THE IMPORTANCE OF INVASIVE EARTHWORMS AS SEED PREDATORS OF COMMON FOREST FLORA OF ONTARIO

By

Colin Cassin

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Ecology and Evolutionary Biology University of Toronto

© Copyright by Colin M Cassin 2015

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THE IMPORTANCE OF INVASIVE EARTHWORMS AS SEED

PREDATORS OF COMMON FOREST FLORA OF ONTARIO

Colin Cassin

Master of Science

Ecology and Evolutionary Biology Department University of Toronto

2015

Abstract Soil seed banks are vital to forest plant community regeneration, having long been viewed as a refuge

for seeds vulnerable to granivory. Here evidence is provided suggesting many seeds entering the seed

bank are subject to previously underestimated rates of granivory via the commonly found invasive

earthworm, Lumbricus terrestris.

Results from an earthworm-addition microcosm experiment suggest nearly 70% of seeds are

removed from the soil surface when exposed one earthworm. Results from a separate granivore-

exclusion field experiment indicate granivory by rodents eclipses that of earthworms under more

natural conditions. When analyzed individually it is clear that different granivores target certain

species of seed over others. This suggests that although rodents are the main driver of seed predation,

earthworms may have the potential to act as an ecological filter, potentially further influencing the

species composition of future forest plant communities by selectively targeting certain seeds, or seed

traits, over others.

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Acknowledgments

Two years ago I set out on the path to study plant interactions along latitudinal gradients,

today I submit a story about seeds and earthworms – funny how things pan out. It has been

an interesting path, full of challenges and opportunities, but I simply could not arrive at this

point without such a special band of colleagues, friends and family.

Thank you to my supervisor, Peter Kotanen. Over the past 2 years you have been a tutor,

therapist, mentor and friend. Your knowledge of natural history seems limitless, and no

graduate student could ask for a more generous and committed supervisor.

Thank you to my best friend, confidant and wife, Katie Robert. You have supported me at

every step, and I am truly grateful.

I am also grateful for the support of an ever-changing cluster of labmates. Thank you

especially to Krystal Nunes for your helpful advice and sympathetic ear. I have also been

fortunate to have two tremendously committed research assistants for during my time at U of

T. Thank you to Leighanne Goodine and Gurpreet Mangat for your enthusiasm and

commitment. No one could count, mark, and retrieve 40,202 seeds with more gusto.

Thank you to my incredibly supportive parents. Your love and encouragement has been

unwavering. Further support and inspiration from Robert Canning, Cara Hernould, and Eric

Sager has been appreciated throughout this journey.

Lastly, I express a great deal of gratitude to Valerie Felicity Frizzle and William Sanford

Nye. If only all young people had such an enthusiastic introduction to the incredible

adventures that science bestows.

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Table of Contents

CONTENTS PAGE NO. CHAPTER 1: AN INTRODUCTION TO THE ISSUES OF GRANIVORY 1.1 Introduction 1 1.2 Objectives 11 1.3 References 13 CHAPTER 2: SEED PREDATION IN TEMPERATE FORESTS 2.1 Introduction 17 2.2 Methodology 22 2.3 Results 32 2.4 Discussion 54 2.5 Conclusions 60 2.6 References 62 CHAPTER 3: GENERAL CONCLUSIONS 3.1 Revisiting & Interpreting Thesis Objectives 66 3.2 Summary of Findings 68 3.3 Directions for Future Research 69 3.4 References 71 APPENDIX A-1 Rationalizations For Seed Selections 72 A-2 Extended Abstract 73 !

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List of Tables

TABLES PAGE NO. TABLE 1 TABLE OF SPECIES AND SEED MASSES USED IN EACH EXPERIMENT

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TABLE 2 RESULTS OF EARTHWORM MICROCOSM EXPERIMENT BY BURIAL DEPTH

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TABLE 3 SPECIES SPECIFIC BREAKDOWN OF EARTHWORM MICROCOSM EXPERIMENT

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TABLE 4 RESULTS OF SUMMER GRANIVORE EXCLUSION EXPERIMENT BY TREATMENT

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TABLE 5 RESULTS OF SUMMER GRANIVORE EXCLUSION EXPERIMENT BY SPECIES

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TABLE 6 RESULTS OF FALL GRANIVORE EXCLUSION EXPERIMENT BY TREATMENT

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TABLE 7 RESULTS OF FALL GRANIVORE EXCLUSION EXPERIMENT BY SPECIES

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TABLE 8 RESULTS FROM SEED MARKING CONTROL EXPERIMENT

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TABLE 9 RESULTS FROM LEAF LITTER REMOVAL CONTROL EXPERIMENT

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TABLE 10 RESULTS FROM SEED BURIAL CONTROL EXPERIMENT

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List of Figures

FIGURES PAGE NO. FIGURE 1 SEED FATE IN MICROCOSM EXPERIMENT

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FIGURE 2 SUMMER GRANIVORE EXCLOSURE EXPERIMENTS SEED RECOVERY RESULTS

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FIGURE 3 FALL GRANIVORE EXCLOSURE EXPERIMENT SEED RECOVERY RESULTS

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FIGURE 4 MARKED AND UNMARKED CONTROL TRIAL RATES OF RECOVERY

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FIGURE 5 RESULTS OF LEAF LITTER REMOVAL EXPERIMENT

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List of Appendices

Appendix PAGE NO. APPENDIX 1 RATIONALIZATIONS FOR THE SELECTION OF EACH SPECIES OF SEED

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APPENDIX 2 EXTENDED ABSTRACT

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Chapter 1 An Introduction to the Issues of Granivory

1.1 INTRODUCTION

Seed & Seedling Ecology The seed and seedling stages are a highly vulnerable phase in the lifecycle of a plant.

As mortality rates during the seed stage are often capable of exceeding 50% (Peters et al.

2003; Hsia and Francl 2009) and many species appear to be seed-limited (Clark et al. 2007),

the acts of dispersal and establishment are critically important for those seeds that ultimately

defy the odds. During the time in which it becomes mature, disperses and eventually reaches

the ultimate fate of mortality or successful germination, a seed will experience an unrelenting

series of selective forces. Although randomness plays a role in determining the final outcome

of a seed, numerous abiotic and biotic factors ultimately govern the likelihood for each seed

to successfully develop into a mature adult capable of reproducing itself.

One such factor, often responsible for tremendously high amounts of seed mortality,

is seed predation, also known as granivory. Granivory can be attributed to an array of agents,

from large grazing ungulates to small scatter-hording sciurids, or even invertebrates and

fungi. Each of these seed-eaters is capable of selecting seeds of particular species, or even

individuals possessing certain traits within a single species. These selective pressures may

potentially have a great impact on the quantity and composition of viable seeds that are

fortunate enough to make it to the germination stage.

In this chapter I will introduce some of the most important factors that contribute to the

biological filter that is seed predation, focusing on both seed and granivores with an

emphasis on temperate forests.

Seed Morphology & Characteristics Seed size has long been viewed as a critical trait in determining the likelihood of seed

predation. Many variations of simple choice seed-addition experiments exist in which

granivores are offered a range of seed sizes. Often seeds with the greatest mass and size are

removed by vertebrates earlier, and in greater quantity than smaller seeds (Jansen et al. 2004;

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McRill and Sagar 1973; Quackenbush et al. 2012). For example, larger seeds of a rain forest

tree (Carapa procera) were removed in greater abundance, and dispersed to greater distances

than seeds of smaller mass by a scatter-hording rodent (Jansen et al. 2004). In contrast,

invertebrates generally are of greater importance in the predation of smaller sized seeds

(Honek et al. 2003; Quackenbush et al. 2012). Despite this they are still able to negatively

impact larger sized seeds, for instance through ovipositing in seeds of large mass (Amsberry

and Maron 2006). Overall, seeds of smaller size tend to suffer relatively lower rates of seed

predation than larger seeds, and some research suggests that some seeds may be small

enough to escape vertebrate predation almost entirely (Reader 1993; Kelrick et al. 1986;

Blaney and Kotanen 2001). However, seed size often is compared in relative, not necessarily

absolute measures, which can cause generalizations (e.g., across different habitats or plant

communities) to be precarious (Radtke 2011).

In addition to seed size, variations in the morphological traits of seeds have long been

observed to contribute to the potential desirability of a particular seed to a granivore. For

instance seeds of the evergreen oak (Quercus ilex) that are more bullet shaped are less likely

to be removed and dispersed by a seed dispersing rodent than those which are longer and

thinner (Munoz et al. 2012). Similarly, Shumway and Koide (1994) found that earthworms

tended to remove seeds with smooth external surfaces in greater quantities than seeds with

rough surfaces. More generally, Quackenbush and colleagues (2012) found that predicting

species of seeds that are at greatest risk of earthworm predation was best achieved when seed

mass was combined with both seed length and width.

The production of specialized external structures can also influence how a seed

interacts with dispersers and granivores. For instance seeds bearing elaiosomes, fleshy

appendages rich in lipids and proteins, are associated with highly specialized ant-mediated

dispersal, or myrmecochory. These appendages are removed (possibly increasing likelihood

of successful germination) as the seeds are dispersed away from the parent plant.

Aphenogaster rudis is one such example of a key North American ant species that employs

myrmecochory (Prior et al. 2014).

Palatability of the seed or its enclosing fruit can also be important. Many animal-

dispersed seeds are toxic, even though the fruit surrounding them may be highly palatable, as

is true in the seeds of Canadian yew, Taxus canadensis (Barnea et al. 1993). This is often

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interpreted as a mechanism to encourage animal dispersal while protecting seeds against

predation.

Regardless of which factor is most influential in determining which seeds will be

subject to higher predation rates than others, it is widely accepted that great differences can

be found in the rates at which different seeds are removed, predated, and/or dispersed. These

differences in probability of granivory lend themselves well to the theory that granivores

likely have a strong potential to act as biological filters in the systems they occupy (e.g.,

Maron et al. 2012). For this reason, understanding the preferences of seed predators may

help guide our understanding of how their activities impact plant communities.

Some studies have highlighted relative differences between seed predation rates in

native and exotic species. For instance, Maron and colleagues (2012) found that although

seed size was the primary factor in moderating seed removal, species provenance exhibited

obvious trends in how likely a seed was to experience rodent predation in a grassland habitat.

Native seeds were removed at considerably higher rates than exotics in a seed-addition

experiment focusing on 39 commonly found grassland species, representing a range in seed

size. Although real differences such as this have been found in how native and exotic seeds

are susceptible to predation it should again be pointed out that seed size often appears to be

the primary determinant in seed removal.

Seed Dispersal Seed dispersal may be simply defined as the movement of a seed away from its

parent plant (Nathan and Muller-Landau 2000). Many different mechanisms are employed to

achieve dispersal, ranging from simple and direct to multifaceted and complex. All dispersal

mechanisms can be classified as either primary dispersal or secondary dispersal. Primary

dispersal is simply the initial process in which a seed is dispersed from its parent plant to its

subsequent new location. Modes of primary dispersal tend to be relatively simple methods

including gravity dispersal and wind dispersal. Secondary dispersal refers to additional

movement away from this initial site of dispersal and is typically mediated by a dispersal

agent, such as a frugivorous bird or a scatter-hording rodent.

Another distinct yet linked way of classifying seed dispersal mechanisms are

described as autochorous and allochorous dispersal mechanisms. Autochorous dispersal

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mechanisms are those which achieve dispersal through a plant’s own means. Common

autochory mechanisms include ballistic dispersal and gravity dispersal. Using ballistic or

self-propelled dispersal, seeds can be released under pressure and ejected great distances

from their parent plant. For example, seeds of the orchid tree (Bauhinia purpurea) are known

to disperse up to 14 meters using ballistic dispersal (Stebbins 1971). Similarly in

temperate habitats, the commonly occurring deciduous forest shrub witch-hazel (Hamamelis

virginiana L.) is a well-known example of a species that disperses seeds via ballistic

dispersal (De Steven 1982). The fruit capsule walls of the shrub become woody and dry

throughout the autumn until the seeds can be mechanically dispersed between September and

November (De Steven 1982). Gravity dispersal is another example of autochory in which

larger more rounded seeds can roll many meters from their parent. One well-known example

of gravity-mediated dispersal can be observed in seeds of coconut trees (Cocos nucifera) as

they disperse numerous meters from their parent plant by gravity-mediated primary dispersal.

Allochorous dispersal mechanisms are more commonly observed in seed dispersal

and describe dispersal achieved through external agents. Common mechanisms of

allochorous dispersal include abiotic factors such as wind and water dispersal, or biotic

dispersal such as bird or rodent dispersers. These methods can be tremendously effective at

dispersing seeds great distances. For instance, many birch (genus: Betula) species utilize

wind dispersal through production of light seeds containing sail-like samaras. These features

allow them to disperse great distances, nearly 500 meters in the case of Betula papyrifera

seeds (Greene 1995). Water dispersal is another effective seed dispersal mechanism for

instance, coconuts and coastal mangrove seeds are known to fall directly into water and be

transported great distances before establishing root growth once the seed reaches an adequate

soil environment (Ward and Brookfield 1992).

Perhaps the most complex dispersal mechanism that plants employ to move their seed

away from a parental plant occurs via animal dispersal agents. This could take the form of

the dispersal of fruit bearing species such as European buckthorn (Rhamnus cathartica)

whose fruits are commonly ingested by birds and moved numerous kilometers from the

parent source (McCay et al. 2008). Another example of the use of animals as dispersal agents

is seen in the caching of oak (Quercus spp.) seeds by small mammals such as the grey

squirrel (Sciurus carolinensis). In collecting, caching, and sometimes failing to return and

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consume acorns, squirrels may be viewed as both a predator and disperser of a variety of oak

species. This example illustrates a perceived grey-area between seed predation and seed

dispersal.

For the seeds that have been dispersed and are fortunate enough to evade predation,

many will ultimately integrate into the soil seed bank. The concept of a soil seed bank is

largely attributed to Darwin (1881), and may be defined as the temporary storage of seeds in

the soil. It has historically been viewed as a location of reduced predation pressure (Hulme

1998), where a seed may persist in a dormant state awaiting more favourable germination

conditions. Current research suggests that there are in fact a number of more specialized

natural enemies that reduce the number of seeds in the seed bank such as earthworms and

pathogens (Eisenhauer et al. 2010; Kotanen & O'Hanlon-Manners 2004; Blaney & Kotanen

2002). Despite their presence, seeds still suffer considerably lower predation when

sequestered within a seed bank than when exposed to surficial granivores (Hulme 1998).

When aboveground conditions change, such as the sudden opening of a forest canopy or

disturbance of a meadow soil, the necessary conditions for germination may be met and a

seed can germinate from the seed bank (Fenner 1985).

