturbed habitats than sexual plants. I then test the prediction that apomictic A. parlinii possess other colonizing traits such as a greater reproductive output and a greater dispersa1 potential. MATERLALS AND METHODS Habitat survey To test for habitat differences between asexual and sexual Antennaria parlinii, 21 populations containhg only fernales (asexual) and 18 populations with both males and females (sexual) throughout Ohio were suweyed (Fig. 3- 1). At each site, I recorded the presence or absence of a canopy as an index of habitat successional stage and noted the general habitat type (roadside, riverside, lakeside, field or cemetery). The majority of populations occurred along roads, and in these populations 1 tested whether asexual and sexual populations were associated with the steepness of the slope on which they occurred (flat, sloped or steep) since populations on a flat or slightly sloped roadside are more likely to be mowed or otherwise disturbed than on a steep slope. Chi-squares analyses were used to test for an association between population type and habitat characteristics. These analyses as well as others reported below were performed using JMP (version 3.1.6, SAS Institute, 1989).

Population survey L To test for differences in reproductive output and dispersa1 ability between asexual and sexual populations, inmictescences were collected from 21 populations in Ohio, two in Ontario and one in Québec. The location of these populations and sampling methods are described in Chapter 2.

Reproductive output - To test whether asexual and sexual populations differed in reproductive output, about 20 inflorescences per population were collected and the nurnber of capitula per inflorescence, florets per capitulum and seeds per floret were determined for each. Seeds Fig. 3-1. Location of Antemaria patlinii populations in Ohio suweyed to test for differences in habitat between asexual (empty circles; n = 21) and sexual (fïlled circles; n = 18) populations. The dark line represents the southem limit of the Wisconsin glaciation. and florets were counted in the centrai capitulum of each inflorescence. Seeds per inflorescence were estimated by multiplying the number of capitula per inflorescence by seeds per capitulum. Nested ANOVA was used to test if these measures of reproductive output differed between asexual and sexual populations. Population type was a fixed effect and population (nested within population type) was a random effect. To meet the assumptions of ANOVA, capitula / inflorescence and seeds / inflorescence were loglrtransformed, and florets/capitulum and seeds/capitulum were square-root transformed. Because sample size varied arnong populations the F-test for population type used a synthetic denominator calculated using the Satterthwaite method (Sokal and Rohlf, 1995, pp. 292-300).

Diaspore measurements - To test if seeds from asexual and sexual plants differed in dispersa1 ability, four traits were measured for a sample of four seeds per plant from 10-28 plants from each of 15 asexual populations (249 plants), and 12-30 plants from each of nuie sexual populations (179 plants; Fig. 3-2). For each diaspore, the nurnber of pappus bristles were counted and the extent to which bristles were barbed (a combination of number and size of barbs) was scored on a scale of 1 to 5. The length of three bristles per pappus, and seed size (width x length) were measured using a Leica WILD M3Z stereo dissecting microscope with a drawing tube mounted over a CalComp DrawingBoard IITM digitizing tablet. Tracings were measured using NM Image program (version 1.59, 1993) with a calibration of 39.55 pixels/mrn and a pixel aspect ratio of 1.19. Not al1 measurements were made for each diaspore so sample sizes Vary among variables. Al1 measurements were done blind to the identity of the plant and population that the seed came from. The ratio of pappus length to seed size was calculated as a measure of Fig. 3.2. Schematic diagram of an Antennaria parlinii diaspore. dispersa1 ability (Sheldon and Burrows, 1973). Nested ANOVA (as above) was

used to test if asexual and sexual populations differed for each of the measured traits. Seed size was logio-transformed to meet the assumptions of ANOVA.

Free-fa11 test

The pappus on Antemaria parlinii seeds acts as a parachute and slows the seed down as it f&. Seeds which fall more slowly presumably have a greater chance of being picked-up by wind and camed away farther and thus have greater dispersa1 potential (Sheldon and Burrows, 1973). I tested whether different pappus characteristics that would appear to increase the size or effectiveness of the parachute affected seed free-fa11 time. Fifty-five diaspores (seed+pappus uni&) were selected from plants with different combinations of the four traits measured above: pappus length, seed size, the nurnber of pappus bristles and barb score. Each diaspore was dropped in still air from a height of 80 an inside a 35 cm diameter cardboard tube. The angle the pappus was open (Fig. 3-2) was measured just before dropping each seed. The other four traits were measured for each diaspore just after the free-fall test. Again measurements were made blind to the identity or free-fa11 tirne of the seed. Simple linear regressions were used to test whether free-fa11 tirne was related to the five diaspore traits. To estimate the effect of each diaspore trait on free-fa11 time independently of the other traits, multiple regression was used. Traits that were significant in the simple regression analysis (pappus-to-seed ratio, angle, barbs) were included in the multiple regression model. There was no significant correlation among the traits (TABLE3-1) so multiple CO-linearitywas not a probiem. 51

TABLE 3-1. Pearson correlations among pappus traits in Antennaria parlinii.

