The Effects of Pollinators and Sex Ratios on the Evolution of Life-History Traits in Aralia Nudicaulis a Thesis Submitted To

The effects of pollinators and sex ratios on the evolution of life-history traits

in Aralia nudicaulis

A Thesis Submitted to the Committee on Graduate Studies in Partial

Fulfillment of the Requirements for the Degree of Master of Science in the

Faculty of Arts and Science

TRENT UNIVERSITY

Peterborough, Ontario, Canada

Environmental and Life Sciences M.Sc. Graduate Program

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1+1 Canada The effects of pollinators and sex ratios on the evolution of life-history traits

in Aralia nudicaulis

Emony Nicholls

ABSTRACT

Aralia nudicaulis is a clonal dioecious herb common to forested ecosystems in eastern

North America. In this study, I had two main objectives: (1) to understand the factors responsible for variation in female frequencies, and (2) to investigate interactions between A. nudicaulis and its pollinators. I investigated variation in female frequencies at

15 sites in Algonquin Park, Ontario. At each site I recorded the age and sex of each flowering ramet. 1 found striking variation in the frequency of females vs. males across the study sites, with the percentage of females ranging from 5 to 95%. Because fruit production involves greater resource costs than pollen production, I predicted that females should be less common where resources such as light are limiting. However, the data were not fully consistent with this expectation and I did not find a significant association between canopy closure and female frequencies. Because of the higher cost of reproduction in females, I further predicted females would have lower rates of survival, and therefore that the oldest plants within populations would be male. Contrary to this expectation, I found that the age distribution was skewed towards older ages for females than for males. Because males provide more rewards for pollinators than females, I predicted that variation in the ratio of females:males across sites would be associated with variation in pollinator abundance. However, in complete contrast to this expectation,

ii I found that pollinators were more abundant in sites with more females, not more males.

Finally, I conducted an experiment to explore the function of infertile staminodes in the pollination of female A nudicaulis, with the expectation that staminodes, which are superficially similar to the pollen-producing stamens produced by males, would be important in pollinator attraction. As expected, pollen receipt by females that had their staminodes removed was substantially lower than for plants with intact staminodes.

Keywords: life history, sex ratio, trade-off, clonal, sexual, reproduction, dioecy, staminode, age, survival, pollinators.

in ACKNOWLEDGMENTS

Most of all, deepest gratitude to Marcel Dorken for his incredible patience, humour and guidance through the whole academic process, I could not have done it without him.

Thank you to my co-conspirators in the field, Eleanor Proctor and Rhiannon Leshyk. And extra thanks to Eleanor for teaching me to clean and identify bees and syrphids. And

Polly for her hard work on the logging roads!

Further thanks to Jordan Ahee for his field work in the staminode experiment, it was invaluable. Thanks to my advisory committee, William Crins and Erica Nol for their help and guidance. Thank you to Cory Sheffield for identifying my bees to the species level and William Crins for identifying my Syrphid flies to the species level.

To NSERC and CANPOLIN for providing funding for this project and hosting of the pollinator identification course.

And finally, thanks to my parents William and Rohaise Nicholls and my brothers

Rohan and Ashley for their encouragement and unfailing support. And to my friends who have cheered me on and supported me through my academic journey, as with everything else.

IV TABLE OF CONTENTS

Abstract ii

Acknowledgements iv

List of figures vii

List of tables viii

CHAPTER 1: General Introduction 1

Clonal and sexual reproduction 2

Pollinators 6

Staminodes 7

Life history of Aralia nudicaulis 8

Objectives 14

CHAPTER 2: Sex ratio variation, pollinators and staminode function in Aralia nudicaulis

Introduction 16

Map of sites 20

MATERIALS AND METHODS 21

Aralia nudicaulis ramet ages and sex ratios 21

Canopy cover 21

Staminode experiment 22

Pollinator sampling 23

Statistical analyses 24

RESULTS 26

Sex ratios and canopy closure 26

v Sex differences in ramet age 26

Pollinator abundance 26

Staminode experiment 27

FIGURES 28

DISCUSSION 33

CHAPTER 3: General Discussion 44

LITERATURE CITED 48

APPENDICES 56

VI List of Figures

Figure 1-1. Schematic of resource distribution in plants. With a limited resource pool, distribution is divided between the three general life-history components seen on the right of the figure. These components are further broken down in the general resource distribution

Figure 1-2. Schematic of resource distribution in male and female plants. Primary sexual characteristics refer to pollen or ovules and secondary characteristics refer to floral displays, scent or nectar. Thicker lines indicate greater resources being invested into that life history trait 6

Figure 1-3. Flowering Aralia nudicaulis ramet 9

Figure 1 -4. Female flower of Aralia nudicaulis, note the staminodes 10

Figure 1-5. Male flower of Aralia nudicaulis 10

Figure 1-6. The most abundant pollinator observed in this study, Toxomerus geminatus on blackberry (Rubus spp.) 11

Figure 2-1. Map with all 15 sites of study in Algonquin Park, Ontario 20

Figure 2-2. Sex ratios of flowering ramets of Aralia nudicaulis across 15 sites in Algonquin Provincial Park during 2009/2010 (indicated as the proportion of total ramets per site). The total number of flowering ramets per site is shown at the top of the bars 28

Figure 2-3. Relationship between the proportion of female ramets of Aralia nudicaulis and canopy closure at 15 sites in Algonquin Provincial Park 29

Figure 2-4. Distribution of ramet ages for female and male shoots of Aralia nudicaulis sampled from 15 sites in Algonquin Provincial Park 30

Figure 2-5. The association between the proportion of female ramets of Aralia nudicaulis and pollinator abundance across nine sites in Algonquin Provincial Park. Abundance data were scaled by dividing the total sample of pollinators by the number of hours of sampling conducted per site. The site indicated with a triangle was identified as an outlier and removed from the analysis 31

Figure 2-6. The effect of staminode removal on pollen loads of female flowers of Aralia nudicaulis 32

vn List of Tables

Appendix 1. Table 1. Summary of location and sex ratio data for 15 populations of Aralia nudicaulis 56

Appendix 1. Table 2. Summary of life-history data for 15 populations of Aralia nudicaulis 57

Appendix 1. Table 3 Pollinator data for 15 populations of Aralia nudicaulis 58

Appendix 2. Table 1. Frequency of males and females among 15 sites in Algonquin Park, Ontario during 2009-2010. G values are given for tests of independence of sex frequencies from the two sexes for each plot. Gy, refers to tests of heterogeneity between sites 59

Appendix 3. Table 1. Summary of pollinator data per site collected in pan traps from nine populations of Aralia nudicaulis 60

Appendix 4. Table 1. Vegetation plots indicating an approximation of percent coverage. Site abbreviations: BAT=Bat Lake Trail, BL=Bud Lake, BP=Big Pines, BREW=Brewer Lake, CHIT=Chit Lake Trail, FLO=Florence, HB=Hemlock Bluff Trail, LOUFL=Louisa Flats, LR=Lost Ray Trail, MAD=Madawaska, MIZZY=Mizzy Lake Trail, PROV=Provoking Portage, STNRD=Station Road, 2RIVINT=Two Rivers Lake Interior, 2RIV=Two Rivers Lake Trail 65

via CHAPTER 1: General Introduction

Perennial plants rely on a finite pool of resources that can be allocated to the various components of their life history (Figure 1-1; Figure 1-2). Distribution of these resources requires trade-offs between the three principle areas of the plant's life-history components: reproduction, survival, and growth (Roff 1992). The trade-offs made within a plant have implications for every aspect of these life-history traits, in turn affecting plant fitness. For example, trade-offs between allocation to survival and reproduction determine whether a plant has a perennial versus annual life history (Salisbury 1942,

Gustafsson 1947). Because the annual-perennial life history axis, at least in part determines the ability of plants to respond to key ecological processes (e.g., disturbance, competition, and stress), such tradeoffs have far reaching implications for the ecology and evolution of plants.

The key components of a plant's life history (reproduction, survival and growth) can be further subdivided into components that are themselves subject to trade-offs (Figure 1-1;

Geber et al. 1999). For example, most perennial plants have two modes of reproduction, sexual and clonal (asexual), that, because they occur more or less simultaneously within plants, draw from the same pool of resources (Figure 1-2; Abrahamson 1980, Thompson and Eckert 2004). Perennials are usually capable of either form of reproduction, with the failure of one mode, the other allows for the plant's persistence (Vallejo-Marin, Dorken and Barrett 2010).

Tradeoffs can also occur among the functions involved in sexual reproduction. For

1 example, most plants are hermaphroditic, and must allocate resources to both female and male gamete production. Even for species with separate sexes (i.e. dioecious plants), tradeoffs play an important role (Figure 1-2) because males and females generally distribute their resources in different ways among the components of life-history (Figure

1-2; Lloyd and Webb 1977). In particular, it is thought that the two sexes have strongly differing costs of reproduction due to their sexual gamete costs, with downstream effects on other components of the life history. For example, males are thought to have lower investment in reproduction because pollen (which contain the male gametes) is cheaper to produce than ovules (which contain the female gametes; Obeso 2002). As a result of males' lower reproductive investment, it is thought that they have more resources to invest in other life-history traits such as growth and survival.

In clonal plants, lower investment in sexual reproduction should therefore free up resources for allocation to clonal expansion. Indeed, male-biased sex ratios (at the shoot level) in clonal plants is commonly observed (Obeso 2002). Moreover, males also tend to have a higher prevalence of flowering, further skewing sex ratios in their favour (Geber et al. 1999). Because the importance of tradeoffs are thought to increase under resource limitation (Stearns 1992), these differences may be further exacerbated in resource limited conditions.

Clonal and Sexual Reproduction

'Reproduction' referred to in figure 1-1 is sexual reproduction and all its associated traits.

The associated traits include both primary and secondary characteristics. Primary sexual

2 characteristics are those most obviously related to reproduction such as sperm and egg, exhibited in plants as pollen and ovules (Darwin 1871, Geber et al. 1999). Secondary characteristics refer to the morphological differences, often sexually dimorphic characteristics including attractant floral traits like the size of petals and sepals, overall flower size and the total number of flowers per inflorescence (Lloyd and Webb 1977,

Geber et al. 1999).

Survival, the second life-history trait depicted in figure 1-1, involves allocation to traits involved in disease and pest resistance, maintenance, and hardiness. Plant age is often used as a proxy measure of survival (Lloyd and Webb 1977, Lovett-Doust and Lovett-

Doust 1988). Growth, by contrast., involves allocation to photosynthetic structures such as leaves, and also to support structures, such as stems and roots. As a result, asexual reproduction is included as a component of growth. Growth of clonal propagules and the spread and branching of rhizomes below ground with ramets or flowering inflorescences above ground are all a part of asexual reproduction.

Trade-offs for resources are strongest between functions that compete for them at the same time (Thompson and Eckert 2004). Competition for resources is commonly seen between the two forms of reproduction. Clonal and sexual reproduction can compete antagonistically for resources, with an increase in sexual reproduction negatively affecting the rate of clonal reproduction (Vallejo-Marin et al. 2010). The competition between the reproductive methods and subsequent trade-offs may constrain evolution by influencing the evolutionary response of populations to environmental unpredictability

3 (Thompson and Eckert 2004). The dual methods of reproduction found in clonal plants confer on them the advantage of choice whenever circumstances and resource base demand flexibility. In conditions not conducive to sexual reproduction, propagation can continue unabated via clonal means. Not surprisingly, clonal plants are commonly found in shaded, nutrient-poor, cold and wet environments (Fischer and van Kleunen 2002).

