Effects of Distance from Invasive Lythrum Salicaria On

Home , Lythrum, Lythrum alatum, Lythrum salicaria, Megachile sculpturalis, Myrica faya

EFFECTS OF DISTANCE FROM INVASIVE LYTHRUM SALICARIA ON

POLLINATOR VISITATION RATE AND REPRODUCTIVE SUCCESS IN NATIVE

LYTHRUM ALATUM

A Thesis

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Anthony Steven Kinyo

August, 2005

EFFECTS OF DISTANCE FROM INVASIVE LYTHRUM SALICARIA ON

POLLINATOR VISITATION RATE AND REPRODUCTIVE SUCCESS IN NATIVE

LYTHRUM ALATUM

Anthony Kinyo

Thesis

Approved: Accepted:

______Advisor Department Chair Dr. Randall J. Mitchell

______Committee Member Dean of the College Dr. Stephen C. Weeks Dr. Charles B. Monroe

______Committee Member Dean of the Graduate School Dr. Peter H. Niewiarowski Dr. George R. Newkome

______Date ii

ACKNOWLEDGEMENTS

Numerous people have helped me during this project and I could not have completed the research without all of their generous support. I would like to begin by thanking Dr. Randy Mitchell for his constant enthusiasm and positive encouragement throughout the research process. Thanks Randy, you have helped me to become a better scientist and researcher and I appreciate all that you have done for me. I would also like to thank my committee members Dr. Steve Weeks and Dr. Peter Niewiarowski, who provided feedback and assistance throughout the research process. Next, I would like to the Cuyahoga Valley National Park, Summit County Metroparks, and Ottawa National

Wildlife Refuge for allowing me to pursue my research on their land. The University of

Akron Biology Department provided financial assistance. Robert Jean, Donald Webb,

Martin Hauser, and John Ascher deserve thanks for helping me to identify the numerous pollinators collected during the experiment. I could not have grown my study plants without the expertise of Dr. Warren Stoutamire, whose knowledge of plant biology is unparalled. I would also like to thank Jeff Dunlap and Jesi Chaney for helping with the field work. Jesi, you helped me get through many trying times and I appreciate you listening to me talk about plants and pollinators incessantly. Last but definitely not least I would like to thank my parents Steve and Tammy Kinyo. Dad, your strong work ethic has inspired me many times and I hope that I can be as great a father to my children as

iii you have been to me. Mom, your constant support has helped me to get through many tough times and I cannot thank you enough for being so caring and loving. Without both of you I wouldn’t be the person I am today, thanks.

TABLE OF CONTENTS

Page

LIST OF TABLES………………………………………………………………...vii

LIST OF FIGURES……………………………………………………………… viii

CHAPTER

I. INTRODUCTION...... 1

II. METHODS...... 4

Study Species and Study System...... 4

Study Area and Experimental Design...... 5

Experimental Plants...... 5

Insect Visitation...... 6

Reproductive Success...... 7

Germination...... 7

Statistical Analyses...... 8

III. RESULTS...... 10

Characterizing the Pollinator Community...... 10

Overall Response...... 10

Visitation...... 10

Reproductive Success...... 12

v Germination...... 12

V. DISCUSSION...... 17

VI. SUMMARY...... 23

REFERENCES...... 24

APPENDICES...... 29

APPENDIX A. TOTAL VISITATION...... 30

APPENDIX B. COMPLETE INDIVIDUAL ANOVAS FOR POLLINATOR VISITATION...... 31

APPENDIX C. COMPLETE INDIVIDUAL ANOVAS FOR SEED SET AND GERMINATION ...... 33

APPENDIX D. GROWTH AND CARE OF LYTHRUM SPECIES...... 34

LIST OF TABLES

Table Page

1. List of pollinator species identified foraging during experimental period...... 13

2. MANOVA for four response variables...... 14

3. Individual ANOVAs...... 14

vii

LIST OF FIGURES

Figure Page

1. Experiment design...... 9

2. Average visitation rate to Lythrum alatum as a function of distance from Lythrum salicaria...... 15

3. Number of seeds per fruit in Lythrum alatum as a function of distance from Lythrum salicaria...... 16

4. Proportion of seeds sired by Lythrum alatum germinated as a function of distance from Lythrum salicaria...... 16

viii

CHAPTER I

INTRODUCTION

Invasive non-native plants have received increasing attention recently due to the negative effects these exotic species can cause on native plants and animals (Mooney and

Drake 1986, Usher 1988, Pimm et al. 1995, Pysek 1995). Aggressive invasive plants often create large monotypic stands with a corresponding decrease in overall plant and animal biodiversity (Thompson et al. 1987, White et al. 1997, Mullin 1998, Van Driesche et al. 2002). Many studies have shown that exotic plants often out-compete native plants for resources such as light (Weihe and Neely 1987), space (Rawinski and Malecki 1984), water (Delph 1986), and nutrients (Vitousek and Walker 1989). However, research into the possible reproductive competitive abilities of invasive plant species has not received as much study (but see Grabas and Laverty 1999, Brown and Mitchell 2001, Chittka and

Schurkens 2001, Brown et al. 2002, Niovi Jones 2004).