These data are from the 55 diaspores used in a free-fa11 test. P-values are in parentheses.

Pappus / Seed Pappus Angle Pappus Number

Pappus Angle t0.177 (0.20)

Pappus Number -0.040 (0.77) -0.156 (0.25)

Barb Score +0.197 (0.149) +O228 (0.09) 4.146 (0.29) To compare differences in dispersal ability between seeds from asexual and sexual populations, the multiple regression equation was used to estimate free-fall time from diaspore characteristics. A pappus angle of 90"

was used for all diaspores because it was the average angle of diaspores in the free-fall test and pappus angle was likely a characteristic of how the seeds were stored rather than what population they came frorn. Nested ANOVA (as above) was used to test for a difference in estimated diaspore free-fa11 time between sexual and asexual populations.

Germination experiment To test whether asexual and sexual seeds differed in viability, the germination of seeds was compared in a common growth chamber environment. Twenty seeds per plant from 15 asexual populations (mean 16 plants per population, range 2-28) and nine sexual populations (mean 20 plants per population, range 11-30) were randomly selected and placed in sealed sterile petri dishes on filter paper moistened with deionized water, and stored for 4 wks at 4'C. Seeds were then germinated in growth chambers at 22'C during 14 h light, and 16'C during 10h dark. Germination was scored daily und few new germinants (~0.5%)were obse~ed(10 days). The difference in total germination between asexual and sexual populations was analyzed using a nested ANOVA (as above). The proportion of seeds germinated was arcsine-transformed ( Y = Z[arcsinfl]) to meet assumptions of ANOVA. RESULTS Habitat difierences between asexual and sexual populations Sexual and asexual populations tended to occur in different habitats in Ohio. Populations under a tree canopy (n = 18) were more often sexual (67%) than asexual (33%), while populations in open areas without a canopy (n = 21)

were more often asexual (71%) than sexual(29%; Fig. 3-3; X2 = 5.66, df = 1, P = 0.017). Cemetery (n = 2), river- and lake-side populations (n = 4) were all sexual, while populations on lawns or in fallow fields (n = 3) were al1 asexual. Roadside populations (n = 30 total), on flat areas (n = 6) and in shallow ditches (n = 13) were usually asexual (73% and 67%, respectively), while those on steep slopes and rocky outcrops (n = 11) were usually semal (75%). The

association between population type and dope was statistically sig~ficant(X2 = 8-04,df = 2, P = 0.018).

Differences in reproductive output between asexual and sexual populations Most measures of reproductive output were greater in asexual than sexual populations (Fig. 3-4; TABLE 3-2). Compared to plants from sexual populations, plants in asexual populations had almost two more capitula per inflorescence (mean f SE = 6.9 I0.33 vs. 5.1 t 0.30), 34% more florets per capitulum (162.9 t 8.4 vs. 123.7 f 6.3), but about the same number of seeds per floret (0.73 t 0.04, range = 0.33-0.94 vs. 0.69 f 0.04, range = 0.48-0.83). OveraU, plants in asexual populations produced 30% more seeds per capitulum (120.2 + 8.4 vs. 83.5 + 4.9) and twice as many seeds per inflorescence (876.3 f 74.5 vs. 439.9 i 43.5). O canopy No canopy

Population type

Fig. 3-3. Number of asexual and sexual populations of Antennaria parlinii populations in habitats with or without a tree canopy. (a) Capitula/inflorescence (b) Florets / capitulum

10 ,- 250 r

- 50 ; 01 Asermal Sexual Asexual Sexual Population type

Fig. 3-4. Components of reproductive output in 15 asexual and nine sexual populations of Antennaria parlinii. Box plots show the distribution of population means. The top edge, middle line and lower edge of the box plots are the upper quartile, median and lower quartile, respectively. The broken line is the mean. The whiskerç extend to maximum and minimum points. Analysis of these data is in TABLE 3-2. TABLE 3-2. Analysis of variation in reproductive output in 15 asexual and nine sexual populations of Antennnria parlinii. Population type was a fixed effect and population (nested within population type) was random. F-tests for population type used (0-g~POpulaoon[PT1+ O-lMSResidual) as a denominator and those for population used MSRsidUal. Means are in Fig. 3-4.

