POLLEN LIMITATION IN SMALL POPULATIONS OF THE

SELF-INCOMPATIBLE PLANT, HYMENOXYS HERBACEA

A Thesis

Presented to

The Faculty of Graduate Séudies

of

The University of Guelph

by

LESLEY G. CAMPBELL

In partial fulfilment of requirements

for the degree of

Master of Science

January, 200 1

O Lesley G. Campbell, 200 1

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ABSTRACT

Pollen Limitation in Small Populations of the Self- Incompatible Plant, Hymenoxys herbacea.

Lesley G. Campbell University of Guelph, 2001

Advis or: Dr. Brian Husband

Fecundity of self-incompaable plants is expected to be limited by poilïnation

(pollen limitation) in small populations due to stochastic loss of matkg types (genetic drift)

and insuffiaent p o k a t o r visitation (Mee effect). I tested these predictions in the rare,

insect-pollinated, self-incompatible plant, HymetloOxy.r herbacea. First, 1 es timated the

magnitude of genetic drift acting on populations by measuring effective size ( Ne). Second,

1 escimated the strength of pollen limitation, pollinator visitation rates and mate diversity in

populations of different size. Effective size ( Ne), estimated using a demographic model,

ranged from 57 to 56,476,700, averaging 43% of the census popuiation size. In most

populations, dPft mas not sufficïently srrong to affect the diversitg of matkg spes.

Percentage seed set varied midely among the 13 populations examined, f?om 27.5O/0

to 66.2%: however, seed production mas poilen lunited in only one case. Mate diversicg

varied arnong populations and was positively correlated mith population size, as theory

predicts; howevee, it did not account for variation in pollen limitation. Contrary to

e-xpectations, pollinator visitation rates dedined with population size and mere not

correlated with pollen Limitation. Overall, these results suggest chat population size impacts

mate diversitg and p o h a t o r behavior but neither effect was strong enough to influence

reproduction in H. herbacea.

My thanks, kt, m u t go to Brian Husband, for his patience, persistence and

encouragement during the entire project Your enthusiasm for explorkg the n a m d wodd

through the process of science is Ïnspinng to me- My project mas k d e d by an NSERC

awarded to BPan and hancial support was also kindly given by the Deparnent of Botany.

Thanks to my three committee memben, Ron Brooks, Doug Larson and Richard Reader,

for their time and helphil comments during the planning and completion of this project-

Thanks also to Jennif'er Windus, Pedro Morin-Palma and Alison Snow for their hdpfd

suggestions to my methodology.

To the best field assistant ever, thank you Chns Hussell, for hiking to the end of the

e h , knowing the s m d derails and singiag at the end of the day.

Many people were instnunental in helping me h d the daisies. Thank you Joe

Johnson, Doug Larson, Jeremy Lundholm, and Michael Oldham for having s h q eyes.

n i d s also goes to a number of organitations who dowed me to work on the daisies that

lived on their land. 1 appreciate the permission and tnist @en to me to do this research

fiom the Cape Croker Band Council, the Federation of Ontario Naturalists, the Bruce

Peninsula National Park and the Bruce Peninsula Provincial Parks. The kind hospidty of

the Bruce PeninsuIa National Park folk was truiy appreciated. Thank you Scott Sutton,

Darlene Upton, Ethan Meleg, Kevin Robinson and h d r e w Promaine. And hnaIly, thanks

goes to Cacherine Onodera for identlfying many of the bugs 1 collected.

T h d s goes to my patents for encouraging my interest in the natural morld and

teaching me to ask questions. Severai other people deserve my gratitude for thek support

and fnendship ove^ the past NO years during the long days of field work and m5ting.

Thank you Megan, Carol Am, Kevin, Man, Paul, Jeremy, Margy and John G. And lasdy,

thanks goes to Chantalle for being the ear, the shoulder and the wings 1 needed.

1 would like to dedicate this thesis to my grandmother, Katherine Campbell, who

initiated my fascination for the rare and the beaud3-

Table of Contents

B S T R A C T

ACKNOWLEDGMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

CHAPTER 1 Introduction

IMPACT OF SMALL POPULATION SXZE

- - LI

... rn

VI

vii

POLLEN LIMITATION

STUDY ORGANISM

RESEARCH OBJECTIVES AND HYPOTHESES

REFERENCES

CHAPTER II Effective population size and genetic drift

in populations of Hymenoxys herbacea.

INTRODUCTION

METHODS

STUDY SITES

CENSUS P O P U T I O N SIZE

EFFECTIVE POPUL.ATTON SIZE MODEL

Estimates of modei parameters

Total number of ramets

Recruitment rates

Survival rates and transition probabilities -

Generation le@

Population selfing rate

Extr@oIation tu oiherpopzdaiiurrr 30

Ela~tin'g anabsir 3 1

POPULATION GENETIC SURVEY 3 1

RESULTS 32

CENSUS POPULATION SIZE 32

EFFECTIVE POPULATION SIZE 33

Estimates of mode/ parameters 33

Total number of ramets 33

Recruitment rates 33

Transition probabilities 33

Generation length 34

Population selfing tate 34

Effective popdation siqe 34

Elastin-iy ana&nk 34

POPULATION GENETIC SURVEY 35

Alielic vanalion 35

Genetic diversity 36

Genetic divernly ivib reqect to popihtion sice 36

DISCUSSION 36

REFERENCES 43

CHAPTER III: Pollen limitation in Hymenoxys herbacea and its

genetic and ecological correlates 60

INTRODUCTION 60

METHODS 63

STUDY SITES 6 3

VARMTION IN SEED PRODUCTION AND POLLEN 63

LIMITATION

SuppIementaI poilinati0n.r

MATE DIVERSITY

POLLINATOR ACTWITY

Po/linatoor diverdy und Mriation rutex

Stkma po(len load

RESUETS

VARIATION IN SEED PRODUCTION AND POLLEN

LIMITATION

Jqt$ementaf poIIinationx

MATE DWERSITY

POLLINATOR ACïrVITY

PoZ/inator ditrern-4 and vLriation rare+

StZgma Pollen ioad

DISCUSSION

REFERENCES

CHAPTER IV: General Discussion

THE MAGNITUDE OF GENETIC DRIFT

VARIATION IN SEED PRODUCTION AND POLLEN

LIMITATION

MATE DIVERSITY AND POLLINATOR ACTlVTTY

THE EFFECT OF POPULATION SIZE

ESmMT1.G POLLEN LMTATION

FUTURE STUDIES

COISSERVATION IhIrLICATIONS

REFERENCES

APPENDIX

DEFINITIONS

List of Tables

Table

Locations of populations of H. herbacea

Summary of 13 H. berbacea populations.

Number of asexual and sexual recnllts in H- berbacea populations.

Transition ma& (1 999-2000) for taro populations of El. herbacea-

Estimation of N, for 13 popuktions of H. herbacea.

Elasticity analysis of Ne estimates of FI. I.e'Nucea.

Leveis of intrapopulation dozyrne variation in H. b e r h a .

Summary of 13 H. herbacea populations

ANOVA of seed sec in H. h e r h a populations.

Mean seed set of 19 populations of H. herbacea.

ANOVAs of population and pokation rreatment effects on seed set.

Regession of Pollen Limitation, mate diversis and insect visitation

versus population sùe and geographic isolation.

List of major insect groups observed in H. herbacea inflorescences.

ANOVAs of insect visitation rates to H. herbacea inflorescences.

ANOVAs of Insect visitor nchness and Shannon-Weaver Diveaiy Index

Effective number of pollen donors in H. herbacea.

List of Figures

Figure

Theoretical relationship of between Pollen -, Pollinator - and Mate -

Limitation and population size.

Photograph of an adult H. hdacea, the organism used in this snidy.

Depiction of EL herbaceds life cycle.

Filled and unmed achenes of H. herbacea.

Number of florets per inflorescence of H. berbocea-

Seed set in open- and supplement-pohted plants.

Seed set of self, cross(within), cross@etween) and open pollinations.

Scattergram of Mate diversity and population size.

Scattergram of Bee visitation rates and population size.

vii

Chapter 1: Introduction

Most plant species are patchily distebuted on the landscape and as such, are

organized into local populations that are finite in size. S m d populations ma7 be subject to

biologid processes not usuaUy associated with krge populations, whidi c m have important

effects on theîr growth (Reiners and Riggs, 1999), persistence (Huenneke, 1991; Newman

and Pilson, 1997) and ulàmatdy, th&- evolution (Lande, 1998). Because of these unique

effects, the study of s m d popdations is necessq not ody to derelop a basic understanding

of population processes in plants but also for idenafying the nSks and appropriate

management actions in conservation. The goal of my research was to examine the effects of

small population size on the reproduction and persistence of the self-k?compaàble plant,

Hymeenoxyr berbacea.

In this general introduction, 1 &st highlight the major ecological and genetic

consequences of small population size and summarize th& potencial impact on plant

reproduction. In particukr, I concentrate on two feanires of s m d populations that may

affect reproduction in self-incompatible plant populations: pollinator limitation and mate

limitation. Nevt, 1 introduce the target of mp research, the self-incompatible plan&

Hymenoxy hedacea. Fin*, 1 o u t h e my research objectives and propose a series of testable

hypotheses conceming the impact of ecological and genetic factors on reproductive success.

IMPACT OF SlMALL POPULATION SIZE

A variey ofecological and genetic processes are assouated with s m d population

size. Belom, 1 focus my discussion on the consequences of the nvo dominant processes:

genebc dtift and the M e e effect.

One of the most snidied consequences of smaU population size is genetic &fidrifr

D e h e d as the random fluctuation in allele 6equenae.s 6eom generation to generauon,

genetic c id i the~reticall~ results in a loss of allele diversis within populations and increased

genetic differentiation among popdations (Lande, 1988; Soulé and bLius, 1998; Schemske et

ai., 1994). Like other fomis of stochasciaty, the magnitude of genetic drift is expected to

increase as population size decreases (Wright, 1939).

hlthough the theoretical effects of drift are weii established, the importance of drift

in namai populations is more controversai. Eridence fiom surveys of protein

polyrnofphisms (electrophoretic) have corroborated the theoretical predictions for drift.

For example, comparative studies of electrophoretic variation have found genetic diversi.

to be lower in rare or geographically restricted plant species than in common, widespread

species (Waller et a(., 1988; Ledig and Co&e7 1983; Lesica et cd, 1988; Kanon et a/., 1988).

In addition, several studies have found signifi~a~lt positive rehtionships between protein

diversicy and popdation size, pamicdadp in species composed of isolated or ephemeral

populations (Barrett and Husband, 1997; Frankham, 1999, Barrett and Kohn, 1991) such

as the tropical tree Pitheceflobium eiiganx (Hall et al, 1996). These results are consistent with

the expected effects of W. However, d these studies have measured variation at genes

b a t are selectively near neuûal and it is less dear whether drift is likeiy to affect genes

affectkg v i d rates of populations (i-e. Schiemp, 1998 versus DiggIe et al., 1998).

There are tmo kinds of evidence that suggest that dEift may affect population

viability- Deneases in isozyme diversity due to s m d population site have been associated

wirh reduced seed set (Fischer and Matthies, 1998; Les et al., 1991; Oubourg and van

Treuren, 1994; Raijman et al, 1994) and hence reduced fimess (Rainey et ai., 1987;

Knowles and Mitton, 1980; Hamrick et al., 1979). It is undear, homever, mhether this

relationship of genetic dnn with fecundiv is due to the hation of deletenous variation or

due to direct impact that genetic diversity itself has on reproductive success. ULtimately,

the process may limit the population's persistence time through a reduced ability to adapt to

changes in its environment (Franklin, 1980; Sodé, 1980; Lande and Barrowclough, 1987).

The second is the effect of drift on the diversity of speùfic genes maïntained b y selecùon

(Le. in heterostylous or SI plants). Studies of Eirhhornia panimiata indicare that interactions

betmeen genetic deft and natural selection cause destabilizaüon of heterostyiy which dows

for the evolution of self-femlization (Barrett et d, 1989).

In theory, the strength of genetic deifc d l be inversdy proportional to the census

population size 0, assuming the population is demographically ideal; that is, it consists of

a constant number of individuals with non-overlapping generations, which randomly mate

and exhibit random variation in f d t y . Due to deviations feom this ideal, two populations

of the same census size may experience very different rates of genetic change Faples,

1991). Therefore, a knowledge of the total number of individuals in a population is

insuffiamt to describe the magnitude of drift affecting populations when these assumptions

are not met To account for this, Wright (1931) developed the concept of effective

population size (Ne), whiçh accounts for such deviations in naturd populations. The

effective size of an acnial population is the number of individuals in a theoretically ideal

population having the same magnitude of random genetic drift as the actual population.

Effective population size usually is lower than the census population size in populations

thae 1) have unequal sex ratios, 2) exhibit non-random variation in fertility arnong parents,

3) are p d y s e l f - f e g ; and 4) experience fluctuations in population sLe (Wright,

1969; Hall e t a/., 1996). In terms of the conservation of s m d populations of organisms, N,

is the most important tool for estimating the rate of l o s of genetic vacation.

Although it is arguably the most important evolutionarg parameter in populations,

effective population size is also one of the most difficult to estimate. This clifficultg stems

from tsvo problems: 1) lack of estimation methods that incorporate the salient life history

features of the organkm and 2) difficulty anaining reliable estimates of demographic

parameters. The dassical theory of N, (summarised, e.g. in Crow, 1954 and Kimura and

Crow, 1963) peaains primarily to monoeuous speues with aon-oveelapping generations

and no a s e d reproduction and is therefore liniited in its appiicabilitg to manp plants.

Recently, models have extended the theory to non-randomly m a ~ g populations with

mked (sexual and a s e s d ) reproduction systems (Orive, 1 993; Yonezawa, 1997). They

accommodate plants that are largely perennial, wîth overlapping generaaons, and thus

populations are composed of plants of different ages or demographic stages.

One potential ecological impact of small population sLe is the Aliee effect. The

Allee effect, h t proposed to describe the effect of low population density on the

popdation gromth rate of s o d a n d s (Allee et al., 1949), postulates that r edukg

population size bdow a threshold number or density of ladividuals M11 cause a decrease in

average fecundicg or viabilicy of individuals and hence reduced population growth (Allee et

al., 1949; Menges, 1991; Lande, 1988; Lamont et aL, 1993; Widen, 1993; b e n , 1996).

This reduction in vital rates may occur because the Iow density of individuals interferes

mith the interactions arnong con-spedics necessary for such population processes as hnding

a mare and defense against predation. The Allee effect is, however, not widely applied to

plants and their ccsocial" interactions. In theoretical modds that incorporate the AiIee

effect, population extinction rates are expected to increase dcamaticdy as density dedines,

and below some densi. threshold, extinction is virtually certain (Kunïn and Imasa, 1996;

Veit and Lewis, 1996). Although supporting evidence has been found to suggest that

density affects population exhction rates, there have been few examples of extinction

thresholds (Kunin and Iwasa, 1996, Groom, 1998). Evidence for the negative impact of

the Allee effea on reproduction and recruitment before the population reaches the

exànction theeshold, however, are much more cornmon (i-e. Ghazoul et a/., 1998; Roll e l

al., 1997; Lamont et al., 1993).

POLLEN LIMITATION

The primary effect of small population size on reproduction in plant populations is

to reduce reproductive success via pollen limitation (Jennersten, 1988; Menges, 199 1 ;

Hendax and Kyhl, 1994; Byers, 1995; Heschel and Paige, 1995; Agen, 1996). Pollen

limitation is a condition in plant populations in which reproductive success is limited bp the

avaikbility of pollen. As a result, the number of offspring produced (fecundiv) is less than

what the avdable number of ovules and resources dl allow (Burd, 1994). Pollen

limitation c m be caused by either the AUee effect or genetic drift-

Pollen limitation may &se due to ecological processes and, speàfically, the Mee

effect, particularlp because plants are dependent on pollen veccors (nind, water, animals) to

ensure pohation and thus fertilization. If the nurnber of pollen grains transfened to a

target individual is insufficient to fertilize ail available ovules, seed production w i u be

reduced. In insect pollinated species, the reduction in the number of offspring produced

due to insuffiuent pollinator visitation is referred to as poha to r limitation. Because small

populations are less conspicuous and offer a smder reward (pollen, nectar), p o b a t o r

visitation rates are expected to decrease, and thus (Allee et a(., 1949; Menges, 1991; Lande,

1988; Lamont et a/., 1993; Widen, 1993; Agren, 199G), pollen limitation should increase in

s m d populations (Figure l.la)(e.g. Schemske, 1980; Rathke, 1983; Sih and Baltus, 1987;

Krannitz and Maun, 1991; Ghazoul et a/., 1998; Schmitt, 1983; Kunin, 1997). This

prediction has been supported by observations of reduced seed set in small populations of

Dianfhm ddt0ide.r (Jemers ta, 1988) and Lythnïm ~alcaoria (Agren, 1996). In both cases the

pattern was attributed to insufficient pollen deposition and hence p o h a t o r limitation.