Waser (1983) defines competition for pollination as any interaction in which co- occurring plant species suffer reduced reproductive success because they share pollinators. This can happen in two ways. First, an invasive plant may reduce the number of pollinator visits to native plants by being more attractive to potential pollinators by having a larger floral display or nectar reward (Heinrich 1979). This decreased visitation rate can reduce pollen transfer between flowering plants and cause a

1 pollen deficiency such that plants are unable to set normal amounts of seed in the presence of the invasive plant species (Chittka and Schurkens 2001, Brown et al. 2002).

Another mechanism by which there may be competition is through interspecific transfer of pollen (IPT). IPT can cause numerous problems for plants including a reduction in seed set due to chemical or physical interference induced by the foreign pollen

(Thompson et al. 1981, Waser 1983, Brown et al. 2002), wasted maternal energy and effort by producing sterile hybrids (Waser 1978), and reduced male fitness due to the deposition and loss of pollen on flowers of a different species.

Competition is not the only possible outcome when two flowering plant species interact. There may also be a facilitative effect where plant species A does not negatively affect the pollinator visitation and reproductive output of species B but it actually increases pollinator visitation and reproductive output of species B (Rathcke 1983). This can also be termed the “magnet species effect” (Thompson 1978). A magnet species may draw pollinators into an area by having a large floral reward, and facilitative interactions with co-occurring plants may arise if the benefits of having more pollinators are greater than any negative effects caused by reproductive interference (Johnson et al. 2003).

Examples of facilitation are rare but a few studies (Thompson 1981, Laverty 1992,

Johnson et al. 2003, Moeller 2004) have demonstrated a facilitative effect. Competitive and facilitative effects may even exist concurrently. There may be an aggregate facilitative affect bringing in more potential pollinators through increased attractiveness to an area and competition among individuals once pollinators are drawn into the area

(Rathcke 1983).

2 Unlike vegetative competition, interactions among plants for pollination need not require the close proximity of the two interacting species. Due to the highly mobile nature of pollinators, interactions among plants for pollinators can occur over some distance. Numerous studies (Stefan-Dewenter and Tscharntke 1999, Klinkhamer at al.

2001, Schulke and Waser 2001, Townsend and Levey 2005) have documented the spatial scale of pollinator activity and how it relates to plant reproductive success but none of these studies examined the effects of a potential competitor on pollinator visitation and reproductive output on a fine spatial scale (<50 meters).

I studied the effects of distance from invasive Lythrum salicaria on visitation rate and reproductive success in its native congener Lythrum alatum. These two species are known to share pollinators (Levin 1970) and to compete for pollinators in sympatry

(Brown et al. 2002). They have similar flowering phenology (July-September) and floral morphology and often occur sympatrically in wetland habitats (Brown and Mitchell 2001,

Kinyo pers. obs.).

I specifically address three questions: (1) Does pollinator visitation to Lythrum alatum differ as a function of distance from Lythrum salicaria? (2) Does reproductive output in L. alatum (measured by seeds/fruit) differ as a function of distance from L. salicaria? (3) Is there a difference in germination rate in L. alatum as a function of distance from L. salicaria? I also characterized the pollinator community to the species level for these two plant species.

CHAPTER II

METHODS

Study Species and Study System

Lythrum salicaria L. (purple loosestrife) is a native of Eurasia and was first reported on the northeast coast of North America in 1814 (Pursh 1814). Lythrum salicaria has successfully invaded many habitats due to its high seed production (Shamsi and Whitehead

1974a, Balogh 1985) and ability to tolerate a wide range of environmental conditions (Shamsi and Whitehead 1974 a, 1974b, 1977). Lythrum salicaria can negatively affect native plants

(Mal et al. 1997, Brown et al. 2002) and native wildlife (Rawinski and Malecki 1984, Mullin

1998) often leading to decreases in floral and faunal diversity of the invaded area (Van

Driesche et al. 2002, Thompson et al. 1987).