Effect (df) Population type (1) Population (22)

Capitula/ 0.36 14.4 (0.001) 6.6 (<0.0001) 0.02 (440) Inflorescence Flore ts / 0.39 14.3 (0.001) 6.4 (<0.0001) 2.87 (403) Capitulum

Seeds / 0.25 0.9 (0.34) 5.9 (<0.0001) 0.04 (402) FIoret

Seeds/ 0.30 11.0 (0.003) 4-8(<0.0001) 5.62 (402) Capitulum

Seeds/ 0.39 22.0 (0.0001) 5.3 (<0.0001) 0.08 (395) Inflorescence Relation between diaspore traits nnd Jree-fa[[ tirne The free-fa11 time of A. parlinii diaspores was significantly correlated with three of four pappus traits (Fig. 3-5). Free-fall time increased with pappus-toseed ratio (r2= 0.36, P c 0.0001), angle of pappus opening (r2 = 0.18, P = 0.001) and barb score (2= 0.14, P = 0.004) but not pappus briçtle nurnber (r2= 0.03, P = 0.199). The multiple regression for the effect of pappus traits on seed free-fall time was statistically significant (TABLE 3-3; R2 = 0.52 P < 0.0001). Partial regression coefficients for pappus-to-seed ratio, pappus angle and barbs were all positive and statistically significant (TABLE 3-3). Since the three diaspore traits were not correlated (TABLE 3-1) variance inflation factors for al1 three traits were approximately 1, indicating low colinearity.

Difierences between asexzcal and sexual populations in diaspore traits Traits which increased seed free-fa11 time were greater in asexual than sexual populations (Fig. 3-6; TABLE 3-4). Seeds in asexual populations were 8% smaller (0.425 f 0.007 mm2)than in sexual populations (0.461 t 0.010 mm2).In contrast, pappus brides were 8 % longer in asexual (7.06 k 0.074 mm) than in sexual populations (6.51 + 0.078 mm). As a result, mean pappus- to-çeed ratio was 16% greater in asenal (17.5 f 0.35) than sexual populations (14.7 i 0.45). Diaspores from asexual populations also had a higher barb score (3.2 k 0.14) than those from sexual populations (2.5 + 0.1 1). The number of pappus bristle per seed, the single trait that did not influence seed free-fa11 time, did not differ sigmfxcantly between population types (asexual: 22.1 t 0.4; sexual: 22.0 f 0.5). Estimates of free-fa11 time were significantly different between population types (Fig. 3-7, TABLE 3-4). Estimated free-fa11 tirne for diaspores from asexual populations (3.70 f 0.04 s) was 10% longer than for seeds from sexual populations (3.32 i 0.05 s). (a) Seed size (mm2) r

(d) Barb score Fm

1.0 0 Asexual Sexual Asexual Sexual Population type

Fig. 3-5. Effect of four diaspore traits on seed free-fall time in Antennaria parlinii (n = 55). See text for description of traits. Analysis of these data is in the text. TABLE 3-3. Multiple regression examining the effect of pappus traits on diaspore free-faIl time in Antennariu parlinii. The three traits were not intercorrelated (TABLE 3-1) so variance inflation factors were al1 approximately 1. For the whole model, R~ = 0.52.

Mode1 3

Residual 51

Variable Estimate Standardized f P Estirnate lntercept 0.7550 O 1.57 0.122

Pappus/seed(P) 0.0797 0.52 5.24 ~0.0001

Pappus angle (P) 0.0112 0.28 2.78 0.0077

Barb Score (p) 0.1726 0.24 2.37 0.022 (a) Pappus/seed ratio (b) Pappus angle c.

-. (c) Barb score (d) Pappus number 7 r

Fig. 3-6. Cornparisons of diaspore traits between 15 asexual and nine sexual populations of Antennaria parlinii. Box plots show the distribution of population means. See Fig. 3-4 for details. Analysis of these data is in TABLE 3-4. TABLE 3-4. Analysis of variation in diaspore traits and estimated free-fa11 time in 15 asexual and nine sexual populations of Antennaria parlinii. Population type was a fixed effect and population (nested within population type) was random. F-tests for population type used (0.9~Opulation[PTl+ 0. lMSResidual)as a denominator and those for population used MSResidual. Means are in Fig. 3-6.