The length to which different species are able to persist in seed banks is highly

variable. These differences can be so great that seed bank species composition is often not

reflected by standing plant communities (Leckie et al. 2000). Some species are able to persist

in soil seed banks for extended periods of time, even capable of germination after decades or

hundreds of years of dormancy (Conn et al. 2006; Bewley et al. 2007). Although some seeds

are able to persist over extended periods of time, it is more common for seeds to exhibit

measurable declines in viability over time (Conn et al. 2006; Bewley et al. 2007). Persistence

over a shorter time is much more commonly observed; typically seeds of temperate forests

persist in seed banks for 1-2 years (Baskin and Baskin 2001). In many species the initial

period of dormancy can often produces little decline in germinability, while notable

reductions can be seen over longer periods of time (Conn et al. 2006).

The utilization of soil seed banks in more applied and practical settings has increased

in recent years. For instance, transplanting donor seed bank material to recipient areas

undergoing ecological restoration has been suggested for use throughout Ontario (Rideau

Valley Conservation Authority 2000). This method has been used with great success the

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lands surrounding the city of Sudbury, Ontario: the expansion of a local highway enabled the

harvesting of a large quantity of topsoil that was then transplanted to areas where alternative

restoration efforts had proved to yield less desirable results (VETAC 2012). This act of

assisted migration not only enriches the locations available seed bank, but also likely further

ameliorates and conditions the soil for a more desired plant community by introducing

beneficial soil-dwelling organisms.

Seed Dispersal Versus Seed Predation

It is easy to view seed predation and dispersal as two mutually exclusive outcomes,

however in reality there are many examples of the ‘grey area’ where these two concepts

overlap. For instance scatter-hording rodents collecting, transporting and burying acorns may

be seen as a clear example of seed predation; however, if the animal was unable to return to

its cache of seeds and the seeds were able to successfully germinate, the differentiation

between these two ideals becomes challenging. Another such example of simultaneous

dispersal and granivory can be seen in the partial predation and dispersal of an endangered,

large-seeded shrub (Myrcianthes coquimbensis) by a collection of native rodents in a Chilean

desert (Loayza et al. 2013).

Mast seeding is the production and dispersal of irregular pulses of seed. This year-

over-year seed production differential has been suggested to partly function as strategy for

reducing the likelihood that a seed will be subject to predation. Recent efforts have noted this

view may be overly simplified, as Schnurr and colleagues (2002) point out that non-

synchronous masting events between species and preference for certain seeds over others

likely complicates the role of masting as a predator control mechanism.

Granivory & Seed Predation Collectively vertebrate granivores are easily the most studied group of seed predators.

A great deal of literature has quantified the role that these seed predators play as the

prominent granivores in both grassland and forested systems (Falls et al. 2007, Maron et al.

2012). Although their relative importance may shift between communities and is subject to

local abundances, they generally tend to be responsible for the largest proportion of seed

removal, especially in larger sized seeds (Fenner 1985). As well, they are often the easiest

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seed predators to observe and manipulate experimentally which likely plays a role in the

existence of remarkably long-term experiments such as the small mammal survey running

continuously in Algonquin Provincial Park for more than 60 years (Falls et al. 2007). The

vertebrate granivore guilds of North American temperate forests are mainly comprised of

small mammals, such as squirrels and mice, and seed eating birds, such as crossbills and jays

(Falls et al. 2007; Richardson et al. 2013).

Temperate insects have historically been less studied as granivores and herbivores.

Recent works have focused on the study of myrmecochory, seed dispersal by ants, as a

potentially important source of seed dispersal and removal. Ants are often attracted to seeds

with elaiosomes. Some evidence suggests that removal of elaiosomes in some species (e.g.

Sanguinaria canadensis, bloodroot) may result in increases in germination rates (Lobstein

and Rockwood 1993). Myrmecochory has been documented in more than 11,000 plant

species across 55 angiosperm families (Lengyel et al. 2009). A well-documented example of

myrmecochory in Ontario can be seen in the dispersal of white trillium (Trillium

grandiflorum) seeds by the keystone ant disperser Aphaenogaster rudis.

Carabid beetles are another example of insects that are capable of seed predation in

Ontario (Honek et al. 2003). Despite being known granivores for more than 100 years

(Forbes 1883), the omnivorous ground beetles of the Carabidae likely have only a small

effect on the seed cycles of Ontario’s forests due to their relatively low density.

Earthworms are yet another group of granivores that have received increased

attention in recent years. Despite being documented as likely seed predators well over 100

years ago (Darwin 1881), many of the details of earthworm seed predation have yet to be

uncovered. For instance, preference for certain seeds over others (Quackenbush et al. 2012)

could very well play an important role in determining which seeds are available to future

plant communities. The role that these invasive species play in seed dynamics is especially

important given their surprisingly ubiquitous distribution across Ontario.

Earthworms As Invasive Species

The Wisconsonian glaciation event is believed to have eradicated nearly all of

Ontario’s native earthworm species by the time it ended, roughly 11,000 years ago (Reynolds

1977). Since then the province of Ontario has been invaded by at least 17 exotic species of

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earthworms (Evers et al. 2012); only 2 of Ontario’s 19 known species of earthworms are

considered to be native to parts of North America (Reynolds 1977). Most species are widely

distributed across the province, yet many species are found to exhibit patchy distributions,

with spatial gaps between populations. These effective invaders are often able to invade

habitats that many would view as too remote for earthworm invasion (Sackett et al. 2014;

Cameron et al. 2007). Both native species occupy aquatic habitats and are less common than

their invasive counterparts (Reynolds 1977), thus resulting in little concern for their ability to

compete with the many terrestrial inhabiting exotic species of earthworms.

Many of the initial invasions are thought to be the result of discarding earthworm-

contaminated soils used in European ship ballasts during the late 1800’s (Reynolds 1977).

Since their introduction exotic earthworms have demonstrated a tremendous capacity to

rapidly expand their invaded range using both anthropogenic and more natural dispersal

mechanisms, spreading to virtually every region of Canada and the continental United States

(Addison 2009). The ability to tolerate a variety of abiotic conditions has aided earthworms

in successful establishment throughout Ontario (Reynolds 1977) in a wide range of terrestrial

habitats including grasslands, and forests (Evers et al. 2012). At least one species of exotic

earthworm occupies virtually every terrestrial region in Ontario south of 52° latitude (Evers

et al. 2012). They have even been found in high abundances even within some of the few

old-growth forests that remain in Ontario. Perhaps the strongest testament to the remarkable

invasion potential of these exotic species is that they now can be found in every province and

territory throughout Canada (Addison 2009; Reynolds 1977).

As the ecology of Ontario’s forests has developed over thousands of years without

the presence of earthworms or a group that effectively mimics their ecological impact, their

reintroduction over the past century has the potential to yield massive changes to the

dynamic processes on which many forests rely. Since their reintroduction to Ontario’s

forests, less than 150 years ago (Reynolds 1977), we have seen these invasive species change

Ontario ecosystems in profound ways and at alarming rates.

Earthworms are classified into 3 functional groups based primarily on their feeding

ecology and burrowing behaviour (Bouché 1977; Addison 2009). The first group is epigeic,

comprised of earthworm species that are typically found at the soil surface and underneath

the leaf litter layer. They tend to be small in size and have relatively high reproductive rates.

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The second group is endogeic, consisting of species that occupy the mineral horizons of the

soil, moving horizontally throughout the soil profile. The final ecological group of

earthworms which exist in Ontario are anecic species. These species feed on detritus and

plant material from the soil surface, and transport it through the soil horizons using

permanent vertical burrows. Each of the 3 ecological groups includes numerous species that

are considered invasive throughout Ontario. These groups differ in their impacts; however,

all have negative impacts on forest systems (Addison 2009).

Earthworms are capable of causing tremendous changes to the habitats they invade,

often producing negative impacts to the systems and processes that occurred prior to their

introduction (Addison 2009; Bohlen et al. 2004b). They have been linked to such

foundational changes in ecosystems that many have classified them as ecosystem engineers

(Holdsworth et al. 2007), or species that create or substantially modify habitat (Jones et al.

1994). Furthermore it has been proposed that earthworms also may facilitate invasions by

other taxa, perhaps providing evidence for an invasional meltdown or invasive facilitated

invasion (Simberloff and Von Holle 1999). For instance, invasive earthworm species have

been suspected to facilitate the invasion of such problematic invasive plants as Rhamnus

cathartica and Alliaria petiolata (Quackenbush et al. 2012).

Impacts of earthworms on invaded ecosystems may occur by a variety of mechanisms

(Addison 2009). Recent studies have documented both biotic and abiotic impacts (Loss et al.

2012; Bohlen et al. 2004; Hale et al. 2005; Sackett et al. 2013) with the potential to inflict

substantial changes to invaded systems.

One way that earthworms drive major change in systems they invade is through

changing the structure and function of the abiotic environment (Holdsworth et al. 2007;

Bohlen et al. 2004a). Their impacts are especially apparent when examining forest soil

layers. Exotic earthworm species consume and degrade the ecologically important leaf litter

layer while homogenizing the organic and mineral horizons of a soil. This process has

profound importance on the availability and movement of nutrients within forest systems.

Previous studies have attributed changes in soil carbon, nitrogen, and phosphorous cycling,

as well as other biogeochemical processes to exotic earthworms (Crumsey et al. 2013).

These results can vary depending on which species and ecological groups of earthworms are

being studied (Bohlen et al. 2004; Hale et al. 2005; Sackett et al. 2013). Changes to these

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forest biogeochemical cycles may also manifest themselves in changes to soil pH, which has

the potential to bring massive change at the community level (Bohlen et al. 2004).

Earthworm-attributed litter layer removal has also been linked to changes in site

hydrology in worm-invaded forests. Intact leaf litter layers and O horizons are vitally

important in northern hardwood forests as they act as buffers between soil and atmosphere,

maintain soil moisture, and moderate temperature fluctuation throughout surficial soils

(Bormann and Likens 1979). Larson et al. (2010) documented just how great the impact of

earthworm-induced litter removal and successive hydrological changes can be when they

identified historic earthworm invasion using dendrochronology in a sugar maple forest. This

potential to impact a forest’s ability to tolerate stressors such as drought may become

increasingly important as climate change continues to impact ecosystems.

Biotic Impacts Of Earthworms

Increasing attention has recently been given to the biotic impacts that invasive

earthworms can have. Large cascading alterations in entire floral and faunal communities has

been linked to exotic earthworm invasion (Dobson and Blossey 2014; Bohlen et al. 2004;

Addison 2009). The most conspicuous biotic impacts of earthworms are changes in the plant

communities of forest floors. Diversity and abundance of herbaceous species such as spring

wildflowers often severely decline in the native understory of invaded forests (Addison

2009). These changes are often so severe as to be visually apparent: uninvaded forest floors

are dominated by herbaceous plants emerging from an intact leaf layer, while severely

invaded sites are dominated by bare soil. These impacts extend to tree seedlings as well: the

abundance and performance of seedlings can be strongly reduced in invaded areas (Griffith

et al. 2013), perhaps reducing canopy tree recruitment and forest regeneration.

These changes in the flora of the forest floor may stem from multiple causes,

including removal of leaf litter, physical disturbance, and changes in soil nutrient status and

structure (Hofsenberger et al. 2011; Larsson et al. 2010; Hale et al. 2005), however, in many

cases, these changes may themselves reflect biotic changes in the community of mycorrhizal

fungi (Lawrence et al. 2003). These increasingly studied soil fungi are critical to nutrient

uptake in many understory plants and tree seedlings, but can be severely reduced in worm-

invaded areas (Lawrence et al. 2003). In particular, one recent study has shown a reduction

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in arbuscular mycorrhizal fungi in earthworm-invaded sites, suggesting earthworms may be

able to impact the ability of dominant forest species to uptake nutrients (Lawrence et al.

2003). Interestingly, the few plant species that seem to prosper in worm-invaded areas are

often non-mycorrhizal (e.g., Carex, Arisaema). The fact that the severely invasive garlic

mustard (Alliaria petiolata) also is non-mycorrhizal may suggest the possibility of an

invasional meltdown situation. These biotic impacts may extend to other taxa as well. For

instance, a study conducted by Loss et al. (2012) showed that the degradation and removal of

the leaf litter layer in hardwood forests by earthworms was strongly linked to declines in

ground nesting birds.

Some recent efforts have been made in an attempt to understand the potential

interactions between earthworms and regeneration in plant communities. Earthworms have

long been known granivores (Darwin 1881), yet their overall role in the seed cycle has

received little attention. A recent series of lab-based approaches have produced results which

document the potential for earthworms to consume and destroy seeds, in addition to showing

preference for certain species of seed over others (Eisenhauer et al. 2010; Quackenbush et al.

2012). For instance, Quackenbush and colleagues (2012) documented species selectivity

within one earthworm species, attributing at least some of the possible preference to seed

size and morphology. This preferential selectivity may be ecologically important, as it could

set the stage for invasive earthworms to act as ecological filters, potentially shifting the

species composition of a soil seed bank. In addition to actively consuming seeds, earthworms

may also be effectively acting as seed predators by moving and burying seeds to depths

which inhibit successful germination altogether.

1.2 OBJECTIVES

This thesis has been written with two primary goals: to document the potential for

earthworms to act as seed predators in a temperate forest, as well as to quantify the actual, or

measured role that earthworms play in temperate forest seed predation, relative to better-

known groups of granivores.

To document the potential that earthworms have to act as seed predators in a

temperate forest I used a seed-addition microcosm experiment. Results from this experiment

will contribute to our understanding of where earthworm-consumed seeds are transported

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within a vertical soil profile, in addition to developing our understanding of preferences in

seed selection by earthworms. To achieve my second goal I used granivore exclusion

experiments to assess the impact of earthworms compared with other granivores. This

approach will provide useful information as to the importance of earthworms in forest

community granivory, while providing insight as to which species are most at risk to

earthworm seed predation. Specific hypotheses and predictions and will be elaborated on in

the following chapter.

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References Amsberry LK, and JL Maron. 2006. Effects of herbivore identity on plant fecundity. Plant

Ecology 187:39-48

Barnea A, JB Harborne, and C Pannell. 1993. What parts of fleshy fruits contain secondary

compounds toxic to birds and why? Biochemical Systematics and Ecology 21:421-

429

Bewley JD, M Black, and P Halmer. 2007. The encyclopedia of seeds: science, technology

and uses. CABI Publishing

Bohlen PJ, S Scheu, CM Hale, MA McLean, S Migge, PM Groffman, and D Parkinson.