Lythrum salicaria is a perennial, herbaceous species growing to heights of 2m

(Thompson et al. 1987). The magenta colored tristylous flowers are fairly large (~17mm diameter) and are arranged in long vertical racemes (Graham 1975, Whitson et al. 2000).

Lythrum salicaria is self-incompatible (Nichols 1987).

Lythrum alatum Pursh (winged loosestrife) is the most widespread native species of

Lythrum in the United States (Graham 1975). Lythrum alatum is a perennial, herbaceous species growing to heights of 1m. The light purple distylous flowers of L. alatum are smaller

(4-13mm) than those of L. salicaria and occur singly or in pairs in the leaf axils (Levin and

4 Kerster 1973, Graham 1975). Flower production, seed production, and pollen production

(Brown and Mitchell 2001, Kinyo pers. obs.) are less than in L. salicaria. The extent of self- incompatibility is unknown in L. alatum but most distylous species are self-incompatible.

Study Area and Experimental Design

Fieldwork was performed during July 2004 at three sites selected from within the

Cuyahoga Valley National Park, Ohio, Summit County (41o16’N, 81o33’ W). All three sites were wet meadows with similar environmental characteristics.

To evaluate the effects of distance from Lythrum salicaria on reproductive success and pollination in Lythrum alatum, at each site I set up a plot of 25 L. salicaria in a 5m x 5m array with ~ 1m spacing between flowering stems. Groups of 4 L. alatum were set up at four distances (0m, 5m, 20m, 50m) from the edge of the established L. salicaria array (Figure 1).

To facilitate reproductive success and legitimate pollen transfer for Lythrum alatum, two long style morphs and 2 short style morphs were placed at each site-treatment.

Experimental Plants

Seeds of Lythrum alatum were obtained from a commercial nursery (Prairie Moon

Nursery, MN) and were sown in the University of Akron greenhouse on March 29, 2004.

Individuals were transplanted to 1-gallon pots when they were 10-20 cm in height. Lythrum salicaria individuals were collected from a large wetland population within the Cuyahoga

Valley National Park at the beginning of the flowering season on July 5 2004 and placed in 3- gallon pots. Lythrum alatum and L. salicaria individuals were randomly chosen from those

5 available and placed at the three field sites on July 9, 2004 with the first day of pollinator observation commencing on July 12, 2004.

Insect Visitation

I observed pollinator visitation at these sites from July 12 - July 21, 2004. Weather was consistent across all 90 of the 15-minute observation periods (78-90 degrees F, sunny-partly cloudy). I rotated time and order of observation at each site randomly.

Observation periods occurred from 1000 hours to 1800 hours and I observed each of the four Lythrum alatum treatments at each site six times and also observed Lythrum salicaria six times at each site. Floral display of each plant was recorded at the beginning of the observation period. This resulted in 18 hours of observation on L. alatum and 4.5 hours of observation on L. salicaria. Visitation data was plant based, taken by observing the number of probes to each individual plant. When more than one pollinator was in an array all probes to each plant visited were accounted. Individual pollinator movement sequences on plants were recorded when possible, but when more than one pollinator was in the array the focus was on the number of probes to each plant and not on movement sequences of individual pollinators within an array.

Probes/flower/hour for each experimental plant was calculated by dividing the total number of probes observed to an individual plant by the average floral display for that plant divided by the total time spent observing that individual plant.

I could readily recognize three main categories of visitors to plants in this experiment (large bees, small bees, and syrphid flies). I collected a sample of the pollinators observed each day after observations were finished. Syrphid flies were not

6 effective pollinators (they collected pollen and seldom contacted stigmas). They were also uncommon (overall mean visitation rate 0.025 probes/fl/15min) therefore I ignore them in subsequent analyses.

Reproductive Success

To allow identification of flowers open during the experimental period I marked

Lythrum alatum stems with a small dot of black paint above the last open flower on each flowering stem prior to being placed into the field. On July 23 after pollinator observations were finished a small white dot of paint was placed below the last open flower on each flowering stem. The experimental L. alatum plants were then brought back to the University of Akron greenhouse to finish fruit maturation. Fruits from between these two marks were collected on August 29, 2004, after development and fruit maturation but before the fruit capsule could open up to release the seeds. Flowers of L. alatum open from the bottom of the stem to the top throughout the flowering season so this allowed for accurate collection of fruits that were available to pollinators during the experimental period. Four fruits were randomly selected from four randomly selected stems on each of the 48 L. alatum individuals used in the experiment for a total of 768 fruits. A dissecting microscope was used to count the number of seeds in each fruit.