Effect (df)

Population type (1) Population (22)

r 2 F (P) F (PI MS~siduai(df)

- - Seed size 0.27 13.8 (0.0011) 3.8 (~0.0001) 0.003 (427)

Pappus length 0.39 21.1 (0.0001) 5.5 (<0.0001) O .25 (404)

Pappus /seed 0.41 23.5 (0.0001) 5.6 (<0.0001) 5.98 (404)

Barb Score 034 20.0 (0.0002) 4.2 (

Pappus nurnber 030 0.07 (0.79) 4.1 (<0.0001) 3.90 (210)

Free-fall tirne 0.51 31.3 (~0.0001) 6.0 (<0.0001) 0.06 (332) 3.0 1 Asexual Sexual Popdation type

Fig. 3.7. Estimates of diaspore free-fall time in still air in 15 asexual and nine sexual populations of Antennaria parlinii. Box plots show the distribution of population means. See Fig. 3-4 for details. Analysis of these data is in TABLE 3-4. Germination differences in nsexual and sexual popdalions The proportion of seeds gerrninating averaged 0.399 I 0.043 in 15 asexual populations and 0.338 t 0.056 in nine sexual populations (Fig. 3-8). A nested ANOVA (whole model: r2 = 0.33, F = 3.05, df = 23,393, P c 0.0001) detected a significant difference in germination among populations with population types (Fig. 3-8a, b; F = 8.66, df = 22,393, P c 0.0001) but not between population types (F = 0.66, df = 1,22, P = 0.42). The rate of germination in both asexual and sexual populations was similar throughout the duration of the experiment (Fig. 3-Sc).

DISCUSSION Habitat differences In addition to having a larger geographical range, 1 found that asexual and sexual populations of A. parlinii, tended to occur in different habitats where their ranges overlap. In Ohio, semials were more likely to occur in treed habitats of later successional stage compared to apomicts. Apomicts were also found in habitats such as roadside ditches and fallow fields that may experience regular disturbances. However, my measures of habitat stability and successional stage were relatively cmde, and a more detailed cornparison of habitat features between sema1 and asexual populations is clearly warranted. Few other studies have examined habitat differences in other species with sexual and asexual individuals, but those that have found that asexual populations occur in ecologically marginal habitats compared to sexual populations. On three Dutch islands, pentaploid asexual Hieracium pilosellu occurred on unçtable sand dunes while tetraploid sexual plants were fond in more stable grassy habitats (Gadella, 1987). However, sexuality is confounded (a) Asexual 0.8 -

2 (b) Sexual Iir 0.8

u -rl (c) All populations -C, 0.8,-

Asexual

LK1- Sexual

Fig. 3-8. Germination of seeds from 15 asexual and nine sexual populations of Antennaria parlinii in a growth chamber environment. In (a) and (b) every line represents an individual population (n = 8337 seeds). In (c)each line represents the mean of population meam and the error bars are f SE. by higher ploidy in H. pilosella making it difficult to directly implicate apornixis as the root cause of this distributional difference. In New Mexican Poa fendleriana, where both asexual and sexual populations occur at the same ploidy level, populations of apomicts were found on more exposed sites than sexual populations (Soreng, 1986).

Reproductive output Greater reproductive output offers two advantages to colonizing species: it increases the chance of some seeds making it to a newly disturbed site (Willson, 1983) and it allows founders to quickly increase their numbers in newly-opened sites (Stebbiw, 1958). My results show that the higher reproductive output in asexual A. parlinii observed by Michaels and Bazzaz (1986) in three Illinois populations is a general feature of the species in Ohio as well. Seed production per inflorescence was two times greater in asexual than in sexual populations. Although there was no difference in average seed set (seeds per floret), the potential for seed production was greater in asexual plants through a greater nurnber of ovules per capitulurn and capitula per inflorescence. Greater reproductive output of apomictic compared to sema1 plants has been reported in at least three genera. In Hieracium pilosella, apomicts produced more capitula, florets and viable seeds per inflorescence than sexual plants (Gadella, 1987). Again the cornparison of sexuals and apomicts in H. pilosella is confounded by differences in ploidy. In species with asexual and sexual individuals at the same ploidy level, asexual Antennaria parvifolia produced significantly more florets and seeds per capitulum than sexual plants (Bierzychudek, 1987b, 1989) and asexual Poa fendleriana had a higher seed set and thus produced more seeds than sexual plants (Soreng, 1986). To date, there are no reports of lower reproductive output of asexual compared to sexual plants. In contrast, animal parthenogens often produce less than half of the number of offspring compared to related sexual species (Lynch, 1984). Since my comparison of reproductive output between sexual and apomictic A. pnrlinii involved parameters measured in natural populations, it is possible that the difference between sex types is due to differences in growing conditions between habitats in which apomictic and sexuals are typically found. However, data from a greenhouse experiment (Chapter 2) suggest that little of the observed difference in reproductive output is due to environmental variance. When grown in a common greenhouse environment plants from apomictic populations still produced more capitula per inflorescence (5.0 f 0.2; n= 91) than sexual plants (3.7 f 0.3; n = 33; t = 3.62, df = 122,l-tailed P = 0.0002). Florets per capitulurn was also greater for asexual (103.7 I 2.72; n = 91) than sexual plants (92.3 + 6.3; n = 17; t = 1.66 df = 106, 1- tailed P = 0.05).