In addition to insuffiaent pollinator visitation, poilen limitation may also arise due

to a lack of compatible pollen donors, or mate limitation. Specifically, in species with

genetically based maMg types, seed production may be lùnited by the quality of pollen

received, even if there is more pollen than ovules. In mate limitation, then, reproduction

will depend on the likelhood of encomtering pollen of a compatible mate, which d l

depend on the frequen y and diversiy of mating types, spatial stxucnice of m a ~ g tppes and

the foraging patterns of polkators (Frankel and Soulé, 1981 ; Wright, 19G9; Vekemans e t

al, 1998). Since small populations are more likely to lose mating ypes due to genetic defg

one expects mate limitation to be inversely proportional to the effective size of the

population (Figure 1.lb).

Mate limitation is most likely to be observed in plants that are self-incompatible.

Self-incompatibiliq is a physiological mechanisrn controlled by one or more genes (de

Nettancourt, 1977). The proàuction of zygotes is controlled bp a rejection reaction between

the s t î p a or style and pollen. If the mio donor plants are of the same mating cgpe, that is,

haring the same self-incompatibility (SI) alleles, f d a t i o n dl not occur (de

Nettancourt, 1977; hfulcahy and hfulcahy, 1985). In sporophytic inconrpatibility s ys tems,

there are multiple SI deles and the diploid pollen donor plant conaols the incompatibiliq

phenotype of the pollen. The limited seed set of many donal, self-incompatible species,

then rnay be a result of geitonogamous pohation within geneticdy uniform patches,

causing mate limitation in local sub-populaüons (III& et a/., 1983; Aspiowd and

Christian, 1992; De Mauro, 1993; Reinhartz and Les, 1994). Once mate divexsity becomes

extremely low, limited seed set rnay be inevitable (Les et a/., 1991; Byers and Meager, 1992;

Byers, 1995; Hnmrick and Godt, 1990).

Athough much theoretical work has been completed on S-allele diversity in

populations (Richman and Kohn, 19%; Byers and Meager, 1 9W), there are relatively few

studies that have examined the relationship between population ske and mate diversi-, and

the magnitude of mate limitation in naturd populations (Byen, 1995). Models by Byers and

Meagher (1992) suggest that M t d overcome the frequency-dependent selecüon acting

to maintain mate diversity only when N, is Iess than 50 individuds @merson, 1939). In

past studies, reduced seed set has been documented in populations of SI species known to

be low in isozyme variation (Les et al., 1991 ; Byers and Meager, 1992; De Mauro, 1993;

HamSck and Godt, 1990). However, the role of mate limitation is only infened and never

tes ted directly (Aspinwall and ChPs tian, 1992; Molano-Flores and Hendox, 199 9; Menges,

1995).

In self-incompatible speues, the effect of mate lunitauon and pollinator limitation

may not be completely independent (Larson and Barrett, 2000). That is, pollinator

limitation may become more pronounced when popdations have low mate diversi ty (Figure

1. lc) (e.g. Menges, 1991; Aizen and Feinsinger, 1994; Byers, 1995; Agren, 1996; Kunin,

1997). This interaction occurs because the pollinator services required for complete

fertilisation will vary depending on the number of compatible mates in the population. For

popdations in which the SI alleles are equally frequent, and their diversity is high (Le. large

populations), most of the pollen d be compatible and a minimum number of grains m u t

be deposited for complete feralisation m e e t al., 1972; Byers and Meager, 1992). On the

0th- hand, individuals kom populations in which SI de le ftequencies are skewed and the

SI dele diversity is low (i.e. s m d populations, where genetic drift is sttonger than large

populations), require more visits fiom pollinators to ensure an equdy hi& probabiliv of

femlization b y compatible mates (Imxie et a/., 1972, B yers and Meager, 1992). Thus

populations, wirh lower mate genotgpe diversity may be more likely to be p o b a t o r limited

as well. Overall then, pollen limitation will be the product of evpected mate and pollinator

iimitacion (Figure 1.1~). While the signScance of pollen limitation is well established in

the plant Iiterature, few studies have related it to population size or separated the two

causes, mate limitation, due to genetic drift, and pollinator limitation, due to the Mlee

effect, in self-incompatible plants.

STUDY ORGANISM

Hymen0xy.r herbocea (EL Greene) Cusick, Asteraceae, is a small, perennial plant

found in prairie, diff or alvar habitats (Cusick, 1991; De Mauro, 1993; Voss, 199G;

Wunderlin, 1971)(Fîgure 1.2). As a rare endemic of the Great Lakes region, H. berbacea is

cunendy known to exist at two natural sites in the United States - Marblehead Quany,

Ohio (De Mauro, 1993) and Mackinac County, Michigan (Voss, 1996). Populations (for a

definition of "populationy7, see Appendix 1) are also found in tmo krger, less disnirbed areas

in Canada - the Bruce Peninsula and southern Manitoulin Island, each of which consists of

several populations (Catling, 1995)(Table 1.1). As populations of H. berbacea often consist

of relatively few individds (for definition of 'cindividual'y or "rametY', see Appendk 1) and

there are few known populations, the survivl of H. herbacea is thought to be at risk

Wor6.n-Palma and Snom, 1997; Reiahara and Les, 1994; Messmore and Knox, 1997). It

is considered a threatened plant n?hin Ohio and the U.S.A. and very rare at both the global

and provinaal (Ontario) level by the National Heritage Information Centre in Canada.

H. berbacea forms low-lying dusters of rosettes (up to 10 an in height) that persist

throughout the &ter and spread via asewd reproduction (for a definition of "asewal

reproduction", see Appendk 1) through rhizomatous grolvth and/or brandiing of the

woody caudeu. ICnown for its conspicuous, solitaq golden inflorescence, H. berbaceds

floral buds fomi the previous fall at the apex of a single rosette. The head is radiate with

pistillate ray norets and perfect disk florets. The mame leaves are dark green, rnoderately

hairy and have a thick cuticle that is interrupted by numerous stornata. In Canada, the

flowerkg season occurs ftom early May to eady Jdy. Seed production is reduced by

herbivores including insects, rabbits, birds and white-tailed deer in remote areas, while

human tra& adds to the damage iacuned in public areas. H. herbacea differs from its

dosest selacive, H. acauli~ var. acazilix, through the lack of glandular hairs on its leaves

(Cusick, 1 99 1).

Most importady, populations of H. herbacea tend to be resmcted in size and

geographically isolated, which makes them nLmrable to the effects of small population

size. One atmbute of H- herhcea, that may Eurther exacerbate the effects of s m d

population size, is its mathg system. H. bmbacea is a sporophytically seIfIfincompatible

plant, and hence an obligate outcrosser (De Mauro, 1993) making it vulnerable to mate

limitation as weli as pollinator limitation.

Within the Amencan populations of EL herbucea, it is unclear whether mate

limitation is occuflng as coaflicting reports on the amount of mate diversity wïthin US.

populations have been found (De Mauro, 1993; Morin-Palma and Snow, 1997). While

some very s m d populations dearly lacked anp diversitg in self-incompatibility alleles (De

Mauro, 1993), a crossing experiment Mthin the largest population s howed that more than

80% of the crosses were compatible (Mor6n-PaIma and Snow, 1997). The magnitude of

pollinator limitation in these populations has not yet been measured. fiowledge of the

genetic and ecological factors influencing reproductive success -dl also be important for

iden-g potential rkks and future management ahons for Canadian populations of H.

herbacea.

RESEARCH OBJECTrVES AND HYPOTHESES

I examined the impact of population size on the reproductive success of the self-

incompatible plant, Hymen0y.r herborea. Specîiically, 1 addressed the followïng four

questions:

1) What is the magnitude of genetic drift in popdations of H. herbacea in Canada?

2) Do H. berbacea populations vary in seed production and are populations pollen

limite d?

3) Can variation in seed production be explained by mate diversity or p o h t o r

activity within populations?

4) 1s the magnitude of polien limitation, mate diversity and pollinator activity

related to population size?

Based on the theory of genetic deift and the AUee effect, 1 wodd expect that the nurnber of

compatible mates and fiequency of pollinator visits wdl decrease in association with s m d

popdation size (Figure 1 a,b). This should result in a dedine in seed production and an

increase in pollen limitation (Figure lc).

REFERENCES

AGREN, J. 1996. Populations size, p o h a t o r limitation, and seed set in the self-

incompatible herb Lytbmru ~a/icaria- Eco/. 77: l?ï9-l79O.

AIZEN, M. A. AND FEINSINGER., P- 1994- Habitat fragmentation, native uisect

pollinators, and feral honey bees in Argentine, "Chaco Serrano". Eco/. A ' . 4:

378-392-

U E E , W- C., EMERSON, A. E., PARK, O-, PARK, T. AND SCHMTDT, K- P. 1949.

Pri'@les of animaI ecoiogv. Philadelph, Pennsylvania. Saunders Press.

ASPINWALL, N. AND CHRIS%"Jy T. 1992. Clonal structure, genotypïc diversity, and

seed production in popuiations of Fd@enduIa mbra (Rosaceae) Lom the northcentral

United States. Am. 1- Bot. 79: 294-299.

BARREïT, S. C. H., MORGAN, M. T., AND HUSBAND, B. C.. 1989. The dissoluuon

of a complex genetic polymorphism: the evolution of self-fertilization in mstglous

El'rhbomrapaninr/ataa n a (Pontedenaceae) . Evol. 43: 1398-141 6.

BARRETTy S. C. H. AND KOHN, J. R. 1991- Genetic and evolutionary consequences of

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Table L1 Locations of each population on the Bruce Peainsula used in the smdy.

- -- - -. - -

1 ru ce hvar Nature Reseme, Bruce Peninsula. Ontario

Population

BC

C U

CCS

CH

CPL

CPS

FW

HL

LC

LFON

NW

-- - -- 1 Cypress Lake, Bruce Peninsuia National Park, Bruce Peninsula, Ontario

Location

Cypress Lake, Bruce Peniasula National Park, Bruce Peninsula, Ontario

Cape Croker Hunting Ground, Bruce Peninsula, Ontario

Cape Croker HunMg Ground, Bruce Peninsiila, Ontario

Cabot Head, Bruce Peninsula, Ontario

Half-way Log Dump, Bruce Peninsula National Parks, Bruce Peninsula, Ontario

Half-way Log Dump, Bruce Peninsula National Parks, Bruce Peninsula, On tario

Cypress Lake, Bruce Peninsula National Park, Bruce Peninsula, Ontario

Cypress Lake, Bruce Peninsula National Park, Bruce Peninsula, Ontario

Cypress Lake, Bruce Peninsula National Park, Bruce Peniasula, Ontario

Bruce rjllvar Nature Reserve, Bruce Peninsula, Ontario

Cypress Lake, Bruce Peninsula National Park, Bruce Perimsuia, Ontario

AR

BL

lm 1 West end, Mannitoulin Island, Ontario

-- - - --

N e z Little Huron Lake, Mannitoulin Island, Onc&o

Burnt Island Harbour, Mannitoulin Island. Ontario

EO LH

1

RT 1 Rocby Trail, Mannitoulin Island, Ontario

- - - -- -

Near Little Huron Lake, hlannitoulin Island, Ontario

Little Huron Lake Road, Mannitoulin Island, Ontario

Figure Ll: Predicted effects of populaaon size on a) pollinator limitation, b) mate

diversity and c) pollen limitation. a) PoIlinator Limitation: As population size deçreases,

small populations become less visible to pollinators and are less able to offer pollinators

s&uent rewards for efficient pollen and nectar fora&iag. Therefore, as population size

decreases, pollinator activity should deaease. This d inuease p o h t o r Limitation,

measured by the number of pollen grains deposited on the stigma, within the popukàoa b)

Mate Limitation: As popdation size decreases, the strength of genetic deEt increases,

kequenues of alleles may become skewed and alleles lost Alleles under non-neutral

selective pressure are more likely to be maintained until popdation 9te becornes extremely

small Ge. 50 individu&) &ers and Meagher, 1992) at which point alleles are lost quickly.

c) Pollen limitation: When the effects of P o h t o r and Mate limitation are combined the

resulting seed set can be drasticdy reduced As popdation size deaeases, pollen limitation

is more severe than either mate or pollinator limitation due to the interaction of the two

component parts.

Pollen Idimitation = (4 O

Cr w

Compatible Polien Grains (%)

Mate Diversity

Number of Pollen Grains Deposited

Poilinator Activity

Figure L2. Photogaph of an addt H. berbacea, the organism used in this study.

Chapter II: Effective Population Size and Genetic

Drift in Populations of Hymenoxy herbacea.

INTRODUCTION

One of the most important evolutionary processes affecthg s m d populations is

genetic drift- Through stochastic fluctuations of dele fiequenaes fiom generauon to

generation, genetic drift d tesuit in random losses of genetic diversity and increased

homozygosity withiu populations, and increased genetic differentiation among popuktions

(Wright, 1931; Sodé and Mius, 1998; Barrett and Kohn, 1991). When speafic alleles, or

the total diversity of alleles, has some hct ional s'gniEicance, either now or in the future,

genetic dPft may lead to reduced fitness and ultimatdy reduced population viability

(Frankel and Sodé, 1981; Schonewald-Cos e t a/., 1983; Sodé, 1986).

The magnitude of genetic dPft in a natural population is best reflected by the

parameter effective population size, Ne. Effeaive population size represents the number of

individuals in a randomly m a ~ g population that has the same rate of random genetic dehr

as in the natural population of interest (Kimura and Crow, 1963). The strength of drift, or

the variance in dele fiequenues within a population, is inversely proportional to Nc

(Frankel and Sodé, 1981; Wright, 1969). Effective population size is usually cstimated in

one of two wvays: 1) by inferring Ne düectly kom the variance in genetic diversity or, more

commonly, 2) by estimaMg the census number, N, and adjusMg it accordlig to the

demographic characteristics that deviate corn an ideal population such as non-random

m a ~ g , uneven sex ratios and fluctuations in popdation ske over rime PVrÏght, 1969; Hd

e t a/., 1996). The dassical theoq of effective population sLe (summarised, e-g., in Crow,

1954 and I h u r a and Crom, 1963) pertains primarily to se-wal, monoeuous speues wich

non-overlapping generations and has considered ody a limited number of demographic

f e a ~ e s in naturd pknt populations. However, these modeh have recendy been =tended

to poppukons of speues mith both asewal and sesual reproduction sys tems whose

generations overlap (Onve, 1993; Yonezawa, 1997).

rUthough the theory of genetic drift has been well developed, documentation of its

relative importance in namal popdations is less complete- The effect of population ske on

the amount of ailozyme genetic diversity within populations has been smdied and, as

predicted, small populations ofien exhibit low allozyme diversity (Jones and Gliddon, 1999;

Young and Brown, 1998; Goodd d d, 1997; Frankham, 1999)- This suggests that genetic

drift can influence the magnitude of genetic dmersity in natural populations. However, loss

of allozyme variation does not necessady imply loss of hmess, as there is lunited evidence

that low dozyme diversitg wiu affect an individu al"^ sumival or reproducrive capautg

(Fischer and Matthies, 1998), nor influence a population's long temi viabilitv. On the other

hand, few smdies have examined the fate of deles that are under selection @ut see Banett

et a(., 1989). One such opportunitg involves alleles that code for mating types of

individuals which must mate disassortatively to produce offspring, such as those associated

with the seIf-incompatibility system of EIymen0xy.r heerbacea.

Genetic Mt may play an important role in the reproductive success and population

viability of the perennial herb, Hymenxy berbacea @.L. Greene) Cusick (Cusick, 1991). The

plant is endemic to the Great Lakes region, and is currendy known fiom approl3mately 30

poputations, which are resmicted in size and isolated fkom other popuktions. This

geographic structure may have a substantial influence on reproduction as maMg in this

species is govemed by a sporophytic self-iacompatibiliqr system. This is a genetically based

mechanism which ensures that successful fertilization occurs only between genotypes

camying diff'ent seIf-incornpatibiliy alleles (De Mauro, 1993). Genetic defi in small,

geographically isolated populations may cause the Ioss of SI alleles and hence ma&g q p e s in

a population, which can s@cantly affect reproduction and ulhately population

viability. Despite its potenaal importance, neither the magnitude of N, nor the strength of

gmetic d d 3 a c ~ g upon H. herbacea populations bas been estimated previously

In this study 1 esamined the magnitude of geneüc drift in populations of H. herbacea

in two wavs: &SC, bg es timating effective population size (NJ and, second, bp quantifjl0.g

the rektionship benveen population size, estimated by the fint objective, and dozyrne

diversity. I used this information to determine the potenrial for genetic drift to influence

m a ~ g type diversity and hence reproduction within 13 populations on the Bruce

Penuisula. 1 estimated effective population size (Ne) for two populations of H. herbacea

using a new demographic approach (Yonezawa, 1997), which incorporates the effects of

ascnal as well as sexual reproduction on the estimate of N,. While the effects of Nc on

neutral genetic diversity are well established, it is more difficult to interpret the significaace

of any given Nc value for self-incompatible plants, since m a ~ g diversitg is maintained by

hequency-dependent selection. As a benchmark, 1 used the results of Byers and Meagher

(1992), who showed that populations with an effective size of less than 50 d be unable

to maintain Luge numbers of mating types due to deft (Byers and Meagher, 1992). This

estimate is s@candy iower than the minimum effective population size of 5000

recommended by Lande (1988) for selectively neurral characters. An elasticity analysis was

then used to determine which life stages conoibute the most (and least) to the magnitude of

genetic drift in H. herbacea (Wood, 1 987).