Clonal structure influences the spatial distribution of flowers and can have an impact on population size, density and sex ratios (Goulson 1999). By influencing patterns of pollen dispersal, clonal structure affects individual mating success (Vallejo-Marin et al. 2010).

Pollinators appear to be influenced by both distances between ramets and plant densities

(Araki 2007, Kunin 1993). Larger distances between flowers are less attractive to pollinators and can lead to pollen limitation in females and low rates of sexual reproduction (Beattie 1976, Kunin 1993). Smaller distances between flowers promote pollinator movements and an increase in fruit set (Beattie 1976, Kunin 1993). Low density populations of plants can experience low pollination success (Kunin 1993, Aizen

1997). For example, in the bumblebee-pollinated gynodioecious herb Glechoma hederacea, fruit set in female clones was found to decrease with increasing distance from the hermphroditic pollen source (Widen and Widen 1990; in Charpentier 2002).

4 Plant Resource Distribution Reproduction

Resource *> Survival Poo! Growth

T Floral Display General Resource Distribution (pollinators) fc Ovules 4 Pollen Resource -*• Survival (age) Poo! ^.Clonal Expansion (rhizomes, ramets) I Photosynthesis Roots (leaf size, height)

5 Male Resource Distribution Pollen

1 Resource ' Survival Pool Photosynthesis Clonal Growth

Female Resource Distribution Fruits, Seeds

Resource* *• Survival Pool Photosynthesis

Clonal Growth

Pollinators

Pollinators act as vectors for the fertilization of the ovules of animal-pollinated plants; a primary role of flowers is to attract them. In particular, flowers appear to act primarily to promote male sexual function in hermaphroditic species (Bateman 1948, Bell 1985).

Indeed, males generally invest more in secondary sexual characteristics than females

(Lloyd and Webb 1977), including pollinator rewards and attractants, and as a result,

6 typically receive more visits from pollinators (Delph et al. 1996). Female fitness is saturated with fewer pollinator visits than males; only a relatively small amount of pollen deposition is required to achieve maximum seed set, whereas male fitness continues to increase with each pollen grain that is disseminated. Also, female flowers often provide fewer rewards for pollinators and have reduced investment in pollinator attraction than males (Bell 1985, Stanton and Galloway 1990, Wilson et al. 1994). Pollinators can often perceive these differences between males and females, preferentially visiting the flowers on male plants (Stang et al. 2009, Beach 1981).

Staminodes

As already noted, females do not generally allocate the same amount of resources to showy floral displays as males but must nevertheless attract pollinators. As a result, there should generally be selection on females to maintain traits normally associated with male function that serve to promote pollinator visitation. In particular, pollinators might exert selection leading to the maintenance of nonfunctional stamens and increase of petal length in females (Ashman 2000). These infertile stamens, or staminodes, might serve to mimic stamens on males, and thereby trick pollinators into visiting the flowers. Although not directly comparable, females of monoecious Begonia involucrata have stigmas that strongly resemble the male anthers, a classic example of deceit when the only reward available to insect visitors is pollen from the male flowers despite both sexes emitting the same sweet scent (Agren and Schemske 1991). Although the production of staminodes presumably entails some resource costs, these are likely to be trivial in comparison with the production of fertile stamens by males, and more than compensated for by enhanced

7 fitness through increased pollinator visitation.

Wild sarsaparilla (Aralia nudicaulis)

Wild sarsaparilla (Aralia nudicaulis) is a long-lived, clonal, dioecious plant found throughout the temperate and boreal woodlands of eastern Canada and the United States.

A. nudicaulis is a clonal rhizomatous herb with slender, branching, underground horizontal axes with shoots surfacing at regular intervals (Bawa et al. 1982). Some vegetative ramets emerge with a flowering stem, which typically consists of three inflorescences with a globose arrangement of greenish-white flowers. Because Aralia is an understory herbaceous plant that flowers and matures fruit after the seasonal development of the leaf canopy, light is thought to be an important limiting resource

(Whitman et al. 1998).

Aralia nudicaulis is an acaulescent perennial. Its leaves and peduncle emerge from a very short stem, both sheathed at the base by dry thin scales, the whole structure arising from a long rhizome (Britton and Brown 1970, Gleason and Cronquist 1963). The shoot or ramet may function independently of its parent or genet via the formation of fibrous secondary roots. Genets can form a circular clone of 10 m or more in diameter due to the branching nature of the rhizomes (Bawa et al. 1982). Foliage leaf scars are left on the erect shoots after each growing season, making aging of the ramets accurate (Bawa et al. 1982). The leaves include an erect petiole with ternate leaves, each divided pinnately into 3-5 foliolate leaflets. Each leaflet is lance-elliptic, ovate to obovate, and finely serrate. There are usually three umbels on peduncles that are much shorter than the petioles. Flowers are

8 small (0.3 cm.), greenish or white with a ring of five petals, five stamens or staminodes and five styles, with insignificant sepals. The styles are very short and appressed in the centre. When in bloom, the petals become strongly recurved. Petals and stamens/staminodes will fall off a couple of days after stigma receptivity in females but will last more than a week in males (Barrett and Helenurm 1981). Males have more flowers per inflorescence than females (Barrett and Helenurm 1981, Bawa et al. 1982).

(Barrett and Helenurm (1981) found male to female ratios of 125.3 : 75.2 flowers per inflorescence, similar to the 119 : 84 found by Bawa et al. (1982). Fruits are purplish- black, globose and five-lobed with five seeds.

Figure 1-3. Flowering Aralia nudicaulis ramet. (Photo credit Marcel Dorken)

9 Figure 1-4. Female flower of Aralia nudicaulis, note the staminodes. (Photo credit

Marcel Dorken)

Figure 1-5. Male flower of Aralia nudicaulis. (Photo credit Marcel Dorken)

10 Figure 1-6. The most abundant pollinator observed in this study, Toxomerus geminatus on blackberry (Rubus spp.). (Photo credit Emony Nicholls)

Evolutionary relationships

Aralia nudicaulis is in the order Apiales, which is divided into two families: Apiaceae and Araliaceae. There is some phylogenetic uncertainty at almost every taxonomic level between the families due to a lack of clarity in the evolutionary trends in morphological characteristics (Plunkett et al. 1996). Currently however, Araliaceae includes 700 species and 70 genera (Plunkett et al. 1996). The family is distinguished by pinnately or palmately compound leaves and flowering heads in large panicles of small flowers.

11 Pollinators

The primary pollinators of Aralia nudicaulis are bees (Hymenoptera:Apoidea) and flower flies (Diptera: Syrphidae; Nicholls, unpublished data). No examination of the potential link between pollinators and sex ratios had been done in any species before. Females begin to flower slightly earlier in the season than males, and they have a greater overall biomass (total dry mass of reproductive ramets; Barrett and Helenurm 1981). Earlier in the flowering season (June), it has been found that there was a significantly higher reproductive effort and absolute biomass of reproductive structures in males than females, likely due to the larger number of flowers on male inflorescences (Barrett and

Helenurm 1981). But within a two-week period, the opposite was found to be true; females' reproductive expenditure increased during June and July as the growth and maturation of fruits occurred, incurring a reproductive cost not experienced by males

(Barrett and Helenurm 1981).

Life history

As has already been mentioned, light is considered to be one of the primary limiting resources for A. nudicaulis, not nitrogen or moisture levels (Whitman et al. 1998). Only one study (Barrett and Helenurm 1981) has examined a link between light availability and its influence on growth and sex ratios. Sex ratios were found to differ between populations growing at roadsides vs. under forest canopies. Forest populations had markedly male skewed sex biases, in contrast to roadsides where the populations were almost equally balanced between the sexes (Barrett and Helenurm 1981). However, there

12 might have been important differences between forest and roadside sites other than light availability (e.g., soil nutrients, soil composition, moisture levels, competition, tree species composition) (Whitman et al. 1998) that were not controlled for in that study. As a result, my approach was to evaluate the association between light availability and sex ratios across a continuous distribution of both variables (rather than a comparison of two discrete population types). When fewer resources are available for growth, populations of

A. nudicaulis are comprised primarily of older ramets (Whitman et al. 1998). Tappeiner et al. (1991) found dense canopy reduced recruitment of understory clonal species.

However, as one moves towards sites with more light and moisture, A. nudicaulis ramet recruitment increases and the population is skewed towards younger growth (Whitman et al. 1998).

Other factors might also contribute to male-biased sex ratios in populations of A. nudicaulis at the ramet level. Opler and Bawa (1978) have suggested higher mortality by female ramets could also contribute to the male bias. Moreover, the shorter juvenile period for male ramets and higher frequencies of flowering over time can also explain male-biased populations (Bawa et al. 1982). These observations imply a higher cost to reproduction by females, and there is evidence for higher resource requirements for seed production and fruit maturation by females - costs not borne by males (Bell 1985, Lloyd and Webb 1977).

A.nudicaulis is an ideal study plant for examining the objectives listed above. First,

Aralia is a clonal dioecious plant with wide variation in its sex ratio across populations

13 (male to female, 0.76-1.10; Barrett and Helenurm 1981). Previous research has suggested that this sex ratio variation should be linked to resource availability but the prediction has not yet been explicitly tested (Willson 1979). Secondly, Aralia is known to attract a range of generalist pollinators (Barrett and Thomson 1982), but a survey of them has never been done. Thirdly, females of A. nudicaulis continue to maintain non-fertile staminodes, allowing for experimental research on their function. Previous research has established that the staminodes of female A. nudicaulis are sterile but an examination of their function has not been done (Barrett and Helenurm 1981).

Objectives

The objective of this thesis was to explore the causes and consequences of sex ratio variation and its effect on pollinator abundance in clonal dioecious plants, using wild sarsaparilla, Aralia nudicaulis, as a study system. Additionally, the function of staminodes as pollinator attractants through mimicry of male stamens was examined.

Objective 1: Sex ratios

In the first part of chapter 2,1 report on an evaluation of male and female frequencies across fifteen populations of A. nudicaulis. In addition, I aged all of the individual flowering ramets as a measure of differences in survival between the sexes. I predicted that male-biased sex ratios would be associated with resource limited environments and that male plants would be generally older in the population than female plants.

Objective 2: Pollinator abundance

14 Males provide more rewards for pollinators in the form of pollen and nectar than do females. As sex ratios can vary widely across populations in clonal dioecious plants, I evaluated whether pollinators were more abundant at sites with more males, i.e., in areas of higher resource abundance for pollinators.

Objective 3: The function of staminodes

Finally, an experiment was performed to determine the function of infertile staminodes found on female flowers. I tested the hypothesis that they function to attract pollinators and that their removal would reduce pollinator visitation.

15 CHAPTER 2: Sex ratio variation, pollinators and staminode function in

Aralia nudicaulis

Resources allocated to reproduction cannot also be allocated to other life history traits, yielding tradeoffs between investment in reproduction and survival and/or growth. For dioecious plants, these tradeoffs should be more evident in the sex with the higher reproductive cost, and almost invariably it is the females that have greater investment in reproduction (Obeso 2002). Accordingly, higher reproductive costs borne by females in comparison with males are associated with sexual dimorphism in other components of the life history, including differences in survival and growth rates between the sexes (Geber et al. 1999). For example, in Rubus chamaemorus, females have lower rhizome production than males, but only if they produce fruits (Agren 1988), suggesting that it is the extra resource cost associated with fruit production that regulates sexual dimorphism in rates of clone formation in this species.