Dis persal abil ity My results suggest a significant association between apomixis, dispersa1 potential and habitat type in Antennaria parlinii. Apomictic plants which tend to occur in more ephemeral habitats had a greater dispersal potential than sexual plants through smaller seeds, longer and more barbed pappus, and a greater pappus-to-seed ratio. The association between ephemeral habitats and dispersa1 ability is also suggested by differences in dispersal potential between populations of sexual plants. The only sexual population whidi occurred in an open "disturbed" habitat (CR) was the sexual population with the largest pappus-to-seed ratio and dispersal potential (outlier in Fig. 3-7). These results support the idea that dispersa1 traits have become associated with apomixis and higher reproductive effort through selection for increased colonizing ability. However, it is worth noting that dispersa1 potential was measured under lab conditions. Consequently, it is impossible to estimate the actual increase in dispersa1 distance afforded by a 10% increase in free-fall time in still air (see Rabinowitz and Rapp, 1981). Aside from selection for colonizing ability in ephemeral habitats, there are at least two alternative explanations for the association between apomixis and seed dispersai potential. Asexual offspring share their parent's genotype and, as a result, may succumb more readily to pathogens that infect the parent (Levi., 1975). This would favour increased dispersa1 potential in asexual over sexual populations (Janzen,1970; Connell, 1971; Howe and Smallwood, 1982). Similarly, selection may favour increased dispersal in asexual populations so îhat asexual offspring can avoid competition with their genetically identical parents and siblings (Young, 1981). Both these alternative hypothesis could be tested in field experiments (e.g., Kelley, 1989a, b). Some of the increased dispersal potential of diaspores produced by apomicts was due to reduced seed size which resulted in a larger pappus-toseed ratio. There are two possible explanations for a difference in seed size in different successional habitats: selection for smaller seeds to increase dispersa1 potential (Fenner, 1987) and/or selection for larger seeds in later successional stages to tolerate shaded habitats (Salisbury 1942; Westoby et al., 1996). Salisbury (1942) found that in the British flora herbaceous species from open, early successional habitats had smaller seeds than herbaceous species of late successional woodland habitats. Significant differences in seed size have also been found within species. In Daucus carota, seed mass in a late- successional habitat was significantly greater than in early successional sites. However, in Claytonin virginica, seed mass was greater in early successional sites (Michaels et al., 1988). Differences in seed free-fall time between habitats within the same speues suggest that smaller seed size is selected to increase dispersa1 ability. There are few studies that compare dispe~dpotential of diaspores from different habitats within a species (Werner & Platt, 1976; Peroni, 1994, Cody and Overton, 1996). Within each of five species of Solidago, seeds from old field habitat had a significantly longer free-fall time than those from later successional praine habitat (Wemer & Platt, 1976). Peroni (1994) also found that diaspores of Acer rubrum from younger habitats had a greater dispersai potential than diaspores from later successional habitats.

Differentiation in seed size may also result from selection for increased cornpetitive ability in semal seeds in more stable and possibly more biotically complex habitats. Although seeds f-rom apomictic A. parlinii are smaller than seeds from sexual plants, germination tests in a common growth chamber environment did not detect any differences in viability between the two types of seeds. However, larger seeds seemed to produced larger seedlings (pers. obs.), which could be an adaptation to survive in a shaded habitat. Michaels and Bazzaz (1986) found that, in A. parlinii, transplanted seedlings from sexual plants, which produce larger seeds, had higher survivorship than apornictic seedlings in a wooded habitat. Like reproductive output, seed size and pappus traits may be influenced by environmental variation arising from differences in habitat types between sema1 and apomictic populations. Matemal effects on seed size and dispersal traits may overshadow differences due to selection for dispersal ability (Silvertown, 1989). For example, seeds of Prunella vulgaris collected from woodland sites were larger than seeds from old field sites (Wh, 1985). When plants collected from these sites were reciprocally transplanted, plants grown in woodland sites still produced si@cantly larger seeds than plants grown in old fields regardless of the origin of the plants. These results indicate that to detect evidence of genetic differences in greater dispersal ability through the production of smaller seeds, seed size would have to be obtained from common garden experiments. However, because pappus and seed size differed in opposite directions between sexual and asexual populations the observed differences in diaspore traits is probably not just a consequence of environmental variation in overall diaspore size. Furthemore, asexual seeds also had more barbs, a trait that increased free fall the, while the only trait that did not affect seed free fall time, the number of pappus brides, did not differ between sexual and asexual populations. Taken together, the results indicate genetic differentiation for diaspore traits between sexual and asexual populations A. parlinii.