Genetic daft was also quantitied, retrospeccively, by estirnahg the relationship

between population size and allozyme diversiy. AlIozymes are commonlp viewed as having

litde or no sdective value and thus provide a useM measure of the s ~ e n g t h of drift, in the

absence of selection. This was accomplished by conducMg an allozyme survey of 13

populations of H. herbacea that differed widely in size. If the effect of drift was reht idy

strong, 1 expected to find a positive relationship between population size and diversis..

METHODS

STUDY SITES

Field smdies mere conducted in t k e e n populations located on the Bruce PeninsuIa

in Bruce County, Ontario (Table 2.1). The populations were chosen not only to represent a

wide range of sizes to ensure that the effects of genetic drift could be detected but also

mere required to meet the followhg uiteria. Populations were: 1) at least 75 m apart; 2)

separated by non-habitat landscape, and; 3) individuals within the populations must

exchange gamets more ofken Mth other individuals michin the population than with other

populations. Of these populations, eight were situated on rodry shorelines, while five

populations were found on dvars, inland Two of these populations (CPL and HL) were

used in the estirnate of Ne nrhile all 13 populations were used in the genetic diversity

analysis.

CENSUS POPULATION SIZE

The census population size was eshated for each population in two ways: &sr as

the total number of reproductive rarnets present (N, ) and second as the total number of

rvnets 0. N, was e s k t e d during a single visit to each population in the first week of

June. As the floral scapes fkom the previous year were severely broken or uarecognkable

afim one year, there was no chance of mistakenly counting them. If the population had less

than approxïmately 2000 inflorescences, ali scapes mere counted For populations greater

&an 2000 plants, N, was estimated by counting the scapes found within one-meter wide

belt transects, which spanned the width of the habitat and mere placed every 20 m. From

this suroey, I esbmated the number of floral scapes per square meter and then multiplied

this by the total area of the population for an e s h a t e of the total nuniber of scapes. The

total area of each population was measured from inka-red a d photographs using the

cornputer program Northem Eclipse v. 5.0 (Empi~ Lmaging, Inc. 1996). To es timare N, 1

conducted a complete census of the number of rosettes in seven populations (shonrn in

Table 2.1) at the beginnlig of May.

Since the magnitude of drift in a population may be influenced by gene flow

betueen populations as well as by the census size (Hd and Clark, 1989; Goodell et al.,

1997), 1 aIso measured the degee to mhich populations were isolated fiom each othcr as

the distance to the nearest H. herbocea population (Geographic Isolation) (Abensperg-Traun

and Smith 1999) (Table 2.1). A Garmin GPS IZXL, 12 channel sptem was used to record

the geog~aphic position of each population (accuracy of i 25m)-

EFFECTIVE P O P U T I O N SIZE MODEL

Ni? 1 estimated effective population sue (NJ and - ratio for two randornly chosen N

populations, HL and CPL (Table 2.1), using a model created for plant species with both

s e - 4 and asexual reproduction systems and with stage structure (Yonezawa, 1997).

FoUowing Yonezawa (1997), effective population size (Ne) was estimated as:

P where N is the number of ramets in the popuktion; a = - , which is the deviation from 2 - P

random outcrossing, where is the population selhng rate; u = 4 - u-, + F, - u, + F, . y, .-

which is the total &action of individuals that survive the year, across all stages, where 6

represents the proportion of individuals within the five plots that belong to the

K demograp hic stage i = 1,2,3; S = (1 - a) + (1 + CX) - -, where k and % are the average and k

variance of the number of sexuaüy produced progeny recruited per ramet;

K (1 + a) - - where c and are the average and variance of the number A = 2 ( 1 + a ) - - - c k

of asexually produced progeny recruited per ramet; 6 is the rate of asexual progeny

production (i-e. the proportion of new recniits that were asexudy denved); and L is the

generation length, which Yonezawa (1997) d e h e d as the mean age in years at which nem

plants produce offspring (asexual or semal). Below 1 describe the methods I used for

e s t h a ~ g each of these demograp hic parameters.

ESTIMATES OF MODEL PARIMETE=

The total number of ramets (N) in each population was detemiined bp a complere

casus of each population as desaibed earlier.

&mitment ratef

To estimate the number of sesual and asexual recruits in each population, 150

ramets were located within 5 randomly positioned plots (30 an x 30 cm) in June 1999 and

then tagged and numbered. In the foIIoMng June (2000), 1 relocated the ragged rarnets and

counted the number of new rames. h y new ramet that was connected to a tagged ramet

was classif5ed as the product of vegetative reproduction (either via branching of the apical

meristem or rhizomatous gronrth). Any new ramet that was not connected to a tagged

ramet mas dassified as the product of sexual reproduction. From this information, 1 then

calcuiated the mean (c) and variance ( y ) of the number of a s 4 recruits per ramer and

the mean (k) and variance (6) of the number of sewal recruits per ramer. The rate of

a s e d recruiment (6) was calculated as the nurnber of asexual recruia divided by the total

number of aew reauits in the plots.

S u m v d ratex and transition probabiIities

To determine the probabiliv ( u ~ ) of a plant changing hom stage-dass I/ to Y, five

additional plots (30 cm x 30 an) were randomly positioned throughout the population.

The plots again induded 150 ramets at each population. Each ramet within the plots mas

tagged and numbered in early June 1999. 1 then dassified each ramet @ne 1999) mithin

the 5 plots into one of thtee demographic stages (pers. c o r n . J. Windus):

1) Juvenile 1 - single rosette, 4-6 leaves;

2) Juvenile 2 - more than G leaves, has not flowered;

3) Adult - reproductive.

Mthough seeds and seedlings are also demographic stages of H. herbacea, these stages did not

exis t in the plots at the time they were censused. This initial survey allowed me to caiculate

the proportion of plants in each stage-dass (4 , 4 , 6 respecuvely). In June 2000,I

censused the marked rmets and scored them again as present/absent and if present, as one

of the tbxee stage classes.

From this two year Surrey, 1 calculated u, the proportion of ramets in each

demographic stage-dass that suroived one pear, plus all transirion probabilities u, for stages

(i = 1, 2, 3 a n d j = 1, 2,3). From this data, 1 &O calculated r< , , the proportion of the

original juvede 1 rarnets ( Jue 1999) that sumived the year, um2, the proportion of the

original juvenile 2 rarnets that surPived the year, and u,, the proportion of the oeginal

reproductive ndults that survived the year. ' u2 ' was calculated, followiug Yonezawa

(1997) as: 4 -K-: + F , - K . ' , + F , -K:

Generation le@

Generation length (L,) was defined by Yonezawa (1997) as the mean age in years ar

which new plants produce offspring (asexual or sexual) and was calculated, over 500

500 500

generations, as: L = xz x - 5 - uj,, f - u;,, where is the probabiliv that a x=i ; x=i j

newbom is in stage j a t age x, where x = 1, 2, ..., 500 years. I extracted estimates of u and

F feom the recniitment and s e v a l studies described earlier.

Popzdation ~e&?ng rate

The population selhg rate (P) was infened fiom the segregation of marker genes

(aiïoqmes) among offspring in open-pollinated progeny anays using the mised m a h g

model of Ritland (1 996). The outcrossing rate (f,) is the mean proportion of offsprhg per

rnother plant sired by cross-fertilization and is related to the selhg rate by P = 1 - t ( t , cl

if any self-fertilisation is o c c ~ g ) .

To estimate t,, families were collected outside of the demographic plots &om 25

randornly chosen rarnen in each population. From each matemal plant, 8 o f f s p ~ g were

groam to the Juvenile 1 stage (at &um) in the Botany Depamnent greenhouse. One

young leaf pec pknt was harvested and ground in 45 ml "Decodon" extraction buffer

(Eckert and Banett, 1994) after fkeezing in liquid niaogen. The homogenate was

cenmfuged for 10 minutes at 4OC and the supernatant \vas applied ro cellulose acetate gels.

An electtic cunent (ZOOV) was run across the gel for 45 to 60 minutes and a stain

consisting of an enzymatic subsaate and a colourimehic dye \vas added to visualize each of

thee polymorphic enzymes (a-ACP, MDH, PGI). The enzyme Acid Phosphatase (a-ACP,

EC 3.1.3.2.), was resolved on morphoiine citrate (pH G.3 buffer, while Malate

Dehydrogenase (MDH, EC I.l.l.W), and Phosphoglucose Isomerase (PGI, 5.3.1.9) were

resolved on es-cittate @H 7.0) buffer systems (Hebert and Beaton, 1989, Manchenko,

1994). Each locus scored in this study (Ap-1, Mdh-1, Pgi-1) consisted of two alleles each,

and these were desîgnated "f' (fast) or "s" (slow) based on th& relative mobilïty.

I used the ma,uimum-Ekelihood estimanon procedure of Ritiand and Jaui (1 981) to

estirnate the outcrossing rate (t,) for each population. The estimation procedure uses

information £iom d three polymorphic loci to generate a single mulàlocus escimate as meU

as a mean single-locus outcrossing rate (t,) which is the average of the three locus-specific

estimates. Matemal genotypes were kifened according to the method of Brown and AUard

(1970). Maximum likelihood estimates were found using Newton-Raphson iteration, with

standard deviations calnikted as the SD of 1000 bootstrap values generated nitb the

progeny anap as the unit of te-sampling. Although a number of different initial tm values

were tried, 1 found that tm=0.5 for the HL population and ~ ~ 0 . 9 for the CPL population

gave the result mith the highest log-likelihood. The se ihg rate was calculated for each

popdation as: P = 1- tm.

Eqolation tu otherpujwIaafions

The effective population size of other H. herbacea populations was determined by

Ne multiplying the respective N values by the - ratio determined fkom the study above. If N

the population had not been censused for N but only the number of reproductive rarnets

N Ne (IV,), a conversion formula was hrst used to convert N,. to N: Ne = N, - avg - - avg -

N r N

N where avg. - is the average ratio of N to N,among 7 popdations (see Table 2.1 for

N r

Ne s p e d c population values) and avg.- is the average ratio of Ne to N, arnong the tnro N

populations, CPL and HL, for which Nc was estimated.

30

To measure the relative effects of each demographic parameter in the N, estimation

mode1 on the value of effective population ske, 1 srudied the response of N, CO small

proportional pemxbations (+lOO/o) in eacch parameter in tum while holding a.U the other

parameters constant If Ne = f (6). when 8 = O,, O,, ..., O, is a vector of n demographic

parameters, Wood (1987) defined: E ( e j ) = - as the elastiuty (E) of Nc to small changes 6,

in the* parameter.

POPULATION GENETIC SURVEY

The magnitude of genetic variability in each of the 13 Bruce Peninsda populations

was assayed using isozyme electrophoresis. From each population, a minimum of 1 5 seeds

fiom 15 randomly selected mothers were collected and grown to the Juvenile 1 stage (at

minimum) in the Botanp Department Greenhouse, Guelph, Ontario. One leafwas

harvested feom each plant and the elecaophoretic procedure desmbed for m a ~ g system

analysis was used. In this case, the genotype of each individual was scored for nine enzyme

loti.

Gels were nui for 45 to 60 minutes at 200 volts and stained for seven isozymes.

The enzymes 6-Phosphogluconate Dehydrogenase (G-PGD, EC 1.1 .1.44), Acid Phosphatase

(a-ACP, EC 3.1.3.2), Diaphorase - NADH (DM-NAOH) and Isocia-are Dehydrogenase

WH, EC 2.1.1.41) were resolved on a morpholine citrate (pH 6.5) buffer system. bialate

Dehydrogenase (MDH, EC 1.1.2.37), Phosphoglucose Isomerase (PGI, EC 5.3.1.9) and

Glucose-6-Phosphate Dehydrogenase (GGPDH, EC 1 -1 -1 -49) were resolved on a tris-citrate

@H 7.0) buffer system (Hebert and Beaton, 1989, Manchenko, 1994). Only those loci that

cxhibi-ied consistent activity, and had dearly imerpretable banding patterns were scored.

When more than one putative locus mas observed for an enzyme, loci were numbered

sequentiallp, mith the most anodally migrating locus designated "one". When polymorphic

loci consisted of nvo alleles each, rhey were designated " f l (fast) or "s" (slow) based on

th& relative mobiliy.

Measurements of population genetic diversity were estirnated using the POPGENE

compter program v.1.31 by Yeh e t a(., (1999): percentage polymorphic loci per population

(P), mean number of deles per locus (A), mean number of deles per polymorphic locus

(rlP), and Nei's (1978) rneasure of expected heterozygosity (H,). I used a multiple

regxssion to examine the relationship between population size and isolation and each of P,

A3-M a d HN.

RESULTS

CENSUS POPULATION SIZE

The average number of floral scapes (N , ) found in Bruce Peninsula populations in

1999 was 97,673-15 (SE = 63,019.8), and ranged from 54 (SC) to 673,672 (LFON)

flowering heads Wle 2.1). The average number of floral scapes found in Bruce Peninsula

populations in 2000 was 101,822.2 (SE = 51,739.60), and ranged hom 3 (SC) to 885-274

(LFON) (Table 2.1). The geometric mean, a better rneasure of central tendency when the

disaibution of values is highly skewed, was 1,443.4 in 1999 and 1,008.2 in 2000. A paired

t-test revealed there was no signi£Ïcant difference between years for the number of

inflorescences per population @aired t-test: t = -0.699, P = 0.498, n = 13). The mean

number of inflorescences in inland populations (1999 = 253,124.3; 2000 = 237,021) did

not diffa £rom coastal populations (1999 = 513.4; 2000 = 423.1) in either year (1999: t =

-1.737, ~ ~ 0 . 1 5 7 , n=13; 2000: t = -1.619, p=0.181, n=13) (Figure 2.2).

The total number of ramets within a population ranged from 134 (SC) to

N l3l,gO5,826 (HI,) for the 12 populations censused (Table 2.1). The average - for the

N r

seven populations was 57.1 (SE=18.43) and ranged from 13 (population CCL) to 149

(population LFON). The degree to which the populations were geographicaliy isokted

ranged fkom 76 m (population FW) to 13167 m (population CH) (Table 2.1).

EFFECTIVE POPULATION SIZE

E~timatex of model parameters

Totd number of ramets

The total number of ramets (N) was 6574 in population CPL and 13756 in

population HL.

Recruitment rates

Of the 150 plants originally tagged, 89 new recruits were found in population CPL

and 84 new recruits were found in population HL. The percemage of these that were

asewal, 6, was 90% in population CPL and 97% in population HL (Table 2.2). The mean

number of new, sevuaIy d&ved ramets per plant, k,(variance, V,) was 0.067 (0.063) in

population CPL and 0.015 (0.014) in population HL. The mean (c) number of asewal

recruits per ramet (vaPance, V,) was 0.62 (0.86) for CPL and 0.61 (0.91) for HL

respectively.

Transition probabilities

Due to vandaiism and human traffic, some of the tags were removed from the

plants, reducïng the number of marked plants in the demographic plots fiom 150 in 1999 to

149 in CPL and 139 in HL in 2000. In 1999, the demographic stage with the most ramets

was Juvenile 2 (kequency = 0.669 in CPL, 0.544 in HL), foliowed bp A d u l ~ (fkequency =

0.243 /O.îW) and then Juvenile 1 (frequency=0.088/0.159) (Table 2.3) (Figure 2.1). The

same trend mas observed in 2000. The sufvival rate averaged 0.990 (CPL: ri =0.986 and

HL: ri ~0.993) for Juvenile 1 individuals (Figure 2. l), 0.966 for Juvede 2 individuals

(CPL: u =0.939 and HL: u ~0.993) and 0.978 for Adult individuals (CPL: u = 0.986 and

HL: u =0.971). The survival rate (zr) of all marked ramets was 0.912 in population CPL

and 0.956 in populaeon HL Fable 2.3). From 1999 ro 2000, Juvenile 1 individuals most

ofien remained as Juvenile 1 (53.4%). O d p f o u percent of Juvenile 1 indimduals changed

to the hdult stage (4%)- Most Juvenile 2 individuah (64.2%) remained as Juvenile 2. Of

ail stages, Juvenile 2 indiriduds were the stage most likeIy to become an Adult (87O/0)).