For dioecious plants that are also clonal, like R. chamaemorus, differences in the production of rhizomes between the sexes lead to biased shoot sex ratios (Barrett et al.

2010). Indeed, male-biased sex ratios at the ramet level (independent vegetative shoots of a clone, identical to the parent) are commonly observed in clonal dioecious plants. For example, in Aralia nudicaulis, male frequencies significantly exceeded 50% in half of the

15 populations surveyed (Barrett and Helenurm 1981). Importantly, this bias in male frequencies was more evident in forested compared to roadside sites. Such habitat effects

16 on sex ratios are commonly observed, with sex ratio biases accentuated in sites with reduced resource availability (Dawson and Ehleringer 1993, Wade et al. 1981), where resource stress might exacerbate differences in the cost of reproduction between the sexes. For A. nudicaulis, which is a forest understory herb, light availability is thought to be a key limiting resource, leading Barrett and Helenurm (1981) to predict a positive association between light availability and female frequencies, which would explain their observation of a difference in the magnitude of the bias in sex ratios between forested and roadside sites. One of the goals of this study was to test this prediction along a continuum of percentage canopy closure, which in turn regulates light availability in temperate forest understories.

Although total reproductive effort is usually greater for females than it is for males, the magnitude of the difference in reproductive expenditures between the sexes varies over the growing season (Geber et al. 1999). During flowering, it is often males that have the greater investment in reproduction (Geber et al. 1999). In particular, males usually produce more and/or larger flowers than females (Ashman 2000, Glaettli and Barrett

2008), and this difference has implications for pollinator attraction. Moreover, males often have greater investment in nectar rewards (Hemborg and Bond 2005), and by default are the only plants in dioecious populations that provide pollen rewards.

Accordingly, males usually receive higher levels of pollinator visitation than females

(Huang et al. 2006, Glaettli and Barrett 2008).

To compensate for reduced pollinator attractiveness, females in some species have

17 evolved mechanisms that appear to function to deceive pollinators by producing structures that closely resemble those found on male flowers. For example, the gynoecia

(i.e., reproductive organs producing ovules and seeds) of female flowers in dioecious

Begonia species can superficially resemble stamens on male flowers, and floral manipulations have demonstrated that this resemblance functions to enhance pollinator visitation (Agren and Schemske 1991). Similarly, female flowers in Rubus chamaemorus closely resemble male flowers, but provide little in the way of nectar rewards (Agren et al. 1986). Because females do not provide pollen rewards but should benefit from multiple pollinator visitation events for successful seed set (Ashman 2000), floral traits that promote resemblance between females and males, and thereby enhanced pollinator attraction, should generally be favoured in animal-pollinated dioecious plants.

In this chapter, I investigated differences in life history attributes between females and males that are predicted to arise from differences in the cost of reproduction. I have used

Aralia nudicaulis as my study species. As already noted, populations sex ratios is male biased and this bias appears to stem from greater reproductive investment by females in comparison with males (Barrett and Helenurm 1981). I begin by testing the prediction made by Barrett and Helenurm (1981) that shoot sex ratios should be associated with light availability in this species. Those authors also demonstrated that males produce more flowers than females, and because they produce pollen while females do not, they are presumably more attractive to pollinators. On the other hand, females produce staminodes that closely resemble the pollen-bearing stamens on male flowers.

Accordingly, I also examined the function of these staminodes by comparing pollen

18 deposition and the fruit set of plants with intact vs. removed staminodes. Finally, because populations of A. nudicaulis can vary strongly in the proportion of plants that are female vs. male, and because pollinators typically preferentially visit males over females, I predicted that there should be greater pollinator abundance at sites with male biased shoot sex ratios.

19 -78 7 -78 6 -78 5 -784 -76 3 45 7 45 7 < , _— 1 0 » nig., »10km

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Figure 2-1. Map with all 15 sites of study in Algonquin Park, Ontario

20 Methods

Aralia nudicaulis ramet ages and sex ratios

I examined the frequency of female vs. male ramets in 15 populations of Aralia nudicaulis in Algonquin Park (Fig. 2-1) during the summers of 2009 and 2010.

Populations were located by searching for patches of A. nudicalis with at least 25 flowering ramets that were separated by at least 500m from another patch of flowering ramets, however, eight of 15 sites had more than 80 flowering ramets (average = 74.6 ±

7.2 S.E.), and 11 of 15 populations were at least one kilometer apart. Data collection commenced at the onset of flowering in May of each year with the labeling of each flowering shoot so that they could be revisited during fruit production in July - August.

In each population, I recorded the sex and age of each flowering ramet. Ramets were sexed by examining whether they produced fertile (pollen producing) stamens (males), or pistils and sterile staminodes (females). The age of each ramet was estimated by counting the number of leaf scars on each stem, with each scar denoting a single year of growth

(following Bawa et al. 1982), yielding measurements from a total sample of 1136 ramets.

Canopy cover

At each of the 15 populations, I estimated the percent canopy cover in July using a convex densiometer (Jennings et al. 1999), to measure light availability at the time of fruit production. Densiometers are primarily useful for comparing the relative canopy closure between sampling locations (Cook et al. 1995). Accordingly, these values are only used in analyses involving comparisons of canopy closure across populations.

21 Estimates of canopy closure were made by first marking the boundaries of the population using stakes. Starting five meters from the eastern edge of the population, I measured the degree of canopy closure along a north-south transect at 5 m intervals until reaching the edge of the population. I took further measurements along additional transects five meters to the west of the preceding transect until reaching the western edge of the population. At a subset of five sites, I also calculated canopy closure directly above each flowering female plant to evaluate the association between fruit set and light availability. At each sampling location in the transect, I took four measurements of canopy closure, one facing in each of the four cardinal directions following the manufacturer's instructions

(Lemmon 1956). The average of these measurements per site are indicated in Appendix

1; Table 2. As densiometers provide a value for open canopy, I subtracted the average value from 100 to estimate the total percentage of canopy closure at each sampling location. All relevant data pertaining to each site including geographical coordinates can be found in Appendix 1; Tables 1&2.

Staminode experiment

To evaluate the function of staminodes on female A nudicaulis, I examined their effect on pollinator visitation and reproductive success by comparing pollen loads on the stigmas of plants with intact vs. removed staminodes. In total, twenty-five female flowering shoots were included and paired in the experiment, yielding 12 control ramets and 12 manipulated ramets. To prevent pollinator visitation prior to the application of the treatments, I constructed domes over each ramet using wire and bridal veil. The domes were placed over the ramets before their flowers opened and were removed when over

22 50% of the flowers were open. Ramets were randomly assigned to the manipulation treatment. For ramets subject to staminode removal, I excised the staminodes from all flowers on the umbel with the most flowers per umbel using forceps. The remaining two umbels were removed entirely from the plant. For control plants, staminodes were left intact, but again, the two umbels with the fewest flowers were removed from the plant.

Each plant was left open to pollinators for 48 hours before taking two randomly chosen flowers to measure pollen loads. I also counted the number of flowers per inflorescence to estimate fruit set.

Pollen loads were counted for all samples by removing the stigmas from two flowers per inflorescence and mounting them on microscope slides using basic fuchsin stain (Beattie

1971). I then counted the number of pollen grains for each of the 5 stigmas per plant using a Nikon Eclipse E200 microscope. Staminode lengths were measured by capturing images of staminodes using a Nikon D5000 dSLR camera mounted on a Nikon SMZ800 dissecting microscope. To measure the lengths of the staminodes in millimeters, staminode lengths (in pixels) were scaled using a sub-millimeter scale bar included in each image.

Pollinator Sampling

Pollinators were collected with the use of coloured (yellow and white) 17 cm diameter pantraps following Taki, Kevan and Ascher (2007). The traps were each filled with dilute soapy water using a ratio of three drops of blue Dawn ® dishwashing liquid to one litre of water. Ten bowls (five of each colour) were arranged in a linear transect through

23 patches of flowering A. nudicaulis, alternating colours and spaced every 60 centimeters, totaling 6 metres. The transects were set out for a minimum of four hours (but up to eight hours) every other day during the flowering period. The pollinators were collected and stored in 95% alcohol until they were washed, pinned and identified.

Statistical analyses

Light levels, age and sex ratios ~ To evaluate differences in the ratio of females:males within and among sites I used replicated goodness-of-fit tests (Sokal and Rohlf 1995), using each site as the unit of replication to group each set of tests.

To evaluate the association between variation in the frequency of females among sites and canopy closure, I used a generalized linear model (GLM) using the glm function in the "stats" library in R (Version 2.12.0, R Development Core Team; Pinheiro et al. 2011).

Because ramet age might also covary with canopy closure (via the effect of light availability on growth rates), it was included as a main effect in the GLM. For this analysis, the proportion of female ramets per site was the dependent (binary) variable, and canopy closure and the median ramet age were the independent variables. To account for overdispersion, the GLM was calculated by specifying a quasibinomial error distribution.

Age of female vs. male ramets ~ To evaluate differences in the age of male vs. female ramets, I formulated a generalized linear mixed model (GLMM) using the lmer function in the "lme4" library in R (Bates and Maechler 2010). Ramet age was the dependent variable and ramet sex was the independent categorical variable. Site was included as a

24 random grouping variable and the model was calculated by specifying Poisson errors. To evaluate differences between the sexes in the overall distribution of ramet ages across all study sites, I conducted a two-sample Kolmogorov-Smirnov test using the ks.test function in the stats library in R.

Pollinator abundance and sex ratios ~ To evaluate the association between pollinator abundance and the frequency of females versus males across sites, I used a linear model of log-transformed pollinator count data per site using the lm function in the "stats" package in R. The pollinator counts were weighted by sampling effort by dividing the number of pollinators collected per site by the number of hours of sampling conducted per site. The data were also log (natural log) transformed to meet assumptions for the linear model. For this analysis, pollinator abundance per site was the dependent variable, and the proportion of female ramets per site was the predictor variable. The results obtained using this analysis were strongly influenced by the outlier site ("Madawaska"), identified using the influence.measures function in R, which estimates the influence of each data point on the slope and intercept of the linear model. Accordingly, this analysis was also conducted by excluding the Madawaska site.

Staminode experiment ~ To evaluate the effect of staminode removal, I conducted a

GLMM using the lmer function in R. Pollen counts per flower were the dependent variable, and the staminode removal treatment was the independent variable. To account for variation between the pairs of plants used for the experimental manipulations and differences between plants, 'pair' and 'plant' were included as a random effect. The model

25 was calculated by specifying Poisson errors.

Results

Sex ratios and canopy closure ~ There was wide variation in the proportion of shoots that were female vs. male across sites (range = 0.05 to 0.95). As has been found in previous studies of A. nudicaulis, some sites were significantly male biased in their ramet sex ratios (five of 15 sites; Appendix 1; Table 1; Figure 2-2). However, even more sites were significantly female biased (seven of 15 sites). This variation in the proportion of female ramets across sites was reflected by the results of the replicated goodness-of-fit tests

(Ghet- X'H = 416.9, P < 0.0001; Appendix 2). However, in contrast to the prediction described above, I found no evidence that this variation was influenced by differences in light availability across sites (GLM: parameter estimate =-0.16 ± 0.08 S.E., t = -1.89, P =

0.08; Figure 2-3). There was also no association between the median age of ramets per site and the sex ratio (GLM: / = -0.151, P = 0.88).