Differences in cornpetifive ability My results support the hypothesis that apomictic plants have adopted a fugitive life-history and have traits which allow thern to maintain themselves in ephemeral habitats. A greater colonizing ability of apomictic A. parlinii may explain their greater geographical range and occurrence in disturbed habitats. However, it does not explain why apomictic plants in Ohio are excluded from woodland habitats. One possibility is that asexual plants in Ohio are excluded from more stable habitats because apomicts are unable to compete against sexual plants. In a senes of experiments in the field and greenhouse Michaels and Bazzaz (1986,1989) found that sexual plants from three Illinois populations allocated more to total biomass, while asexual plants allocated more to reproductive biomass and had a higher stolon death rate. When grown together, sexuals also tended to outcompete apomicts (Michaels, 1986). However, in these experiments, sexual plants were tetraploid while asexual plants were hexaploid (H. Michaels, pers. corn.) and thus these results may not be representative of the species in general since sexuals and asexuals are mainly hexaploids in most parts of the range, including Ohio (Bayer, 1980,1984; Bayer and Stebbins, 1981). I made casual observations of six populations in Ontario and Québec, where only asexual populations occur, and all seemed to occupy habitats where sexual populations would typically be found in Ohio. The presence of apomich in more stable woodland habitats in Ontario and Québec, where sexuals are absent, is consistent with the hypothesis that they are competitively excluded from these habitats in Ohio. Apomictic plants may be excluded from stable and complex habitats because they are incapable of dealing with biotic agents (Levin, 1975; Glesener and Tilman, 1978). 1 found that seed predation was more common in asexual populations of A. pnrlinii than in sexual populations. However, this difference in predation is complicated by differences in habitat, since it is impossible to tell whether higher predation in asexual populations is because they are more susceptible to predation or that theçe occur in habitats where seed predators are more common. Because both apomictic and sexual populations occur close-by in Ohio, reciprocal transplant experiments could be use to determine whether asexual plants are more susceptible than sexual plants to biotic pressures such as predators and pathogens in stable woodland habitats. CHAPTER 4: General Discussion and Conclusions

Main findings Populations of Antennaria parlinii in Ohio are strongly differentiated in sexuality and there is little variation in sexuality within populations. Female populations appear to be wholly apomictic while mixed-sex populations are predominantly sexual, and partial apomixis is not common. Although the view that variation in sexuality should be considered a continuum from cornplete sexuality to cornplete apomixis is gaining popularity among botanists (Nogler, 1984; Bayer et al., 1990), my results agree with theoretical models which predict that populations should be predominantly sexual or asexual (Charlesworth, 1980; Marshall and Brown, 1981). In Ohio, apornictic populations tended to occur in disturbed habitats that are probably ephemeral, while sexual plants occurred in woodland habitats that would appear to be more stable. Apornicts had a greater reproductive output than sexuals, setting twice as many seeds per inflorescence, and producing diaspores with traits which shouid increase dispersa1 potential. This combination of apomixis, high reproductive output and high seed dispersa1 potential may explain why apomicts are much more common in ephemeral habitats than sexual plants.

Partial apomixis Although partial apomkis does not commonly occur in A. parlinii, low ievels of sexuality may still be important in introducing genetic variation in predominantly asexual lineages (Lynch and Gabriel, 1983). To test for partial apomixis, pollination experiments using genetic markers should be used to estimate the relative amount of asexual and sexual seed production within individual plants. Molecular genetic tediniques such as RAPD or microsatellite markers would have to be used since hexaploidy in A. parlinii makes segregation at allozyme loci diffidt to extensive detect due to gene duplication (R. Bayer, pers. corn.).