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forests. Frontiers in Ecology and Environment 2:427-435

Bormann FH, and GE Likens. 1979. Pattern and process in a forested ecosystem. New York:

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Cameron EK, EM Bayne, and MJ Clapperton. 2007. Human-facilitated invasion of exotic

earthworms into northern boreal forests. Ecoscience 14:482-490

Clark CJ, JR Poulsen, DJ Levey, and CW Osenberg. 2007. Are plant populations seed

limited? A critique and meta-analysis of seed addition experiments. The American

Naturalist 170:128-142

Conn JS, KL Beattie, and A Blanchard. 2006. Seed viability and dormancy of 17 weed

species after 19.7 years of burial in Alaska. Weed Science 54:464-470

Crumsey JM, JM Le Moine, Y Capowiez, MM Goodsitt, SC Larson, GW Kling, and LJ

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soil carbon dynamics in a sandy temperate forest. Ecology 94:2827-2837

Darwin C. 1881. The formation of vegetable mould through the action of worms.

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De Steven D. 1982. Seed production and seed mortality in a temperate forest shrub (witch

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establishment in deciduous forests of north-eastern North America. Journal of

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Falls JB, EA Falls and JM Fryxell. 2007. Fluctuations of deer mice in Ontario in relation to

seed crops. Ecological Monographs 77:19-32

Fenner M. 1985. Seed ecology. Chapman and Hall, London

Griz LMS and ICS Machado. 2001. Fruiting phenology and seed dispersal syndromes in

caatinga, a tropical dry forest in the northeast of Brazil. Journal of Tropical

Ecology 17:303-321

Honek A, Z Martinkova and V Jarosik. 2003. Ground beetles (Carabidae) as seed predators.

European Journal of Entomology 100:531-544

Jansen PA, F Bongers, and L Hemerik. 2004. Seed mass and mast seeding enhance

dispersal by a neotropical scatter-hording rodent. 74:569-589

Kelrick MI, JA MacMahon, RR Parmenter, and DV Sisson. 1986. Native seed preferences

of shrub-steppe rodents, birds, and ants: the relationships of seed attributes and seed

use. Oecologia 68:327-337

Larson ER, KF Kipfmueller, CM Hale, LE Frelich, and PB Reich. 2010. Tree rings detect

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Lawrence B, MC Fisk, TJ Fahey, and ER Suarez. 2003. Influence of nonnative earthworms

on mycorrhizal colonization of sugar maple (Acer saccharum). New Phytologist

157:145-153

Leckie S, M Vellend, G Bell, MJ Waterway, and MJ Lechowicz. 2000. The seed-bank in an

old-growth, temperate deciduous forest. Canadian Journal of Botany 78:181-192

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global diversification in flowering plants. PLOS One 4:1-6

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five ant dispersed seeds. Virginia Journal of Science 44:59-72

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Maron JL, DE Pearson, T Potter, and YK Ortega. 2012. Seed size and provenance mediate

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of Ecology 100:1492-1500

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determinants and consequences for recruitment. TREE 15:278-285

McCay TS, DH McCay, and JL Czajka. 2009. Deposition of exotic bird-dispersed seeds

into three habitats of a fragmented landscape in the northeastern United States. Plant

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Munoz A, R Bonal, and JM Espelta. 2012. Responses of a scatter-hording rodent to seed

morphology: links between seed variability. Animal Behaviour 84:1435-1442

Prior K, K Saxena and M Frederickson. 2014. Seed handling behaviour of native and

invasive seed-dispersing ants differentially influence seedling emergence in an

introduced plant. Ecological Entomology 39:66-74

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Radtke TM. 2011. Granivore seed-size preferences. Seed Science Research 21:81-83

Reader RJ. 1993. Control of seedling emergence by ground cover and seed predation in

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115:863-873

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vegetation for plant salvage or habitat restoration projects. Rideau Valley

Conservation, Ottawa

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soils. Canadian Journal of Forest Research 42:375-381

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rodent populations and tree seed survival. Oikos 96:402-410

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Chapter 2 Seed Predation in Temperate Forests

2.1 INTRODUCTION

Once a seed has ripened it will disperse away from its parent plant. The seed then

enters a phase is known as the post-dispersal period. This phase can be a critically important

to determining a seed's ultimate fate, as it is typically when seed’s are most vulnerable to

predation (Chambers and MacMahon 1994; Fenner 1985). Some of the most dominant tree

species, such as maples (genus: Acer) and oaks (genus: Quercus) suffer post dispersal

predation of >80%, and sometimes nearly 100% (Meiners 2005; Myster and Pickett 1993).

Despite the fact that sexually reproductive adults of some species are capable of

producing literally billions of seeds over their lifetime, a recent meta-analysis of seed-

addition experiments has shown that many communities are indeed seed-limited (Clark et al.

2007). This could suggest that seed loss has important consequences for the structure of plant

communities. This lost seed is an important food source to faunal communities, exhibited by

its ability to help drive small mammal abundance in temperate hardwood forests (Schnurr et

al. 2002; Falls et al. 2007). In a 36-year study in a temperate hardwood forest, Falls and

colleagues (2007) recorded a number of trends in deer mice populations directly after peak

seed crops. These trends included spikes in population sizes and increases in body mass in

mast years compared to lower seed production years.

Major Granivores

The seed predators of temperate forests fall in two main groups: aboveground

predators and belowground predators. Aboveground seed predators consist mainly of small

mammals such as mice and squirrels, as well as birds such as finches and sparrows (Hsia and

Francl 2009; McCay et al. 2009). The effects they have on seed ecology are relatively well

studied compared to their belowground counterparts.

Certainly one of the most influential groups of post-dispersal seed predators consists

of small mammals. Their collective roles as seed predators and dispersers are well

established in temperate forests. Small mammals such as grey squirrels (Sciurus

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carolinensis), eastern chipmunks (Tamias striatus), and a number of species of deer mice

(genus: Peromyscus) are common and widespread throughout North American forests and

widely viewed as the seed predators responsible for the greatest volume of seed removal

(Falls et al. 2007; Hsia and Francl 2009). They can inflict substantial rates of seed removal

during both pre- and post-dispersal stages (Peters et al. 2009; Hsia and Francl 2009). For

example, in a 3-year observational study quantifying seed loss to white spruce (Picea glauca)

by cone caching red squirrels (Tamiasciurus hudsonicus), the authors estimated substantial

seed losses to these abundant granivores (Peters et al. 2003). Between 48% and 55% of

cones were lost to predation by red squirrels. This loss is considerable when paired with the

authors’ expectations that nearly all seeds that escape red squirrel granivory to be lost to

secondary seed predators such as red-backed voles (Clethrionomys gapperi) and deer mice

(Peromyscus maniculatus) (Peters et al. 2003).

Despite the large quantities of seed that small mammals can remove, they can also act

as critically important seed dispersal agents (Hadj-Chikh 1996). These dispersers tend to

prefer medium to larger sized seeds (Jansen et al. 2004), such as acorn caching by scatter-

hording rodents. This form of dispersal can be particularly effective at dispersing great

distances, as noted by Jansen and colleagues (2004) in a study on rodent dispersal of the

large seeded tree Carapa procera. The study tracked the fate of 3000 seeds, many of which

were dispersed and cached at distances >100 meters from the original source. Seed caches

regularly go unexploited and can result in microsites with favourable germination conditions

(Vander Wall 1995; Jansen et al. 2004). Dispersal to such distances away from the parent

plant has been observed to greatly reduce density-dependent mortality (Vander Wall 2001) as

originally proposed by the Janzen-Connell hypothesis (Janzen 1970; Connell 1971).

Birds are particularly effective seed dispersal agents, yet they are often believed to be

responsible for a lower proportion of temperate forest granivory than the small mammal

guild of seed predators (Pizo and Vieira 2004)). Some common temperate forest bird species

such as jays, finches, and others are known to be granivores (Richardson et al. 2013) and

dispersers to a variety of oak (Quercus spp.) and other high-reward seeds. Although cases of

forest birds as granivores are well documented, the role of birds role in forest community

seed removal is likely overshadowed by abundance and efficiency of small mammals. Their

collective value as dispersers in forest systems, however, is valuable to a number of trees that

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rely on them to disperse seed great distances, at spatial scales often unattainable for most

small mammal seed predators (Vander Wall 2005).

Some carabid beetles are known seed predators (Honek et al. 2003), but a very

limited amount of research has been conducted regarding their ability to act as substantial

seed predators in temperate forest systems. The ability for these beetles to remove a

substantial volume of seed in temperate forests is likely constrained by their limited

abundance (relative to other granivores) and sometimes generalist foraging nature (Honek et

al. 2003). Likewise, while harvester ants are key seed predators in deserts and semi-arid

systems (Mull 2003), such species are absent in most temperate areas of North America.

Instead, most ants in Ontario forests act as dispersers of herbaceous species that produce

elaiosomes, or oil-rich structures that act as rewards to ant dispersers (often found in spring

wildflowers in Ontario) (Prior et al. 2014).

In contrast, earthworms may play an important but underappreciated role as seed

predators. Despite the fact that they occur with tremendous abundance and have been

identified as granivores for over 100 years (Darwin 1881), their role as granivores remains

understudied. Emerging literature on earthworms suggests that they may be rather important

seed predators, especially considering their remarkable invasion success throughout virtually

all of North America (Eisenhauer et al. 2010; Quackenbush et al. 2012).

A handful of recent studies have demonstrated the ability for earthworms to act as

potentially important seed predators. Hopfensperger et al. (2011) collected soil cores from

two forest transects and found that fewer seeds germinated from soil cores collected from

areas containing adult Lumbricus species than sites that contained no adult earthworms.

Other experiments have specifically examined earthworms as seed predators in petri dishes

and other artificial environments. Quackenbush et al. (2012) were even able to document

species selectivity within one Lumbricus species, attributing at least some of the possible

preference to seed size and morphology. In a series of simple choice experiments, seeds of

Alliaria petiolata were preferentially selected over a number of other species of seeds

(Quackenbush et al. 2012). Earthworm ingestion is not necessarily fatal to a seed, as it may

be egested along with the worm's castings. However, many seeds are killed, either directly by

digestion, or indirectly by burial to depths from which successful germination is impossible.

The impact of egestion on a seed is believed to be variable among species (Quackenbush et

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al. 2012).

Despite these examples, surprisingly few studies have examined the ecological

implications of earthworm seed predation, and nearly all have focused on agricultural

systems (Lee 1985). This is unfortunate given the abundance of exotic earthworms in North

American forest ecosystems (Reynolds 1977). Could some of the negative impacts worms

have on understory plants be the results of seed predation? How do these compare with

effects of other seed predators, such as rodents? In this study I conducted a field and

microcosm experiments in a mixed forest in Southern Ontario, using the seeds from a

number of commonly occurring forest plants and one of the most common and largest

species of invasive earthworm to further investigate this issue. Looking at both the direct

(consumption and digestion) and indirect (burial) removal of seed from temperate forests will

help us better understand the impact that these abundant invaders will continue to have on

the landscapes they invade.

Objectives And Hypotheses The primary goal of this study is to advance current understanding of the impacts that

invasive earthworms have regarding community-level seed predation in temperate forests. To

do this, I have conducted a series of experiments to quantify 1) the potential that earthworms

have to act as important seed predators and 2) the actual, or measured impact that these

invasive species have, relative to better-studied guilds of seed predators.

The first experiment will examine the extent to which earthworms are capable of seed

predation in isolation from other seed predators. Using a seed-addition microcosm

experiment I aim to determine the following:

Question 1. When isolated from other granivores are earthworms capable of removing a

substantial quantity of seed from the soil surface?

Hypothesis 1. In the presence of earthworms a significant amount of seeds are removed from

the soil surface.

Question 2. What happens to those seeds removed from the soil surface? What proportion is

removed outright (digested), buried to a depth that likely enables germination, or buried to a

depth which likely inhibits germination?

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Hypothesis 2. Earthworms are capable of significantly reducing the amount of seeds

available to germinate through outright removal or burial to a depth that likely inhibits

germination.

Question 3. Do earthworms remove certain species of seed over others? Do traits such as

seed mass or species provenance seem to guide preferences?

Hypothesis 3. Certain species of seed are removed from the soil surface at greater rates than

others. For example, seeds of smaller mass will be removed from germinable depths at

greater frequency than seeds of larger mass.

The second experiment outlined in this chapter will attempt to place earthworm-seed

predation in context of other guilds of seed predation in forest systems. Using a seed-

addition, predator-exclosure approach I will attempt to answer the following questions:

Question 4. What proportion of seed predation can be attributed to earthworms, relative to

other guilds of seed predators?

Hypothesis 4. As previous studies have identified, a large proportion of seed predation is

attributed to aboveground seed predators such as small mammals. I expect a smaller, but

considerable proportion of seed loss will be attributed to earthworms.

Question 5. Are different species of seed removed at different rates? If so, what traits may

help explain these preferences? Seed mass? Species provenance?

Hypothesis 5. Larger species of seeds will be removed in greatest abundance. We may also

see native seeds being removed in greater abundance than their exotic congeneric relatives.

Lastly, to ensure accuracy and precision of the methods employed in this study, and

ensure reliable interpretation in their results, I will outline a series of controls which help to

determine if my methods may be introducing biases into my results. Specifically, I will test

whether predation rates on my seeds are affected by UV-marking and the presence of leaf

litter, and whether seeds removed by earthworms may nonetheless be capable of successfully

emerging from burial.

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2.2 METHODOLOGY

Site Information Experimental work was conducted at the University of Toronto’s Koffler Scientific

Reserve at Jokers Hill (KSR) (website: www.ksr.utoronto.ca), King Township, Ontario

(44°02’25” N79°32’00” W). This property falls within the Mixedwood Plains Ecozone

(Ecoregion 6E), as defined by the Ontario Ministry of Natural Resources and Forests. The

Ministry has further classified the property and its surrounding conservation lands as an Area

of Natural and Scientific Interest (ANSI). Furthermore, the property sits within one of the

province’s more significant landforms, the Oak Ridges Moraine.

Although the KSR property is comprised of a diverse range of habitats from aquatic

to terrestrial, this portion of research was conducted exclusively within the forested section

of the property. Nearly all forest stands are mature secondary growth, often dominated by

Acer saccharum, Fagus grandifolia, Tsuga canadensis and Quercus rubra. These stands

connect to surrounding stands of mixed forest and form a network of natural land cover that

is uncommon throughout much of Southern Ontario. The soils within the study site are

similar to many of the forested areas on the Moraine. They typically consist of a thin (<5cm)

layer of detritus, sitting atop an organic horizon often less than 10 cm. These organic layers

sit atop many meters of nearly pure sand.