Germination

To assess the viability of these seeds as a function of distance from Lythrum salicaria I set up an experiment to test for the germination rate. Seeds were given a cold period of one month at 8 degrees C after harvesting. One fruit from each of the 4 stems

7 sampled for seed set was randomly selected in the germination experiment. I randomly chose ten seeds from each of the 4 fruits from each plant. The 40 seeds from each plant were placed in-between moistened filter paper in a 9.0 cm petri dish on March 12 2005 under 2 HD metal halide lamps set on 12:12 light cycle. Seeds were kept moist throughout the experiment and emergence of new growth was recorded on a regular basis by presence of green cotyledon. I ran the experiment for 2 months to give all of the seeds a chance to germinate.

Statistical Analyses

Because response variables (visitation rate by each pollinator type, seeds/fruit, and germination rate) were all measured on the same plants they may be correlated with one another. Therefore, I analyzed these data using MANOVA of plant means to test for effects of site, treatment, and their interaction. We considered all of these effects to be fixed, and used type III sums squares in hypothesis tests. When the MANOVA indicated a significant effect I used individual univariate ANOVAs to help understand the overall patterns of response among response variables (Zar 1999).

50m

20m

5m L S

Figure 1. Experiment design. L and S refer to long and short morphs of Lythrum alatum. Squares are 25 Lythrum salicaria. This design was set up at all three study sites.

CHAPTER III

RESULTS

Characterizing the Pollinator Community

I was able to identify eighteen pollinator species out of 96 specimens collected during the experimental period (Table 1). All were seen or collected on both Lythrum alatum and Lythrum salicaria with the exception of the single Megachile sculpturalis

Smith I collected.

Overall Response

MANOVA indicated significant effects of treatment, site, and their interaction on the overall response by plants (Table 2). I then used individual univariate ANOVAs to examine the response variables separately and report the results below.

Visitation

During 18 hours of observation of Lythrum alatum I recorded 2886 floral probes.

Small bees were the main pollinators of L. alatum comprising 66% of the total floral probes observed. Large bees made up 21% of the total floral probes observed and other pollinators (syrphid flies and lepidopterans) comprised 13% of the total floral probes observed.

10 During 4.5 hours of observation of Lythrum salicaria I observed a total of 2418 floral probes. The pollinator composition for L. salicaria was comprised mainly of large bees, which accounted for 78% of all floral probes observed. Small bees made up 19% of total observed floral probes and other pollinators (syrphid flies and lepidopterans) comprised 3 % of the total observed floral probes. Overall visitation rate to L. salicaria was high (0.68 probes/flower/15minutes; large bees = 0.53 probes/flower/15minutes; small bees = 0.13 probes/flower/15minutes).

Different types of pollinators responded differently to distance treatments, although the overall total visitation rate did not vary with treatment (Figure 2, F3,36 =1.94, P<0.15).

There was a strong site effect for overall visitation rate (F2,36 =16.75, P<0.0001) but the site x treatment interaction was not significant (F6,36 =1.11, P<0.4). However, when pollinators were divided into large bees and small bees there was a significant treatment effect (Table 3, Figure 2). The large bee visitation rate declined significantly with distance from L. salicaria while small bees had their lowest visitation rate in the mixed species plot, exhibiting an increasing trend in visitation rate as distance from L. salicaria increased. Large bees showed no site effects but did show a strong site x treatment interaction. Despite this interaction the general pattern was maintained at all sites as visitation by large bees generally declined with distance from L. salicaria. Small bees exhibited a strong site effect due to the low abundance of small bees at site 3 (only 18% as many probes/flr/15min as other sites); there was no site x treatment interaction.

Pollinators only rarely moved between the species. Only 67 interspecific plant transitions (20% of all interplant moves) were observed during the 9 hours of observation of L. alatum and L. salicaria in the mixed species plot. Of these 67 interspecific

11 transitions, 75 % were moves from L. alatum to L. salicaria. Although observed interspecific movements were rare, I believe that this number slightly underestimates the actual number. During the observation of pollinators visiting plants, the focus was on probes to each plant and not on following individual pollinators. Because of this level of observation some interspecific transitions were probably not accounted for.

Reproductive success

Seed set in Lythrum alatum did not differ as a function of distance from Lythrum salicaria (Figure 3, Table 3). The number of seeds per fruit was highly variable ranging from a maximum of 81 seeds per fruit to a few fruits that produced zero seed set. There was a strong site effect but no site x treatment interaction. Sites differed significantly by as much as

37 %.