Adults in 1999 most ofien reverted to the Juvenile 2 stage the following year (71.4O/0) and

resulted in a diange in Nc of 66%. Next to u2, u (proportion of individuals that survive

the year) had the largest impact on N,. An increase of 10% resulted in a change in N, of

55%. The t k d most influentkd parameter, 6 (the proportion of individuak in the plots

that are at the Juvenile 2 stage), changed Ne by 52% when F, was increased by 10%.

Finally S (calculated £rom values of selfmg rate, se,wal reproduction rate and variance)

changed Nc by 46% when it was increased by 10%.

In HL - Elasticity analysis revealed that, again, parameter u, .- (proportion of

Juvenile 2 ramets that survive the year) had the largest impact on N,. An inaease of 1 O%

resulted in a change in N, of 118%. Next to u, .- , u2 had the largest impact on Ne. An

increase of 10% resulted in a change in Ne of 88Oh. The thitd most influentid parameter,

5 (the propomon of individuals in the plots that are at the Juvede 2 stage), changed Ne

by 7Z0/o when F2 was increased by 10%. Fiady, u, (proportion of Adult ramets that

survive the year), changed N, by 59% when it mas increased by 10°/o.

POPULATION GENETTC SURVEY

A/Iekc vanation

From the seven enzymes (a-ACP, MDH, PGI, G-6-PDH, IDH, PGD and DIA)

sampled, 9 loci were scored: a-Aq-7, Mdh-7, Pgi-1, G-6-pd.b-1, Idh-1, Pgd-1, Pgd-2, Dia-i,

Dia-2. All the enzyme loci exhibited consistent banding patterns mith the same sub-uait

structure for each enzyme thxoughout all the populations. A total of 14 alleles mere

observed across aLl populations and the total number of alleles obsemed per population

ranged Erom 9 (populations SC and CCS) to 13 (populations CH and FW). Three loci were

polyxno'phic in most populations (A@, Mdh-1, Pgi-1) while only one locus (A@-1) was

polyrnorphic in all populations. Ali polporphic loci had tmo deles. Populations CH and

F W each had one unique allele that was not round in m y other population.

Genetit divcdy

M of the thirteen populations of H. berbacea had variation in at least one locus,

with an average of 1.33 alleles per locus (Table 2.6). The percentage of polymorphic loci

ranged fiom 11 -1 1°/o to 44.44%, with a population average of 30.77%.

Nei's (1978) measure of expected heterozygosity (H,) varied among populations,

from -0.288 (CPS) to +0.46 (CH), with no value > 0.5 (Table 2-61, and averaged 0.2035

(SE = 0.0791).

From a multiple regression, 1 found no signïfïcant relationship between population

size and geographic isolation and anp of the above genetic diversity variables (Table 2.6).

DISCUSSION

This study was designed to determine the magnitude of genetic dPft operathg in

populations of Hymengyr berhacea. Effective population size, es timated in m o populations,

averaged 4343.4 individuals, 43% smaller than a simple census of individuals would

indicate. In an allozyrne survey, there was no apparent relationship between population

size and population genetic diversitg. Here 1 examine the accuracy of these estimates,

consider their potencial impact on mate diversity and discuss the implications for

conservation-

Effective population size of the twelve H. herbacea populations averaged

5,357,466.5 (geomemc mean = 21,993.9) and ranged from 57.4 to 56,476,699-8

individuals @le 2.4). An effective sLe of at leas t 5000 individuals is generdy

recommended to avoid loss of neutrd genetic variation and evolutionary potential (Lande

1988). Only four of the twelve H. berbacea populations had (esàmated or extrapolated)

values lower than this d e of thumb suggesting that most popuiations are not at risk of

losing neutrd genetic variation (Table 2.4). In order to produce o f f s p ~ g in a population in

a self-incompatible plant speaes, it is important to maintain matiug type diversig. Self-

incompatibility (SI) alleles are maintained through fiequencg-dependent selection and hence

are less vulnerable to the effects of genetic drift- Byers and Meagher (1992) determined

that geneac Mt has severe effects on m a ~ g type diversitg only once the population is

smder than 50 individuals. The smdest population (SC) on the Bruce Peninsula diat we

saw has an effective size 57 effective individu& which indicates that genetic drift may not

have a great impact on self-incompatibdity alleles, except in the smdest population in diis

study-

Values of Ne in this study are similar in magnitude to the only other study of

effective population sLe that considers both a s e d and senial reproduction conducted by

Yonezawa (2000) on Fn'tih'a~a canz.xhatcensiz, a pprimarily donally reproducing p k o t The

Nc of F- cam~cbatcensir ranged korn 3274 to 5329, in the two populations exarnined, mhich

Ne is similar to the range for H. herbocea (2830.9 to 5855.9)). However, the - ratio for H. N

Ne herbacea (rnean: - = 0.43) was higher than chat of F. cc~m~chatcen.u~ (mean: %=0.255). N N

The differences between the speaes may be explained by demographic characteristics.

When cornparkg the two species, more indmiduals in F. cam~cbatcemir remain non-

reproductive over a one year interval (Yonezawa 2000). Mortaiity ody occurred in the

first, non-reproductive stage of F. camcbatcemir and mas relatively high (22.4%) mhile

death of H. b e r h a individuals occurred at aU stages and was on average low (7.1 %).

Finally, sgm6cant.y more incUviduals of H. berbocea (91.2O00) are at a reproductive stage

(either asexual or sexual reproduction) than individuals of F. cam~chatcemir (6.5O00)

(Yonezawa 2000). In the elasticitg analysis @able 2.5), the propomon of individuah in the

reproductive stage is estremely important as a change in 4 by 10% resulted in a change ia

N, by at least 66%. These demographic differences map esplain the differences in effective

population size between the nvo speues.

Ne Theoretical studies have explored the possible range of - (Nunney 199 1, 1993, N

1996) and have conduded that it should be dose to 0.5 in populations assurned to be ideal

(range = 0.25 to 0.75) except for flucniations in population size. If fluctuations in

population size are accounted for, this is reduced to an average of 0.43. In contrast with

this theoretical expectation, a review of 38 empiPcal estimates by Frankham (1995)

Ne revealed that the - ratio for in demographic snidies of plant speues averaged 0.32 and N

ranged h m 0.008 to 0.68. The ratio of H. herbacea (0.43) f d s within the range N

desuibed b y Frankham (1 995). Frankham (1 993, however, does not explore the

Ne demographic rasons for the differences in - ratios arnong plant species. One important N

demographic characteristic considered by Yonezawa's mode1 was the effect of clonal

reproduction. Few studies have examined the effect of donal reproduction on N, but

N, published results of - ratios of donai organîsms tend to be lower than those published for N

non-donal organisms (Orive, 1993; Olfelt e t al'., 1998; Richman and Kohn, 1999). As the

ratio for H. herbacea was higher than many of the published results, it is possible that other

demographic characteristics such as life history and breeding systems may also influence NE.

The N, estimates kom this study may be iduenced by other demographic factors

not taken into consideration, spe&cally yearly variation in population size. Fluctuations in

Ne population size map result in very s m d values of - and low N, in animal populations N

(Vucetich et al., 1997). This has been shown to be important also in plant species, i.e.

Eichhomiupanic/data, a colonizing speues, mhere Husband and Barrett (1992) found that

temporal variation in population size reduced N, to 50°/o of N, while O ther demographic

factors had much smder effect on Ne. As my estimate is calculated ftom onlv one vear's

data, 1 may be drastically overestimating long-term Ne. This \dl result in an

underestirnation of the rate of genetic drift and perhaps an overestimation of the probability

of population persistence (Vucetich et ai., 1997; Newman and Pilson, 1997). However, the

perennial nature of H. herbacea helps to maintain constant population sizes unlike those seen

in E. p a d a t a populations whose population sizes fluctuate rapidly. Fiuctuations in

population size over the iwo years in which populations were rnoairored were not

sigdïcantly different than zero. This suggests that yeaely fluctuations in N in H. hwbacea

may not have a large influence on effective population size.

Nc was estimated for two populations of H. herbacea using the demographic mode1

Nc by Yonezawa (1997). 1 then used the estimated - ratio to estimate N, in 10 other N

populations on the Bruce Peninsula for which we have census population size data (Table

Ne 2.4). These are reasonable estimates of N, assuming that the - ratio semains cons tant N

Ne across ail census popdation sizes. However, some studies have shown that - gets srnder N

with an increased population size. This suggests that my estirnates of N rnay be an

Ne underestimate of - in s m d populations and an overesàmate in large populations. N

Homever, I believe this is not likdy to be a problem in H. herbacea for three reasons. First,

Ne the two estimates of the - ratios were rernarkably simiriir. Secondly, outcrossing N

N, populations, such as those of H. herbacea, tend to exhibit less variation in - among N

populations than s e h g populauons (Schoen and Brown, 1991). Thiedly, the 3 ratios in N

Ne larger populations are generally smaller than - in smaller populations (Husband and N

Barrett, 1992; Pray et a/., 1996; Nozawa, 1970; Wade, 1980) prirnarily because larger

populations are more iikelp CO eevpetience fluctuations in population size (Nei and Gram,

1984, Frankharn, 1995). However, H. berbacea populations did not fluctuate sigaificandy

in population size over the two years.

The accufacy of the N, estimates may also be inhenced by the method of

estimation 1 used (i-e. ecological vs. genetic). Ecological rnethods for estimating effective

population size, based on various demographic and reproductive variables have been used

quite frequently and compilations of these e sha tes have been undertaken for various

plant groups ( H e ~ o o d , 1986). Hom accurately these measures predict the genetic

behavior of populations, however, is diffïcult to evaluate because of the Iimited number of

studies involving both ecological and genetic approaches @ut see Begon et al., 1980;

Husband and Barrett, 1992). In one such cornparison, Husband and Banett (1 992) found

that demographic estimates of effective population size were between 2 and 500 cimes

largex than genetic estimates. Although the ecologically based Ne values of H. herbacea

populations were high, if they were reduced by even a factor of 10, many of the

populations mould be considered at danger of losing important genetic dmenity, including

SI allele diversity. Thus ecologicdy based estimates should be conoborated mith genetic

rneasures to more accurateiy e s h a t e the effect of genetic drift on the maintenance of SI

alleles.

In general, populations of H. berbacea have moderate levels of i s o z p e variation,

with a population average of 30.8% polymorphic loci. The mean of Nei's measure of

expected heterozygosity was 0.20. My measures of genetic diversity Mthin H. herbacea

populations were similar to that in other perennial, endemic, outcrossing, animal-pollinated

pknts (HamPck, 1990). As H. herbacea is a self-incompatible plant and heoce obhgate

outcrosser, populations are predicted to be relatively undifferentiated nrith respect to

genetic vaBation at the population level. Asexual reproduction and perennialiy map

maintain genetic diversity during periods of non-random mating and uneven mating type

ratios. Every population had aUelic variation in at least one locus (mean = 1.33

alIeles/locus) however some populations had very Little.

If genetic d.rîft in s m d populations mas strong, one mould expect to see a relation

between popdation site and isoqme diversiv. Several plant studies have found a

signifïcant positive relationship between population size and % polymorphic loci, number

of deles per polymorphic loci and/or gene diversity (i-e. expected heterozygosîty)

(Ellstrand and Elam, 1993; Prober and Brown, 1994; Raijman et a[., 1994; Sun, 19%). En

H. berba~ea~ however, no such relationship was observed. The lack of slgnificant relationship

between population genetic diversity and census or effective population sizes in H. hcrlacea

suggests that dPft is not an important force in these popuiations or that its importance is

masked by other processes. Fluctuations in population size, for example, may be an

important feanire of this species. As a result popuiations may not have reached a deift-

migration equilibrium and the estimates of N based on one year's observations wiu not

reflect the dPft experienced in that population. In addition, populations of H. berbarea, in

general, are not extremely isolated, wbich may mean that populations d not become

geneticaily diffetentiated even if N, is low, due to the homogenizing effects of migration.

Finally, esàmates of N, may sugges t that rnos t populations are large and thus drift may no t

be su Çficiently s trong to affect isozyme diversitp.

Should land managers be concemed about the strength genetic drift and the loss

diversitg within popuiations of H. herbacea? The theoretical results desmbed by Byers and

Meagher (1992) recommend that sigdïcantly smaller popuiation sizes than those seen in

the populations of H. herbncca we surveyed will maintain self-incompatibility mating types.

Therefore the SI allele diversity of most populations in this smdy d be unaffected bp

genetic dPfc. Compared to the theoretical minimum population size (5000) that is

considered to have minimal amounts of Mt, only four popuiations are srnder than this

and the neutral genetic diversis. w i t h populations of H- herbacea on the Bruce Peninsula

may be slighdy affected by genetic fi. Homever, as genetic diversitg in most populations

is at a moderate level and is not predictable fiom population size, ir is unnecessary to take

measures to maintain or increase this diversity,

In conservation efforts, it is imperative that programs target the mos t influentid and

vuluerable life stage of the organism in order to most efficiendy preserî-e the species

(Schemske et a/., 1994). In this study, using elasticis analpsis, 1 found that the Juvenile 2

M e stage (more than G ieaves, no inflorescence) was the most prevalent plant stage in the

populations and its rate of sumival (u-,) was very influentid on the effective population

size. Therefore, if N, becomes extremely low due to habitat fragmentation, consemation

efforts that increase the sumival rate of this iife stage will Wtely be the most successful.

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Table 2.1. Summary of 13 Hymenoxy berbacea popdations on the Bruce Peninsula,

Ontario, surveyed for popdation ske (N, - total number of inflorescences and N - total

number of ramets), geographic isolation (m) and habitat location (C=coas tal, I=inland).

Populations indicated with * indicate popdations discovered in 1999. N/A = data not

Population size Habitat Typc

C

- .-

Geograp hic ( Population

isolation (m)

Geometric Mean I

Table 2.2. nie number of asexuai and sexual recmits produced, over the period of one

year, within sub-plots (containing a total of 150 ramets initially) of popdations HL and

CPL of H. herbacea on the Bruce Peninsda, Canada, 6 = rate of asexual recruitment of H.

herbacea ramets.

Population

CPL

HL,

Ase.waI Recruits

80

82

Sexual Recruits

9

2

6

0.90

0-97

Table 2.3. The transition matnx over one year (1999-2000) for populations CPL and HL

of H. herbacea from the Bruce Peninsula, Canada Juvenile 1 individuals are composed of a

single rosette with 4-6 leaves, JuveniIe 2 individuals have more than 6 Ieaves and have not

flowered, and Adult individuals are reproductive. The top value in each box represents the

number of individu& that were initially stage i in 1999 and changed into stage j in 2000.

The lower value in brackets in each box represents the proportion of individuais that were

initially stage i in 1999 and changed into stage j in 2000.

Juvenile 2 Juvenile 1 Juvenile 2 Adult

4

(O. 1 1)

Juvede 2

Adult

Dead

(O. 1 1)

Table 2.4. Estimates of N, for 13 popdations of El- hehcea from the Bruce Peninsula,

Canada. N, = number of inflorescences in the population, N = number of rarnets in the

population, N, = effective number of individuals in the population. Subscnpts iodicate

which method was used to estimate N, : 1) dire+ fiom the effefective population size

Ne mode1 by Yonezawa (1997); 2) by multiplying N by die - ratio; and 3) by multiplying N

pz-

Avexage 1 -

Table 2.5. Elasticity analysis of effective population size using pararneter estimates

fiom two populations of Hymenoxys herbacea, CPL and HL, fiom the Bruce Peninsula,

Canada. The values represent the degree to which N, changed with a proportional change

(i.e. 10%) in the value of each parameter in the modei. N = number of ramets in the

population; us, = proportion of original juvenile 1 ramets that survived one year; u , -- =

proportion of original juvenile 2 ramets that survived one year; u, = proportion of

original adult r a t s that survived one year; 4 = proportion of individuals in Juvenile 1

stage class; F, = proportion of individuals in Juvenile 2 stage class; F, = proportion of

individuals in the Adult stage class; c = mean number of asexual recruits per ramet; y=

variance of the number of asexmi recruits per ramet; k = mean number of sexual recruits

per ramet; V, = variance of the number of sexual recruits per ramet; 6 = the rate of asexual

recruilment; L = mean age, in years, at which plants produce offspring (asexual or sexual);

1 Variable -10% (CPL) +IO% (CPL) - r

-

-

-

-

- I

-

-10% @-IL)

L

a

A

S

u2

K

+IO% (HL)

-

1.2344

1.5540

0.7656

O -446 O

1.8885

1.401 6

0.1115

0.5984

Table 2.6. Levels of intrapopulation dlozyme variation in Hymenoxys herbacea. When

15 individuals of one population could not be scored, the population was left out of the

andysis. P = Percentage of polymorphic loci; A = Mean number of alleles / locus; AP =

Mean nurnber of polymorphic alleles per locus; H, = Nei's measure o f expected

heterozygosity .