Species Information A set of 23 locally occurring and ecologically important woody and herbaceous forest

species, including a variety of seed sizes and both natives and exotics, were selected for use

in the experiments detailed in this chapter (see Table 1). Attempts were made to select

species that do not contain elaiosomes to mitigate any effect of myrmecochory.

A pair of summer and fall granivore-exclusion field experiments utilized the greatest

richness of seeds, 21 and 22 species respectively. Both trials used highly similar sets of

species, however a few changes were necessary due to seed availability; efforts were made to

replace unavailable species of seed with ecologically similar (seed size, native/exotic, etc.)

species. The earthworm seed predation microcosm experiment utilized a subset of 6 species

(Table 1) also used in the granivore exclusion experiments. The 3 methodological control

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experiments outlined in this chapter used either 5 or 6 species (Table 1) of seed also

previously utilized in the granivore exclusion experiments.

All seeds used in each experiment were marked using day-glow highlighter pens with

inconspicuous UV-fluorescent dyes. The effectiveness of the dye-marking method did not

diminish over the duration of the experiment. Seeds exposed to field conditions for more

than 18 months were still able to be recovered using the UV-marking method.

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Table 1 Native and exotic species used in the granivore exclusion, earthworm granivory, and 3 method control experiments, as

ordered by seed mass. Seed mass represents the mean weight of 200 individual seeds randomly selected from the same seed stock

used in each experiment. “*” indicates use of the noted species in each experiment.

Origin Species Mean Seed Mass (mg)

Earthworm Microcosm

Field Experiment

Summer Exclosure

Field Experiment

Fall Exclosure

Field Experiment

Leaf Litter Field

Experiment

Seed Marking

Field Experiment

Germination from Depth Greenhouse Experiment

Betula papyrifera 0.3 * * Betula alleghaniensis 1.1 * * * * Larix laricina 1.7 * * Tsuga canadensis 2.6 * * Lonicera canadensis 3.0 * * * * * Pinus banksiana 3.8 * * Pinus strobus 10.8 * * * * * * Native Acer rubrum 12.9 * * Maianthemum racemosum 28.5 * * Fraxinus americana 56.3 * * Prunus serotina 76.6 * * * * * * Acer saccharum 92.7 * * * * * * Tilia americana 117.8 * * Carya cordiformis 2905.0 * * * Carya ovata 4331.0 * Quercus rubra 4790.0 * * * Betula pendula 0.1 * Alliaria petiolata 2.6 * * * * Exotic Pinus sylvestris 6.2 * * Berberis thunbergii 17.0 * * * Robinia pseudoacacia 19.4 * * Rhamnus cathartica 23.1 * * Acer platanoides 97.7 * *

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Experimental Design

Earthworm Microcosm Field-Experiment A microcosm experiment was established in a 10m x 10m plot of mixed forest (>25%

deciduous and <75% coniferous by canopy) at the Koffler Scientific Reserve in the first

week of September 2013. The plot had a nearly level grade and was dominated by mature A.

saccharum, Pinus strobus, and Tsuga canadensis while containing <5% herbaceous ground

cover. The litter layer within the plot was intact and nearly continuous throughout the entire

plot.

PVC tubing was used to create sixty (length 30cm, diameter 15cm) earthworm

microcosms. Each tube was filled with soil that was sieved from the site using a 1cm sieve to

remove any large earthworms and coarse debris. Fiberglass window screening was fixed to

the bottom of each tube to permit drainage. Tubes were secured into the soil by partially

burying each microcosm (approximate depth 15cm).

Earthworms were purchased from a local fishing bait distribution company

(nationalbait.com), and confirmed to be common nightcrawlers, Lumbricus terrestris. L.

terrestris was selected as the most appropriate earthworm species due to its high abundance

relative to other exotic species of earthworms at this site (Choi 2011; Krajewski and Haroon

unpublished data), its use in other related research (Quackenbush et al. 2012; Eisenhauer and

Scheu 2008), and its large size; this anecic species is among the largest species of

earthworms in Ontario (Reynolds 1977), making it a potential predator of seeds over a large

range of sizes.

After purging for 24 hours on moist filter paper, a single earthworm was added to 54

of the 60 microcosms. Those tubes that received an earthworm were designated as treatment

units, while the 6 tubes that did not received an earthworm were designated as control units.

All tubes were left undisturbed for 7 days to allow added earthworms to adjust to their

environments. Fifteen seeds from 1 of 6 different possible species were then added to each of

these 54 tubes, resulting in 9 randomly selected replicates per seed species; the species of

seed used in this experiment were all locally occurring species, including four native trees,

one exotic shrub, and one herbaceous invader (Table 1; Appendix 1). Each of the remaining

6 control tubes received 10 seeds from each of the 6 species (60 seeds/tube) to estimate rates

of seed burial in the absence of worms.

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After seed addition, the top of each tube was covered with fiberglass screening that

was secured with a rubber band to allow for easy visual inspection throughout the course of

the experiment. All seeds used in both control and worm treated tubes were marked using

day-glow highlighter pens with inconspicuous UV-fluorescent dyes, in order to facilitate

recovery; trials indicated UV-marking did not affect rates of seed removal (See section

2.2.3.3.1).

Two weeks after seed addition the microcosms were removed from the field and

transported to the lab. The soil-filled tubes were randomized then stored in two 4°C

refrigerators for no longer than 5 days as processing took place. Cold storage was used to

slow down the metabolic rate of the earthworms and reduce the likelihood of further seed

movement. Individual tubes were then removed from the refrigerator and processed in a

randomized manner. Upon removal from soil worms appeared lethargic until they warmed to

room temperature suggesting activity during refrigerator storage was limited. Soil was

carefully excavated from each tube in predetermined layers (<1cm, 1-3cm, 3-5cm, etc.) and

examined for seeds using a powerful ultraviolet flashlight (HQRP 390nM UV LED). Once

the soil from each microcosm had been exhaustively searched, it was spread evenly (~3cm

depth) within a standard greenhouse flat (28cm x 54cm x 6cm) and transported to a growth

chamber. Soils were then exposed to 12-hour day/night cycles and watered daily to ensure

any unrecovered seeds did not remain in the soil. Soil was disturbed 4 weeks after initial

placement in the growth chamber ensuring any potential seeds were exposed to the soil

surface. Soils remained in the growth chamber for a total of 6 weeks. Only one single seed

(B. thunbergii) was uncovered during the growth chamber control. All analyses were

conducted with and without its inclusion, yielding virtually identical results. For simplicity,

it has been omitted from all reported analysis.

Relative Role Of Earthworms & Granivores

Summer Trial

This experiment was designed to monitor and compare removal rates of different

species of seeds by belowground seed predators. Twelve sites were selected throughout the

property’s mixed hardwood forest. Each site consisted of a 10m x 10m plot whose corners

were marked using coloured flagging tape. Care was taken to ensure sites were spaced

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>100m apart from the next closest site, and a minimum of 25m from the closest footpath.

Each site contained 2 replicates of 2 different 0.1m2 (diameter 0.36m) treatment units

fastened to the ground with three 15cm ground staples: an exclosure designed to exclude

access by belowground seed predators (primarily earthworms), and a control that permitted

access to both above- and below-ground seed predators. These open-top cylindrical

exclosures covered the same footprint (height ~3cm) as control units. Cylindrical frames

were constructed using 1.25cm2 hardware cloth and plastic zip ties. The floor and sides of

these units were then lined with fiberglass window screen to prevent small insects and other

belowground seed predators from penetrating the exclosure. The upper 3cm of the leaf litter

and O horizon was carefully removed and placed into the exclosure unit, making the opened

exclosure top flush with the surrounding leaf litter. Thin metal wire was used to delineate

0.1m2 circular control units. These were placed atop the leaf litter layer that was manually

disturbed to mimic the disturbance applied to the belowground treatment. Exclosures were

installed on 12 July 2013 and left undisturbed for 2 weeks to allow for naturalization to their

environment.

On 29 July 2013, early in the annual cycle of seed dispersal and 2 weeks after plots

were established, seeds were added to each experimental unit. A set of 21 species (13 native

and 8 exotics) was selected based on a range in seed size, native vs. exotic status, and local

abundance (Table 1). For each experimental unit 12 seeds from each of the 21 species were

counted and marked with an ultraviolet fluorescing highlighter, and then added to their

respective experimental unit. To establish how granivory changes over time, one replicate of

each treatment type was randomly selected for collection 2 weeks after seed addition (12

August). The remaining units were collected 4 weeks after seed addition (26 August);

previous work at this site suggested that this would be sufficient time for significant seed

removal to have occurred.

Fall Trial

The twelve 10m x 10m plots used in the summer seed removal trial were also used in

the fall trial. The experimental design for the summer exclosure experiment was replicated

during the fall exclosure experiment, with the addition of an aboveground exclosure

treatment and a slight modification to the seed species used (see Table 1). A full factorial

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design including above and below exclusion within one treatment unit was briefly

considered, however was not pursued due to the considerable processing time the design

would have required. Aboveground exclosure units were constructed using 1.25cm2

hardware cloth that was formed into a roofed but bottomless cylinder (height 0.25m,

diameter 0.36m) using plastic zip ties. These exclosures were then fitted with a 10cm wide

strip of metal flashing around the perimeter of each cage, buried flush to the soil surface

using a spade. The flashing was necessary to prevent burrowing by small rodents, as became

apparent during summer trials. Experimental units were placed in the field on 22 August

2013. Seed addition occurred on 5 September 2013, well into the annual cycle of seed

dispersal. The first sample harvest (1 unit from each treatment at each site) was collected 2

weeks after seed addition, on 19 September. The second harvest then occurred 4 weeks after

seed addition, on 3 October 2013.

Wildlife Cameras

During both summer and fall granivory experiments, motion-sensitive trail cameras

(Primos® Truth Cam Blackout model) were placed at 5 randomly selected sites prior to seed

addition. These cameras took photos both in daylight and at night, using an invisible infrared

flash. The cameras remained in place for the duration of the experiment to provide

qualitative information on potential seed predators and impact of human disturbance.

Wildlife camera footage indicated that exclosures were regularly visited by both diurnal

(e.g., Eastern grey squirrels, Sciurus caroliniensis, red squirrel, Tamiasciurus hudsonicus,

and Eastern chipmunk, Tamias striatus) and nocturnal (deer mice, Peromyscus sp., and

Southern flying squirrel, Glaucomys volans) rodent seed predators.

Sampling of Summer and Fall Exclosure Experiments To collect aboveground exclosure units, cages and ground staples were manually

removed. The aboveground material and top 3cm of soil was then excavated and collected

for further analysis in lab. Control units were collected in a similar way. Contents of

belowground exclusion treatments were removed from the ground and soil and detritus

contents were emptied into collection bags. Samples were then returned to the lab and stored

in a -4°C freezer until further processing. To process samples, bags were emptied onto a tray

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and left to thaw and dry at room temperature for >4 hours. Soil was then visually scanned

with an UV LED flashlight (HQRP Professional®) to search for seeds. Number of whole

seeds were then recorded and scored as ‘viable’, while any highlighted seed fragments were

set aside and later pieced together to estimate the number of seeds represented by fragments.

Seed Marking Control Experiment

This experiment was designed to test whether the UV marking used on experimental

seeds affected their removal rate. The marked and unmarked control trial was conducted in a

separate area of the same mixed deciduous forest. Plots from either experiment were spaced

no closer than 200m from each other.

Six sites were established within a mixed deciduous forest. At each site 2 circular

quadrats (area = 0.1m2) were placed approximately 5 meters from each other. The leaf litter

within each quadrat was disturbed to simulate the effects of litter disturbance outlined in the

previous experiments. Each quadrat was randomly selected to be treated with either marked

or unmarked seeds of 5 native species (C. cordiformis, A. saccharum, P. serotina, P. strobus,

L. canadensis). These species were selected based on a range in seed size, local abundance,

and ecological importance (Table 1, Appendix 1). Eight seeds from each species were either

marked using a UV fluorescent highlighter or remained unmarked, and then added to their

respective treatment quadrat at each of the 6 sites (5 species x 8 seeds = 40 seeds/quadrat).

Quadrats were then visually monitored daily for the next 8 days to estimate seed removal.

Care was taken to not disturb the plots until the end of the experiment. After 8 days the

surface litter and upper 3cm of soil was excavated and placed in a plastic bag; a short

exposure period was used since data indicated most seeds were removed in only a few days.

Samples were then frozen in a -4°C freezer until they could be visually searched using either

a UV flashlight or LED spotlight, depending on treatment type.

Leaf Litter & Seed Predation Control Experiment Aside from directly consuming seeds, earthworms may increase seed predation by

rodents by removing protective leaf cover. This experiment was designed to test whether the

removal of litter from the forest floor by earthworms affected rates of seed removal. The

litter and no-litter control trial was conducted in yet another area of the same patch of mixed

!

!

!

30!

deciduous forest that was used in previously mentioned experiments. Plots from this

experiment were no closer than 200m from any other plot from any other experiment.

Ten 5x5m sites were selected within a patch of mixed deciduous forest. Sites were no

less than 100m from each other and no less than 25m from the nearest footpath. Two 0.1m2

circular quadrats were fashioned using metal wire and placed approximately 5 meters from

each other. One quadrat was designated the no-litter treatment and placed in the center of a

1m x 1m area that was raked clear of any surficial detritus. In the other plot, the surface leaf

litter was carefully removed, slightly disturbed and replaced to mimic disturbance of other

treatments. Quadrats were fixed to the soil using 3 ground staples.

Six native and one exotic (Table 1) forest plant species were selected based a range in

seed size and local abundance. 10 individual seeds from each of the 7 species were marked

using a UV fluorescent highlighter and added to each quadrat. Seven days after seed addition

the upper 3cm of soil and any detritus layer was collected and placed in a plastic bag.

Samples were then returned to the lab and stored in a -4°C freezer until further processing.

To process samples, bags were emptied onto a tray and left to thaw and dry at room

temperature for >4 hours. Soil was then visually scanned with an UV LED flashlight to

search for seeds. Number of whole seeds were then recorded and scored as ‘viable’, while

any highlighted seed fragments were set aside and later pieced together to estimate the

number of seeds represented by fragments.

Germination From Burial Depth Control Experiment

A greenhouse control study was conducted to determine the maximum depth at which

each species could successfully germinate. Twelve seeds of 6 species (see Table 1) were

buried in each pot varying depths: (1) soil surface, (2) 3cm below surface, (3) 5 cm below

surface, and (4) 7 cm below soil surface. Pots were filled with a standard Promix potting soil.