Sex differences in ramet age ~ Female ramets were significantly older than male ramets

(GLMM: Wald's Z= -5.8, P < 0.0001, N sites = 15, N shoots = 1131). On average, female ramets were 14%o older than male ramets; the average ramet age for females was

5.6 years, compared to 4.9 years for males. The oldest ramet (41 y) in the sample was female; the oldest male shoot was substantially younger (28 y). The distribution of ramet ages was significantly shifted towards older ages for females vs. males (two-sample

Kolmogorov-Smirnov test: D = 0.10, P < 0.005; Figure 2-4).

Pollinator abundance ~ A total of 345 pollinators was caught in white and yellow pan

26 traps across the 9 sites sampled. Most of the pollinators in the sample were flower flies

(Syrphidae; 73%, or 252 of 345 insects). The remaining pollinators were bees (Apidae;

27%, or 93 of 345 insects). The most abundant species of Syrphidae was Toxomerus geminatus, comprising 84 individuals (24.3% of the collected Syrphids). Over 90% of the bees were represented by just two families: Halicddae and Andrenidae, with Halictidae as the most abundant (52% of the total bees in the sample; Appendix 3).

Contrary to my expectations, the abundance of pollinators was positively associated with the proportion of female shoots of A. nudicaulis (Figure 2-5), but only if the outlier site

(Madawaska) was excluded from the analysis (with Madawaska: parameter estimate =

1.99 ± 0.93 S.E., / = 2.14, P = 0.07; without Madawaska: parameter estimate = 3.10 ±

0.71 S.E., / = 4.37, P < 0.01, r=0.069). The Madawaska site was identified as having a statistically significant influence on the outcome of the linear model (influence coefficient for Madawaska: -1.11; range of coefficients for the remaining sites: -0.58 -

0.51). The positive relationship between pollinator abundances and female frequencies was driven by the number of flies per site (Flies: linear model parameter estimate = 0.83

± 0.25 S.E., / = 3.32, P < 0.05; Bees: linear model parameter estimate = 0.32 ± 0.34 S.E., t = 0.95, P = 0.38).

Staminode experiment ~ Staminode removal significantly reduced the amount of pollen deposited on stigmas (GLMM: Wald's Z = -2.2, P < 0.05, N pairs = 12; Figure 2-6).

Intact plants received more than three times as many pollen grains as did plants for which the staminodes had been removed (average number of pollen grains per stigma per plant for intact plants = 18.9, for plants that had their staminodes removed = 5.0).

27 FIGURES

66 93 44 70 87 81 26 27 113 90 110 40 95 96 73 1.00n

CD Male 1.0.75 Female CO E & 0.50 gc g.0.25 a. 0.00 (0d)(!)(BltW=»OJ >T3 „ o CO CD CtnO CmO r-C CmO —? ™ .*— r- •—. iv or\ i_ rn =5 CO ffi E H 0-E 2 a en CD _i I— CD CO X - 5 a C T3 CO m tor= - o co o S W D)6 o D (11 i_ CO CO > TO >CD or X c/5 a: o o .5 5

Figure 2-2. Sex ratios of flowering ramets of Aralia nudicaulis across 15 sites in

Algonquin Provincial Park during 2009/2010 (indicated as the proportion of total ramets per site). The total number of flowering ramets per site is shown at the top of the bars.

28 o

E • •• CD in 0> o CD E o CD O c g CM J o n=15 o. P=0.08 o o A o 1 1 1 r— '50 60 70 80 90 100 Percent canopy closure

Figure 2-3. Relationship between the proportion of female ramets of Aralia nudicaulis and canopy closure at 15 sites in Algonquin Provincial Park.

29 80-

60-

40-

20-

0- c 3 O O 30-

60-

40-

20-

0- t 20 30 4C Age

Figure 2-4. Distribution of ramet ages for female and male shoots of Aralia nudicaulis sampled from 15 sites in Algonquin Provincial Park.

30 1-1 • CD • O cz co "O o- £= • Z> X) • • CO i_ 1- o • "co

o 2- Q. • • n=8 3- 1- —i— —i 1 . . 1 T 1 P<0.005 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of female ramets

31 JU-

t/J 1 20- o

0 §- 10-

I n=48 o- 1 P<0.05 Intact Removed Treatment

Figure 2-6. The effect of staminode removal on pollen loads of female flowers of Aralia nudicaulis.

32 DISCUSSION

This study has revealed substantial variation in ramet-level sex ratios across populations of Aralia nudicaulis. However, my data provide little support for the hypothesis by

Barrett and Helenurm (1981) that this variation is tied to light availability. My survey of ramet ages also indicated that female ramets are, on average, older than male ramets.

Although this finding ran counter to my prediction of greater male longevity, the result can be explained by differences between the sexes in their rates of ramet formation and the age at which flowering begins. Surprisingly, I found a strong association between the proportion of female ramets across sites and pollinator abundances. Finally, the staminode-removal experiment revealed a strong effect of staminode removal on the amount of pollen deposited on female stigmas, suggesting that they are important for pollinator attraction. Each of these findings is discussed in greater detail below.

A previous study indicated that variation in the frequencies of females might be tied to light availability (Barrett and Helenurm 1981). Specifically, female frequencies were shown to be higher at roadside than forest interior sites, leading the authors of that study to predict a positive association between light availability and female frequencies.

Although my results were consistent with their prediction (in that the slope of the line relating the proportion of females to canopy cover was negative), they were not statistically significant and this lack of statistical support may simply reflect an insufficient number of sites in my sample and, therefore, insufficient power to reject the

33 null hypothesis of no association. Alternatively, something else might have driven the pattern observed by Barrett and Helenurm (1981). Disturbance has been shown to be associated with increased nitrogen concentrations in plants growing in forested ecosystems (Manninen et al. 2009). Therefore, if nitrogen also limits the growth of A. nudicaulis, and particularly if it limits the growth of females more than males, disturbances associated with roads might have contributed to the observation of higher female frequencies along roadsides.

Differences in the proportion of female vs. male ramets across sites might also be driven by differences in the age structure of the populations at those sites. Among younger age classes of A. nudicaulis, male ramets flower more frequently than females (Bawa et al.

1982). My data are only partially consistent with this suggestion. I included the median ramet age per site as a factor in the analysis of the association between canopy closure and ramet-level sex ratios and the inclusion of ramet age improved the model fit (data not shown). The lack of a significant association between the median ramet age and relative female frequencies might again simply reflect a lack of statistical power, which, by virtue of the fact that sex-ratios are properties of entire populations, is limited by the number of sites from which sex ratios were observed, rather than the number of ramets that were aged.

My data, and the predictions made by Barrett and Helenurm (1981), run counter to observations made for another clonal dioecious herb, Mercurialis perennis. As in A. nudicaulis, populations of M. perennis tend to be male biased (Vandepitte et al. 2009a and references therein). However, unlike A. nudicaulis, M. perennis populations become

34 increasingly male biased with greater light availability (Vandepitte et al. 2009b). Both species are found almost exclusively under forest understories, but A nudicaulis can also be found in disturbed and logged forests (e.g., regenerating clear-cut forests; Corns and

La Roi 1976, cited in Flanagan and Bain 1988), whereas M. perennis is an old-growth indicator species that is most commonly found in undisturbed forests (Kenderes &

Standovar 2003, Vandepitte et al. 2009b); their broadly similar ecologies make these contrasting patterns difficult to reconcile. The results for M. perennis were interpreted in light of the difference in the cost of reproduction between females and males: females had higher investment in seeds in sites with greater light availability, potentially exacerbating differences in the cost of reproduction between the sexes and further enhancing ramet production by males (Vandepitte et al. 2009b). It is not yet known whether allocation to fruits by female A. nudicaulis also increases in sites with greater light, and therefore whether the cost of reproduction for females varies across this gradient.

The study's focus on ramets rather than genets might, at least in part, explain why the majority of populations surveyed here were significantly biased in their sex ratios.

Replicated goodness-of-fit tests assume that sampling units are independent (Sokal and

Rohlf 1995), an assumption that is clearly violated in a clonal plant (unless genets can be identified a priori). If sites were represented by a small number of genets, each with numerous ramets, then statistically biased sex ratios are much more likely to be found than they would be for a comparable number of shoots sampled from a non-clonal dioecious plant (i.e., the number of ramets of each sex do not reflect the number of genets of each sex, artificially increasing the likelihood of detecting a significantly skewed sex

35 ratio). Thus, my inference that ramet-level sex ratios were biased at some sites should not be taken as evidence for biased sex ratios at the genet level at each site. Moreover, my data do not provide evidence for a sex-ratio bias across sites; I found a roughly equal distribution of sites with a majority of females vs. a majority of males.

Female ramets of A. nudicaulis were older than male ramets, with the median age for females 14% older than that for males (5.6 years for females vs. 4.9 years for males). In general, male plants are thought to have higher rates of survival via reduced costs of reproduction in comparison with females (Obeso 2002, Geber et al. 1999, Bell 1980).

Indeed, if males have higher rates of clonal growth than females, it is possible to find younger males on average than females even if survival rates are higher for males.

However, my data are not consistent with this; the distribution for female ramets is shifted towards higher ages across the entire range observed. Indeed, the proportion of female flowering ramets aged 11 years or higher was 13.6%, while 10.5% were male; this in accordance with Bawa et al.'s (1982) study which found the age of 11 yielded significant differences in flowering frequencies between the sexes. This finding cannot simply be explained by differences in the cost of reproduction. An alternate explanation for differences in life history traits between females and males is that they arise from divergent selection rather than as a byproduct of differences in the cost of reproduction

(Dorken and Drunen 2010). In particular, increases in the number of reproductive ramets via clonal propagation might, under certain circumstances, enhance fitness gains through male function but decrease it through female function. If so, males should clone at a higher rate, while females are predicted to have higher resource budgets per ramet, enabling greater investment in reproduction. Reproductive effort in plants often scales

36 with plant size (Weiner 2004); however, reproduction in polycarpic clonal plants such as

A. nudicaulis, might be less strongly tied to the size of individual ramets, which are not particularly variable, and more closely associated with the availability of stored resources, and these stored resources should also enhance survival. These expectations fit with my observations. Faster rates of clonal expansion should result in a preponderance of young male ramets, and higher survival rates for female ramets should lead to a preponderance of older female ramets, both of which were observed here. Concordant observations have been made for American eelgrass (Vallisneria americana), an aquatic perennial; despite significantly male-biased sex ratios for this plant, it was found that females have significantly more leaves and greater biomass than males (Lovett-Doust and

Laporte 1991), which should in turn lead to higher survival rates for females compared to males.

My site with the lowest proportion of female ramets also had a relatively open forest canopy and high pollinator abundance and was consistently divergent from my other sites with respect to patterns between sex ratios and these other two variables. This divergence was expressed by substantial values for the influence coefficients for regressions involving these variables. This site had recently been logged (2006/2007), and so the current environmental conditions at the site do not reflect those that would have prevailed during the growth of the genets within the population over the previous decades.

Moreover, the plants appeared to suffer damage from the higher light levels at this site.

Of the 92 shoots at the site, 51 of them suffered some necrosis in the leaf tissue, which was followed by inflorescence and fruit abortion. Confirmation of these inferences would require tracking the fates of ramets following logging, a study that would appear to be

37 needed not just for A. nudicaulis but also for other understory forest perennials.