Differences in reproductive oufput and dispersal potential Many workers have pointed out that uniparental reproduction may explain the wide ecological and geographical distribution of asexual species (Stebbinç, 1950; Lynch, 1984; Michaels and Bazzaz, 1986; Bierzychudek, 1987a). However, few studies have specifically compared other facets of colonizing ability between asexual and sema1 taxa. The few studies to date have found greater reproductive output in apomictic plants compared to sexual plants (Michaels and Bazzaz, 1986; Soreng, 1986; Gadella, 1987; Bierzychudek, 1989). Midiaels and Bazzaz (1989) also found that asexual A. parlinii allocated more to reproductive biomass than sexual plants in all environments except for low light habitats. Diaspore characters that increase seed dispersal distance may also contribute to colonizing ability and thus a wider geographical and ecological distribution. Michaels and Bazzaz (1986) found that apomictic Antennaria parZinii seeds were smaller suggesting a greater dispersal potential. My results support their hypothesis by showing that apomicts possess other diaspore traits that might enhance dispersal. Because many species of apomicts occur in the Asteraceae and have special dispersal structures, cornparison of dispersa1 potential between sexuals and apomicts could easily be done in other taxa. In Antennaria, A. parvijblia and A. media also include both asexual and sexual individuals, and the correlation between dispersal potential and reproductive mode could be tested in these taxa. Habitat differentiation In Ohio, I found that asexual popdations tended to occur in more dishirbed habitats than sexual populations but my characterization of habitat differences was crude and more detailed comparisom should be conducted to confirm rny results and maybe identify the specific habitat axes that differ between asexual and sexual populations. I also observed that apomictic populations of A. parlinii in Ontario and Québec did not occur in the same habitats as in Ohio. In Ontario, where there are no sexual populations, apomicts occurred in woodland habitats which suggests that they are competitively excluded from these habitats in Ohio by sexual individuals. The absence of apomicts from woodland habitats in Ohio may be because they are not as good as sexuals in dealing with biotic challenges such as pests and pathogens that characterize more complex habitats (Levin, 1975; Glesener and Tilman, 1978). Antennariu parlinii is an ideal system for examining differences in competitive interactions between sexual and apomictic plants because both

reproductive types cmbe derived from seeds and have the same ploidy level. In Ohio, apomictic and sexual populations of Antennaria parlinii occur within a few km of each other but in different habitats. Reciprocal transplants

could be used to assess the cost of asexual reproduction in the potentially more biotically complex woodland habitats. Similarly, sexual plants could be transplanted in habitats where apomicts suffer high seed predation rates (Chapter 3) to test if they are more resktant to pests. Michaels and Bazzaz conducted a series of experirnents comparing the competitive ability of apomictic and sexual plants from three Illinois populations of A. parlinii (Michaels, 1986; Michaels and Bazzaz, 1986, 1989). When transplanted into wooded habitats, seedlings from asexual plants had a higher mortality than those from sexual plants (Michaels and Bazzaz, 1986). In the greenhouse, sexual plants seemed to be more cornpetitive, had a higher growth rate, greater allocation to biomass, lower mortality and performed better in low light conditions than apomicts (Michaels, 1986; Michaels and Bazzaz, 1986, 1989). However, apomictics in their study were hexaploids while sexual plants were tetraploids (H. Michaels, pers. corn.). Because ploidy may significantly affect growth and life history (Levin, 1983), and since both sexual and apomictic A. parlinii are predominantiy hexaploids throughout most of its range (Bayer, 1980,1984; Bayer and Stebbins, 1981), we do not know to what extent the differences in performance between semals and apomicts observed in their study are representative of the species as a whole.

Evolutionary origin of associations between sexuality, reproductive output, seed dispersal and habitat distribution In Ohio populations of A. parlinii, four different characters are linked: sexuality, reproductive output, seed dispersal and habitat type. The simplest evolutionary mode1 for habitat and life-history differentiation between sexualç and apomicts is that hybridization produced a gene pool that varied in terms of semality, reproductive output, and seed dispersa1 (see Stebbins, 1941). Then, disruptive selection on the three traits in different habitats (i.e., disturbed vs. stable) resulted in the observed trait correlations. This differentiation may have occurred at specific periods in the evolutionary history of A. parlinii, such as during post-glacial colonization (see Bayer and Stebbins, 1983). The phylogenetic origin of the various lineages that make up A. parlinii may also have contributed to the differences in ecology and life- history between sexual and apomictic populations. Antennaria parlinii 75

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APPENDIX 1: Location and habitat characteristics of Antennaria parlinii populations involved in natural seed set, field pollinator exclusion and greenhouse pollination experiments.

AL: Ohio, Delaware Co.; Berlin twp.; south of route 36, parking area at northeast end of Alum Creek Lake; steep diff over lake, wooded area.

CC: Ohio, Lorain Co., Brownhelm twp.; Vermillion rd., 200 m north of Middle Ridge road, east slope of Chance Creek; wooded area.

CM: Ohio, Paulding Co., Carryall twp.; south side of route 192, east of road 49, 1.4 km east of road 45 (Trernbley), north of the Maumee River, Slough cemetary; in lawn.

CR: Ohio, Hocking Co., Benton twp.; Hocking State Forest, junction of route 374 and Chape1 Ridge road; in grass near parking lot.

GN: Ohio, Guemsey Co., Londonderry W.;Route 22,2.4 km South of Smyrna, East side of road; steep, wooded, mossy bank.