Germination

Germination rate in L. alatum showed a strong trend of increasing as a function of distance from L. salicaria (Figure 4, Table 3). There were strong site effects with treatment and site x treatment interaction on the verge of statistical significance. Sites differed significantly by as much as 18%.

12 Table 1. List of pollinator species identified foraging during experimental period. X denotes presence and O denotes absence.

Species Lythrum alatum Lythrum salicaria

Large Bees

Bombus bimaculatus X X Bombus impatiens X X Bombus perplexus X X Bombus vagans X X Apis mellifera X X Megachile sculpturalis X O

Small Bees

Megachile companulae X X Agapostemon virescens X X Agapostemon sericeus X X Augochlora pura pura X X Lasioglossum (Dialictus) rohweri X X Lasioglossum (Lasioglossum) leucozonium X X Ceratina (Zadontomerus) calcareta X X Ceratina (Zadontomerus) dupla dupla X X Ceratina (Zadontomerus) strenua X X

Syrphid Flies

Toxomerus geminatus X X Toxomerus marginatus X X Eristalis tenax X X

Table 2. MANOVA for four response variables. Response variables tested include large bee visitatation rate, small bee visitation rate, seeds per fruit, and germination rate in Lythrum alatum. Treatment refers to distance from Lythrum salicaria.

Source df Num. df Den. Pillai’s Trace P

Site 8 68 0.771 <0.0001 Treatment 12 105 0.834 0.0004 Site x Trt. 24 144 0.919 0.0194

Table 3- Individual ANOVAs. Analyzed visitation by large bees and small bees, number of seeds produced per fruit by individual experimental plants, and proportion of seeds sired by experimental plants that germinated. Treatment refers to distance from Lythrum salicaria.

Large Bees Small Bees Seeds/Fruit % Germination R2=0.536 R2=0.535 R2=0.236 R2=0.278

Source df P P P P

Site 2 0.7149 <0.0001 0.0001 0.0295 Treatment 3 <0.0001 0.0227 0.9595 0.0624 Site x Trt. 6 0.0043 0.4545 0.8913 0.0637 Error 36

14 0.4

0.3

Small bees 0.2 Large bees

0.1 Probes / FLR 15 min Probes 0 0 1020304050 Distance from Lythrum salicaria (m)

Figure 2. Average visitation rate to Lythrum alatum as a function of distance from Lythrum salicaria. Least squares means.

15 40

10 Number seedsNumber per fruit

0 0 1020304050 Distance from Lythrum salicaria (m)

Figure 3. Number of seeds per fruit in Lythrum alatum as a function of distance from Lythrum salicaria. Least squares means.

0.8

0.7

0.6

Proportion of seeds germinated Proportion of 0.5 0 1020304050 Distance from Lythrum salicaria (m)

Figure 4. Proportion of seeds sired by Lythrum alatum germinated as a function of distance from Lythrum salicaria. Least squares means.

CHAPTER IV

DISCUSSION

This study evaluated the effects of distance from Lythrum salicaria on pollinator visitation and reproductive success in Lythrum alatum. The types of pollinators (large bees and small bees) responded in opposing ways to the distance treatments and these differences in foraging were shown to have important consequences for offspring viability, as shown in this study by the decreased germination rate of L. alatum seeds produced by study plants within 5m from L. salicaria.

A primary result of this study is that the different types of bees on L. alatum responded quite differently to the distance treatments. Large bees had their highest visitation rate in the mixed species plot with a decreasing trend as a function of distance from L. salicaria, while small bees had their lowest visitation rate in the mixed plot.

These results suggest that the increased presence of the large bees in the mixed species plot may have influenced the foraging decisions of the small bees such that they avoided the mixed species plot due to the high abundance of larger bees. This is consistent with previous work (Morse 1977, Roubik 1978) that documented competition among bees at available flower patches. These effects are likely to be transient and bees may readily shift their foraging behavior to avoid competition (Roubik et al. 1986).