Sexual reproduction for dioecious plants requires pollen transfer from male anthers to female stigmas. In Aralia nudicaulis, this action is performed by generalist pollinators in the form of bees and flies (Appendix 3). Interestingly, no bumblebees (i.e., Bombus) were caught, and were rarely observed in my study sites. This is in contrast to Barrett and

Thomson (1982), who observed the major pollinators to be Bombus, with bees in the families Andrenidae and Halictidae, and syrphid flies being in the minority. However,

Barrett and Thompson (1982) did no direct trapping of the insect visitors to A. nudicaulis and conducted their research in a different part of the species range where plants were growing in a different habitat type (their site was dominated by spruce and fir trees, whereas my sampling occurred in mixed deciduous forest). Moreover, I assumed that the insects collected in the pan traps were representative of those visiting A. nudicaulis at my study sites, and this assumption is not likely to be completely upheld. For example, Cane et al. (2001) found pan traps not to offer wholly accurate samples reflective of the native bee fauna visiting Larrea tridentata. Moreover, Campbell and Hanula (2007) found more

Hymenoptera were caught in pan traps than Diptera in a study across three temperate forest sites, with both preferring white over yellow pan traps. Disney et al (1982) also found the family Syrphidae to be more attracted to white pan traps, than were other

Diptera. Despite the difference though, Hanula (unpublished data) in Campbell and

Hanula (2007) found pan traps in forests captured most of the bee species that were collected by net while visiting flowers in nearby roadside habitats.

38 Although the specific feeding and nesting habits of most of the pollinators in my sample are not known, they are most likely to be generalists (e.g., Toxomerus; Vockeroth 1992).

Accordingly, many of the pollinators captured at my sites may have been visiting other co-flowering species in addition to, or instead of, A. nudicaulis. Vegetation surveys conducted at my sites indicate that Oxalis acetosella, Maianthemum canadense, and

Cornus canadensis, all plants with small white flowers that can be food sources for the insects in my sample, occurred at densities similar to those of the A. nudicaulis plants at some sites (Appendix 4). Despite the relative abundance of pollinators in the pan traps perhaps differing from those actually visiting A nudicaulis, all of the species sampled are potential pollinators , and most likely do visit Aralia flowers. This study is also the first study to sample pollinators within Aralia clones (Appendix 3; Table 1). A combination of passive and active sampling with sweep nets would have been the best option, but would also have reduced the number of sites that could have been sampled.

In general, the male flowers of dioecious plants provide more rewards for pollinators than do female flowers because they produce both pollen and nectar (Niesenbaum et al. 1999).

Accordingly, males, or plants with male function, often receive higher rates of visitation than females (e.g., Sagittaria latifolia, Glaettli and Barrett 2008; Fragaria virginana,

Case and Ashman 2009). For this reason, I had predicted that sites with more male ramets would have a higher abundance of pollinators. Unexpectedly, I found more pollinators at sites with higher female frequencies and this pattern appeared to be driven by greater abundances of syrphid flies at sites with more females. Although small solitary bees of such as those observed in this study tend to forage across distances of 100 m or less

39 (Rader et al. 2011; and see Greenleaf et al. 2007), syrphid flies can transport viable pollen over distances of 400 m (Rader et al. 2011). Because these longer distances roughly correspond with those between flowering patches of A. nudicaulis in the study region, syrphid flies might have the option to choose where to forage. Indeed, given a choice, syrphid flies are capable of choosing sites with more floral resources (MacLeod 1999).

This raises the possibility that females provide more nectar rewards than males and the measurement of nectar in female vs. male plants of A. nudicaulis would help resolve why we found more pollinators at sites with more females.

Staminodes are morphologically similar to stamens, although reduced in size, and perform a variety of functions, including the attraction and positioning of pollinators

(Walker-Larsen and Harder 2000, Decraene and Smets 2001). My data shows that the presence of staminodes enhances pollen receipt, and from this I infer that staminodes are actively maintained by selection. Staminodes have been shown to serve similar functions in other plants. In the hermaphroditic plant Jacaranda oxyphylla, for example, staminode removal was associated with decreased pollen receipt, but did not affect pollen removal, suggesting that they serve to enhance female function in this species (Guimares et al.

2008). For other plants, staminodes serve to position pollinators to maximize the efficiency of pollen transfer (Decraene and Smets 2001). However, for A. nudicaulis, positioning seems an unlikely function; its flowers are borne on globose umbels and I have observed pollinators approaching them from a variety of directions, often by crawling from flower to flower. Instead, increased pollen receipt is probably the result of pollinator attraction by the staminodes in A. nudicaulis. However, my data do not reveal

40 on their own whether the attraction of pollinators is via visual or chemical cues, or if it

occurs because staminodes provide pollinator rewards (i.e., pollen). However, given that

the staminodes render females visually similar to males, at least part of their attractive

function is likely to occur via visual cues. If so, A. nudicaulis would be similar to plants with 'food deception mimicry' (Little 1983, Agren and Schemske 1991). Such plants provide the illusion of rewards (in this case, pollen), attracting pollinators without

incurring the cost of producing actual rewards. For example, the monoecious neotropical herb Begonia involucrata produces female flowers with a branched, dark yellow stigma that resembles the anthers of the male flowers (Agren and Schemske 1991). This form of mimicry, sometimes called deceit pollination, attracts pollinators to rewardless flowers, and is maintained by natural selection via its positive effects on female function (Little

1983, Willson and Agren 1989, Schemske and Agren 1995).

The flowers of A. nudicaulis are radially symmetrical, presenting an open umbel and

offering no obvious nectar guides, but the arrangement of the staminodes encircling the central style may indicate to pollinators the appropriate structural 'set up' they are

seeking (Little 1983). If pollinators depend on their visual senses to locate food sources, a similar floral spectral pattern would be expected, as would scents or odours in olfactory-dependent pollinators (Little 1983). Bumblebees have been found to require both visual and olfactory cues to incite their landing (Lunau 1991). There is no information on the production of chemical cues in A. nudicaulis like scent but it is possible they invest more in it than males to attract pollinators. This is entirely possible as there are examples of females producing more rewards than males in some areas;

41 Coccoloba padiformi, Cordia collococa, Simarouba glauca, Trichilia cuneata, Triplaris americana are all dioecious plants where the females produce more nectar than males

(Bawa and Opler 1975, Opler and Bawa 1978). As for visual cues, Corff et al(1998), examined the response of Begonia's principle pollinator, Trigona fulviventris, to two species with different shapes and tepal areas. They found B. tonduzii, whose female flowers differ in shape but not area to the males, to be twice as frequently visited as B. urophylla, whose female flowers are similar in shape to those of males but of a different area. They concluded that the shape of the flower helps more in deception of pollinators than tepal area.

The investigations conducted in this thesis have provided some surprising, but insightful results into the biology of A. nudicaulis. In general, female response has been unexpected in connection to light levels as well as to survival rates, living to an older age than males. Both of these results run contrary to theories regarding trade-offs; it was hypothesized that females would have fewer resources to devote to growth and clonal expansion than males. Fewer resources might also mean lower rates of survival for females, but I found that female ramets were older than male ramets, and this finding cannot simply be explained by differences in the rate of ramet formation between the sexes. The third unpredicted response came from higher rates of pollinator abundance in sites with higher female frequencies. Higher rewards for pollinators were predicted to draw them towards more male sites but instead, the opposite was found. Reasons for this outcome are not clear but the pollinators may have been constrained by their foraging distances and the observed pattern may have been driven by some unmeasured variable.

42 Staminodes clearly play a role in pollinator attraction to female A. nudicaulis, apparently because they render female inflorescences visually similar to the presumably more rewarding inflorescences produced by males.

43 GENERAL DISCUSSION

Main findings

Is there an association between canopy cover and female frequencies?

The results offer partial support for Barrett and Helenurm's (1981) finding of light levels shifting the sex ratio from one of male bias to one of an equal balance between the sexes.

Instead of a balanced ratio though, I found a slight female bias in areas of high light levels, (albeit not statistically significant). With light as a vital resource for plants and one of a limited nature in forested environments, the sexes respond accordingly in their respective manners. The female bias was unexpected. According to theories on resource distribution (Lloyd and Webb 1977) between the sexes, clonal dioecious females have fewer resources to commit to growth of both vegetative and flowering ramets, so it is a surprise that they surpassed male genets in growth with such small levels of light increases. Perhaps female genets are more efficient with their resource use, using the increase in light levels to greater effect than male plants (Geber et al. 1999).

This study focused on patterns occurring at the ramet level, raising the possibility that my results do not reflect what is going on at the genet level, and therefore the level at which selection operates. Because I did not use molecular markers and was therefore not able to identify genets, my sampling strategy was to include as many sites with flowering ramets as I could find. Using this approach, sites, rather than shoots, were the unit of replication for most of the statistical tests conducted in this study. As a result, any lack of independence among ramets within sites (i.e., because they were part of the same genet

44 and/or shared similar growth conditions) should not have influenced statistical inferences made in this thesis (although the discussion of the results of the replicated goodness-of-fit tests). Perhaps future work could include providing labelled limited resources to both male and female clonal dioecious plants under stress and tracking which components of life history received them.

Is there a difference in age between male vs female ramets?

This question offers further insight into resource allocation between the sexes in clonal dioecious plants. Survival is regarded as one of the three principle components of general resource allocation, measured by age of the ramet. I found female ramets were significantly older than males, and a greater number of female ramets lived past their teen years and into their twenties. Males were found predominantly in younger age classes, suggesting that males were investing more in the growth of new ramets instead of maintaining their existing ramets. This inference runs counter to the prediction that females should have lower survival due to their larger investment in sexual reproduction.

For some plants, females are more efficient in their resource use than males (Geber et al.

1999), so perhaps could allocate more resources to survival and reproduction than males.

For a more robust experiment in the future, tracking the fates of the ramets would be beneficial. Examining their survival rate as they age, as well as rates of clonal and sexual reproduction, would offer a more complete picture of the factors influencing ramet age and sex ratios in A. nudicaulis.

Is there an association between the frequency of males and pollinator abundance?

A key observation in this study was that there was a positive association between the

45 relative frequency of females and pollinator abundance. I had initially expected to find the opposite pattern - with more pollinators in sites with more males, and therefore more floral resources. As far as I know, this is the first time that this association has been examined, and so guidance from other studies on why the pattern was observed is not available. It may simply be that pollinators, particularly the small-bodied bees and flies that were common in my study sites have small foraging distances and could not choose between sites, with more vs. fewer males. If so, then the observed pattern can most easily be explained by the occurrence of some as yet unidentified variable that causes both high female frequencies and pollinator abundances. The obvious link is light availability, but as already noted, light was not significantly associated with either sex ratios or pollinator abundances. Regardless, the association between light availability and female frequencies was nearly statistically significant and may have been detectable with a larger number of sites. Pollinator abundance and activity was highest during the sunniest parts of the day, late morning and into early afternoon in apple orchards (Vicens and Bosch 2000).

Pollinators in forested systems were found in greater abundance and diversity in forest gaps with higher light levels, as opposed to the darker interior forest (Proctor, unpublished thesis 2011). The density of the patches could also help explain pollinator abundance. Dense populations of flowering ramets including males, even with higher female frequencies would attract pollinators, however, there was also no association between plant densities and pollinator abundances across my sites. Alternatively, females may be offering more rewards than previously thought; higher nectar production by females vs. males would explain why the pollinators were to be found in those sites.

Unfortunately, nectar volumes were too small to quantify accurately. For future work, I

46 would highlight the possibility that light is the shared variable that links high female frequencies and pollinator abundances.

Is there an association between staminode presence and pollen loads on female ramets?