Each pot was replicated 3 times for each of the 6 species (72 pots total). Pots were kept in a

greenhouse and exposed to mean temperatures of ~21°C and approximately 12-hour

day/night cycles. All pots were watered twice weekly or as needed. Pots were monitored for

90 days, and any seedling emergence was recorded.

!

!

!

31!

Statistical Analysis

All analyses presented in Chapter 2 were conducted using JMP 11 (SAS Inc. 2012).

When applicable, the number of recovered seeds was converted into a proportion which was

then transformed using the arcsine squareroot transformation in an attempt to better meet the

assumptions of Analysis of Variance (ANOVA). In the earthworm microcosm experiment

treatment and control microcosms were both analyzed using ANOVAs and subsequent

Tukey-Kramer HSD tests. ANOVAs were performed separately for each burial depth (<1cm,

<5cm, >5cm, and removed seeds). This strategy effectively separates seeds into germinable,

possibly germinable, and unlikely germinable and outright removal; a repeated-measures test

was not used since these categories are non-independent. ANOVA assumptions were not met

from seeds recovered at certain depths (e.g., assumption of normality in <1cm from soil

surface in worm exposed treatment) and therefore related results should be interpreted with

caution. To remedy those instances of compromised ANOVA assumptions at some burial

depths, results from a non-parametric Kruskal-Wallis ranked-sums test are also reported;

these have generally produced very similar results to parametric tests.

For analysis of the exclosure experiments (both summer and fall), transformed data

were analyzed using a randomized block factorial ANOVA (Kirk 1995) using a Restricted

Maximum Likelihood (REML) approach. "Species" was treated as a fixed effect (as the

species used were a nonrandom set deliberately chosen to include a representative variety of

species, see Appendix 1 for more information) and "treatment" (Summer: protected from

worms, or open and Fall: protected from worms, rodents, or open) was treated as a crossed

fixed effect. "Site" was used as a random blocking factor; "plot" was not directly included in

the model, since plot effects are equivalent to the residual site x species x treatment

interactions. In subsequent analyses each species also was analyzed separately, using a

randomized block model ("treatment" as a fixed factor, and "site" as a randomized block).

Tukey-Kramer HSD tests were used to further explore significant relationships. ANOVA

assumptions failed to be met in some cases (e.g. assumption of normality was poorly met for

numerous seed species) and as such results from nonparametric tests may best be interpreted

with caution. Since ANOVA assumptions often were poorly met in some cases, results for

each species also were analyzed with Wilcoxon Rank-Sum tests (X2 approximation), with

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!

!

32!

"site" as a blocking factor; results were generally very similar or slightly stronger than for

parametric statistics and have also been included.

Results for the UV control and litter experiment were analyzed using the same REML

ANOVA models outlined in the granivore exclosure experiments. For both of these

experiments, a randomized block factorial model was used (treatment x species); "site" was

treated as a blocking factor, and "treatment" and "species" were fixed; again, "plot" is

equivalent to the site x treatment interaction.

2.3 RESULTS Earthworm Microcosm Experiment Results

Overall Removal of Seeds

Effectively all (99.9%) of the 1093 seeds that were recovered from the microcosm

experiment were discovered using visual inspection aided by ultraviolet light. Although

germination trials may tend to be biased by nature, only a single seed (<0.1%) was recovered

by germination from soil incubated in the growth chamber. Consequently, the following

discussion includes only visually detected seeds.

The recovery rate for all species combined recovered <1cm from the soil surface was

27.2%±4.1% (all values are mean ± SEM) (Table 3). Nearly all of the seeds missing from the

top cm of soil were likely removed by earthworms: recovery from the top cm of control tubes

without worm addition was nearly 100% (97.8%±0.7%) for all species combined (Table 3;

Figure 1).

Results were similar for seeds recovered from the top 5cm of soil, though recovery

rates were increased, reflecting burial deeper than 1 cm (Figure 1). When all species are

combined, only 45.7%±4.3% (Table 3) of seeds remained within 5cm of the soil surface

when exposed to earthworms, while 98.0%±0.7% (Table 3) of seeds in control tubes were

recovered from the upper 5cm.

Comparatively a relatively small number of seeds were recovered at depths below

5cm than at shallower depth profiles. In the presence of earthworms only 23.8%±3.7% of

seeds were recovered buried >5cm deep while 0% were recovered in control tubes at the

same depth (Table 3).

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!

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33!

Finally, in the presence of earthworms, a total of 30.5%±5.0% of seeds were removed

outright and assumed digested (Table 3). Control tubes, which did not contain earthworms,

yielded a negligible loss of seeds (2%±0.7%) likely attributable to experimental error and

cracks in the soil surface.

Seed Species Preferences

All species of seed used in this experiment experienced surficial removal at some

rate, however considerable differences were apparent between species especially when

considering the depth at which the seeds were recovered (Table 3, Figure 1). For instance,

ANOVA and post-hoc results indicate significant differences among species in rates of

removal from the soil surface (<1cm: F5,48 = 9.9009, p = <0.001) (Tables 2, 3). Data poorly

met ANOVA assumptions; however Kruskal-Wallis tests produced essentially identical

results overall [X25 =24.9968, p = 0.0001]. Tukey HSD tests found P. strobus seeds were

recovered in significantly (p = < 0.0001) greater abundance (73.3%±5.7%) on the soil

surface than all other species used in this study, providing evidence for preferential feeding

habits by earthworms (Table 3). Each of the remaining species were recovered in much

lower rates; for example, <10% of A. petiolata seed was recovered within the upper 1cm;

however, there were no significant differences among other species (Table 3, Figure 1).

Recovery rates for seeds buried <5cm again differed significantly (p < 0.0001) among

species (Tables 2, 3). Kruskal-Wallis tests produced essentially identical results overall [X!!

=29.2198, p = <0.0001]. Again, white pine appears to suffer the least damage compared to

the other 5 species, as 85% of seeds were recovered within the upper 5cm of the soil profile

(Table 3, Figure 1). More generally, >50% of the two largest-seeded species were recovered

within the upper 5cm of the soil profile, compared to <25% of the while the 2 smallest-

seeded species. Tukey-Kramer results showed that again P. strobus was recovered in

significantly greater abundance than nearly all other species (p < 0.0001), again providing

evidence for preferential feeding (Table 3). Tukey-Kramer results provide further evidence

for preferential feeding with respect to A. petiolata, whose seed was removed in significantly

(p < 0.005) greater abundance than nearly all other species (Table 3, Figure 1).

Results for seeds recovered at depths >5cm again indicated significant (p < 0.0001)

differences exist among species (Tables 2, 3). Kruskal-Wallis tests produced essentially

!

!

!

34!

identical results overall [X25 =31.0968, p = <0.0001]. Seeds of the 3 smallest species were

recovered in very low abundance, compared to the 3 largest species (Figure 1).

Finally, results indicate significant (p < 0.0001) differences among species regarding

number of seeds digested and ultimately removed by earthworms (Tables 2, 3). Kruskal-

Wallis tests produced essentially identical results overall [X25 =42.6842, p = <0.0001]. Once

again Tukey- Kramer analyses indicate the two smallest seeded species were removed at

significantly (p < 0.0001) greater rates than all other species of seed (Tables 2, 3). More

specifically Alliaria and Betula were removed at 89% and 68% respectively, while the four

larger species were removed at rates lower than 15% (Table 3). This suggests the two

smallest species of seeds are largely responsible for increasing the mean rate of seed removal

across all species to 30.5% (Table 3).

! 35!

Table 2 Results of ANOVA testing the proportion of seeds recovered at notable ecological

fates after being exposed to earthworms (n=9). Significant results in bold. Depth <1cm <5cm >5cm Removed DF F Ratio Prob>F DF F Ratio Prob>F DF F Ratio Prob>F DF F Ratio Prob>F Species 5 9.9009 <0.0001 5 11.0060 <0.0001 5 12.6412 <0.0001 5 69.5863 <0.0001 Error 48 48 48 48 Total 53 53 53 53

! 36!

Table 3 Results of Lumbricus terrestris seed predation microcosm experiment for each species: mean proportion of seeds

recovered ±SEM (n=9 replicates per species for worm addition treatment, n=6) for controls) for 6 species at various ecologically

meaningful depths. Significant results are shown in bold. Species are listed in ascending order of mean seed mass. Also shown are

results of separate Tukey-Kramer tests for each worm treatment at each depth (species at that depth sharing the same letter do not

differ significantly: p > 0.05).

Treatment Species Surface: Buried <1cm

Tukey-Kramer

Germinable: Buried <5cm

Tukey-Kramer

Buried at Depth: Buried >5cm

Tukey-Kramer

Removed: Seed Not

Recovered

Tukey-Kramer

Worm Betula alleghaniensis 0.185±0.052 B 0.252±0.038 BC 0.067±0.031 B 0.681±0.051 B Addition Alliaria petiolata 0.089±0.038 B 0.111±0.046 C 0 ±0 B 0.889±0.046 A

Pinus strobus 0.733±0.057 A 0.852±0.047 A 0.074±0.034 B 0.074±0.026 CD Berberis thunbergii 0.296±0.117 B 0.467±0.102 B 0.4±0.086 A 0.133±0.037 C Prunus serotina 0.230±0.057 B 0.504±0.083 B 0.496±0.083 A 0±0 D Acer saccharum 0.096±0.073 B 0.556±0.103 AB 0.393±0.101 A 0.052±0.024 CD

Combined Sp. Mean 0.272±0.041 0.457±0.043 0.238±0.037 0.305±0.050 Worm-free Betula alleghaniensis 0.956±0.022 A 0.956±0.022 A 0±0 A 0.044±0.022 A

Controls Alliaria petiolata 0.944±0.027 A 0.956±0.022 A 0±0 A 0.044±0.022 A Pinus strobus 1.000±0.000 A 1.000±0.000 A 0±0 A 0±0 A Berberis thunbergii 0.978±0.022 A 0.978±0.022 A 0±0 A 0.022±0.022 A Prunus serotina 1.000±0.000 A 1.000±0.000 A 0±0 A 0±0 A Acer saccharum 0.989±0.011 A 0.989±0.011 A 0±0 A 0.011±0.011 A

Combined Sp. Mean 0.978±0.008 0.98±0.007 0±0 0.02±0.007

! 37!

Figure 1 Mean proportion of seeds recovered ± SEM, at various depth profiles within

microcosms exposed to (worm addition) (n=9) and protected from (controls) (n=6) the

granivorous earthworm Lumbricus terrestris. In part A, species at each depth sharing the

same letter do not significantly differ; in part B, no significant differences among species

were detected Seed species are ordered by increasing seed size.

0.0

0.1

0.20.3

0.40.5

0.6

0.70.8

0.91.0

Prop

ortio

n R

ecov

ered

B

BB

Betu

la a

llegh

anie

nsis

Allia

ria p

etio

lata

Pinu

s st

robu

s

Berb

eris

thun

berg

ii

Prun

us s

erot

ina

Acer

sac

char

um

0.00.1

0.2

0.30.4

0.50.6

0.7

0.80.9

1.0

Prop

ortio

n R

ecov

ered

A) Worm addition

B) Controls

<1cm

<5cm

>5cm

Not recovered

B

B

BC

B C

B

A

A

A

BCD

B

B

A

C

B

B A

D

B

AB

A

CD

!

!

!

38!

Granivore Guild Experiment Results

Summer Trial

For the summer trial of the granivory guild experiment, exclusion of belowground

seed predators was compared to control plots in which seed was available to both

belowground and aboveground seed predators. At the 2-week sampling all fixed factors were

significant (p<0.05) in the full ANOVA model (Table 4). More seeds were recovered from

plots that excluded belowground granivores than from control plots. The model displays a

significant interaction between treatment and species, indicating that the effect of treatment

differs between species (Table 4). When analyzed separately differences between treatments

at the species level became apparent (Table 5). Individual F-tests indicate that after 2 weeks

of exposure to predation, 9 of the 21 species had significantly (p<0.005) greater recovery

when belowground seed predators were excluded (Table 5). Wilcoxon Rank-Sum tests also

identified each of these species as significantly different between treatments; in addition,

results for Pinus banksiana were marginally significant (p=0.049). In all cases, recovery was

higher in below-ground exclosure treatments than controls (Table 5).

After 4 weeks of exposure to granivores both exclusion treatment (p=0.0007) and

species (p<0.0001) were found to have significant effects, while the interaction between was

not significant (Table 4). As expected, many species showed recovery rates that are lower

than those found after just 2-weeks exposure to granivores; however, only one species

(Betula allegheniensis) differed among treatments according to parametric statistical tests,

likely because higher background rates of removal by vertebrate seed predators obscured any

patterns (Figure 2). In addition to this example, Wilcoxon Rank-Sum tests identified

differences among treatments for Larix laricina (P = 0.007), Maianthemum racemosum (P =

0.020), Berberis thungberii (P = 0.044), Betula pendula (P = 0.048). In all cases, recovery

was higher in below-ground exclosure treatments than controls (Table 5, Figure 2).

! 39!

Table 4 Results of comprehensive randomized block factorial ANOVAs from summer

granivore exclosure experiment (n=12). Site used as a blocking factor; "Treatment" =

Control, Above-ground exclosure, or Below-ground exclosure; "Species" = species tested

(Table 1). Significant results (p < 0.05) are indicated in bold.

Sampling Week 2 Week 4 DF F P DF F P Treatment 1,451 38.37 <0.0001 1,451 11.54 0.0007 Species 20,451 47.89 <0.0001 20,451 45.55 <0.0001 Treatment x Species 20,451 2.12 0.0035 20,451 0.87 0.6244

!

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!

40!

Table 5 Results of randomized block ANOVAs for recovery of seeds of each species.

Significant results (p<0.05) are indicated in bold. Also shown are results of F-tests

comparing controls (C) and below-ground exclosures (treatment "B"); "-" means no

significant result was detected (p>0.05).