17 The high numbers of large bees observed in the mixed species plot is not surprising given that previous research suggests that the presence of a high nectar producing plant, such as L. salicaria, increases visitation rates and the numbers of flowers visited on an inflorescence (Laverty 1992, Johnson et al. 2003)

An interesting result of this study is that large bees seem to discriminate among patches of plants on a scale of greater than 5 meters. The mixed and 5 m treatments show no significant difference in visitation rate while the 20 m and 50 m plots have an extremely low visitation rate by large bees. It appears as if large bees can discriminate between patches of plants over this fine spatial scale. These results agree with the research of Klinkhamer et al. (2001) in which they showed that Bombus sp. could discriminate among intraspecific patches of plants with different nectar production rates

(NPR). However, contradictory to the results of Klinkhamer et al. (2001), and consistent with the results of Thompson (1988), large bees did discriminate among plants having a high NPR (L. salicaria) and a low NPR (L. alatum) within a patch. This is evident by the much higher visitation rate to L. salicaria (0.531 probes / flr / 15min) in the mixed species plot compared to 0.139 probes / flr / 15min to L. alatum in the mixed species plot.

The intraspecific studies described above have many similarities to the experimental design I employed but it is possible that the different floral morphology of the two species is also acting as a cue to rewards. The large foraging range of large bees

(Osborne et al. 1999, Beekman and Ratnieks 2000) allows them to be quite discriminating among patches on such a small spatial scale without hindering their ability to forage efficiently. Small bees, however, forage on a much smaller scale (Stefan-

18 Dewenter and Tscharntke 1999, Gathmann and Tscharntke 2002) and their foraging choices are therefore much more limited.

This difference in foraging behavior of large bees and small bees could have important consequences relating to the ecological outcome of the interaction between two closely related plant species. Several studies (Chittka and Schurkens 2001, Brown et al.

2002, Bell et al. 2005) have documented a reduction in seed set of a focal species in the presence of a competitor. The reduction in seed set in these studies is usually attributed to either a reduction in visit quantity or visit quality by pollinators.

In this study there was no overall reduction in visit quantity to L. alatum in the presence of L. salicaria but when divided by pollinator types (large bees and small bees) the effects are antagonistic. The increased large bee visitation to L. alatum in the mixed species plot implies a “magnet species effect” in which L. salicaria attracts more large bees in the area while the reduction in small bee visits to L. alatum in the presence of L. salicaria implies a competitive effect of L. salicaria on visitation in L. alatum. It is because of these two inversely correlated visitation patterns that there is no overall effect of treatment on visitation. However, the differing responses of the types of pollinators to the distance treatment could lead to different ecological outcomes when pollinator types differ in abundance. One can speculate that in an area with few small bees and mostly large bees that unless L. alatum could persist in close proximity to L. salicaria, which is unlikely due to the tendency of L. salicaria to form dense monospecific stands, over time the reduction in visit quantity to L. alatum would be enough to eliminate the population due to decreased individual recruitment. Conversely, L. alatum would do better in a

19 situation where small bees are the main pollinators in an area, even if L. salicaria were common.

One may speculate as to the efficiency of pollen transfer by the different pollinator types. It is well known that pollen adheres to the hairy bodies of bumblebees and honeybees and I observed numerous specimens during this study that had visible Lythrum pollen on their bodies. However, the level to which small bees transfer pollen between flower visits in these species is not yet understood.

It follows that if the small bees were not very efficient at transporting pollen that the

20m and 50m plots of L. alatum would show a reduction in seed set due to pollen limitation since small bees performed nearly all of the pollination at these distances. This would be the case if visits were limiting. Also, if large bees were moving between L. salicaria and L. alatum frequently in the mixed and 5m plots one would expect to see a reduced seed set due to the transfer of foreign L. salicaria pollen onto stigmas of L. alatum. But neither of these expectations were met. Surprisingly, there was no effect of treatment on seed set in L. alatum. Previous work by Brown et al. (2002) showed a reduction in seed set in L. alatum when in the presence of L. salicaria. The fact that seed set in the mixed treatment during this study was not reduced is puzzling and deserves some explanation. One possibility is that interspecific transitions were too rare to cause

IPT. Indeed, during my study only 20% of the plant-plant transitions by pollinators in the mixed species plot were between species and 75% of those transitions were from L. alatum to L. salicaria. Although this number most likely slightly underestimates the actual number of interspecific transitions observed due to the plant based observation I employed, it is nowhere near the 33-65% interspecific movements that were reported by

20 Brown et al. (2002). Another contributing factor may be that the pollinator composition was quite different during the two studies. Brown et al. (2002) report that during year 1 of their study 50% of the visitors were large bees (Bombus sp and Apis mellifera) while

50% of the visitors were others (classified by Brown et al. (2002) as syrphid flies, moths, butterflies, and miscellaneous flies) while in year 2 they reported 95% of the visitors as large bees and only 5% as others. There is no mention of the abundance of the small bees that I saw during my study. This difference in pollinator composition may be due to differences in study sites. My study was conducted in the wild meadows of a national park whereas Brown et al. (2002) conducted their study in a wetland on the Kent State