HS: Ohio, Hocking Co., Benton twp.; Hocking State Forest, route 56, 100 m east of Ash Cave parking lot; north side of road, shailow ditch along wooded area.

IN: Ohio, Lake Co., Leroy twp.; Indian Point, Lake Co rnetroparks, end of hicking trail; edge of south facing cliff above river.

JL:Ohio, Ashtabula Co., Harpersfield twp.; 4 km east of Lake/Ashtabula Co line. John Linehan property, 5834 route 307; north side of Grand River; steep wooded slopes/river slumps.

SR: Ohio, Fairfield Co., Madison twp.; Snortin Ridge road, east side of road, 2.2 km south of Clear Creek Rd.; wooded, steep roadside slope.

Female populations

AF: Ohio, Delaware Co., Berlin twp., Africa Road, South of Cheshire road., both sides of road; in grassy, shallow ditch.

BR: Ohio, Lorain Co., Carlisle twp.; 4174û-41741 Buttemut Ridge road; north side of road, grassy slope. FG: Québec, Gatineau Co., Nez Chelsey, Across the road from Les Fougères restaurant, on steep, wooded roadside bank.

FR: Ohio, Lake Co., Madison twp.; Ford Road, west of Loveland road; shallow ditch in lawn-

GC: Ohio, Hamson Co., German twp.; near Germano cemetary; south-west facing slope, east side of road, steep grassy slope.

GR: Ohio, Huron Co., Greenwich twp.; Route 13, north of 224, east side of road, 200 rn north of route 224, south of railroad tracks; in grassy, shallow ditch at edge of a field.

HU: Ohio, Fairfield Co-, Madison Twp.; 6289 Revenge road., north of Beck's road, east and west side of road, next to "Hugh's Cycle", steep grassy bank.

IF: Ohio, Lucas Co., Spencer twp.; Irwin Prairie Nature Preserve, south of Bancroft street-, West of hi.road, 200 rn from parking on east and west sides of boardwalk. Remains of flood plain prairies.

KL: Ontario, Frontenac Co., Kellerville Twp., Route 11,600 m south of Simpson Rd.; rocky roadside on edge of forest.

KT: Ohio, Lucas Co., Spencer twp.; Kitty Todd Nature Conservancy Presewe, north of Old State Line road, east of Shwamberger Road; road side, sandy waste area.

00: Ohio, Lucas Co., Swanton twp.; Oak Openings, Toledo metro parks, south side of road along trees in sandy soi1 on Bat surface.

PF: Ohio, Lorain Co., Pittsfield twp,; Pittsfield, on route 303,500 m West of Route 58; south side of road in lawn.

QU: Ontario, Leeds & Grenville Co., Queen's University Biology Station, near Lake Openicon, grassy area near trees, on lawn, and on wooded ridge.

SC: Ohio, Auglaize Co., Noble twp.; east side of Route 66, south of Six Mile Creek; West facing shallow slope in ditch, along trees.

SM: Ohio, Mercer Co., Center twp.; north of St-Mary's; Route 33,500 m west of Rice road; shallow ditch along field. APPENDIX II: Summary of mean seed set in 9 mixed-sex populations of Arifemw'upnrliriii under different treatments in field and greenhouse experiments.

FIELD GREENHOUSE Open-pollina ted Bagged Unpollinated Pollina ted

Population Sex ratio N 11 rrieanf SD rt meani SD ri meankSD ri meanf SD

APPENDIX IV: Sumrnary of mean measures of reproductive output in nine ded-sex and 15 female populations of Antemarin parlinii : C/I = Capitula per inflorescence, F/C = Florets per capitulum, S/C = Seeds per capitulum, and S/I = Seeds per inflorescence.

Mixed-sex populations

Female populations AF 19 6.1 (1.6) BR 10 5.9 (1.0) FG 16 6.3 (1.8) FR 15 5.6 (2.1) GC 14 8.1 (1.5) GR 18 8.8 (2.5) HU 21 7.2 (1.8) IP 26 5.0 (1.4) KL 16 6.1 (3.3) KT 28 7.7 (2.8) 00 20 6.3 (2.0) PF 19 7.7 (1.3) Qu 18 5.7 (2.2) SC 20 7.6 (2.2) SM 18 9.4(3.0) APPENDIX V: Summary of mean measures of diaspore characters in nine mixed-sex and 15 fernale populations of Antennaria parlinii: SS = seed çize, PL = pappus length, P/S = pappu-to-seed ratio, P# = number of pappus bristies, BS = barb score, %G = percent germination.

POP n SS (SD) PL (SD) P/S (SD) P# (SD) BS (SD) %G (SD) Mixed-sex populations

Female populations