2 Weeks 4 Weeks

Origin Species F1,11 P Treatments F1,11 P Treatments Native

Acer rubrum 0.13 0.7236 - 0.58 0.4632 - Acer saccharum 0.00 1.0000 - 1.00 0.3388 - Betula alleghaniensis 10.09 0.0088* B>C 6.21 0.0300* B>C Betula papyrifera 2.37 0.1520 - 1.55 0.2395 - Carya cordiformis . . - . . - Fraxinus americana 1.00 0.3388 - 1.39 0.2632 - Larix laricina 23.81 0.0005* B>C 3.93 0.0728* - Lonicera canadensis 5.39 0.0405* B>C 2.75 0.1257 - Maianthemum racemosum 1.30 0.2785 - 3.23 0.0996* - Pinus banksiana 2.34 0.1544* - 1.16 0.3045 - Pinus strobus 6.68 0.0254* B>C 0.09 0.7737 - Prunus serotina 1.00 0.3388 - 1.00 0.3388 - Quercus rubra . . - . . - Tilia americana 0.52 0.4849 - 0.01 0.9107 - Tsuga canadensis 9.62 0.0101* B>C 1.73 0.2146 -

Exotic

Acer platanoides 0.00 1.0000 - . . - Berberis thunbergii 7.63 0.0185* B>C 3.72 0.0800* - Betula pendula 10.52 0.0078* B>C 1.71 0.2177* - Pinus sylvestris 0.16 0.6951 - 1.24 0.2888 - Rhamnus cathartica 7.61 0.0186* B>C 0.66 0.4336 - Robinia pseudoacacia 14.68 0.0028* B>C 0.0044 0.9483 -

* Significant in Wilcoxon Rank-Sum tests (P<0.05)

! 41!

Figure 2 Mean proportion of seeds recovered ± SEM, after 2 and 4 weeks exposure to

various granivore treatments in a summer exclosure experiment (n=12). Treatments: "Below"

= below-ground exclosure, "Control" = no protection. For both natives and exotics, species

are ordered by increasing seed size.

Natives Exotics

Betu

la p

apyr

ifera

Betu

la a

llegh

anie

nsis

Larix

laric

ina

Tsug

a ca

nade

nsis

Loni

cera

can

aden

sis

Pinu

s ba

nksi

ana

Pinu

s st

robu

s

Acer

rubr

um

Mai

anth

emum

race

mos

um

Frax

inus

am

eric

ana

Prun

us s

erot

ina

Acer

sac

char

um

Tilia

am

eric

ana

Car

ya c

ordi

form

is

Que

rcus

rubr

a

Betu

la p

endu

la

Pinu

s sy

lves

tris

Rob

inia

pse

udoa

caci

a

Berb

eris

thun

berg

ii

Rha

mnu

s ca

thar

tica

Acer

pla

tano

ides

0.0

0.2

0.4

0.6

0.8

1.0

A) Week 2

B) Week 4

0.0

0.2

0.4

0.6

0.8

1.0Proportion

Proportion

Below

Control

!

!

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42!

Fall Trial

For the fall trial, an above-ground exclosure treatment was added to the worm

exclusion treatment. At the first (2-week) sampling, all fixed factors were significant in the

full ANOVA model (Table 6). Seeds were most often recovered at the lowest rate in exposed

control plots, and at the highest rate in protected above-ground plots; recovery from plots

protected from worms was often close to that of the controls (Figure 3). Species differed

significantly (p < 0.0001) in their responses, and a significant (p < 0.0001) interaction

indicated that the effect of the exclosures depended on species identity (Table 6). When

species were considered separately (Table 7), 15 differed significantly among treatments

according to analysis of variance; Wilcoxon Rank-Sum Tests added Pinus banksiana (P =

0.016) and Tsuga canadensis (P = 0.024). In all cases, this was because above-ground

protection improved recovery over one or more other treatments (Figure 3). Rates of

recovery from below-ground protected treatments seldom exceeded recovery of controls,

though in some cases (Berberis thunbergii, Pinus sylvestris, Rhamnus cathartica) it was

indistinguishable from the above-ground exclosure treatment. There was no obvious

tendency (X21 = 0.03, p = 0.86) for natives (10/16 species = 63%) to be more vulnerable to

predation than exotics (4/6 species = 66%). Smaller-seeded species (Betula, Tsuga, Alliaria)

tended to have lower rates of removal (Figure 3).

Results at the second (4-week) sampling were similar, though as expected, rates of

recovery were lower (Figure 3). Again, all fixed factors were significant in the full ANOVA

(Table 6). Seeds were generally recovered at the lowest rate in exposed control plots and the

highest rate in above-ground exclosures (Figure 3). Again, species also differed significantly

in their responses, and a significant interaction (p < 0.0001) indicated that the effect of the

exclosures depended on species. When species were considered separately (Table 7), for 14

species, ANOVA detected a significant (p<0.0001) difference among treatments; Wilcoxon

tests added Tsuga canadensis (P = 0.029) and Alliaria petiolata (P = 0.046), even though one

site needed to be dropped from the analysis due to a lost treatment resulting from a fallen

tree. In most, this was because above-ground protection improved recovery over one or more

other treatments; however, this effect was indistinguishable from the below-ground

exclosures in several species (e.g. Maianthemum racemosum, Pinus banksiana, Pinus

!

!

!

43!

sylvestris), and in the case of Lonicera canadensis, below-ground protection increased

recovery relative to controls (Figure 3). Natives tended to be more vulnerable to predation

(12/16 species = 75%) than exotics (2/6 species = 33%), though this tendency was not quite

significant (X21 = 3.27, p = 0.07. Again, smaller-seeded species (Betula, Tsuga, Alliaria)

tended to have lower rates of removal (Table 7).

It is possible that the effects of earthworms may have been statistically concealed by

the much stronger effect of the above-ground exclosures, not used in the first trial. When this

treatment was deleted from the parametric analyses, differences between controls and below-

ground exclosures were detected at 2 weeks for Betula papyrifera (P = 0.0499) and at 4

weeks for Lonicera canadensis (P = 0.0024), Pinus banksiana (P = 0.0314) and Tsuga

canadensis (P = 0.0147). Wilcoxon tests similarly detected significant differences between

open and below-ground exclosure treatments for Betula papyrifera (P = 0.0034) and Pinus

sylvestris (0.0477) at 2 weeks, and for Fraxinus americana (0.0171), Lonicera canadensis (P

= 0.0002), Pinus banksiana (0.0022), Alliaria petiolata (P = 0.0102) and Tsuga canadensis

(P = 0.0024) after 4 weeks. For Alliaria and Betula, recovery was actually higher in controls,

likely reflecting random error; in the other 5 cases it was lower, consistent with mild

earthworm impacts. Treating these analyses as one-tailed Z-tests (recovery from below-

ground exclusion > control, one-tailed Wilcoxon Rank Sum p < 0.05) slightly increased the

number of significant results, adding Tsuga canadensis and Rhamnus cathartica (summer

trial, week 4), Larix laricina and Lonicera canadensis, and Tsuga canadensis (but losing

Betula papyrifera) (fall trial, week 2) and Tilia americana, Berberis thunbergii, and

Rhamnus cathartica (but losing Alliaria petiolata) (fall trial, week 4).

! 44!

Table 6 Results of comprehensive randomized block factorial ANOVAs with site as a

blocking factor; "Treatment" = Control, Above-ground exclosure, or Below-ground

exclosure; "Species" = species tested (Table 1) (n=12). Significant results (p<0.05) are

indicated in bold.

Sampling Week 2 Week 4 DF F P DF F P Treatment 2,715 422.88 <0.0001 2,695.6 289.22 <0.0001 Species 21,715 15.88 <0.0001 21,693.1 16.75 <0.0001 Treatment x Species 42,715 19.40 <0.0001 42,693.1 14.22 <0.0001

! 45!

Figure 3 Mean proportion of seeds recovered ± SEM, after 2 and 4 weeks exposure to

various granivore treatments in a fall exclosure experiment (n=12). Treatments: "Above" =

above-ground exclosure, "Below" = below-ground exclosure, "Control" = no protection. For

both natives and exotics, species are ordered by increasing seed size.

Natives Exotics

Betu

la p

apyr

ifera

Betu

la a

llegh

anie

nsis

Larix

laric

ina

Tsug

a ca

nade

nsis

Loni

cera

can

aden

sis

Pinu

s ba

nksi

ana

Pinu

s st

robu

s

Acer

rubr

um

Mai

anth

emum

race

mos

um

Frax

inus

am

eric

ana

Prun

us s

erot

ina

Acer

sac

char

um

Tilia

am

eric

ana

Car

ya c

ordi

form

is

Car

ya o

vata

Que

rcus

rubr

a

Allia

ria p

etio

lata

Pinu

s sy

lves

tris

Rob

inia

pse

udoa

caci

a

Berb

eris

thun

berg

ii

Rha

mnu

s ca

thar

tica

Acer

pla

tano

ides

0.0

0.2

0.4

0.6

0.8

1.0

A) Week 2

B) Week 4

0.0

0.2

0.4

0.6

0.8

1.0Proportion

Proportion

Above

Below

Control

! 46!

Table 7 Results of randomized block ANOVAs for recovery of seeds of each species.

Significant results (p<0.05) are indicated in bold. Also shown are results of Tukey HSD tests

comparing controls (C), above-ground exclosures (treatment "A"), and below-ground

exclosures (treatment "B"); "-" means no significant result was detected (p>0.05). Results

failed to converge for Betula allegheniensis; however, means were very similar across all

three treatments.

2 Weeks 4 Weeks

Origin Species F2,22 P Treatments F2,21 P Treatments Native

Acer rubrum 18.53 <0.0001* A>B,C 4.79 0.0190* A>B,C Acer saccharum 77.86 <0.0001* A>B,C 20.18 <0.0001* A>B,C Betula alleghaniensis 0.49 0.6189 - - - - Betula papyrifera 3.98 0.0334* - 0.033 0.9677 - Carya cordiformis 4015.44 <0.0001* A>B,C 115.66 <0.0001* - Carya ovata 4015.44 <0.0001* A>B,C 111.67 <0.0001* A>B,C Fraxinus americana 23.51 <0.0001* A>B,C 17.35 <0.0001* A>B,C Larix laricina 0.49 0.6218 - 2.26 0.1290 - Lonicera canadensis 0.88 0.4275 - 7.22 0.0040* A,B>C Maianthemum racemosum 1.313 0.2894 - 5.13 0.0151* A>C Pinus banksiana 2.55 0.1010* - 3.82 0.0379* A>C Pinus strobus 16.05 <0.0001* A>B,C 6.77 0.0052* A>B,C Prunus serotina 72.43 <0.0001* A>B,C 79.05 <0.0001* A>B,C Quercus rubra 4015.4 <0.0001* A>B,C 108.45 <0.0001* A>B,C Tilia americana 147.87 <0.0001* A>B,C 46.26 <0.0001* A>B,C Tsuga canadensis 1.7528 0.1966* - 2.70 0.0899* -

Exotic

Acer platanoides 112.25 <0.0001* A>B,C 20.79 <0.0001* A>B,C Alliaria petiolata 0.79 0.4672 - 1.62 0.2209* - Berberis thunbergii 4.81 0.0185* A>C 1.22 0.3159 - Pinus sylvestris 4.73 0.0196* A>C 5.00 0.0164* A>C Rhamnus cathartica 6.14 0.0076* A>C 2.03 0.1550 - Robinia pseudoacacia 1.39 0.2690 - 2.28 0.1264 -

* Significant in Wilcoxon Rank-Sum tests (P<0.05)

! 47!

Method Control Experiments

Seed Marking Control Experiment Results

Analysis of variance confirmed that neither seed losses nor the number of fragmented

seeds was affected by the marking treatment (p > 0.15) (Table 8). The fraction of seeds

removed by seed predators was very high (Figure 4), resulting in the inability to homogenize

variances. However, it was clear that UV marking did not present a deterrent to seed

predators.

Again significant differences in removal rates between all species were apparent (p <

0.05). For instance, viable seeds of the relatively small-seeded Lonicera canadensis were

recovered more than the other, larger-seeded species (Quercus rubra, Prunus serotina, Acer

saccharum, Carya cordiformis) (Figure 4).

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Table 8 Results from randomized block factorial ANOVA for UV marking on seed removal

(n=6). For the analysis site was used as a blocking factor; "Treatment" = Marked or

Unmarked; "Species" = species tested (Table 1). Significant results (p<0.05) are indicated in

bold.

Viable Seeds Remaining DF F P Treatment 1,45 1.16 0.287 Species 4,45 3.03 0.027 Treatment x Species 4,45 0.56 0.693

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Figure 4 Proportion of viable seeds remaining ± SEM, for marked and unmarked seeds, after

7 days exposure to granivores (n=6). Treatments: "marked" = UV-marked, "Unmarked" =

unmarked. Species are ordered by increasing seed size.

Loni

cera

can

aden

sis

Pinu

s st

robu

s

Prun

us s

erot

ina

Acer

sac

char

um

Car

ya c

ordi

form

is

0.0

0.2

0.4

0.6

0.8

1.0MarkedUnmarked

Proportion

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50!

Leaf Litter & Granivory Experiment Results

Analysis of variance indicated that neither seed losses nor the number of fragmented

seeds was affected by the litter treatment; as well, there were no significant interactions

(Table 9). In contrast, there were differences in removal rate between species (Figure 5). As

found in the summer and fall granivore exclosure experiments (Figures 2, 3), removal rates

were lowest in smaller-seeded species (Betula alleghaniensis, Alliaria petiolata, Lonicera

canadensis) compared to larger-seeded species (Quercus rubra, Prunus serotina, Acer

saccharum) (Figure 5).

Germination From Burial Depth Experiment Results

Of the 6 species used in the germination from depth experiment, only Pinus strobus,

Berberis thunbergii and Betula alleghaniensis were successfully germinated at any depth

(Table 10). The inability to successfully germinate Acer saccharum, Prunus serotina, and

Alliaria petiolata can likely be attributed to a failure to properly address specific

pregermination requirements (e.g. scarification, cold dormancies, etc.) for each species.

In each of the 3 species that were successfully germinated (Pinus, Berberis, and

Betula), germination rates were greatest for seeds buried <1cm from the soil surface. As

burial depth increased, rates of successful germination decreased for all species (Table 10).

Both Berberis and Betula were unable to successfully germinate from depths greater than

3cm. Pinus was able to successfully germinate as deep as 7cm, however germination rates

from depths >5cm were minor. Only 2.8% of seeds buried at 7cm germinated compared with

80.5% at the soil surface (Table 10).

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Table 9 Effects of litter on seed removal (n=10). Results of randomized block factorial

ANOVAs with site as a blocking factor; “Treatment”=Litter present or Litter Removed,

“Species = species tested (Table 1). Significant results (p<0.05) are indicated in bold.

Viable Seeds Remaining Fragments DF F P DF F P Treatment 1,117 0.00 0.985 1,117 1.60 0.208 Species 6,117 152.60 <0.0001 6,117 156.68 <0.0001 Treatment x Species 6,117 0.81 0.561 6,117 1.70 0.127

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Figure 5 Mean proportions of viable seeds remaining ± SEM, in plots with and without leaf

litter layers (n=10). Treatments: "Litter" = litter present, "No Litter" = litter removed. Species

are ordered by increasing seed size.