Number of alleles observed

0.0827 (O. 1748)

CCS 9 0.0876 (O. 1799)

CPS 10

I

O. i OS5 (O. 1969)

1.1 OS2 (O. 1656)

Figue 2.1. Life-cyde graph for H~en0xy.r hdacea populations (CPL/HL). F,=the

nurnber of newboms produced per piant in stage uq=probability that a plant in stagej

deveiops to stage iin the next y-, Di=Fra&on of plants in the demographic stage i,

1 - ut = the probabillq that plants of stage i die in the next year. Nomendanire follows

that of Yonezawa (2000).

I -rr ,= 0.15410.045 Juvenile 1

Dl= 0.08810.159

- Transition -- * Recruitrnent --(> Death

Chapter III: Pollen limitation in Hymeelzooxy herbacea

and its genetic and ecological correlates.

INTRODUCTION

Pollen limitation is a condition in plant populations in which plant fecundiv is

limited by the availabilitg of pollen, and results in fewer o d e s being fertiLized than are

available- It is traditionally measured as the difference in fenindiq between open

pollinated flowers and those receiving a pollen supplement It has been documented in

62% of d angiosperm species examine< and often varies spatially and temporally within a

single species (Burd, 1994). Given ia broad taxonomie dismbution, the specinc ecological

and genetic mechanisms underlying pollen limitation are likely diverse. However, we

currently have a poor understanding of the conditions in which pollen limitation d l &se

(Larson and Baerett, 2000). One ckcumstance in which poilen iimitation ma? be Wcely is in

small populations, where it rnay be caused by two mechanisms, one ecological and one

genetic.

Pollen limitation rnay occur in small populations due to a reduction in pollinator

Visitation, termed here pollinator limitation. With few individuals, a s m d population will

have a smder floral display and will produce fewer floral renrards (i-e. nectar, pollen) than

large populations. Hence, small populations may be less able to attract and main&

potential pollinators than large populations (Mee et a[., 1949; Lamont et a/., 1393; Widen,

1993; Agen, 1996; Sih and Baltus, 1987). AU else being equd, having fewer poilhators

nrill result in fewer insect visits per flower, less opportunity for pollen transfer among plants

and hence lomer seed production. With pollinator limitation, pollen may be in adequate

supply in the population but the pollen vectors are insuffiaent to transfer the quantities of

poilen necessary to femiule aU ovules parson and Barrett, 2000).

Pollen limitation in srnail populations may also arise k o u g h a second and related

process; a Iack of cross-compatible mates (referred to here as mate limitation). In dioecious

and self-incompatible plant speues, reproductive success is contingent upon the kequenq

of compatible matkg %es in a population. However, in s d populations, genetic drift

can cause sexes or mating types to become skewed in fiequency or Iost altogether (Frankel

and Soulé, 1981, Wright? 1969; De Mauto, 1993; Aspinwd and Christian, 1992, Widen,

1993; Les et a/., 1991)- In dioecious speues, the loss of males will result in inadequate

pollen production for complete seed set In self-incompatible (SI) speues, loss of some

X n a ~ g types and the conesponding dominance of another rnating ype renders most

pollinations among individuals ineffectual. In this case, pollen may be deposited in nurnbers

in excess of o d e s ; however mosS if not al, wiu be incompatible and will result io

ineffectual polliaations.

The two mechanisms of pollen limitation, mate and p o h t o r limitation, are not

always independent due to the way that pollen limitation is measured (ie. fecundity afkr

open pollination versus open pollination plus pollen supplement) . For populations in

which SI mating -es are equally fiequent and diversiy is hrgh (i-e. large populations),

most of the pollen will be compatible and a minimum number of poIlen grains must be

deposited for fertilization of the rnajonty of ovules. Under these circums~nces a

population may be limited by the abundance of polinators but not mates. On the other

hand, populations with skewed SI matïng type fiequenaes and low mating type diversis

(Le. small populations) mill requke more visits from pollinators to ensure an equally high

probability of encountering compatible mates. Thus? small populations will not only have

lower mate diversitg but d be more likely to be pollinator h t e d than large populations,

which are more visible and diverse. Few studies have compared the magnitude of pollen

limitation to population size @ut see Agen, 1996) and to my kaodedge none have

attempted to disentangle these uaderlying causes of limitation.

Hymenoxyx herbacea (E.L. Greene) Cusick is a herbaceous perennial that is endemic to

the Great Lakes region and commonlp found in prairie, cliff or alvar habitats (Cusick, 1991;

De Mauro, 1993; Voss, 1996; Wunderlin, 1971). Known for its conspicuous, solita.ry

golden inflorescence that blossoms fkom eady May to Juiy in Canada, H. herlacea also forms

low-lying dusters of rosettes that persist throughout the arinter and spread vegetatively.

herbacea may be a good candidate for pollen limitation for three reasons. First, it is insect

pollinated, f l o w e ~ g in the sp&g when few other speaes are flowering in the atea DuSng

the unpredictable weather of spring, poha tor s e c e is ofien enatic and may be

inadequate for complete seed set Second, H. herb~cea is self-incompatible. Self-

incompatibility is a genetically controlled breeding s ystem that preven ts self- fertillation and

ensures that seeds are produced only after mating between iodividuals that carry different SI

deles (ie. diffkrent m a ~ g ypes). This raises the possibility that p h t s are pollen limited

even if pohators are abundant Thid, H. berbacea consis ts of small, isolated populations.

Nthough rarnets are u s d y locally abundant, only a s m d propomon of ramets flower in

any given year. Therefore it seerns likely that the effects of s m d population site rnay

enhance pollen limitation via poollinator and mate Ilnication. Pollen limitation due to a iack

of mates bas been described in a population of 30 individu& in the U.S but mas not

apparent in another population of more chan a d o n ffowering individuals (De Mauro,

1993). However, due to the hgh conservation concern for the speaes, a broader analpsis of

pollen limitation and its causes is required.

In this shidy 1 examined the relationship berneen population size and reproduction

in the self-incompatible pknc Hymenaxys berbacea. 1 expect pollen limitation to increase as

population size decreases, due to either pollinator or mate limitation or both. Specificdy, 1

addressed the following four questions. First, what proportion of ovules manire into seed

and are populations pollen limired? Second, do popdations diE& in mate diversity and to

what extent does this contribute to poilen limitation? ThLd, do populations differ in

poha to r activity and to what extent does this contribute to pollen limitation? Findy, is

the magnitude of pollen limitation, mate diversity and poha tor activiq related to

popdation size?

METHODS

STUDY SITES

Thirteen populations, located on the Bruce Peninsula in Bmce County, Ontaeo and

six populations, located on Manitoulin Island, Ontario were used in chis snidy Fable 3.1).

Population sizes and habitats of the Manitoulin Island populations were not measured. Of

the 13 Bruce Peninsula populations, eight were situated on rocSr shorelines and designated

as coastd, while 6ve populations were found on dvars, inland. The sites covered the

species' entire geographic range in Canada and were chosen to represent a wide range of

sizes (N \J 3 to 885,274) to ensure that the effects of population size codd be detected

(Imne, 1972; Fischer and Mattbies, 1998). Population size mas previously estimated (see

Chapter 2 for methods) for each location as the total number of reproductive ramets, since

this best reflects the number of potential mates and the amount of pollen available for

fercilization in any single year (Table 3.1). It was also estunated as N,, which best reflects

the strength of genetic drift ac&g on the diversity of m a ~ g types. As the magnitude of

pollen limitation in a population may be influenced by prorcimity to other populations

(Hard and Cl& 1997), 1 also measured the distance to the nearest population for each site

(Geograp hic Isolation) p b l e 3.1).

VARIATION IN SEED PRODUCTION AND POLLEN LIMITATION

To examine vatiation among popdations in reproductive success, mean seed

production was estimated for unmanipulated inflorescences in each of 13 Bruce Peninsula

populations and 6 Manitoulin Island populations d d g the spring of 1999, and the 13

Bmce Peninsula populations again during the spPng of 2000. In 1999, twenty-five

inflorescences were randomly selected and marked in each population prior to anthesis. In

2000, twenm-five inflorescences were again randomly selected and marked in each

population prior to anthesis and, mithin each capitulum, 5 florets were marked (non-toxic,

permanent rnarker) and the adjacent flores were removed so the target florets codd be

relocated. A k anthesis, when the seeds had matuîed, fifieen randornly chosen florets in

1999 and five fiorets in 2000 in each capirulum were selected and scored as med (Le. with

mature seed) or unfilled (mature seed absent). Filled seed were easily identined as being

round, hard and da& achenes, whereas unfïJled achenes were flattened, sofk, and white

(Figure 3.1). Preliminary germination tests showed that unfilled seeds were 100% non-

viable.

Reproductive success was reported as seed set, which is the number of mature seeds

expressed as a proportion of the number of ovules examined Seed set was arc sine

txansfomied for all analyses in order to improve the nomiality of the residuals. Mean seed

set was calculated for each population and region @ruce Peainsula and Manitoulia Island)

and differences among means were tested with a Nested -VA (Sokal and Rohlf, 1989)

using SPSS software (1 999)).

To estimate total seed production per head, seed set mas multiplied by the mean

number of florets per capitulm. The average nurnber of florets was estimated in 15

randomly chosen inflorescences fiom each Bruce Peninsula population. A one-way

ANOVA was perfomied to test for differences in the number of florets per capitulum

between populations and a t-test was perfomied to detect differences in the number of

florets per capitulum between habitats.

Szq@ementaI PoL.knafions

To determine the strength of pollen limitation, tsvo pollination txeatments were

perfomied within each of the thïrteen popuiations of H. herbacea on the Bruce Peninsula in

spring 2000: open pohation (described in previous section) and open pokation plus

pollen supplement Twenty-five pairs of floral scapes were randomly located within each

population. For each inflorescence, I randomly located and marked (non-toxic marker) a

group of five receptive stigmas and removed the adjacent florets so the florets could be

relocated. The k s t inflorescence of each pair was Iefi unmanipukted and e-xposed to

nahird pollinator activity (open pohation). The florets in the second inflorescence of

each pair were also exposed to poULiators. Supplemental pollliation was meant to increase

the quantity of pollen as mell as the diversity of m a ~ g qpes conmbuting to the pollen. In

addition, 1 added five anthers' worth of pollen from each of five randomly chosen pollen

donor plants (with replacement) to the five florets. In srnall populations, pollen donor

plants were sometimes used more than once because of the limited number of inflorescences

available. In both pollination treatments, the inflorescences were left uncovered, to allow

for conhuai insect visitation. Approximately tmo weeks afier pollinations were

conducted, the inflorescences were bagged in nylon mesh to prevent seed loss. Three weeks

later the florets mere collected, and scored as 6Ued or unhlled. Seed set was caiculated as

previously described-

Mean seed set for both pollination treatments was calculated for each population.

A two-way ANOVA was used to test for diffaences in seed set between p o k t i o n

treatments, populations and th& interaction. To test for pollen Luniration in each

population, linear contrasts were used to compare the mean seed set in open pollinations to

that in the pollen supplement treatment.

If plants have received an adequat~ amount of compatible pollen, then seed set in

open pollinated flowers should be equal to seed set in the pollen supplement treatment If

seed set in the pollen supplement Peatment exceeds that of the open-pollliation aeatment,

plana are pollen limited. To express the magnitude of pollen limitation ushg a single

measme, 1 used a Poilen Limitation Index (P), which mas calculated as:

Supplemental pollination seed set - Open pollinated seed set P =

Supplemental pollination seed set

The Pollen Limitation Index ranges from O, indicating no pollen limitation, to 1,

which represents complete pollen limitation. 1 imposed a lower bound of zero for the

index since a negative result (greater fertiliy from open than supplemental polliaation

treamients)is likely due to experimental or Type 1 statistical error (Young and Young, 1992;

Burd, 1994) and is not informative in the curent contest (Larson and Barrett, 2000).

To test the prediction that the magnitude of pollen limitation is related to

population size, 1 used a multipk tegression Nith Pollen Limitation Indes (P) as the

dependent variable and population size and geographic isolation as independent variables.

To detemiine the potential for mate limitation, a series of controiled pollinations

were performed on each of 25-30 individuals per population in the Botany Department

greenhouse, University of Gudph durhg Winter 1999-2000 ushg plants from the 13 Bmce

Peninsula populations. Three pollliation treatments were performed:

1) Self-pollination - pollen was transferred fiom anthen to stigmas of the same

capitulum;

2) Cross (within population) pollination - polien was aansfmed fiom one randomly

chosen patemal plant fiom within the population;

3) Cross (berneen population) pollination - pollen was transfened kom one

randomly selected patemal plant h m arnong the remahkg populations

SeLf-poUinations were conducted to c o h that H. herbacea is self-incompatible

and to esthate the seed set e-xpected in incompatible crosses. Cross(within)-pollinations

were conducted to escimate mate divesitg wîthin populations. The propomon of crosses

that are compatible wiii reflect the probabilitg of encoutering a compatible mate within

the population, whîch is linearly relared to the diversitg of SI matïng types.

Cross@etween)-po1ILiations were conducted to d e t e e the proportion of compatible

mates in the sueroundhg populations within the Bruce Peninsula.

Eight offspring fiom each of 25 randomly selected mothers Gom each population

were grown under greenhouse conditions. The hnt pknt of each progeny anay to flower

was used as the matemal plant for all pollinations. Each pollination treatment was

replicated on five florets within each inflorescence. For the within- and benveen-

population crosses, a different patemal parent was selected for each reupient inflorescence;

however, replicate florets of a given treatment received pollen from the same patemal

plant Plants used as pollen donors were also used as matemal parents. To distliguish the

three pollination treatments, pollinated florets were rnarked with wearher resistant paint

applied to the petals of neighbouring ray florets.

Seed production in each cross was summarized in two different ways: 1) mean seed

set, and 2) the proportion of crosses that are compatible, where any cross r e s u l ~ g in

greater than 5% seed set was compatible, a condition set by De Mauro (1993) while

working with H. herbacea populations. Both meanires reflect the efficacy of the pollination;

however, the latter measure is preferable for estimating mate diversitg as it identi£ïes,

unarnbiguously, which p ollinations involved compatible ma ting types and is less in fluenced

by diffaences in resource avaikbility arnong matemal plants.

The statis tical analysis was designed to assess the overail effects of the experimental

polfiuations, across ail populations, and to evduate the following specitic p h e d

cornparisons at the popdations levd

1) Self-versus Cross(Within)-pollination - If Cross(Within)-poIlination seed

production was simikr to thac of self-pollinations, then the number of

compatible mates within the population is low and most individuals have

the same mating type.

2) Self-versus Cross(l3emeen)-pohation - If CrossQ3enveen)-pollinations seed

production was similar to that in self-pollinations, then individuals fiom

other populations tend to have the same maMg type as the focal population.

3) Cross (Wii4G.n)- versus Cross (Belmeen)-pollination - If Cross pithin)-pokation

seed production was si& to that in Cross(I3emeen)-pohations, then the

diversitg of compatible mating types from the sumounding populations is

similar to the diversity of compatible m a h g types within the population.

4) Cross(Within)- versus Open-pollination - If Cross(Within)-polfiuation seed

production was sUnilac to the seed set of Open-pohation, then the mate

diversity received from a single pollen donor within the population is a good

approximation of the amount of diversicy received &om open-pobation.

Using seed set as a measurement of mate diversity, pollinations were compared using

a two-way ANOVA. Planned comparisons iu each population were performed using ILiear

conaasts. In order to avoid Type 1 enor, a was reduced to 9.86 x 104as suggested by Sokal

1 - and R o m (1981) using the Dunn-Sidak equation: O! = 1 - (1 - a)' for k=52 compaüsons.

Using the percent compatible crosses as a measure of mate diversiq, pollinations were

compared using contingency table anaiysis, &st, in all the popuhtions combined and then,

for individuals popuiaaons.

Mate dmersity within populations was measured as the proportion of compatible

crosses as this is the most direct and intuitive measure. This measure reaects the probabiliry

of encountering a compatible mate wiaithin the population regardless of resouces avaikble

to produce the seed or genic sterility withitl the population.