University campus. During year 1 of their study the reduction in seed set was 12% less than in 1998. They argue that these differences may be due to changes in plant culture conditions but it is also possible that the differences are due to the vastly different pollinator communities observed during year 1 and year 2 of their study and the foraging behavior of those different types of pollinators in relation to the two plant species. The proportion of probes to L. alatum in the mixed species treatment of my study is similar to that observed in year 1 by Brown et al. (2002) with 43% of the observed probes by small bees, 42% by large bees, and 15 % by others. It is quite possible that the difference in pollinator community along with the different foraging patterns observed during my study produced an effect in the mixed L. alatum treatment somewhat different from that observed in the study by Brown et al. (2002).

There may also have been an effect on the size and weight of the seeds produced by

L. alatum. I counted seeds whether they were small or large equally and did not weigh the fruit capsule due to their tiny size. Because some seeds were lost during the counting

21 process in became impossible to analyze this data after seeds were counted from each fruit. However, if L. alatum individuals near L. salicaria produced as many seeds as those further away but the quality of those seeds was reduced an effect may be shown in the viability of those seeds.

The effect of treatment on germination rate was very close to statistical significance

(P= 0.063) and the site x treatment interaction was also very close to statistical significance (P= 0.064). I performed a regression analysis treating distance as a continuous effect and the results were significant for site (P=0.04) and treatment (P=0.03) but no interaction was found. An increasing trend was observed in germination rate of L. alatum as a function of distance from L. salicaria across all three sites implying that although seed set was not affected by treatment there may be an effect on viability. A

12% increase in viability was recorded in the 20m and 50m plots relative to the 0m and

5m plots.

Previous work by Anderson and Ascher (1993) showed that crosses between L. alatum and L. salicaria produce seeds with 40% ± 24 (mean ± SD) germination rate and isozyme work by Strefeler et al. (1996) shows that introgression may have already occurred between L. alatum and L. salicaria. I attempted to grow the germinated seedlings from the mixed species and 5 m plot to determine if any individuals had characteristics similar to both species as reported by Anderson and Ascher (1994).

Unfortunately due to their high mortality relative to germinated seedlings from the 20m and 50 m plots I was not able to determine if any hybrids had formed.

CHAPTER V

SUMMARY

The purpose of this study was to ascertain whether there was an effect of distance from Lythrum salicaria on pollinator visitation, seed production, and viability of seeds produced in Lythrum alatum. It was determined that overall visitation rate to L. alatum did not differ with distance from L. salicaria but that different pollinator types (small bees and large bees) responded in significantly different ways to the distance treatment.

Large bees exhibited a trend of decreased visitation rate as a function of distance from L. salicaria while small bees exhibited a trend of increased visitation rate as a function of distance from L. salicaria. No effect was detected in seed production (measured by seeds/fruit/plant) in L. alatum as a function of distance from L. salicaria but an effect was seen in the viability of seeds produced by experimental plants. This effect was indicated by the decreased germination rate of seeds sired by L. alatum study plants within 5 m of

L. salicaria. It was also demonstrated that the two plant species share pollinators.

There are numerous areas left to explore within this system including pollinator- based observation to obtain a better feel for pollinator movement patterns while foraging.

Pollen transfer studies on small bees could reveal how much pollen they transfer between visits and how much pollen they deposit on the plants stigma surface while probing for nectar. Hand pollination studies may be able to determine viability of Lythrum alatum seeds sired by mixtures of differing qualities of pollen. 23

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APPENDICES

APPENDIX A

TOTAL VISITATION

0.5

n 0.4

0.3

0.2

Probes / FLR 15 mi 0.1

0 0 1020304050 Distance from Lythrum salicaria (m)

Total visitation to Lythrum alatum as a function of distance from Lythrum salicaria across all three sites including all pollinator types. Least squares means.

APPENDIX B

COMPLETE INDIVIDUAL ANOVAS FOR POLLINATOR VISITATION

I used univariate ANOVA to analyze the visitation data. Site, treatment, and the site x treatment interaction were considered fixed effects and I used type III sums squares. Visitation data is in units of probes / flower / 15 minutes.