Betu

la a

llegh

anie

nsis

Allia

ria p

etio

lata

Loni

cera

can

aden

sis

Pinu

s st

robu

s

Prun

us s

erot

ina

Acer

sac

char

um

Que

rcus

rubr

a

0.00.10.20.30.40.50.60.7

0.80.91.0

LitterNo Litter

Prop

ortio

n re

mai

ning

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Table 10 Proportion of seeds successful in germinating from various burial depths in a

greenhouse control study (n=3). Species are listed in order of decreasing mean seed mass

(See Table 1).

Species Prop. of Surface

Germination (±SEM)

Prop. of 3 cm Buried Germination

(±SEM)

Prop. of 5 cm Burial Germination

(±SEM)

Prop. of 7cm Burial

Germination (±SEM)

Acer saccharum 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 Prunus serotina 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 Pinus strobus 0.806±0.073 0.583±0.048 0.306±0.073 0.028±0.028 Berberis thunbergii 0.056±0.023 0.056±0.023 0.0±0.0 0.0±0.0 Alliaria petiolata 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 Betula alleghaniensis 0.222±0.028 0.083±0.0 0.0±0.0 0.0±0.0

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2.4 DISCUSSION

Potential Of Earthworms As Granivores Earthworms are potentially significant agents of mortality for seeds of forest species.

When all plant species used in the microcosm experiments are combined, 72.8% of seeds

placed on the soil surface were removed when exposed to earthworms. Separately, each of

the 6 species used in this experiment suffered considerable increases in removal when

exposed to earthworms relative to their respective worm-free controls. These losses occurred

over the relatively short period of only two weeks, suggesting that earthworms could be

removing a remarkably large proportion of seeds over longer periods of time. These rates of

seed loss are comparable with losses observed in seed predation studies (Eisenhauer and

Scheu 2008; Quackenbush et al. 2012).

Seed removal from the soil surface is not necessarily fatal. Seeds remaining at or near

the soil surface almost certainly have the potential to successfully germinate, although they

may be exposed to higher rates of predation by rodents and birds compared to buried seeds

(Chambers and MacMahon 1994). Results for seeds transported to greater depths are more

complicated. First, some seeds were fragmented or vanished entirely (>30%). As

experimental controls indicate seed detection methods were highly effective (<2% of seeds

escaped detection), it is assumed that vanished seeds suffered a fatal outcome. It is probable

vanished seeds were ingested, pulverized within the digestive tract, and ultimately destroyed

by earthworms, as seen in other studies (Quackenbush et al. 2012). In other cases it is

possible seeds were ingested, transported, and excreted by earthworms. Interpretation on how

ingestion and excretion impacts subsequent germination rates is unclear, being shown to both

positively and negatively impact germinability on different seeds (Eisenhauer et al. 2009).

Transportation and burial at depth perhaps has a clearer impact on the likelihood of

germination, as many species are unable to germinate from depths at which seeds are often

deposited by earthworms.

Of the remaining buried seeds, the chance of survival likely depends strongly on

burial depth. Few species likely emerge from depths >5cm, as indicated by the growth

chamber experiment; most of these likely are ecologically dead, at least unless they are

further transported to the soil surface once again. Many smaller seeds, such as those of

yellow birch, are only capable of germinating at the soil surface due to specific requirements

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such as light (Burns and Honkala 1990). In other cases, burying a seed more than 1cm deep

in soil can lead to exponential declines in ability to successfully germinate since they likely

contain insufficient resources to enable seedlings to reach the soil surface (Burns and

Honkala 1990). This again may be particularly problematic for small-seeded species such as

yellow birch, whose ability to germinate successfully even when only buried by leaf litter is

negligible; more than 80% of yellow birch seeds in this experiment appear to be unavailable

for germination when in the presence of earthworms. Garlic mustard is another small seeded

species that appears to suffer a similar fate, as 91.1%±3.8% of seeds were removed from the

soil surface, and thus largely unavailable for germination.

In contrast, some larger-seeded species, notably white pine, may be able to rarely

emerge from greater depths: in the greenhouse experiment, < 3% of seed emerged from

>5cm burial depth. Burial greater than 5cm is likely fatal for the remaining species used in

this study as zero individuals were able to successfully germinate from such depths. Thus for

the purpose of this study, it is reasonable to assume that most seed buried more than 5cm

below the soil surface is effectively removed and unavailable for germination. Though the

possibility some seeds may ultimately emerge clouds the interpretation of my results, similar

issues apply to seed-caching rodents such as squirrels (Hadj-Chikh et al. 1996).

In the microcosm experiment it was clear that earthworms preferentially removed

some species over others. On one hand, white pine appears to be considerably less prone to

removal than other species: as 73.3% of seeds remained on the soil surface. This lack of

removal suggests this species of seed may be relatively immune to earthworm seed predation

compared to other species of seed. Previous studies have linked decreases in seed palatability

to earthworms to factors such as seed size, morphology, and plant functional group

(Eisenhauer and Scheu 2008, Quackenbush et al. 2012). Coupled with the ability of seeds

that do become buried to germinate from deeper depths, these results suggest white pine may

be relatively resistant to earthworm granivory.

Rates of removal were much higher for the remaining species, even the relatively

large seeded P. serotina, B. thunbergii, and A. saccharum. Nearly 50% of seed from each of

these species was either buried below a depth that allows successful germination, or assumed

digested. The relatively large size of these seeds, particularly P. serotina, and A. saccharum,

may suggest that earthworms are capable of directly or indirectly removing at least some

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quantity of the majority of species commonly found in temperate hardwood forests. Results

are even more striking for the small-seeded A. petiolata and B. alleghaniensis, which suffer

tremendously high rates (88.9% and 74.8% respectively) of effective seed removal from

germinable depths. This reduction in the amount of available seed for smaller seeded species

could perhaps influence the number of mature individuals in future plant communities.

It is notable that the problematic invader A. petiolata was removed in significantly

greater abundance than all other species. This agrees with the results of Quackenbuch et al.

(2012), who also found that Alliaria was highly palatable to introduced earthworms. This

may suggest that, rather than producing invasional meltdown (Simberloff and Von Holle

1999), earthworms may actually decease the success of this invasive plant. However, this is

not true for all invaders: B. thunbergii experienced much lower rates of removal, while the

native B. alleghaniensis displays similarly large rates of predation.

Relative Role Of Earthworms & Other Granivores

Twenty-one species of seed were used to assess the relative role of belowground

predators in the summer granivore exclusion experiment. Samples collected after 2 weeks of

exposure to granivores show that excluding belowground seed predators yielded a significant

increase in the fraction of seeds recovered. Therefore, in the absence of belowground

granivores, such as L. terrestris, seeds stand a better chance at survival, which indicates that

this suite of granivores may play a more influential role in community seed dynamics than

previously thought. This relationship appears to hold true over a longer period of time as

samples collected after 4 weeks of exposure to granivory continued to show reduced but

significantly greater overall seed recovery when L. terrestris was excluded, though there

were few species-level effects. Although a handful of studies have shown that L. terrestris is

indeed capable of ingesting, egesting, and even digesting some species of seed (Quackenbush

et al. 2012, Eisenhauer and Scheu 2008), evidence of seed removal by earthworms has not

previously been demonstrated in a realistic field experiment.

Belowground granivores such as L. terrestris clearly exerted significant (p < 0.0001)

preferential feeding on some species over others in this experiment. After 2 weeks, nearly

half (9 of 21 species) were removed at significantly different (p < 0.05) rates between

treatments. The ability of L. terrestris to preferentially feed on some species over others has

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also been demonstrated before using a series of simple choice experiments comparing garlic

mustard to 6 other herbaceous species (Quackenbush et al. 2012). In this study L. terrestris

ingested A. petiolata at significantly (p < 0.05) greater rate than 3 of 6 other species tested.

How these preferences can influence and manipulate soil seed banks has yet to be directly

evaluated.

In the fall trial of this experiment the addition of an aboveground exclusion allowed

effects of worms to be compared with other seed predators. Using a suite of 21 different

species of seed it was evident that generally aboveground granivores remove a considerably

greater quantity of seed than belowground seed predators, although after a greater length of

exposure to seed predators the discrepancies between granivore guilds became less

pronounced. When exposed exclusively to belowground seed predators 35.0%±2.02% of

seeds were removed after an exposure period of 2 weeks. However plots exposed to

aboveground seed predators and control units (exposed to above & belowground predators)

exhibited nearly identical levels of seed removal (75.0%±1.79% and 76.6%±1.76%

respectively). These results point to the importance of aboveground predators such as

numerous species of rodents as the prominent granivores in the system, likely attributable to

the speed and efficiency at which they remove recently dispersed seed.

After 4-weeks of exposure to seed predators, it appears that belowground granivores

played an increasingly important role, removing 47.0%±2.29% of seeds when they are the

exclusive seed predators. As expected, aboveground predators and control units also saw

increases in the amount of seeds removed, with removal rates of 79.5%±2.29% and

84.2%±1.65% respectively, again pointing to their importance at removing large quantities of

seed.

To some extent, the lack of strong earthworm effect at the fall sampling may be a

result of very strong rodent effect statistically masking weaker worm predation. This

interpretation is supported by the fact that deleting the above-ground exclosure treatment

from the analyses added several significant results. Despite this, it is clear that seed predation

by rodents was far more frequent, stronger, and easier to detect than these relatively subtle

earthworm effects.

Collectively these findings suggest aboveground granivores are especially effective at

removing seeds that have been recently dispersed, especially those seeds that are medium to

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large in size. It appears that belowground granivores, likely mainly consisting of earthworms

in this system, may play a more important role in the removal of seeds of smaller mass. This

is supported by the low recovery rates of smaller species such as Betula alleghensis and

Lonicera canadensis in treatments exposed to earthworms compared with other treatments.

Earthworms likely play a measurable but relatively small role in seed predation on a

community scale in this forest system. Generally speaking, temperate forests tend to host

species with reasonably large seeds compared to other habitats, many of which are quickly

targeted by aboveground granivores such as rodents and birds. This combined with the fact

that earthworm populations tend to exhibit decreased activity towards the end of the summer

months (Edwards and Bohlen 1996), when seed dispersal is beginning but when small

mammal populations tend to peak (Falls et al. 2007), suggests that earthworms are likely

responsible for relatively little seed removal from a community perspective. These life

history traits may be at least partially responsible for an apparent decrease in earthworm seed

removal in the fall trial compared with the summer trial. The relatively small role that

earthworms appear to play in forest seed predation may not hold true in other systems,

especially those with a different range in seed size and different patterns of dispersal.

Although both aboveground and belowground seed predator guilds clearly remove

seeds at different rates, they are similar in that they clearly attack different species of seed at

different rates. Stark differences between different species of seed are evident after both

shorter (2 week) and longer (4 week) exposure to seed predators. A number of trends begin

to emerge when similar species are grouped together into different functional groups. For

instance, when looking at congeneric sets of species within the same granivore treatment,

highly comparable seed removal rates are evident. The 3 Pinus species (P. strobus, sylvestris

and banksiana) used in this study were subject to comparatively similar rates of recovery in

aboveground excluded (37.5%±10.93%, 30.6%±8.72%, and 13.9%±5.16%), belowground

excluded (9.1%±4.69%, 12.1%±4.26% and 6.1%±2.54%), and control treatments

(3.5%±1.91%, 6.9%±2.68% and 1.4%±0.94%). The relatively similar rates of granivory

between members of a genus was also observed in the Acer, Betula and Carya genera.

Of course other likely important factors confound with genera, such as the size and

nutritional value of a seed. A few interesting trends arise when looking at the role of seed

size on granivory. For instance, when exposed to aboveground granivores, larger sized seeds

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such as those of the genera Quercus, Carya, and Acer tend to suffer remarkably high rates of

seed removal. In fact each of the largest 8 species of seed used in this experiment were

subject to >90% removal when exposed to aboveground granivores. Conversely, smaller and

less resource-rich seeds tended to escape high rates of removal from aboveground predators.

In this experiment only 2 of the smallest 7 species of seeds experienced predation rates

greater than 30%.

Interestingly, earthworms appear to be driving similar seed selection trends to those

observed in the earthworm microcosm experiment. In the aboveground exclusion treatments

that were subject only to belowground seed predators such as earthworms, many of the larger

seeds were recovered at remarkably high proportions. This may suggest that belowground

predators have a relatively minor impact on larger seeded genera such as Acer, Quercus, and

Carya. Conversely, these belowground seed predators again appear to be driving the removal

of smaller seeded genera such as Alliaria, Larix and Pinus, where earthworms were

attributed with the removal of >60% of seeds.

Potential Biases

All seeds that were used in each of these experiments detailed were marked using an

ultraviolet fluorescent dye to aide in seed recovery. ANOVA results indicate the number of

recovered seeds and seed fragments recovered are not significantly different between marked

and unmarked treatments. Also, as expected, differences in removal rates between species

were significant (p < 0.05). These findings suggest that the methods utilized in seed marking

in this experiment have a non-significant impact on the interpretation and application of

these findings to naturally occurring, unmarked seed.

The leaf-litter removal experiment provided remarkably similar results between

control and litter-removed treatments. These similarities were unexpected and quite

surprising to discover. Intuitively it would appear logical for the removal of the leaf litter

layer to expose seeds to increased rates of granivory, however these results were unfounded

in this study. An increased sample size, range of seeds (including seeds subject to lower

removal rates than those used) and duration may have increased the success of this study.

The germination from depth experiment provided useful insight into the interpretation

of the earthworm microcosm results. As expected, burial at increasing depth generally leads

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to reductions in germinability. Although differences between species of seeds were quite

strong, such as B. papyrifera's inability to germinate when buried below 1cm depth and P.

strobus’ ability to in rare cases germinate buried below 5cm, the species of seeds used are

largely unable to germinate successfully when buried at depths of 5cm and greater. This

suggests that it is reasonable to assume that seeds buried below 5cm of soil are effectively

unavailable for germination, and for the purpose of interpretation for this study, effectively

removed from the soil seed bank.

2.5 CONCLUSIONS

In the presence of earthworms seeds appear to be restricted to 1 of 4 possible fates.

They may (1) remain at the soil surface, (2) be buried at a shallow depth that does not

eliminate the possibility for successful germination, (3) be buried at a depth that restricts

successful germination, or (4) removed outright and likely digested.

The results of this experiment clearly indicate that when in the presence of

earthworms seeds potentially can be removed from the soil surface in substantial quantities.

All but one species used in my microcosm experiment saw more than 70% of their seeds

removed from the soil surface when in the presence of earthworms. Despite these

tremendous rates, many seeds (>50% from 4 of 6 species) were found to be buried within the

upper 5cm of the soil profile, suggesting that the overall reduction in fitness may not be as

pronounced as originally believed. Still 4 of the 6 species of seed used in this experiment

maintained less than 60% of their original quantity of seed within a germinable depth of soil.