To io~amine the contribution that mate diversity makes to pollen limitanon, 1

regressed mate diversitg (proportion of compatible crosses withia a population) against the

Pollen Limitation Index. Secondly, 1 perfomed a multiple regression of mate diversiq

(propoflon of compatible within population crosses) versus log (population sïze) and

geographic isolation to examine the hypothesis that mate diversity will decrease with

decreasing population size and inaeasing geographic isolation. As mate diversity wdl

necessarily be O when population sLe is O, 1 restricted the intercept of the regression to O.

POLLINATOR ACTIVITY

Pollinator Diverri.. and VZjrtation Rates

To determine the potential for pollinator limitation, I estirnaced the diversitp and

abundance of insect visitors svittiin each of the 13 populations on the Bruce Peninsuk.

Dueiog the 2000 field season, four plots (2m x 2m) were randomly placed within each

population. Each plot was observed for insect activity during typical peak activig periods

for insects, between 10:30 a.m. and 3:00 p.m. Observations were made in a minimum of

four 30 minute periods randomly dispersed throughout the flowering season. As the density

of the flowers within the observation plot may influence the number of insect visitors

amacted to the area, the number of open inflorescences in the observation plot were

counted each tirne flower visitors were observed and =pressed on a per mZ basis. For each

observation penod, each time an insect Ianded on the capitulum, it was recorded as a visit

and the kind of insect was noted. Visitauon was suffiuently low as to allow all visits to be

recorded. One speümen of each kind of insect visitor was caught with a net, killed and

mounted for identïkation. Insects were identified to f d p using the key Lom the

Introduction to the Study of Insects (Borror et al., 1981). From these data, four response

vaeiables were estimated: number of visits per plant pes observation period, visitor

diversitg, relative abundance of visitors, and richness at a f d y level-

The number of visits per plant per observation period was calculated as (the number

of visits received per 30 minute observation period)/(the number of plants in a plot). The

diversity of flower vis i to~ was quantified using the Shannon-Weaver Diversitg Index

(H): X = -x pi log, pi, where pi is the propomon of each species (r) in the tord sample of

individu&. This index is a logarithmic measuee of diversity, which gives less weight to rare

speaes than to common ones. Relative abundance was calculated as the number ofvisits by

an insect group, in propomon to the total number oE visits observed in the plot per 30-

minute period. Richness at a f d y level was measured as the number of insect farnilies

that visited the plot mithin the average observation period.

Sttgma P o l h Load

To test whethee small populaaons were pollen iimited, due to a lack of pollen

deposited by pollinators, 1 estimated the mean stigma pollen load in srnail and large

populations. I coilected 15 receptive stigmas, during the 2000 field season, fiom each of 25

plants in 3 large populations and 15 plants in 4 small populations. S+a collections for ail

populations were done w i h a two-day period during the peak of the f l o a e ~ g season.

These days were suany mith no precipitation at least 3 days prior to the date of sbgma

collection. Stigmas were chosen Zrom flores that had been open for 3 days, the stigma

lobes were M l y reflexed/open, had lost the majoriy of their self-pollen load and were lÎght

tan in colour. Stigmas were mounted in a drop of glycerol on a glass microscope slide and

imbiediately sealed with nail polish. I counted the total number of pollen grains per stigma,

using hght microscopy. The plants from *ch the stigmas were collected were marked and,

afier Qowenclg, the inflorescence ws bagged in nylon to collect the seed Three weeks

after anthesis, once the seed had dried, m e e n florets were randomly selected in each

capinilum and scored as filied or unfilled and seed set detemiined. Mean seed set was

calculated for each population. 1 used a p a k d t-tes t to examine the difference in stigma

poilen load and seed set among small and large populations.

RESULTS

VARIATION IN SEED PRODUCTION 2WD POLLEN LIMITATION

Petcentage seed set in 1999, averaged aaoss regions, was 42.9 Oh (SE = 0.057,

n=19 populations, 607 inflorescences) and ranged among populations from 27.5%

(Population LH) to 66.2% (Population CCS). Mean seed set of populations Lom the Bruce

Peninsula (48.G%, SE=O.O29, n= 13 populations) was no t sigdïcantly different fiom

populations of Manitoutin Island (37.3%, SE=0.024, n=6 populations) (Nested ANOVA,

Region Effect: =2.1 G8, p=O.Z126) (Table 3.2). Within each region, in 1999, significant

diffaences for percentage seed set were found among populations (Nested ANOVA,

Populations[regions] Effect Fl,5,=3.21, p = 0.0000) Vable 3.2). Population mean seed

set ranged from 30.7% (population CPS) to 66.2 O h (population CCS) within Bruce

Peninsula populations and fiom 27.5 %(LH) and 44.9 % (BL) in Manitoulin Island

populations (Table 33). Percentage seed set in 2000 averaged 47.4% (SE=0.03, range =

1845%) on the Bruce Peninsula and there were sisnificant differences in seed set among

populations (Table 3.3). Seed set in 2000 dîd not differ signiticantly fiom 1999 seed set

(paixed t-test: t=-0.701, p = 0.498, df=l 1). Seed set within populations did not differ

stgitficantly between years, except in population LFON (seed set increased sipiEcantly in

2000) and population CPL (seed set decreased significantly in 2000).

The number of florets per inflorescence for Bruce Deninsula populations in 1999,

averaged 87.2 (SE = 1.77) and ranged from 3 8 to 150 (Figue 3.2). There were significant

differences in the number of florets per capidum among popdations (ANOVA: FLZ182 =

10.97, p= 0.000) and populations from coastal regions had significantly more norets than

those inland (t-tesc t = 2.849, df= 10, p = 0.017). Based on estimates of seed set and the

number of florets per capitulum, the mean number of seeds produced pet in£iorescence in

1999 was 42.6 and ranged from 23.8 (Population LFON) to 59 (Population CCS).

Sqp/emeniaZ Po//inafions

Mean percentage seed set for the pollen-supplernented plants was 47.5% (SE =

0.02) and ranged h m 35% (Population CPS) to 59% (Population LFON). Mean seed set

for open-pollinated florets was 47.4% (SE = 0.03) and ranged from 18% (Population CPL)

to 65% (Population CCS). Averaged across all populations, there mas no sigaificant

difference bemeen the mean percentage seed set for the pollen-supplemented and open-

poihated Gorets (Two-way N O V A (Treamient): F,, , =O.Il, p = 0.75) Fable 3 - 4 4 . At

the population level, one of the 12 populations had sqydicantly greater seed set after

pollen-supplementation (41°/o) than open-poilination (18%)(CPL: FliLIS= 5.07, p =

0.02) (Figure 3.3). The Pollen Limitation Index (P), averaged across ail 12 populations, was

0.02 (SE = 0.06) and ranged fiom (-0.30) to (0.54). In four of the 12 populations, open-

pollinations had greater seed set than the supplemental-poIlinations. After constraining all

values of P to 0, the Pollen Limitation Index averaged 0.08 (SE=0.04). In a multiple

regression, the Polien Limitation Index was not significantly rekted to either population

size (Regression: F,8=0.46, p = 0.52) or geographic isolation (Regression: F,,8=0.03, p =

0.86)Vable 3.5).

MATEDIVERSI?"Y

Averaged across aii populations, mean seed set was 0.78 % (SE = 0.005) for self-

p o h a t e d flowers (range = O - 6.32%), 14.6% (SE = 0.021) for cross(within)-pollinated

flowers (ranged = 3.75% - 29.2%), 19.2O/0 (SE = 0.021) for cross@etween)-pollinated

flowers (range = 10% - 33%) and 48.6% (SE = 0.03) for open-pohated flowers (range =

18% - 65%). The Two-way ANOVA of percent seed set indicated that there was a

sqpificant difference among pollination treatments and a significant interaction among the

main effects (population and teeatment); however there was no significant difference

among populations Fable 3.4.B). Linear contrasts revealed that seed set in cross(within)-

pohations was not significantly diffaent than that for self-pollinations in five populations

(Populations CCL, CPL, CPS, HL and SC)(Figue 3.4). Seed set from cross@etween)-

polinations was significantly different than self-pollinations in 8 populations (Populations

BC, CCS, CH, CPL, FW, LC, LFON, SFON)(Figure 3.4). Seed set in cross@etween)-

pohat ion and cross(arithli)-polination were not siaiificantly different in any population

(Figure 3.4). Seed set in open-pohation was significantly greater than cross(within)-

polhation treatments in ali but two populations (Population CPS and WON) (Figue 3.4).

The mean proportion of compatible crosses, across a i l populations was 5.7% (SE =

0.02) for self-pollinated florets (range = O to 27'/0), 33.5% (SE = 0.04) for cross (wïthin)-

p o h t e d florets (range = 17% to 58%) and 43% (SE=0.04) for cross (berneen)-

pollinated norets (range = 16% to 64%). There was a sgruficant difference in the

proporcion of compatible crosses among populations for self-pollinated norets (Chi-square

= 23.8, p = 0.0215, df = 12) and cmss(mithin)-pollinated florets (Chi-square = 23.7, p =

0.02, df = 12) but not among populations of cross@enveen)-pohated flowers (Chi-square

= 17.5, p = 0.13, df = 12). The contingency table analysis showed that the proportion of

compatible crosses across all populations diffeed significantly between self-pollinated and

c.ross(within)-pollioated florets (Chi-square = 72.3, p ~0.0001, df = l), self-pollinated and

cross@etween)-pollinated flowers (Chi-square = 108, p~0.0001, dE = 1) and cross(wirhin)-

and cross@etween)-pollinated flowers (Chi-square = 4.71, p = 0.03, df = 1). In a

population by popuiation analysis of the cross(within)- versus cross(between)-pollinations,

only one population (population NW) had a sïgdïcantly lower proportion of compatible

crosses within populations than between populations. In a population by population

analysis of the cross(mithin)- versus self-pollinations, eight of the thirteen populations had

si@candy higher proportion of compatible crosses Li the cross(T;tithin)-poIlinations than

the self-pollinations. Nine of the thirteen populations had significandy higher proportion of

compatible crosses in the cross @enveen)-pohations than self-pokations.

Mate diversity within populations averaged 33.5% (SE = 0.04) and ranged from

17% to 58%. The measure of mate diversiy within populations was not rekted to pollen

lLnitation (Regression: n=12, FI ,,, =1.11, R ~ = 0.1 0, p = 0.32)(Figure 3.5). In addition,

mate diversity mithin populations was no t rela ted to geographic isolation (Regression: F, ,, , ~0.453, p=O.GG), however, it was sigruficantly positively related to population size

(Regression: F ,.,, = 5.76, p = 0.0001, R~ = 0.883)~able 3.5).

POLLINATOR ACTWITY

Po/Iinator DiverSig and Virilorion Rater

1 watched poilinator plots for 114 half-hou sessions, a total of 57 hours of

observation. Over the 57 hours of observations, 2013 insect visits were recorded. The

mean number of in£Iorescences per square meter within the pollinator observation plot mas

10.9 (SE=0.61) and ranged hom 2.5 to 27.3 flowers per meter squared. Density was

wealdy conelated with population size (Correktion: rz0.58, p=0.0815, n=10). However,

densis. did have a sigdicant impact on visitation rates (Regression: F1,,,=24.9, p=O) and

most specïfïcaily fly visitation rates (Ft,l12=22.2, p=O) but not bee visitation rates

~3.12, p=0.08). Densiy also had an effect on visitor richness (F,,,,,=4.92, ~ ~ 0 . 0 3 ) but no

effect on the Shannon-Weaver Diversiy Index (F,,,12=0.20, p=O.Gl).

A total of 2 Orders (Arachnida and Insecta) and 8 families of insects mere

represented in the poha to r sumey ïnduding: Hymenoptera, Diptera, Lepidoptera,

Neuroptera, Homoptera, Hemiptera, Coleoptera, Orthoptera (Table 3.6). As I did not

observe spiders moving from one inflorescence to another, 1 excluded them as potential

poUinators and did not idenafy them past the levd of Order. Visitor richness averaged

2.68 different insects per 30 minute observation session, differed sigdicantly among the

twelve populations (ANOVA: FII,,,,=10.8, p=O), ranging from 0.86 to 5.17 different

insects per 30 minute observation session Fable 3.8). Visitor Richness was not condated

Mth the Poilen Limitation Index (Regression: F,,=2.69, ~"0.14) (Figure 3.6). In a

multiple regression, Visitor Richness \vas not signi£ïcandy related to either geographic

related to population size or geographic isolation in multiple regressions.

Stigma Po& L a d

The mean number of pollen grains per scigma was 96-71 (SE = 8.04) and ranged

£iom 78.20 (Popuktion CH) to 138.87 (Population LC). The mean seed set that

coxesponded with these innorescences was 50.4Vo (SE=0.03) and ranged &om 34Oh

(Population CPL) to 63% (Popuktion CCS). An rWOVA revealed that there were

si@cant differences arnong populations in the number of pollen grains per stigma

(2WOVA: FI, = 2.838, p = 0.014) and the percemage seed set (ANOVA: = 2.556, p

= 0.025). Mean number of pollen grains/stigma (SE) was 94.19 (13.38) for large

populations and 98.60 (11.59) for small populations. There was, however, no sgnZcant

diffeence in pollen grains benveen large and srnall populations (t-test: ~ 0 . 2 5 0 , p = 0.813,

df = 5).

DISCUSSION

Seed set in openly poiiiuated flowers was 43%, on average, and varied widely

among the 19 populations examined fiom Bruce Peninsula and Manitoulin Island (range =

27.5% to 66.2%)- This estimate of the propomon of viable seed produced by open

pohation was similar to a previous estimate of 46.9% reported by De Mauro (1993) for a

population in Ohio. Both values indicate that a large proportion of all ovules (5G0/0) did

not mature into seed This may be caused be a number of ecological and genetic factors,

induding: 1) resource availability, 2) genk sterility, or 3) insuffiCient compatible pollen

being deposited on stigrnas. The focus of this study was to test the role of pollination,

which seems likely gïven that H. hedncea is self-incompatible, its populations are physically

disuete and relatively small.

Pollen limitation, measured as the propomonal increase in seed set due ro the

addition of pollen to open-pollinated flotets, was negligible when averaged across aU 12

populations eïamined (mean = 0.08). In other words, overall, addbg more pollen had no

effect on seed set. However, pollen limitation did v q from O to 0.54 and seed set in

supplemental pollinations was significantly higher in one of the 12 populations. Mthough,

variation in seed set among populations cannot be M y accounted for by pollen limitation

there appears to be some potenaal for it to occur in H. berbacea.

The overd measure of pollen limitation was extremely srnd (l? = 0.08) cornpared

to other reports of pollen limitation. In an extremely large US. population of H. herbacea

(Mvlarblehead Quarry), pollen supplementation elevated seed set only 7% above open

pollination, a difference which was not significant (t-test: ~ 0 . 2 3 , p>O.S, df=73). The

low pollen limitation 1 observed in Canadian populations is consistent nrith the dam from

the U. S. population (De Mauro, 1993). However, De Mauro's estimate is consemative,

as the poIlinations were the product of only one pollen donor (not pollen supplementations)

and only induded pollinarions De Mauro had dassihed as compatible. Pollen limitation in

H. herbacea is extremely low compared to mean pollen limitation calculated in a s w e y of

other nngiospenn species (0.40)&arson and Barrett, 2000). In fact, H herbacea had

u n u s d y low pollen limitation for a self-incompatible plant, which tend to have higher

pollen limitation (mean poilen limitation = 0.59 (k 0.04)) than self-compatible plants

(mean pollen limitation = 0.31 (2 0.03)). Within the SI taxa examined by Larson and

Banett (2000), some speaes did have pollen limitation values less than 0.2; howerer, there

is no discussion of the taxonomie and ecoIogical features of these plants. In H. herbacea, the

effect of the SI m a ~ g system on pollen limitation may be masked by other characteristics

Specihcally, H. berbaceu has an unspecialized floral morphology typical of Asteraceae.

Larson and Barren (2000) e&ed the effect of 'sperialized' versus 'unspecialized' floral

morphology and found that unspeaalized speaes can have very low pollen limitation (C0.2)

although the mean pollen limitation for uaspecialized species was fairly high (0.38). Clearlp,

H. herbacea is unusual in its ability to acquke sufficient compatible pollen under different

ecological conditions.

To bette1 understand the ecological and genetic basis for variation in pollen

limitation, 1 attempted to esamine its tnro components: 1) mate diversit)., which influences

the percentage of pollen that is compatible; and 2) pollinator visitation, which govems the

amount of pollen deposited on each stigma.

Mate diversity, measuted as the propomon of compatible crosses Mthin a

population, ranged from 17% to 58% per population and averaged 33.5%. Roughly

speaking, 33.5% of ail crosses were compatible. This value is lower than the proportion of

compatible crosses in a study of H. herbacea by De Mauro (1993), mhere 58% of the nithin

population crosses were compatible. Mate dive+ was sûongly coneia ted Mth

population size. My observation foilows the prediction chat as popuiation size decreases,

the strength of genetic M t increases and mating type diversity is lost Exaemely s m d

populations of H. herbacea may lose all nate dmersity, reducing seed production to zero.