The results of this experiment support the hypothesis that staminodes function to attract pollinators to females. Staminodes are infertile, offering no reward to visiting insects, but appear to mimic stamens on males. Despite the cost to females, this vestigial secondary sexual characteristic is vital to the pollination of their stigmas and continues to be maintained. This is congruent with other plants/trees found with other forms of mimicry designed to fool pollinators into visiting their flowers. This work is novel in being the only manipulative experiment done on staminodes in a dioecious plant and contributes to the literature on floral dimorphism and mimicry in plants. Further work would include the experiment being repeated with a higher number of plants at a larger number of sites.

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55 Appendix 1. Table 1. Summary of location and sex ratio data for 15 populations of Aralia nudicaulis.

Site UTM Latlitude/ Site Description Females Males Total Sex Ratio Coordinates Longitude (fem/total) Bat Lake 17T0693793 45.587566, -78.515638 5051227 trail side- spruce 23 47 70 0.3286 Big Pines 17T703296 45.583027,-78.394021 5051024 trail side- spruce 16 11 27 0.5925 Brewer 17T0693697 45.589787,-78.516771 5051471 road edge site-east facing 69 4 73 0.9452 Bud Lake 17T711329 45.572145,-78.291571 5050080 trail side-portage 79 16 95 0.8316 Chit Lake 17T0693598 45.599949,-78.517592 5052598 trail side- north facing 9 84 93 0.0968 Florence 17T0701449 45.455231,-78.423541 5036752 logged forest clearing 9 44 53 0.1698 Hemlock Bluff 17T691351 45.572963,-78.54758 5049528 trail side-east facing 34 53 87 0.3908 Jordan's site 17T693739 45.582509,-78.516553 5050663 open roadside NA NA 26 NA Lost Ray 17T692719 45.589276,-78.529331 5051384 trail side-portage 66 24 90 0.7765 Louisa Flats 17T0701066 45.462589,-78.428104 5037558 logged forest clearing 33 48 81 0.4074 Madawaska 17T711647 45.327948,-78.299184 5022931 logged forest clearing 3 63 66 0.0454 Mizzy Trail 17T680110 45.550234,-78.692581 5046667 trail side 13 13 26 0.5 Provoking 17T696225 45.570895,-78.485205 5049449 trail side 67 46 113 0.5929 Station Rd. 17T0693689 45.588872,-78.516914 5051369 open roadside 90 20 110 0.8182 Two Rivers 17T0697405 45.573835,-78.46995 Interior 5049813 mature forest 33 7 40 0.825 Two Rivers 17T0697541 45.574776,-78.468165 Trail 5049922 trail side- old railbed 80 16 96 0.8333

56 Appendix 1. Table 2. Summary of life-history data for 15 populations of Aralia nudicaulis. Site Age Median Age Maximum Flowering Non-flowering Shoots Flowering Density Ratio Canopy Closure (%) Density Bat Lake 5 24 13 125 0.0942 80.79 Big Pines 4 17 NA NA NA 77.88 Brewer 3 9 22 220 0.0909 67.43 Bud Lake 5 16 NA NA NA 80.12 Chit Lake 8 28 8 107 0.0696 90.38 Florence 3 15 10 111 0.0826 80.54 Hemlock Bluff 3 17 NA NA NA 71.04 Jordan's site NA NA NA NA NA 53.27 Lost Ray 4 24 NA NA NA 74.48 Louisa Flats 3 16 10 141 0.0662 73.03 Madawaska 3 11 21 203 0.0938 73.89 Mizzy Trail 3.5 24 NA NA NA 74.29 Provoking 3 12 NA NA NA 80.734 Station Rd. 4 22 1 125 0.0079 70.86 Two Rivers Interior 5 13 NA NA NA 80.15 Two Rivers Trail 7 41 7 167 0.0402 81.69

57 Appendix 1. Table 3 Pollinator data for 15 populations of Aralia nudicaulis

Site Pollinator Pollinator Pollinator

Abundance (#) Bowl Hours Abund /Hour

Bat Lake 12 36 83 0 3801

Big Pines NA NA NA

Brewer 15 29 33 0 5455

Bud Lake NA NA NA

Chit Lake 4 41 25 0 0970

Florence 3 28 58 0 1749

Hemlock Blufl NA NA NA

Jordan's site NA NA NA

Lost Ray NA NA NA

Louisa Flats 16 35 33 04812

Madawaska 15 11 5 1 3043

Mi/zy Trail NA NA NA

Provoking 13 1925 0 6753

Station Rd 136 51 58 2 6367

Two Rivers Interior 37 37 0 9730

Two Rivers Trail 94 46 66 2 1860 Appendix 2. Table 1. Frequency of males and females among 15 sites in Algonquin Park, Ontario during 2009-2010. G values are

given for tests of independence of sex frequencies from the two sexes for each plot. G/, refers to tests of heterogeneity between sites.

Site Male Female n G Significant Bat Lake 0.67 0.33 70 8.397 ** Bie Pines 0.41 0.59 27 0.931 Brewer 0.06 0.95 73 70.18 *** Bud Lake 0.17 0.83 95 45.55 *** Chit Lake 0.90 0.1 93 69,78 *** Florence 0.83 0.17 53 25.18 *** Hemlock Bluff 0.61 0.39 87 4.183 * Lost Rav 0.27 0.73 90 20.38 *** Louisa Flats 0.59 0.41 81 2.793 Madawaska 0.95 0.05 66 67.08 *** Mizzv Trail 0.5 0.5 26 0 Provoking 0.41 0.59 113 3.925 * Station Road 0.18 0.82 110 48.18 *** Two Rivers Interior 0.18 0.83 40 18.35 *** Two Rivers Trail 0.17 0.83 96 46.57 ***

Gu 416.8

*P<0.05 **P<0.01 ***P<0.001

59 Appendix 3. Table 1. Summar! y of po linator data per site collected ir i pan traps from nine populations of Aralia nudicaulis. Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Toxomerus Platycheirus Brachyopa Brachyopa Brachyopa Lejops Lejota geminatus spp. perplexa dacckei ferruginea anausis cyanea

Bat Lake 4 0 0 0 0 0 0 Brewer 0 0 0 2 0 0 0 Chit Lake 3 0 0 0 0 0 0 Florence 0 0 0 0 0 0 0 Louisa Flats 6 0 0 0 0 0 0 Madawaska 2 0 0 0 0 0 0 Provoking 12 0 0 0 0 0 0 Station Road 25 1 1 0 2 1 0 Two Rivers Interior 30 0 0 0 0 0 0 Two Rivers Trail 2 0 3 5 3 1 1

Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Myolepta Sericomyia Sericomyia Sericomyia Chalcosyrphus Xylota Xylota nigra bifasciata chrysotoxoides militaris piger annulifera quadrimaculata

Bat Lake 0 0 0 1 0 1 1 Brewer 0 0 2 0 0 1 0 Chit Lake 0 0 0 0 0 0 0 Florence 0 0 0 0 0 0 3 Louisa Flats 0 1 0 0 0 0 0 Madawaska 0 0 1 0 1 0 1 Provoking 0 0 0 0 0 0 0 Station Road 0 0 1 0 1 2 5 Two Rivers Interior 0 0 0 0 0 0 0 Two Rivers Trail 2 1 35 2 0 5 2

60 Appendix 3. Table 1. Summary of pollinator data per site collected in pan traps from nine populations of Aralia nudicaulis.

Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Xylota Xylota Xylota Xylota Cheilosia Melanostoma Chrysogaster hinei atlantica subfasciata confusa rita mcllinum antitheus

Bat Lake 0 0 0 1 0 0 0 Brewer 0 0 1 3 0 0 0 Chit Lake 0 0 0 0 0 0 0 Florence 0 0 0 0 0 0 0 Louisa Flats 1 2 0 1 0 0 0 Madawaska 0 0 0 0 0 1 0 Provoking 0 0 0 0 0 0 0 Station Road 2 0 0 1 1 1 2 Two Rivers Interior 0 1 3 0 0 0 0 Two Rivers Trail 0 0 2 5 0 1 0

Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Neoascia Neoascia Parasyrphus Parasyrphus Syrphus Temnostoma Temnostoma sandsi globosa (semiinteruptus genualis rectus cxcentrica venustrum or new spp) Bat Lake 0 0 0 0 0 1 0 Brewer 0 0 0 0 0 0 0 Chit Lake 0 0 0 0 0 0 0 Florence 0 0 0 0 0 0 0 Louisa Flats 0 0 0 0 0 0 0 Madawaska 0 0 1 0 0 4 0 Provoking 0 0 0 0 0 0 0 Station Road 3 1 0 1 0 3 0 Two Rivers Interior 0 0 0 0 1 0 0 Two Rivers Trail 0 0 0 0 0 1 1

61 Appendix 3. Table 1. Summary of po linator data per site collected ini pan traps from nine populations of Aralia nudicaulis. Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Syrphidae Temnostoma Orthonevra Orthonevra Sphegina Sphegina Sphegina Sphegina balyras nitida pulchclla lobata flavimana keeniana flavomaculata

Bat Lake 0 0 0 0 0 0 0 Brewer 0 0 0 1 3 0 0 Chit Lake 0 0 0 0 0 0 0 Florence 0 0 0 0 0 0 0 Louisa Flats 0 0 0 0 0 0 0 Madawaska 0 0 0 2 0 0 0 Provoking 0 0 0 0 0 0 0 Station Road 0 1 7 0 0 1 0 Two Rivers Interior 0 0 0 1 0 0 0 Two Rivers Trail 2 0 0 0 1 2 1

Syrphidae Syrphidae Syrphidae Syrphidae Andrena Andrena Andrena Sphegina Sphegina Eupeodcs spp. eriginiae vicina distans campunulata rafinventris spp. (lost heads)

Bat Lake 0 0 0 0 0 0 0 Brewer 1 0 0 0 0 0 0 Chit Lake 1 0 0 0 0 0 0 Florence 0 0 0 0 0 0 0 Louisa Flats 0 0 0 0 0 1 1 Madawaska 0 0 0 0 0 0 0 Provoking 0 0 0 1 0 0 0 Station Road 0 1 0 1 12 1 0 Two Rivers Interior 0 0 1 0 0 0 0 Two Rivers Trail 2 2 0 2 0 0 0

Appendix 3. Table 1. Summary of pollinator data per site collected in pan traps from nine populations of Aralia nudicaulis.