I also found evidence that earthworms are capable of removing different species of

seed at significantly different rates. This supports the conclusion that earthworms are capable

of exhibiting preferential selection as granivores (Quackenbush et al. 2012; Eisenhauer et al.

2010). In contrast, I found that in a more realistic setting that earthworms are eclipsed as

granivores by more efficient seed predators, namely small rodents (Garett and Graber 1994;

Falls et al. 2007; Schnurr et al. 2002). Earthworms were still capable of removing seeds with

certain traits however, and may be viewed as size-restricted specialists.

Some of the interesting trends in earthworm seed preference would benefit from

further study. For instance, how earthworm seed selection manifests itself over time to

influence the structure and composition of a plant community would be a fascinating angle

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for research. Specifically a duration of 2-3 growing seasons may help us better understand

longer-term implications of earthworm activity on soil seed bank processes.

Broader Implications The results of this study provide evidence that invasive earthworms may have direct

impacts on invaded plant communities through seed consumption, as well as indirect impacts

via soil modification (Larsson et al. 2010; Bohlen et al. 2004a). These impacts are additional

to depredations by rodents, and similarly may involve both negative (mortality) and positive

(protection) components. These pressures likely differ among species of seed, depending on

their palatability and capacity to emerge from depth, and perhaps suggest that earthworms

may act as ecological filters in their potential ability to restrict success of certain species.

Earthworms are known to have negative effects on many understory plants, though

the mechanisms are not entirely clear. Many explanations have been proposed (Eisenhauer

and Scheu 2008; Griffith et al. 20013), notably impacts on mycorrhizae and complex

consequences of removal of the litter layer (Lawrence et al. 2003; Hopfensperger et al.

2011). Although Darwin was aware of the ability of earthworms to act as seed predators as

long ago as 1881, this aspect of earthworm ecology has been largely overlooked in recent

ecological studies. The results of this study suggest that although earthworms are certainly

capable of removing remarkable quantities of seed under controlled conditions, they often

appear to be eclipsed as seed predators at the community level in temperate forests where

rodents are common and seeds are larger than those in other habitat types. In less rodent-

dominated systems, they may be capable of playing a greater role in seed predation,

especially for small-seeded species.

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Larson ER, KF Kipfmueller, CM Hale, LE Frelich, and PB Reich. 2010. Tree rings detect

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Lawrence B, MC Fisk, TJ Fahey, and ER Suarez. 2003. Influence of nonnative earthworms

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Chapter 3 General Conclusions

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3.1 REVISITING & INTERPRETING THESIS OBJECTIVES

The negative impacts inflicted by exotic earthworms on the forests of Ontario have

been well documented in the literature. The extent to which they negatively impact the

structure and function of Ontario ecosystems has been so severe that they are deemed

ecological engineers (Jones et al. 1994, Holdsworth et al. 2007) for their ability to alter the

structure and function of the habitats they invade. Their presence has been observed to alter

numerous foundational aspects of North American habitats including geochemical cycling,

and plant and animal diversity (Sackett et al. 2012; Addison 2009; Bohlen et al. 2004).

Increasing focus has been given to the biotic consequences of these invasive species.

A few studies (Eisenhauer et al. 2010; Quackenbush et al. 2012) have noted the ability of

earthworms to act as seed predators across a range of habitats. Other seed predators such as

mice and squirrels have been observed to act as biotic filters, preferentially consuming

certain species of seed over others. With this in mind those concerned with the impacts that

these invasive species may be inflicting on forests of North America could use a deeper

understanding on the potentially similar role that these invasive earthworms play in the

availability of seeds in forest systems.

This thesis has two core stated goals: to better understand the potential that invasive

earthworms have to act as seed predators, and to measure the actual role that they play in

seed predation, compared to other more well documented granivores. To achieve the first

goal, I used a microcosm experiment to address 3 questions that collectively outline the

potential that invasive earthworms hold to act as important seed predators in the forests of

Ontario.

Question 1. When isolated from other granivores are earthworms capable of removing a

substantial quantity of seed from the soil surface?

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Outcome. Six species of seeds were added to the soil surface in microcosms that contained a

single earthworm. The earthworms removed a significant proportion of seeds in five of the

six species. Irrespective of seed species, only 27.8% of seeds placed on the soil surface

remained after 7 days of exposure to an earthworm. When seeds were applied to control

mesocosms lacking earthworms, 97.8% of seeds were recovered from the soil surface over

the same time period.

Question 2. What happens to those seeds removed from the soil surface? What proportion is

removed outright (digested)? Buried to a depth enabling germinable? Buried to a depth

inhibiting germination?

Outcome. Most (45.7%) of the seeds that are removed from the soil surface can be recovered

within 1-5cm from the soil surface. Nearly all species of seed used in this study are capable

of successfully germinating at this depth. Roughly one quarter (23.8%) of seeds were

recovered at depths between 5cm and 30cm. Burial at this depth would effectively render

most of these species of seed ecologically “dead” with very little chance at successfully

germinating. Perhaps most importantly, in the presence of earthworms, nearly one-third

(30.5%) of seeds are removed entirely. These seeds are likely fully digested by earthworms.

This number appears to be at least partially driven by particularly high losses in smaller-

seeded species such as Betula and Alliaria.

Question 3. Do earthworms remove certain species of seed over others? Do traits such as

seed mass or species provenance seem to guide preferences?

Outcome. Although all species of seed are removed from the soil surface in significant

numbers compared to microcosms containing no earthworms, considerable differences

between species are clear. For instance, earthworms remove >90% (91.1%) of Alliaria

petiolata from the soil surface, compared to only 26.7% of Pinus strobus. Although only 6

species of seeds were used in this experiment, it is clear that species with a smaller seed mass

suffer from higher rates of earthworm granivory than those of larger mass. Since only 6

species were used, disentangling the effect of species provenance is challenging.

The second of the two core stated goals of this thesis is to quantify the relative

importance of invasive earthworms compared with more established seed predators. To

answer this issue I designed a seed-addition granivore-exclusion field experiment.

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Question 4. What proportion of seed predation can be attributed to earthworms, relative to

other guilds of seed predators?

Outcome. Seeds from more than 20 species of forest plants were added to a series of

exclosures which inhibited predation from either aboveground granivores (squirrels, mice,

birds, etc.) or belowground granivores (mainly earthworms). Results for different species

were highly variable between treatments, suggesting that different groups of granivores

target different sized seeds. Despite the differences between species, it is clear that plots

targeted by aboveground predators yielded significantly fewer recovered seeds than plots

subject to only earthworm predation. These findings were largely driven by medium to large

seeded species which were efficiently removed by aboveground granivores. Trends in

smaller seeded species were less clear, however for a number of species plots excluding

earthworms had a greater proportion of seeds recovered than those exposed to earthworms.

This may suggest that in at least some cases, earthworms are contributing a measurable effect

to seed removal. However, the trends in medium and large sized seeds strongly suggest that

rodents are primarily responsible for driving predation in many species.

Question 5. Are different species of seed removed at different rates? If so, what traits may

help explain these preferences? Seed mass? Species provenance?

Outcome. As mentioned previously seed removal was highly variable between both species

of seeds and granivore treatments; all species of seed tended to respond to granivores in

different ways. With that said, seed size appears to play a large role in predicting which

granivore guilds will remove a seed. Larger genera, such as Carya and Quercus, were often

removed at rates approaching 100% when exposed to aboveground granivores. Earthworms

often seem to prefer seeds that are less-desirable to efficient above-ground predators, such as

seeds of smaller mass.

3.2 SUMMARY OF FINDINGS

A small handful of studies (Eisenhauer et al. 2010; Quackenbush et al. 2012) have

pointed to earthworms as seed predators, and even made reference to potential traits that may

increase or decrease a seed's desirability to a granivorous earthworm. These results show that

indeed in an isolated environment, such as a microcosm or petri dish, earthworms are capable

of removing a remarkable quantity of seeds off the soil surface. They are also capable of

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exhibiting strong preferences, removing certain species of seeds in much greater quantity

than others (Quackenbush et al. 2012). At least some of these preferences can be explained

by seed size, although a more complete list of seeds representing realistic ranges in size

would be useful. Despite these interesting patterns in earthworm granivory, results from the

granivore exclusion experiment clearly indicate that earthworms likely provide a rather small

influence on granivory at the community level; aboveground granivores clearly remove seed

in considerably greater rates within Ontario forests.

A different system that still contains similarly abundant earthworm populations yet is

home to a considerably less prominent aboveground seed predator population could

potentially provide a situation where earthworms are a dominant granivore. Perhaps a

disturbance rich, possibly novel ecosystem, such as an active agricultural habitat may

provide such conditions. The findings of this thesis however demonstrate that although

exotic earthworms are a serious threat as an invasive species to Ontario’s temperate forests,

they likely have a modest impact on seed predation in a temperate forest and for most species

are unlikely to influence forest seedbank dynamics in a meaningful way.

3.3 DIRECTIONS FOR FUTURE RESEARCH During the development, execution, and analysis of this research project, a number of

interesting questions have arisen that I was unable to answer within the scope of this project.

Addressing these issues could perhaps lead to useful developments in our understanding of

this study system.

Earthworms have clearly demonstrated the ability to significantly reduce seed

availability in isolation, but appear to be easily eclipsed by more effective seed predators in

temperate forest systems. Could their effectiveness as a major seed predator be depressed in

this habitat compared to others? How do differences between earthworm densities in

different habitats factor into these conclusions? Could their impacts on seed survival likely

to be greater in a habitat with smaller seeds such as a meadow or old-field? Are their impacts

greater in habitats where granivorous rodents are scarce? Similar methods and approaches

could be used to answer these questions and more, to better understand the depth that these

invasive species impact the temperate region of North America.

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Very little is also known about the long-term implications for those seeds that are

transported vertically through the soil seed bank. Although it can be a very challenging

system to experimentally manipulate, a developed understanding of the long-term effects of

seed burial would certainly help us understand the scope of earthworm/seed dynamics.

Finally, recent efforts have also been made to better understand the role that ants of

temperate North America play in community level seed predation. Ants tend to prefer

smaller seeds, especially those with elaisomes, perhaps enabling both earthworms and ants to

function as competitors for certain species of seed.

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References Addison JA. 2009. Distribution and impacts of invasive earthworms in Canadian forest

ecosystems. Biological Invasions 11:59-79

Bohlen PJ, S Scheu, CM Hale, MA McLean, S Migge, PM Groffman, and D Parkinson.

2004. Non-native invasive earthworms as agents of change in northern temperate

forests. Frontiers in Ecology and the Environment 2:427-435

Eisenhauer N, and S Scheu. 2008. Invasibility of experimental grassland communities: the

role of earthworms, plant functional group identity and seed size. Oikos 117:1026-

1036

Holdsworth AR, LE Frelich, and PB Reich. 2007. Regional extent of an ecosystem engineer:

earthworm invasion in northern hardwood forests. Ecological Applications 17:1666-

1677

Jones CG, JH Lawton and M Shachak. 1994. Organisms as ecosystem engineers. Oikos

69:373-386

Quackenbush P, RA Butler, NC Emery, MA Jenkins, EJ Kladivko and KD Gibson. 2012.

Lumbricus terrestris prefers to consume garlic mustard (Alliaria petiolata) seeds.

Invasive Plant Science and Management 5:148-154

Sackett TE, SM Smith, and N Basiliko. 2014. Exotic earthworm distribution in a mixed- use

northern temperate forest region: influence of disturbance type, development age, and

soils. Canadian Journal of Forest Research 42:375-381

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Appendix APPENDIX 1 Rationalizations for the selection of each species of seed.

Acer platanoides Exotic Exotic congener of native maples Acer rubrum Native Widespread deciduous tree Acer saccharum Native Common (often dominant) deciduous tree species Alliaria petiolata Exotic Problematic herbaceous invader, possibly preferred by worms Berberis thunbergii Exotic Problematic invasive shrub Betula alleghaniensis Native Small seeded deciduous tree Betula pendula Exotic Exotic congener of native birch Betula papyrifera Native Common, small seeded deciduous tree Carya cordiformis Native Large-seeded nut tree Carya ovata Native Large-seeded nut tree Fraxinus americana Native Common deciduous tree Larix laricina Native Small seeded deciduous tree Lonicera canadensis Native Widespread deciduous shrub Maianthemum racemosum Native Native herbaceous groundcover in mixed forests Pinus banksiana Native Native congener of exotic pine Pinus strobus Native Common coniferous tree species in mixed forests Pinus sylvestris Exotic Exotic congener of other native pines Prunus serotina Native Common deciduous tree species, seeds an important food source Quercus rubra Native Large-seeded nut tree Rhamnus cathartica Exotic Problematic invasive shrub/tree Robinia pseudoacacia Exotic Larger-seeded exotic tree Tilia americana Native Widespread deciduous tree Tsuga canadensis Native Small-seeded native conifer tree

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APPENDIX 2 Extended abstract.

THE IMPORTANCE OF INVASIVE EARTHWORMS AS SEED PREDATORS OF COMMON FOREST FLORA OF ONTARIO

Colin M Cassin

Master of Science

Ecology and Evolutionary Biology Department University of Toronto

2015 ABSTRACT Soil seedbanks play a vital role in forest plant communities, having long been viewed as a refuge for

seeds that are vulnerable to aboveground seed predators. This study provides evidence that seeds

entering the soil seedbank may be subject to previously underestimated rates of granivory by a

common species of invasive earthworm, Lumbricus terrestris. We report that nearly 55% of seeds

from 6 ecologically important forest species were either digested or buried below a germinable depth

when in the presence of earthworms in a microcosm experiment. L. terrestris was also found to

preferentially remove certain species of seed over others, for instance smaller sized seeds were often

removed in higher abundances than larger sized seeds. The common forest invader Garlic Mustard,

Alliaria petiolata, was subject to the highest removal rates in this study, as nearly 90% of seeds were

effectively destroyed by earthworms.

In contrast, results from a field exclusion experiment indicate that seed predation by rodents may

eclipse that of earthworms under natural conditions, for most temperate forest seeds. While seed

predation by rodents was high in mid to large seeded species, earthworms produced a greater relative

effect in seeds of smaller mass.

These findings suggest that although rodents are the main driver of seed predation, earthworms may

have the potential to act as an additional layer of ecological filter, and potentially further influence

the species composition of future forest plant communities by selectively targeting certain seeds, or

seed traits, over others. Although the effects may be small, the application of these findings may

prove useful to management of forests where threat of earthworm invasion exists.