Overall visitation rate

Source DF Sum of Squares F P Site 2 0.7565616.75 <.0001 Treatment 3 0.13164 1.94 0.14 Site x Treatment 6 0.15062 1.11 0.3748 Error 36 0.81284

Large bees

Source DF Sum of Squares F P Site 2 0.00199 0.34 0.7149 Treatment 3 0.12187 13.83 <.0001 Site x Treatment 6 0.06850 3.89 0.0043 Error 36 0.10574

Small bees

Source DF Sum of Squares F P Site 2 0.714355524.2214 <.0001 Treatment 3 0.15901184 3.5944 0.0227 Site x Treatment 6 0.08649069 0.9775 0.4545 Error 36 0.53087

Syrphid flies

Source DF Sum of Squares F P Site 2 0.010353947.0013 0.0027 Treatment 3 0.00182315 0.8219 0.4904 Site x Treatment 6 0.00336723 0.759 0.6067 Error 36 0.02661945

APPENDIX C

COMPLETE INDIVIDUAL ANOVAS FOR SEED SET AND GERMINATION

Seed set

Source DF Sum of Squares F P Site 2 1542.6789011.48 0.0001 Treatment 3 20.15400 0.10 0.9595 Site x Treatment 6 150.40320 0.37 0.8913 Error 36 2419.69340

Germination

Source DF Sum of Squares F P Site 2 226.29167 3.89 0.0295 Treatment 3 232.41667 2.67 0.0624 Site x Treatment 6 386.70833 2.22 0.0637 Error 36 1046.50000

Statistics below are germination data treating distance as a continuous effect.

Germination

Source DF Sum of Squares F P Site 2 226.29167 3.25 0.0486 Treatment 1 165.64643 4.76 0.0348 Site x Treatment 2 38.75017 0.56 0.5772 Error 42 1461.22840

APPENDIX D

GROWTH AND CARE OF LYTHRUM SPECIES

Lythrum alatum is somewhat difficult to grow from seed because of its requirements for moist but not wet soil. I started the seeds in planting flats using Premier pro mix-bx sphagnum peat moss mixed with miracle-gro potting soil and a small amount of sand.

The soil mix was composed of 75 % peat moss, 23% potting soil and 2% sand. The seeds should be placed in the flat with soil that has already been moistened by using a mister and sifting through the soil. Place the seeds on top of the soil mix in the flat. Do not bury them, as Lythrum alatum seeds need light to germinate. I covered the flat with a plastic flat size lid (obtained from any commercial greenhouse for minimal cost). This kept moisture in and decreased the need for a mist bench as some people use when growing plants that require constant moisture and cannot withstand small periods of drought. Once the plants were 5-10 cm in height I transplanted them to small 4-inch potting containers. This allowed the plant to form a nice root mass and will lead to a high percentage of successful transplants into the larger 1 gallon pots once they reach 15-20 cm in height. I started fertilizing once I transplanted the plants to the small 4-inch pots using low dosage 20:20:20 fertilizer once every 2 weeks. When the plants began to flower I gave them a 10:60:10 fertilizer at full strength once every 2 weeks. Once plants get to be around 35 cm in height they may begin to fall over. I put a fan in the

34 greenhouse to facilitate the swaying action plants experience in outside environments and this seemed to strengthen the stems a bit. I also put 3' wooden bamboo stakes next to each plant and tied the plant to the stake with very thin, 24 - gauge wire. It is necessary to mist the plants at least once per day and preferably twice a day.

It is very important to time the planting seeds such that they are flowering when the

Lythrum salicaria is flowering in the wild (July and August). I recommend planting two sets of seeds at different times to give a range of time when you will have flowering plants. I planted seeds in mid March and at the end of March and they were all flowering by mid June. I recommend planting seeds at the end of March and in the middle to end of

April. The greenhouse in which the experimental plants were being grown had an increase in temperature where the average temperature in the greenhouse was over 95 degrees for one week. This increase in temperature dramatically influenced the flowering of plants being grown such that many of them flowered too early and were not able to be used in the experiment. An ambient temperature of less than 80 degrees is preferred for optimal growth and timing of flowering.

Lythrum salicaria is much easier to maintain. Transplants from the field root well in most soils and their only requirement is to keep them constantly supplied with water.

They are even less drought tolerant than L. alatum, so it is best to keep the potted plants in containers of water.

When placing individual plants into the field be cautious of rodents and deer as they are very fond of both of these species. Rodents will often dig through the pot and destroy the root structures while deer prefer to eat the flowering stems of both species. I constructed a simple fence made of white string, which I wrapped around the perimeter

35 of each group of plants. This simple fence worked extremely well in deterring deer. I also sprayed ROPEL brand rodent and deer repellant around the edges of the fence and near the base of the plants at the field sites.