62 Andrena Andrena Andrena Andrena Andrena Halictus Halictus melanochroa nasonii clarkella rufosignata erythronii confusus rubicundus

Bat Lake 0 1 0 0 0 0 0 Brewer 0 0 0 0 0 0 0 Chit Lake 0 0 0 0 0 0 0 Florence 0 0 0 0 0 0 0 Louisa Flats 0 0 0 1 0 0 0 Madawaska 1 0 0 0 0 0 0 Provoking 0 0 0 0 0 0 0 Station Road 0 1 2 8 9 1 1 Two Rivers Interior 0 0 0 0 0 0 0 Two Rivers Trail 0 0 0 0 0 0 0

Lasioglossum Lasioglossum Lasioglossum Lasioglossum Lasioglossum Lasioglossum Lasioglossum planatum cressonii leicocomu nigroviride versans novascotiae lacvissimum

Bat Lake 1 0 0 0 1 0 0 Brewer 0 0 1 0 0 0 0 Chit Lake 0 0 0 0 0 0 0 Florence 0 0 0 0 0 0 0 Louisa Flats 0 0 0 0 0 0 1 Madawaska 0 1 1 1 0 0 0 Provoking 0 0 0 0 0 0 0 Station Road 13 2 0 0 1 3 13 Two Rivers Interior 0 0 0 0 0 0 0 Two Rivers Trail 0 4 0 0 0 0 0

63 Appendix 3. Table 1. !Summar y of po linator data per site collected ir i pan traps from nine populations of Aralia nudicaulis. Lasioglossum Lasioglossum Lasioglossum Andrenidae Hylaeus Hylaeus Total sample divergcns foxii qucbcccnsc nomada annulus modestus

Bat Lake 0 0 0 0 0 0 12 Brewer 0 0 0 0 0 0 15 Chit Lake 0 0 0 0 0 0 4 Florence 0 0 0 0 0 0 3 Louisa Flats 0 0 1 0 0 0 16 Madawaska 0 0 0 0 0 0 15 Provoking 0 0 0 0 0 0 13 Station Road 1 0 0 3 0 0 136 Two Rivers Interior 0 0 0 0 0 0 37 Two Rivers Trail 0 2 0 0 2 2 94

64 Appendix 4. Table 1. Vegetation plots indicating an approximation of percent coverage. Site abbreviations: BAT=Bat Lake Trail, BL=Bud Lake, BP=Big Pines, BREW=Brewer Lake, CHIT=Chit Lake Trail, FLO=Florence, HB=Hemlock Bluff Trail, LOUFL=Louisa Flats, LR=Lost Ray Trail, MAD=Madawaska, MIZZY^Mizzy Lake Trail, PROV=Provoking Portage, STNRD=Station Road, 2RIVINT=Two Rivers Lake Interior, 2RIV=Two Rivers Lake Trail. Average Site Common Name Latin name Plotl Plot 2 Plot 3 Plot 4 Plot 5 BAT Wild Sarsaparilla Aralia nudicaulis 15 0 0 0 0 3 BAT Fly Honeysuckle Lonicera canadensis 12 0 0 0 0 2.4 BAT Balsam Fir Abies balsamea 0 3 5 0 13 4.2 BAT Blue Bead Lily Clintonia borealis 0 7 6 0 0 2.6 BAT Red Pine seedling Pinus resinosa 0 18 0 0.5 0 3.7 BAT Velvet Leaf Blueberry Vaccinium myrfilloides 0 0 3 0 0 0.6 BAT Canada Mayflower Maianthemum canadense 0 0 2 3 15 4 BAT Trembling aspen Populus tremuloides 0 0 0 1 0 0.2 BAT Large leaf Aster Aster macrophyllus 0 0 0 0 6 1.2 BAT Bracken Fern Pteridium aquilinum 0 0 0 0 25 5 BAT Bunchberry Conius Canadensis 0 0 0 0 10 2 BAT Starflower Trientalis borealis 0 0 0 0 5 1 BAT Ground Pine Lycopodium dendroideum 0 0 0 0 12 2.4 BAT Beaked Hazel Corylus cornuta 0 0 0 0 30 6 BAT Wild Raisin Viburnum cassinoides 0 0 0 0 9 1.8

BL Red Maple Acer rubrum 26 22 7 75 0 26 BL Wild Sarsaparilla Aralia nudicaulis 18 15 20 25 30 21.6 BL Striped Maple Acer pensylvanicum 3 0 0 0 0 0.6 BL Canada Mayflower Maianthemum canadense 1 2 0 0 0 0.6 BL Large Coralroot Corallorhiza maculata 1 0 0 0 0 0.2 BL Bunchberry Conius canadensis 2 10 0 0 0 2.4 BL Wood Sorrel Oxalis acetosella 0 80 0 70 2 30.4 BL Blue Bead Lily Clintonia borealis 0 12 30 0 0 8.4 BL Indian cucumber Root Medeola virginiana 0 0 17 0 0 3.4 BL Starflower Trientalis borealis 0 0 5 1 0 1.2 BL Sugar Maple Acer saccharum 0 0 10 0 30 8

65 Appendix 4. Table 1. Vegetation plots indicating an approximation of percent coverage. 1 Average Site Common Name Latin name Plotl Plot 2 Plot 3 Plot 4 Plot 5 BL Spinulose Wood Fern Dryopteris carthusiana 0 0 0 0 15 3

BP Blue Bead Lily Clintonia borealis 8 12 6 0 3 5.8 BP Sugar Maple Acer saccharum 6 3 0 50 0 I 11.8 BP Wood Millet-grass Milium effusum 3 0 0 25 0 1 5.6 BP Indian Pipe Monotropa uniflora 1 0 0 0 0 0.2 BP Canada Mayflower Maianthemum canadense 2 1 2 0 0 1 BP Trembling aspen Populus tremuloides 1 1 0 2 2 1.2 BP Wild Sarsaparilla Aralia nudicaulis 2 0 25 4 0 6.2 BP Spinulose Wood Fern Dryopteris carthusiana 20 0 0 0 0 4 BP Wood Sorrel Oxalis acetosella 1 25 30 4 0 12 BP Balsam Fir Abies balsamea 1 2 1 35 0 7.8 BP Drooping Wood Sedge Carex arctata 4 3 1 0 12 4 BP Bunchberry Cornus canadensis 0 13 6 2 4 5 BP Red Maple Acer rubrum 0 16 0 2 2 4 BP Twintlower Linnaea borealis 0 4 0 7 0 2.2 BP Fly Honeysuckle Lonicera canadensis 0 9 0 0 0 1.8 BP Bracken Fern Pteridium aquilinum 0 0 85 0 0 17 BP Starflower Trientalis borealis 0 0 2 0 0 0.4 BP False Solomon's Seal Smilacina racemosa 0 0 9 0 0 1.8 BP Partridgeberry Mitchella repens 0 0 25 0 50 15 BP Large-leaf Aster Aster macrophyllus 0 0 2 0 0 0.4 BP Spinulose Wood Fern Dryopteris carthusiana 0 0 0 2 0 0.4 BP Ground Pine Lycopodium dendroideum 0 0 0 1 0 0.2 BP Rose Twisted Stalk Streptopus roseus 0 0 0 2 0 0.4 BP White Birch Betula papyrifera 0 0 0 0 2 0.4

66 Appendix 4. Table 1. Vegetation plots indicating an approximation of percent coverage. Average Site Common Name Latin name Plotl Plot 2 Plot 3 Plot 4 Plot 5 BREW Trembling aspen Populus tremuloides 5 0 0 0 0 1 BREW Sensitive Fern Onoclea sensibilis 8 0 0 16 0 4.8 BREW Sugar Maple Acer saccharum 27 35 30 17 20 25.8 BREW Rough-stemmed Goldenrod Solidago rugosa 27 0 0 22 6 11 BREW Rough Hawk weed Hieracium gronovii 11 0 0 0 8 3.8 BREW Narrow-leaved Goldenrod Euthamia graminifolia 3 0 0 2.5 0 1.1 BREW Bushy Pasture Spear Grass Poa saltuensis 50 0 0 0 0 10 BREW Ox-eye Daisy Chrysanthemum leucanthemum 10 0 0 0 0 2 BREW Starflower Trientalis borealis 0 3 6 0 0 1.8 BREW Red Trillium Trillium erectum 0 6 0 0 0 1.2 BREW White Spruce Picea glauca 0 0 0 100 0 20 BREW White Birch Betula papyrifcra 0 0 0 3 0 0.6 BREW Red Raspberry Rubus idaeus 0 0 0 7 12 3.8 BREW Wild Sarsaparilla Aralia nudicaulis 0 0 0 12 30 8.4 BREW Woodland Horsetail Equisetum sylvaticum 0 0 0 0 25 5 BREW Canada Mayflower Maianthemum canadense 0 0 0 0 4 0.8 BREW Quackgrass Elymus repens 0 0 0 0 j> 0.6 BREW Wild Strawberry Fragaria virginiana 0 0 0 0 "t 0.4

CHIT Rose Twisted Stalk Streptopus roscus 11 2 0 0 5 3.6 CHIT Balsam Fir Abies balsamea 35 0 0 30 0 13 CHIT Canada Mayflower Maianthemum canadense 7 0 0 0 22 5.8 CHIT Striped Maple Acer pensylvanicum 7 13 35 35 6 19.2 CHIT White Birch Betula papyrifera 0 0 100 0 0 20 CHIT Wild Sarsaparilla Aralia nudicaulis 0 8 8 0 6 4.4 CHIT Starflower Trientalis borealis 0 2 1 0 2 1 CHIT Wintergreen Gaultheria procumbens 0 10 0 9 0 3.8

67 Appendix 4. Table 1. Vegetation plots indicating an approximation of percent coverage. Average Site Common Name Latin name Plotl Plot 2 Plot 3 Plot 4 Plot 5 CHIT Beaked Hazel Corylus cornuta 0 0 25 0 0 5 CHIT Eastern White Cedar Thuja occidentalis 0 0 0 100 0 20 CHIT Spinulose Wood Fern Dryopteris carthusiana 0 0 0 6 0 1.2 CHIT Goldthread Coptis groenlandica 0 0 0 6 0 1.2 CHIT One sided Pyrola Orthilia secunda 0 0 0 0 2 0.4

FLO Striped Maple Acer pcnsylvanicum 60 0 0 0 0 12 FLO Sugar Maple Acer saccharum 12 2 8 20 6 9.6 FLO Starflower Trientalis borealis 15 0 2 0 0 3.4 FLO Canada Mayflower Maianthemum canadense 2.5 2 0 1 0 1.1 FLO Mountain Maple Acer spicatum 0.5 70 0 1 0 14.3 FLO Spinulose Wood Fern Dryopteris carthusiana 0 3 0.5 5 40 9.7 FLO White Birch Betula papyrifera 0 0 0.5 0 1 0.3 FLO Red Raspberry Rubus idaeus 0 0 0 15 0 3 FLO Wild Sarsaparilla Aralia nudicaulis 0 0 0 3.5 0 0.7 FLO Wood Sorrel Oxalis acetosella 0 0 0 0.5 6 1.3 FLO Hemlock Tsuga canadensis 0 0 0 0.5 0 0.1 FLO Hobblebush Viburnum lantanoides 0 0 0 3 0 0.6 FLO Drooping Wood Sedge Carex arctata 0 0 0 6 0 1.2

HB Wild Sarsaparilla Aralia nudicaulis 25 12.5 40 11 0 17.7 HB Spinulose Wood Fern Dryopteris carthusiana 20 2 0 0 7 5.8 HB Hobblebush Viburnum lantanoides 10 0 0 0 0 2 HB Wood Sorrel Oxalis acetosella 6 35 0 0 6 9.4 HB Sugar Maple Acer saccharum 0 12 30 5 0 9.4 HB Balsam Fir Abies balsamea 0 11 8 0 0 3.8 HB Striped Maple Acer pensylvanicum 0 3 0 0 0 0.6 HB Red Maple Acer rubrum 0 18 25 0 I 8.8 HB Starflower Trientalis borealis 0 0 0 1 1 0.4