Mate divezsity was, however, not conelated with pollen limitation, not a surprising result as

pollen limitation was low in most populations. Mate diversitg withli populations may not

be low enough yet to have an effect on seed production withiu current H. herbacea

populations.

The measure of mate diversity used in this smdy ma7 not reflect the neighbourhood

of maMg types available to mos t florets. Insect visitors generally move short distances

(Harder and Wilson, 1998; Pleasants, 1991). For populations of donal species, in mhich

genets are spaaally spuctured, most pollinations are geitonogarnous, ive. belmeen flowers of

the same genet. If the plant is self-incompatible, these pollliations miu be ineffective

(Thein e t d, 1985, Aspinwall and Christian, 1992, De Mauro, 1993, R e i n h m and Les,

1994). Thus, the estimation of mate diversitg within populations, via hand pollinations,

likely overestknated the amount of compatible pollen received by each stigma. 1 would

expect this bias to exaggerate the degree of pouen limitation due to low mate diversity.

My measure of mate diversity within popdations is a measure of the fiequenq of

encountering compatible mates mithin the entire population, if the population is assumed to

have an equal opportunity to mate with each individual of the population. This, homever,

may be misleading as it is not a measure of the actual diversiry of pollen deposited on the

stigma or the number of pollen donors conmbuting to each stigma. The effective number

of pollen donon per stigma can be estimated for H. herbacea, using an equation modifïed

fiom Charlemorth and Chadesworth (1987), originaily used to estirnate mating systems.

Using es timates of seed set in pollïnations with a maximum of five donors and seed set in

p o h t i o n s with a single donor, I e s h t e d the effective number of donon in open

pollliated flowers u s i q

within(multip1e pollen donors) seed set - open seed set

within(multip1e pollen donors) seed set - within(single pollen donor)seed set

The effective number of pollen donors pollinating a p h t ranged fiom 0.093 (CPL) to 7.88

&C) donon and averaged 4.87 among the twelve populations (Table 3.9). This is suffident

to ovexcome the low frequency of compatible mating types in most populations.

The percentage of compatible crosses in pollinations between populations was

signïfïcantly higher than that within populations overd but only significant within 1 of 12

popuiations of H. berbaceu. This suggests that mate diversitg differs little among popuiations

in the region. Rather, most of the mate diversitg throughout the region is represented

within each populations. 1 found that the US. population was mate limited relative to the

mate diversirp within the U.S. region (t-test: ~ 3 . 6 7 6 , p<0.01, df=9), when I used the data

£rom De Mauro (1993) for within and berneen population crosses. However, the mate

diversitg must be relatively high within the US. population in order to produce such a high

percent seed set (mean=41°/o) as a result of within population crosses. There may be an

overd Iack of mating type diversity within the Bruce Peninsula region in cornparison to the

matiug type diversis of the U. S. population at Marblehead Quany. Populations of H.

herbacea in Bruce PenLisula are hypothesized to be relatively young and possibly of

inuoduced ongin (US. Fish and Wildlife S e ~ c e , 1990) which may explain the relatively

low diversity in mating types mithin the region. However, if H. berbncea has only evis ted in

Bruce Couny for the past 35 years, the spread of the plant has been quite rapid as there are

at least 15 h o w n popukuons found there now.

Pollinator visitation was highly variable among populations (range = O to 5.33 visits

per plant per 30 minute p&od) and averaged 0.66 visits per plant per 30 minute period. In

general, poha tor tisitation is susceptible to the vagaies of the environment, induding

temperature, wind, and precipitation and the visitation by insects to H. herbacea was no

exception. In yean with more extreme weather conditions, e s p e d y those not conducive

to pollen dehiscence, pollinator activity may affect pollen limitation more ckastîcally than in

years with good weather conditions for insect amvitp.

The insect visitors of H. herboca are a diverse group, a common feature of the insect

visitors of many plants that flower in early spPng mein et al, 1983; Godley and Smith,

1981). However, it is likely that not ail insects were effective pollen vectors. It was

obvious to my eye that the bees canied the most pollen, however, 1 did not cokc t data on

how effective each visitor was at transfkning pollen to stigmas. Other studies of H. hedacea

suggest that bees (Apidae, Xylocopidae and Halictidae) are important insect visitors (De

Mauro, 1993). Data on pollinator Visitation versus pollen limitation indicated that there

mas a sigdicant relationship for bee taxa but not fly taxa, the other common group of

insect visitors. This suggests that 1) bees are a5tica.l pollen vectors of H. dmbacea and 2)

populations that do not have bees visiting the plants may ewperience pollen limitation.

Stigma pollen bads of H. herbacea were exaemely hgh (mean = 97 grains/stigma) in

all populations considering that there is oniy one ovule per o v q in each fioret of H.

herbucea. Some of this pollen will be seif-pollen from neighbouring florets or even the

materna1 Iloret, itself and may hide any relationship between pollen deposition and pollen

limitauon. In self-incompatible plants, the number of pollen grains on the stïgma is not a

good indicator of pollen limitation as the compatibiliq of these pollen grains is unknomn

from a simple couat-

1 expected that population size would influence pollen limitation and its two

components based on the theory of geneUc drift and the hUee effect; however, 1 found no

patterns relating popdation size to polien limitation in this study. Appareatly plants are

receiviag enough compatible pollen, even in the smallest populations of this study. There

are two reasons for the la& of relationship betmeen populaaon size and pollen limitation.

Firçt, perhaps small population size does not affect mate diversi. and polhator activity as

predicted. Second, o u small population sizes may not be s m d enough to be mate limited

or pollinator limited. There is evidence in this study that goes against the former postdate

and supports the latter expianation. When mate diversity is e sha ted by within population

crosses, the amount of mate dmersity within a population was significantly rekted to

population size. There is obviously SI aiiele diversitg implications with s d population

size. However, most populations are not so limited in mate diversity that reproduction is

reduced drastically. The population sizes of H. herbuceu have, in geneeal, not reached a

threshold levei, below which, it is impossible to maintain SI alleies.

Additionally, pollinator visitation rates actudy increase as population sizes

decrease This nins counter to theoretical expectations. Recent snidies of pollen limitation

have tended to emphasize the negative effects of s d population size on f d t y because

of the effects of reduced floral displays on pollinator visitation rates (Jementen, 1988,

Agren, 1996, Alexandersson and Agren, 1996). However, large populations, where floral

resources are in excess relative to the number of pollinators available, may also experience

reduced f d t p (Fga and Nilsson, 1994; Larson and Banett, 1999). In out study,

individuals in a large popdations had a lower probability of being visited than an individual

in smder sized populations, and thus not necessdy ensured of high potentiai for out-

crosskg and reproduction. In order to see pollinator limitation, population sizes seemligly

need to be much smaller.

Hymenoxys herbacea is a rare endemic of the Great Lakes and has been a target of

conservation initiatives in both the United States and Canada. For maoy plant speues,

increasing rates of habitat loss and fragmentation wiU lead to greater degrees of population

isolation and, perhaps, to lowered pollination success within such isolates (Groom, 1 998).

From this smdy we have shown that most of the tmelve Bruce Penïnsula populations are

not suffering fiom any type of pollen limitation and that pollen limitation cannot be

predicted by population size, mate dmersitp or pollinator activity. Populations, in general,

have enough p o h a t o r activity and mate diversicg to ensure that seed set is not pollen

limited. However, population size plays an important role in pouen &taoon via mate

divenity and p o b a t o r activity. Natural areas managers must be awre that should the size

of these populations of H. betbacea become smaller, pollen limitation codd become a chreat

to th& persistence-

REFERENCES

AGREN, J. 1996. Populations size, pollinator limitation, and seed set in the self-

incompatible herb Lythmm ~aIic& Eco/. 77: 1779-1 790.

ALEXANDERSSON, R. AND AGREN, J- 1996. Population size, p o h a t o r visitation,

and Enut production in the deceptive orchid Ca@so bdbosa. OecoL 107: 533-540.

iULEE, W. C., EMERSON, A- E., PARK, O., PARK, T. AND SCHMIDT, K. P. 1949-

Pn'n@ieg of animai ecou~ . Philadelphia, Pemsylvania. Saunders Press.

A S P I N W U , N. AND CHRISTIAN, T- 1992. Clonal structure, genotypic diversi., and

seed production in populations of FiI'enduLa mbra (Rosaceae) from the northcentral

United States. Am. J- Bot. 79: 294-299

BORROR, D. J., DELONG, D. M. and TRIPLEHORN, C. A. 1981. A n intrvduction to the

xfu4 of insecfi Philadelphia, Pe~sylvania. Saunders Press.

BURD, M. 1994. Bateman's principle and plant reproduction: the role of pollen limitation

in fruit and seed set. Bot. Rev. 60: 83-139.

CHARLESWORTH, D. AND CHARLESWORTH, B. 1987. Inbreeding depression and

its evolutionary consequences- Ann. Reu. EcoL 5ju-t. 18: 237-268.

CUSICK, A. W. 199 1. Hymeno~s berbaceu (Asteraceae): an endemic species of the Great

Lakes region. Rhod. 93: 238-241.

DE MAUROY M. M. 1993. Relationship of breeding system to rarity in the Lakeside Daisy

{Hymenoxys acas/liJ- var. ghbra) . Comem Biol. 7 :S42-5 5 0.

FISCHER, M. AND MATTHIES, D. 1998. Effects of population size on performance in

the rare plant GentianeLia gemanica. J- Ecol. 86: 195-204.

FRANKEL, O. H. Ï\ND SOULÉ, M. E. 1981. Consenation andEuo/llloa Cambridge.

Cambridge University Press.

FRITZ, A. -L. AND NILSSON, L. A. 1994. Hom pohator-mediated m a ~ g varies nrith

population size in plants. Oecol. 100: 451-462.

GODLEY, E. J. AND SMITH, D. H. 1981. Breeding systems in New ZeaIand plants. 5.

Pmdowintera colorata (Winteraceae). AT. 2. J. Bot. 19: 151 -1 56.

GROOM, M. J. 1998. AlIee effects iirnit population viabiliv of an annual plant R.n. Nat.

15 1: 487-49 6.

HARTL, D. L. AND CLARK, A. G. 1997. Ptfn@Iex ofpopuIationgenetics- Sunderland, MA.

Sinauer Assoc.

-ER, L. D. AND WILSON, W. G. 1998. Theoreticai consequences of

heterogeneous transport conditions for pollen dispersal by animals. EcoL 79:

2789-2807.

IMRIE, B. C., KIRKMAN, C. J. AND ROSS, D. R 1972. Cornputer simulation of a

sporop hytic self-incompatibility breeding sy s tem. AZIIL j. BioL Sn. 25: 343-349.

JENNERSTEN, 0. 1988. Pollinatîon in Dianthzts dehoides (CargophyUaceae): Effecrs of

habitat fragmentation on visitation and seed set Consem. BioL 2: 359-366.

M O N T , B. B., KLINKHMER, P. G. L, AND WITKOWSKI, E. T. F. 1993.

Population fragmentation may reduce fertility to zero in BankRagoodii - a

demonspation of the ALlee effect. Oec~Iogr'a~ 94: 446-450.

LARSON, B. M. H. AND BARRETT, S. C. H. 2000. A comparative analysis of pollen

limitation in flowering plants. Bi01 J. fim. Soc. 69: 503-520.

LARSON, B. M. H. A N D BARRETT, S- C. H. 1999. The ecology of poilen limitation in

buzz-polhated Rbe-xia vhginica (Meiastomataceae). J. Ecd 87: 371-38 1.

LES, D. H., REINHARTZ, J. A. AND ESSELMAN, E. J. 1991. Genetic consequences of

rarity in Rrterfùrcatus (As teraceae), a direatened, self-incompatible plant. EvoL 45:

1641-1650.

PLEASANTS, J. M. 199 1. Evidence for short-distance dispersal of pollinia in Arc/Epias

gnaaca L. Fam. EcoL 5: 75-82.

REINMRTZ, J. A. AND LES, D. H. 1994. Botdeneck-induced dissolution of self-

incompatibility and breeding s ys tem consequences in AxterfZ/rcutz~x (As teraceae) .

Am- J. Bot. 81: 446-455-

SIH, A. AND BALTUS, M. 1987. Patch size, p o k a t o r beharior, and p o h a t o r iimitation

in Camip. Eco/. 68: 1679-1690.

SOKAL, R. R. AND ROHLF, F. J. 1981. Biometq. Srjcrh edition. New York, New Yo&

U.S.A. Freeman.

SPSS INC- 1999. SPSS, v.10 for Windows. Illinois.

THEIN, L. B., WHITE, D. A. AND YATSU, L. Y. 1983. The reproductive biology of a

relict-Ifltcium fian'danum Ellis. Am. J. Bot. 70: 7 1 9-727.

U.S. FISH AND WILDLIFE SERVICE. 1990. RecoveypZanfor the Zakeniie daig

(Hymenoxy~ a c a ~ h var. gfabra). Twin Cities, M N . US. Fish and Wildlife Service.

VOSS, E. G. 1996. Michigan Flora. Part ID. Dicots (;r)yroZaceae - Cunrpoxitae). P p. 378.

Cranbrook Institute of Science Bulletin 61 and University of Michigan HerbaSum.

WIDEN, B. 1993. Demographic and genetic effects on reproduction as related to

population size in a rare, p e r e d herb, Jeneno integnj.,/iz~.r (Asteraceae). BioL J.

Enn. Soc. 50: 179-195.

WRIGHT, S. 1969. Evolution and the Genetics of Populations. In The the09 ofgene

freg~enne~ v. II. Chicago, IL. University of Chicago Press.

WUNDERLIN, R. P. 1971. Contributions to an Illiaois flora. No. 4. Compositae II.

(Tribe Heliantheae, Part 1 - Hymenoy~, Hymenopqtpm, and Polymnia). Tram IZL Srare

Acad. Sci. 64: 317-327.

YOUNG, H. J. AND YOUNG, T. P. 1992. Alternative outcornes of natutal and

experimental high pollen loads. Ecol. 73: 639-647.

Table 3.1. Summay of 13 Hymenoys heniiacea popuiations, on the Bruce Peninsula,

Ontario, sweyed for population size (N, - total number of inflorescences), geographic

isolation (m) and habitat type (C = coasd, 1 = inland). Popdztions indicated with *

iadicate populations discovered in 1999-2000,

Geographic isolation (ml

Habitat Type Population size I Population

LFON

eomemc Mean

Table 3.2. S u m m q of ANOVA of seed set of Hymenoxys hwbacea populations. Data from

1999 mas from two regions (Bruce Penînsula, Manitoulin Island) and hence a nested

ANOVA was used while, in 2000, only Bruce Peninsula populations were sampled Seed

set data was arc sine transfomied in order to improve the normahv of the residuals. *** ,

p< 0.0001; *, p <0.05.

Source of Vatiation I df h?lS I F

1999

2000

Region

Population [pegion]

Enor

Population

Error

1

18

584

12

287

0.52

0.3 7

0.1 1

0.49

0.15

1-65

3.21***

3.20***

Table 3.3. Mean seed set (%) in open pollinated florets from 19 populations of H. herbacea

on the Bruce Peninsula (B) and Manitoulin Island (M) across two years. N/A = data not

availa ble.

Population r-- Location % Seed Set (SE) % Seed Set (SE) 1999 2000

Table 3.4. Two-way ANOVA of experimental pouliauons of Hyme?zo-y berbocea. A.

Two-way ANOVA testing for the effects of population and pollination treatment (open-,

supplemental p o b t i o n ) on percentage seed set in 12 H. berbacea populations. Percent

seed set was arc sine transfomied to increase the n o d t y of the residuals. B. Two-way

ANOVA testkg for the rffects of popuiation, pollination treatment (open, self,

aoss(within) and cross@etween)) and their interaction on percentage seed set on 12

populations of H. herbacea from the Bruce Peninsda, Ontario. Percent seed set was arc sine

transfomied to inuease the nonnality of the residuals. *, pcO.05. ***, p c 0.0001.

1 1 Source ldf I M S 1 F Ratio A.

B.

Population

Treatment

Population * Treatment

Population

Pollination Treatment

11

1

I I

12

3

0.63

0.02

3.83*

0.11

O - I G

0.40

20.99

1.07

1.98

11 0.60***

Table 3.5. Multiple regressions of Pollen Limitation Index? mate diversity and Lisect

visitation rates a@st log@opulation size) and geographic isolation, for H. herbacea

popdations fiom the Bruce Penins*, Ontario. *, p< 0.05.