68 Appendix 4. Table 1. Vegetation plots indicating an approximation of percent coverage. Average Site Common Name Latin name Plotl Plot 2 Plot 3 Plot 4 Plots HB Fly Honeysuckle Loniccra canadensis 0 0 0 15 0 3 HB Bunchberry Conius canadensis 0 0 0 2 0 0.4 HB Indian cucumber Root Medeola virginiana 0 0 0 0 2 0.4 HB Red Raspberry Rubus idaeus 0 0 0 0 4 0.8 HB Rose Twisted Stalk Streptopus roseus 0 0 0 0 2 0.4 HB Drooping Wood Sedge Carex arctata 0 0 0 0 3 0.6 HB Wood Millet-grass Milium effusum 0 0 0 0 18 3.6 HB Beaked Hazel Corylus cornuta 0 0 0 0 5 1 HB Twinflower Linnaea borealis 0 0 0 0 2 0.4 HB Yellow Birch Betula alleghaniensis 0 0 0 0 1 0.2 HB Hemlock Tsuga canadensis 0 0 3 20 0 4.6 HB Canada Mayflower Maianthemum canadense 0 0 0 0 1 0.2

LOUFL Wild Sarsaparilla Aralia nudicaulis 25 0 0 4 6 7 LOUFL Ground Pine Lycopodium dendroideum 2 0 0 18 3 4.6 LOUFL Sugar Maple Acer saccharum 20 30 15 6 10 16.2 LOUFL Starflower Trientalis borealis 2 0 20 0 3 5 LOUFL Spinulose Wood Fern Dryopteris carthusiana 0 0 12 0 0 2.4 LOUFL Red Raspberry Rubus idaeus 0 50 15 0 0 13 LOUFL Drooping Wood Sedge Carex arctata 0 12 0 0 0 2.4 LOUFL Wood Millet-grass Milium effusum 0 6 0 0 0 1.2 LOUFL Hemlock Tsuga canadensis 0 0 1 0 0 0.2 LOUFL Indian cucumber Root Medeola virginiana 0 0 0 2 0 0.4 LOUFL American Beech Fagus grandifolia 0 0 0 0 2 0.4

LR Wild Sarsaparilla Aralia nudicaulis 5 45 50 4 25 25.8 LR Spinulose Wood Fern Dryopteris carthusiana 12 0 0 0 0 2.4

69 Appendix 4. Table 1. Vegetation plots indicating an approximation of percent coverage. Average Site Common Name Latin name Plotl Plot 2 Plot 3 Plot 4 Plot 5 LR Red Maple Acer rubrum 1 8 20 4 25 11.6 LR Trembling aspen Populus tremuloidcs 0 0 2 0 0 0.4 LR Starflower Trientalis borealis 0 0 1 0 0 0.2 LR Bunchberry Cornus canadensis 0 60 80 0 40 36 LR Bracken Fern Pteridium aquilinum 0 30 0 0 17 9.4 LR Sugar Maple Acer saccharum 0 15 0 70 18 20.6 LR Drooping Wood Sedge Carex arctata 0 12 2 0 0 2.8 LR One sided Pyrola Orthilia secunda 0 0 0 2 0 0.4 LR Canada Mayflower Maianthemum canadense 0 1 0 3 0 0.8

MAD Hooked Spur Violet Viola adunca 80 0 0 0 0 16 MAD Red Raspberry Rubus idaeus 12 0 60 4 85 32.2 MAD Sugar Maple Acer saccharum 8 30 0 35 0 14.6 MAD Bladder Sedge Carex intumescens 20 0 2 0 0 4.4 MAD Black Bindweed Polygonum convolvulus 8 0.5 2 4 8 4.5 MAD Wild Sarsparilla Aralia nudicaulis 0 50 17.5 50 2.5 24 MAD White Birch Betula papyrifera 0 0 1 0.5 0 0.3 MAD Chokecherry Prunus virginiana 0 0 0 6 0 1.2

MIZZY Red Maple Acer rubrum 32 0 27 35 0 18.8 MIZZY Ground Pine Lycopodium dendroideum 6 32 20 38 11 21.4 MIZZY Spinulose Wood Fern Dryopteris carthusiana 45 0 35 0 18 19.6 MIZZY Hemlock Tsuga canadensis 23 0 0 5 0 5.6 MIZZY Red Raspberry Rubus idaeus 20 0 0 0 0 4 MIZZY Wood Sorrel Oxalis acetosella 8 0 0 0 0 1.6 MIZZY Yellow Birch Betula alleghaniensis 40 0 0 6 0 9.2 MIZZY Wood Millet-grass Milium effusum 2 0 3 0 0 1 MIZZY Southern Ground Cedar Diphasiastrum digitatum 0 0 20 0 0 4 MIZZY Wild Sarsaparilla Aralia nudicaulis 0 50 15 0 9 14.8

70 Appendix 4. Table 1. Vegetation plots indicating an approximation of percent coverage. Average Site Common Name Latin name Plotl Plot 2 Plot 3 Plot 4 Plot 5 MIZZY Sugar Maple Acer saccharum 0 30 8 23 0 12.2 MIZZY Indian cucumber Root Medeola virginiana 0 0 2 0 0 0.4 MIZZY False Solomon's Seal Smilacina racemosa 0 4 0 0 0 0.8 MIZZY Drooping Wood Sedge Carex arctata 0 5 0 0 0 1 MIZZY Hobblebush Viburnum lantanoides 0 0 0 0 100 20 MIZZY Early low blueberry Vaccinium angustifolium 0 0 0 0 2 0.4

PROV Northern Beech Fern Phegopteris connectilis 40 0 35 0 15 PROV Wild Lettuce Lactuca canadensis 15 0 0 0 3 PROV Heal-all Prunella vulgaris 2 0 0 0 0.4 PROV Wood Millet-grass Milium effusum 20 35 0 0 11 PROV Fringed Brome Grass Bromus ciliatus 0 0 12 0 2.4 PROV Jewelweed lmpatiens capensis 0 45 0 0 9 PROV Common Sedge Carex communis 0 75 0 20 19 PROV Starflower Trientalis borealis 0 0 1 0 0.2 PROV Tall Buttercup Ranunculus acris 0 3 0 0 0.6 PROV Sensitive Fern Onoclea sensibilis 0 25 0 0 5 PROV Spinulose Wood Fern Dryopteris carthusiana 0 15 0 0 3 PROV Drooping Wood Sedge Carex arctata 0 0 2 0 0.4 PROV Wild Sarsaparilla Aralia nudicaulis 0 0 35 0 7.6 PROV Indian cucumber Root Medeola virginiana 0 0 45 0 9 PROV Bracken Fern Pteridium aquilinum 0 0 0 60 55 23 PROV Red Raspberry Rubus idaeus 0 0 0 27 5.4 PROV Large-leaf Aster Aster macrophyllus 0 0 0 70 14.4 PROV Fragrant Bedstraw Galium triflorum 0 0 0 4 1.6 PROV Fly Honeysuckle Lonicera canadensis 0 0 0 12 2.4 PROV Wild Strawberry Fragaria virginiana 0 0 0 2 0.4 PROV Balsam Fir Abies balsamea 0 0 0 2 1.4

71 Appendix A. Table 1. Vegetation plots indie;ttin g an approximation of percent coverage. Average Site Common Name Latin name Plotl Plot 2 Plot 3 Plot 4 Plot 5 PROV Sugar Maple Acer saccharum 0 0 0 5 3 1.6 PROV Beaked Hazel Corylus cornuta 0 0 0 0 3 0.6 PROV Quackgrass Elymus repens 0 0 0 0 40 8 PROV Canada Wood Betony Pedicularis canadensis 0 0 0 0 2 0.4 PROV Poverty Oat Grass Danthonia spicata 0 0 0 0 7 1.4 PROV Rough Hawkweed Hieracium gronovii 0 0 0 0 3 0.6 PROV Spreading Dogbane Apocynum androsaemifolium 0 0 0 0 2 0.4

STNRD Beaked Hazel Corylus cornuta 75 0 50 70 0 39 STNRD Sugar Maple Acer saccharum 20 1 0 0 4 5 STNRD Large leaf Aster Aster macrophyllus 6 0 25 0 0 6.2 STNRD Fly Honeysuckle Lonicera canadensis 2.5 0 0 0 0 0.5 STNRD Velvet leaf Blueberry Vaccinium myrtilloides 2 0 9 0 0 2.2 STNRD Wild Sarsaparilla Aralia nudicaulis 0 0 0 12 0 2.4 STNRD Balsam Fir Abies balsamea 0 35 0 20 2 11.4 STNRD Starflower Trientalis borealis 0 7 0 0 0 1.4 STNRD Canada Mayflower Maianthemum canadense 0 6 0 0 7.5 2.7 STNRD Bracken Fern Pteridium aquilinum 0 7 20 0 30 11.4 STNRD Common Sedge Carex communis 0 0 0 0 25 5 STNRD White Pine Pinus strobus 0 0 0 0 1 0.2 STNRD Bunchberry Cornus canadensis 0 0 0 0 1 0.2 STNRD Quackgrass Elymus repens 0 0 0 0 3 0.6 STNRD White Spruce Picea glauca 0 0 0 0 1 0.2 STNRD Twinflower Linnaea borealis 0 0 0 0 2 0.4

2RIVINT Wild Sarsaparilla Aralia nudicaulis 25 45 0 7 3 16 2RJVINT Sugar Maple Acer saccharum 6 0 18 0 40 12.8

72 Appendix A. Table 1. Vegetation plots indicating an approximation of percent coverage. Average Site Common Name Latin name Plotl Plot 2 Plot 3 Plot 4 Plots 2RJV1NT Spinulose Wood Fern Dryopteris carthusiana 85 0 30 0 0 23 2RIVINT Oak Fern Gymnocarpium dryopteris 25 0 11 3 0 7.8 2RIVINT Red Maple Acer rubrum 0 6 0 0 0 1.2 2RIVINT Balsam Fir Abies balsamea 0 3 0 0 0 0.6 2RIVINT Indian Cucumber Root Medeola virginiana 0 2 3 0 4 1.8 2RIVINT Red Trillium Trillium erectum 0 0 3 0 0 0.6 2RIVINT Beaked Hazel Corylus cornuta 0 0 100 0 0 20 2RIV1NT Ground Pine Lycopodium dendroideum 0 0 3 0 0 0.6 2RIVINT Striped Maple Acer pensylvanicum 0 0 0 90 0 18 2R1VINT American Beech Fagus grandifolia 0 0 0 23 0 4.6 2RIVINT Foamflower Tiarella cordifolia 0 0 0 0 25 5 2RIVINT Canada Mayflower Maianthemum canadensis 0 0 0 0 13 2.6 2RIVINT False Solomon's Seal Smilacina racemosa 0 0 0 0 6 1.2 2RIV1NT Yellow Birch Betula alleghaniensis 0 0 0 0 30 6

2RIV Red Raspberry Rubus idaeus 25 2 4 11 0 8.4 2RJV Wild Sarsaparilla Aralia nudicaulis 15 6 0 45 20 17.2 2R1V Starflower Trientalis borealis 11 3 3 0 0 3.4 2RIV Balsam Fir Abies balsamea 9 0 100 55 0 32.8 2RIV Mountain Ash Sorbus decora 4 0 0 0 0 0.8 2RIV Beaked Hazel Corylus cornuta 50 0 17 0 0 13.4 2RIV Hemlock Tsuga canadensis 0 0 0 25 0 5 2RIV Meadowsweet Spiraea alba 0 0 0 0 3 0.6 2RIV Rough Hawkweed Hieracium gronovii 0 2.5 0 0 0 0.5 2RIV Common Plantain Plantago major 0 4 0 0 0 0.8 2RIV Sugar Maple Acer saccharum 0 7 0 0 0 1.4 2RIV Quackgrass Elymus repens 0 30 0 0 80 22 2RIV Beaked Hazel Corylus comuta 0 17 0 0 0 3.4 2RIV Bunchberry Cornus canadensis 0 0 0 0 12 2.4