Dependent Variable

Pollen limitation

'source ,

Log (Population Size)

Isolation

Isolation

Mate Diversity Log (Population Six )

log (Population Size)

Pollinator Visitation Isolation

Table 3.6. List of major insect groups observed, populations in which they occuned, and

overd mean rektive abundance of the insen groups in populations of H. herbacea on the

Bmce Peninsula, Ontario.

l Hyrnenop tera

Homoprera r- Coleop tera t--

Observed in following Popuktions

LC, CPL, BC 0.0096

Overail Meaa Relative Abundanc

CCL, CCS, CH, CPL, CPS, LC, WON, SFON, NW

0.1093

AU 12 popdations 0.9171

LFON, CH O

LC, CCS, CCL, LFON, SFON, BC, CH

0.0127

Table 3.7. Summary of the ANOVAs on ciifferences of insect visitation rates ( ïotal ,

Hymenoptera, Diptera) among populations of H. herbocea fiom the Bruce Peainsula,

Ontario. ***, p <0.001.

Total Visitation Rate

Hymenoptera Visitation Rate

Diptera Visitation Rates n

Source

Population

Population

Error

Population

Error

Table 3.8. Surnmary of one-way ANOVAs tesàng for ciifferences among populations with

respect to richness and Shannon-Weaver Diversity of insect visitors to H. berbacea from the

Bruce Peninsula, Ontario. *** represents p <0.001,

Shannon-Weaver Diversiy Index

Source 1 df 1 MS FRatio

Population) 11 1 23.55

Total 1 113 1 I

Total 112

Table 3.9. The effective number of pouen donors contributing pollen per floret for 12

populations of H. berbacea on the Bruce Peninsula, Ontario. The number of donors was

calculated using the equation:

Cross (mulaple pollen donors) seed set - open seed set

Cross (multiple pollen donors) seed set - Cross (single pollen donor) seed set

modXed fiom Charleswoah and Charlemorth (1987). The seed set data used for this

analysis was f?om the crosses describrd for the pollen limitation and mate diversitp hand

pollinations desuibed eatlier.

1 Population 1 Effective Number of 1

l CPL 1 O - 9 2 1

BC

I CPS

Donors (D)

4.24

1 SFON l 3.70 I

LFON 4.68

Figure 3.1. Photograph of filled and u d e d achenes of H. hwbacea- The achene on the

right is not hue4 datkened or rounded. The achene on the left is fille4 ddrened and

xouaded. The pauit on the top of the achenes is an ïndicator of the cross we and not

naturally occUmngcumng

Figure 3.2. Mean number of florets (+ SE) per idorescence for each of the twelve

populations of H. berbacea on the Bruce Peniasula, Ontario. Values for aU populations are

the mean of 15 plants. Populations with the same letter are not signifïcantly différent based

on a Tukey's post-hoc test

Population

Figure 3.3. Mean (+SE) seed set in open- and supplemented-pobtion treatments for 13

populations of H- herbacea fiom the Bruce Peninsula, Ontario. There is a signifïcant

difference between the two txeamimts in population CPL based on hear conaasts. Values

for all populations are the mean of 25 plants except in populations CPS @=Il) and CPL

(N=16). Values shown above are &om non-tram fomed data, however, dwïng analysis,

seed set was arc sine transformed in order to increase the notmality of the data.

NOdS

/X\N

NOd?

31

'TH

Figure 3.4. Mean seed set (+SE) in self-, cross(within)-, cross@etween)- and open-

pollinated plants for 12 popuiations of N. hw&acea from the Bruce Peninsula, Ontario.

Values for all populations are the mean of 25 plants except in populations CCL (N=20) and

CPS (N=15)*

SC-

LFON-

SFON- CH - BC- F W - CPL- LC - HL -

CPS - CCS -

Self

aD Open

Cross (Within)

Cross (Between)

Figure 3.5. Scattergram of mate diversiy versus popuktion size for 12 populations of £3.

berbacea on the Bruce Peninsula, OntaPo. Mate dmersity is equal to the proportion of

crosses, in pollinations with single poiien donors, that are compatible (> 5% seed set)

Log (Population Size)

Figure 3.6. Scattergram of Bee visitation tates versus population size in 12 populations of

H- bherbacea on the Bruce Painsula, Ontario.

2 3 4 5

Log (Population Size)

Chap ter IV: General Discussion

The goal of this research was to describe the impact of population size on

reproductive success of the rare, self-incompatible plant, Hymenyr herbacea. In doing so, 1

asked four specifïc questions:

1) What is the magnitude of genetic drift acting on m a ~ g type diversiy in

populations of H. hwbacea in Canada?

2) Do H. herbacea populations vary in seed production and are populations pollen

Ilnited?

3) Cm variation in pollen limitation be explained by mate diversiq or pollinator

activity within populations?

4) 1s the magnitude of pollen Iïmitation, mate diversity and pollinator activity

related to population size?

In this chapter 1 would like to consider these questions in Iight of my research hdLigs,

examine a historical problem with estimating pollen Limitation that my work raises, and

offer future research directions that would bu-ease our understanding of population size

and pollen lunitarion- Finaliy, 1 wiu discuss the implications of my findiags for the

conservation management of H. hehcea.

THE MAGNITUDE OF GENETIC DRIFT

Geneac d&, measured as the effeche size (Ne) of H. herbacea populations,

averaged 4343 in the two populations in which it was estimated dlectly, and, based on an

N, - ratio of 0.43 , ranged from 57 to 56,476,670 iadividuals in 10 populations on the N

Bruce Peninsula. O d y one population was sufficiently srnaIl@-e. ZE 50) to cause the

s tochastic loss of genetic rnating =es, required for seed production. Only 3 of the 12 were

less than 5000, a population size small enough for dgfc to have a detectable impact on the

diversity of neuual genes (Lande 1988). The Iom magnitude of drift was consistent with

the absence of any relationship between population size (N) and diversity observed in an

isozyme survey of 12 populations. Although the strength of genetic drift is hypothesized to

increase as population sire decreases, we did not h d a relationship among population size

and geneüc diversity rneasures. Therefore most populations were suffiuently large to

rninimize the effect of genetic drift on isozyme and m a ~ g type diversity.

VARIATION IN SEED PRODUCTION AND POLLEN LIMITATION

Populations of H. d e h a differed in seed production, ranging fiom 27.5%

(population LH) to 66.2% (population CCS) seed set These populations, fiom Manitoulin

Island and Bruce Peninsula, had similar seed set to that measured in Ohio (De Mauro 1993).

The pollen limitation index ranged fiom O to 0.54 among these same populations but only

one of these values was different £rom zero. This suggested that, in general, low seed set

was not caused by the lack of suffiuent compatible pollen.

MATE DTVERSITY AND POLLINATOR ACTIVI.TY

Mate diversitg, measured as the proportion of pollen donors that are compatible,

averaged 0.34 (SE = 0.02) aaoss the 12 populations e~amined and ranged feom 0.17 to

0.58- These values suggest that the fiequency of compaable mating types is quite low in

most populations but that no population consists of onlp a single m a ~ g type. Despite

these low values, mate diversity was not correkted with the pollen limitation index and

therefore is having little effect on seed production. Presumably mate diversity is above a

critical threshold, necessarg for maximum f ' a t i o n . Total insect visitation averaged 0.62

visits/plant/fO minute observation p&od and ranged fiom 0.03 to 2.82. As a subset of

insect visitation, bee visitation averaged 0.06 and ranged fiom O to 0.36. Total insect

visitation was not related to the magnitude of pollen limitation among populations,

however, a negative relationship did exist for bees alone. This implies that bees are the

flower visitor of H. herbacea most effective at collecting and depositing pollen on H.

herbacea inflorescences.

THE EFFECT OF POPULATION SIZE

Neither pollen limitation nor pohator visitaaon rates were related to the size of

H. berbocea populations and hence, do not support the predictions based on the Allee effect.

In the mate dmersiy study, however, we found that mate diversitg was rekted to

popuhuon size, which is consistent with the theory of genetic dnft However, as predicted

by the N, study, none of the populations had such low mate diversitg as to cause mate

limitation, a signifïcant reduction in seed set due to a low diversity of compatible mates.

ESTIMATING POLLEN LIMITATION

One t b g that mas dear upon c o m p l e ~ g this study was that there are p o t e n d

problems assoaated with conventional methods of estimating pollen limitation.

Traditionally pollen limitation is measured as the increase in seed set afker pollen

supplementation and is @en by:

pollen supplemented seed set - open p o h a t e d seed set PoUen limitation = . Implicit in this

pollen supplemented seed set

approach is the assumption that the pollen addition: 1) mimics an increase in the number of

pollinator visits; and 2) does not alter the diversitg or quality of the poilen fÏom what

would be delivered by the insect When measuring pollen Limitation in a seIfIfincompatible

plant, however, 1 became awaee that any addition of pollen to the flower Mil involve a

change not only in quantitg but also quality compared to what rhe bee would deïver. A

change in quality of pollen can indude the addition of pollen from different mathg *es in

SI species or, in self-compatible species, pollen donors that may differ in their compatibility,

"th the pollen recipient. Such changes are Uely to arise as in most studies of pollen

limitation, pollen is collected fiom 1 to many donors by the experimencer with linle or no

knowledge of the number of donors naturally conmbukg pollen to the average stigma or

the distance from which it is dispersed. Such changes could have a large influence on

esha tes of pollen limitation, as seed production is largeiy influenced by the matemal x

patemal combinations involved (b1arshaI.l and Ellstclnd 1988).

To disentangle the effects of changing the qunlity and quantity of pollen added to

stigmas it is necessary to measure:

1) pollinator limitation - pollen limitation due co the number of pollen grains

. deposited; and

2) mate limitation - poUen limitation due to the diversitg or compatibiliq of pollen

grains deposited.

To measure pollinator limitation, one must determine how much poiien is naturally

deposited and then add more pollen while holding the quality of pollen constant. The

p011Liation limitation index would be caiculated as:

Cross(Increased number of pollen grains) seed set- open seed set Pohator limitation =

Cross(Increased number of pollen grains) seed set

To measure mate limitation, one must maximize the diversitg of pollen while holding the

number of pollen gains deposited constant- The mate limitation index wodd then be

calcuIated as:

Cross(Inueased pollen diversitg) seed set - open seed set Mate Limitation = - For both of

Cross (Increased pollen diversity) seed set

these equations, one must have two pieces of information: 1) the number of poiien grains

deposited on a stigma under natural conditions and 2) the diversitg (number of pollen

donors) of pollen graias deposited on a stigma under naturai conditions. M e a s e g the

first variable is relatively sttaightforward and would involve counting the number of poilen

grains on a random collection of stigmas from the population. However, it is more difficult

to estimate the diversity of pollen grains on ope* pohated s w s . The most accurate

method to esbmate the number of pollen donors would be to survey the diversity of

patemal genotypes of achenes mithin a capitulum using a genetic analysis.

If genetic analyses is not an option, however, one may dso estimate the diversitg of

pollen donors via hand pohations. Three pollination treamients are requled: open-

pohation, cross (multiple pollen donors)-pohtion and cross (single pollen donor) -

pollination. Open-pohated plants are e-xposed to th& namal pollen vectors under

normal population conditions. Cross(multip1e)-polhations involve the addition of polien

&orn multiple pollen donors. The number of pollen donors involved must be higher than

what is received by open-poilïnated plants whkh codd v q dependkg on the type of

breeding sys tem (outcrossing, selhng, mixed, etc,) of the plant Cross (single)-pohated

phnts receive pollen fiom a single pollen donor. The number of pollen donors is then

Cross(multip1e) seed set - open seed set calculated as: D = p - (1 - ) where p =

Cross(multip1e) seed set - Cross(sing1e) seed set

the number of pollen donors used in the Cross (multiple)-pollination aeamient.

Here I have attempted to separate the effects of pollinator limitation and mate

limitation to aeate a dearer pictue of the mechaaisms bebind pollen limitation. It is

important to recognke, in measmkg pollen limitation, that it is not purely a result of either

ecological or genetic mechanisms but most Wcdy a combination of the nvo.

There is still much work to be done to bette understand the effect of population

size on reproductive success in s m d populations of H. herbacea. Because of its idluence on

genetic diversity, effective population size is tradiaondy estimated in populations for

N e conservation management reasons. To do this, land managers o h apply an - ratio, N

calculated from other populations or speaes, to these managed popuktions. Homevee the

Ne - values may not be representative. To have more accurate escimares of Ne for H. N

herhcea across the range of population sizes, very large and very small populations should be

used in the initial estimates of 3. This will give H herbacn conservation management N

teams a greater ability to pinpoint popuktions in danger of losing genetic diversiy.

A second area of research that requires attention concems pollen limitation. Sm&

popuktions are theoretically more 1ib:ely to be pollen limited than large populations;

however, the srnaIlest popuktions 1 obsemed were not pollen Ilnited. This may reflect the

biology of the partic& species. On the other hand, populations may not have been smail

enough in this study to detect an effect Theeefore 1 would suggest using smder population

sizes of H. herbacea to test the theones of genetic &fi and AIlee effect If it is not possible

to find smder natural populations, one could consmict populations of different sizes and

nurnber of compatible mates. Findy, pollen lLnitation has not traditiondy separated

limitations imposed by mate compatibility and poUinator activity nrithïn populations. Only

after 1 had completed this project, did i more fully understand how to better measure mate

and p o b t o r limitation. As H. herbacea is an ideal orgdnsm to measure mate and

pollinator Limitation simultaneously, it would be both usefid and interestkg to apply this

new approach-

CONSERVATION IMPLICATIONS

Hymenoxys herbacea is a rare endemic of the Great Lakes and as such its persistence is

of concem to both Canadian and US. conservation authorities. The po t end for Limitation

of reproduction due to population size is obviously of great devance for shaping

conservation management strategies for the plant and its populations. From my effective

N e population ske es tirnation, population managers may use the - ratio of 0.43 to roughly N

estimate effective size of other H. berbacea populations. As Byers and Meagher (1992) have

suggested, mating type diversiry will be eroded by genetic drift once the effective number of

individuals drops below 50. Therefore, managers should be concerned about populations

smaller than approximately 1 17 rameû.

In general, seed production in 12 populations of H. berbacea fiom the Bruce

Peainsula was not affected by mate limitation. Both effective population size estimates and

hand pollinations indicated that there is enough mate diversitg within populations to ensure

that reproductive success is sufficient Homever only 34Oh of crosses mere compatible

which is lower than thac observed in US. populations (58%). Moreove., from the benveen

population crosses, it seems as if mate di ver si^ at a regional level is lower on the Bruce

Peninsula populations than it is in the US. Therefore, if inmeaskg mate diversity in Bruce

Peninsuk populations becomes a priority, it would be most efficient to reuuit diversitg

from US. or Manitoulin Island populations than recruiting Gom within the Bruce Peninsula

region. Poha tor activity is unpredictable and changes with the vagaries of weather.

PoUuiator Visitation may be more likely to affect pouen limitation, especially since bees, the

most influentid pollinators, are heterogeneous in their distribution.

From this study, I can condude that the reproductive success of most populations

of H. herbocea (ranging fkom moderately srnail to reasonable large) has not been limited by

the quaLity or quantiq of pollen deposited. Full seed set in pollen supplemeated plants,

however, was never obserped in a Canadian inflorescence. This may indicate that resources

or other factors may be more important for ensuring reproductive success of H. herbatea in

Canada.

REFERENCES

BYERS, D. L. AND MEAGHER, T. R 1992. Mate availabiiity in s m d populations of

plant spedes Mth homomorp hic sporop hytic self-inc~rnpatibilit~. Hered. 68: 353-

359.

DE MAUROY M. M. 1993. Relationship of breeding system to rarity in the Lakeside Dais'

(Hymenoxy~ ocai& var. giabra) . Consem. BioL 7: 542-550.

LANDE, R. 1988. Genetics and Demography in Biological Conservation. Ecol. 75: 584-

GOG.

MARSHALL, D. E. AND EI.T.STRAND, N- C. 1988. Effective mate choice in wild

radish. Evidence for selective seed abortion and its mechanism. Am. NaL U1:

739-756.

WCETICH, J. A., WAITE, T. A. AND NUNNEY, L. 1997. Fluctuating population size

and the ratio of effective to census population size. Em[. 51. 2017-2021.

DEFINITION O F TERMS

AsexuaI Repmductoon - Any form of multiplication of earnets that produces self-sustainkg,

self-replicating rosettes of H. berbacta- In H. berbacea thL indudes rosettes produced from

branchiug of the woody caudex and underground rhizomes.

Indim'duaL - (=tamet) Any self-sustaining, self-replicating rosette of H. herbacea. In H.

herbacea, these plants have two or more leaves and a,xikq roots.

Popuiatiun - A duster of individuals that interact more dosely with one another than any

other duster of individuals. The duster of individuals must be separated by 75m of non-

habitat landscape.

Ramef - See dehnition of Individual above