Relative Contribution of Sexual and Asexual Reproduction

Home , Lysimachia, Lysimachia terrestris, Remusatia

RELATIVE CONTRIBUTION OF SEXUAL AND ASEXUAL REPRODUCTION

IN LYSIMACHIA TERRESTRIS

Victoria C. Brown

Thesis submitted in partial fulfillment of the

requirements for the Degree of

Bachelor of Science with

Honours in Biology

Acadia University

April, 2017

This thesis by Victoria C. Brown

is accepted in its present form by the

Department of Biology

As satisfying the thesis requirements for the degree of

Bachelor of Science with Honours

Approved by the Thesis Supervisors

Dr. Rodger Evans ______

Date

Dr. Kirk Hillier ______

Date

Approved by the Head of Department

Dr. Brian Wilson ______

Date

Approved by the Honours Committee

Dr. Jun Yang ______

Date

I, Victoria Brown, grant permission to the University Librarian at Acadia University to reproduce, loan or distribute copies of my thesis in microform, paper or electronic formats on a non- profit basis. I, however, retain the copyright in my thesis.

______

Signature of Author

______

Date

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ACKNOWLEDGMENTS

I would like to express my gratitude to Dr. Rodger Evans and Dr. Kirk Hillier for sharing their knowledge and providing valuable resources that facilitated the completion of my Honours thesis. I would also like to thank Dr. Cory Sheffield and The Friends of the Royal Saskatchewan Museum for project funding as well as Emily Evans and Erica Gillis for their welcomed support in laboratory.

Table of Contents

Acknowledgments……………………………………………………………...……..…..iv

Table of Contents……………………………………………………………………..…. .v

List of Tables ………………………………………………………………………...... vii

List of Figures …………………………………...…………………………………...... viii

Abstract………………………………………………………………..……………...... x

Introduction……………………………………………………………………….…..….1

Lysimachia terrestris…………………………………………………………..…..1

Bulbils and asexual versus sexual reproduction …………………………….….…2

Pollination ecology and Macropis nuda ……………………………………..……5

Objectives………………………..………………………………….……………..6

Materials and Methods

Collection Methods …………………………………...……………………..…....6

Light and scanning electron microscopy techniques………………………...….....8

Statistical Analysis: Pollinator Exclusion treatments…………………...…...….....9

Results

Floral and Bulbil Development……………………………..…………………..…9

Pollinator Exclusion……………………………………………………….……..20

Discussion……………………………………………………………………..…………26

Stages of Floral Morphological Development…………………………….....…..26

Bulbil Developmental Stages………………..…………………………..…...…..27

Pollinator Exclusion…………………………………………………………..….27

Literature Cited …………………………………………………….………………...... 31

Appendix A: Mean, standard deviation, standard error and raw data: Number of fruits

and number of bulbils……………………………………..…………...... ……..34

Appendix B: Mean standard deviation, standard error and raw data: Number of fruits and

number of bulbils..…………………...………………………...…………...…….35

Appendix C: Comparisons for all pairs using Tukey- Kramer HSD: Ordered Difference

Report: Number of Fruits (Highway 101)……………………...……………..….40

Appendix D: Comparisons for all pairs using Tukey- Kramer HSD: Ordered Difference

Report: Number of Bulbils (Highway 101)………………...………...………….41

Appendix E: Comparisons for all pairs using Tukey- Kramer HSD: Ordered Difference

Report: Bulbil length (Highway 101).………………...……………………....….42

Appendix F: Comparisons for all pairs using Tukey- Kramer HSD: Ordered Difference

Report: Mean number of seeds per flower (Highway 101) ………………...... ….43

Appendix G: ANOVA single factor analysis for Levene’s test……………...……...…..44

List of Tables

Table 1. Mean number of fruits, number of bulbils, bulbil length and number of seeds per flower for treated Lysimachia terrestris off Highway 101 in Middleton Nova Scotia and at the Harriet Irving Botanical Gardens in Wolfville, Nova Scotia……………...... 20

Table 2. Summary: Mean number of fruits (Highway 101)……………………..………21

Table 3. Summary: Mean number of bulbils (Highway 101)………………...………… 21

Table 4. Summary: Bulbil length (cm) (Highway 101)……………………………….…22

Table 5. Summary: Number of seeds per flower (Highway 101)…… ………………… 23

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List of Figures

Figure 1A. Lysimachia terrestris inflorescence (Middleton, Nova Scotia)……………….1

Figure 1B. Bulbil formation at leaf axils of Lysimachia terrestris……….……………...…1

Figure 2. Scanning electron microscopy image succession of morphological

development of Lysimachia terrestris flower…………………………………...... 11

Figure 3. Scanning electron microscopy image of Lysimachia terrestris seed capsule...... 12

Figure 4. A: Scanning electron microscopy images of unfertilized ovules of Lysimachia

terrestris and developing seed……………………………………………….……12

Figure 5. Scanning electron microscopy images of bulbil development………...…..……13

Figure 6. Scanning electron microscopy images of bulbil vascular cambium comprising

proto-xylem (px) and proto-phloem (pp)……………..…………………….…….14

Figure 7. Longitudinal paraffin sections (10 µm) illustrating progressive morphological

development of Lysimachia terrestris flower………………...……….……….….15

Figure 8. Floral ontogeny of Lysimachia terrestris- paraffin transverse sections

(10 µm)…………………………………………………………………………...16

Figure 9: Paraffin sections of Lysimachia terrestris seed capsule (10 µm)…..………....17

Figure 10. Paraffin longitudinal section (10 µm) of seed capsule of Lysimachia

terrestris...... 17

Figure 11. Ontogenic development of Lysimachia terrestris bulbil (paraffin transverse

sections)……………………………..……………….…………………...………18

Figure 12. Progressive development of early bulbil primordia ……………………..…..19

Figure 13: Progressive morphological development of Lysimachia terrestris bulbils.....19

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Figure 14: Graph of mean length of bulbils amongst treated Lysimachia terrestris at the

Highway 101 site at Middleton, Nova Scotia and at the Harriet Irving Botanical

Gardens in Wolfville, Nova Scotia. Plants treated with pollinator exclusion bags

had zero, two, six, twelve or all flowers uncovered………………….……..……24

Figure 15. Line graph of mean length of bulbils versus number of bulbils amongst treated

Lysimachia terrestris at the Highway 101 site at Middleton, Nova Scotia and at

the Harriet Irving Botanical Gardens in Wolfville, Nova Scotia…………...……24

Figure 16. Graph of the total number of fruits from by Lysimachia terrestris treated with

pollinator exclusion bags at the Highway 101 site at Middleton, Nova Scotia and

at the Harriet Irving Botanical Gardens in Wolfville, Nova Scotia. Treated plant

groups had zero, two, six, twelve and all flowers uncovered………………….…25

Figure 17. Line graph of the number of bulbils developed by treated Lysimachia

terrestris versus number of fruits produced at the Highway 101 site at Middleton,

Nova Scotia and at the Harriet Irving Botanical Gardens in Wolfville, Nova

Scotia. Zero, two, four, six, twelve and all flowers were left uncovered among the

treated plants…………………….…………………………………….………….25

Abstract

Lysimachia terrestris (Myrsinaceae) is one of few species within the genus Lysimachia known to asexually reproduce via bulbil formation. It is unknown if pollination and subsequent fruit and seed production inhibits or reduces bulbil formation in L. terrestris.

To examine whether there is a tradeoff between asexual and sexual modes of reproduction in this species, populations were investigated in Middleton and Wolfville, Nova Scotia.

Impact upon bulbil production was monitored through application of pollinator exclusion bags prior to anthesis to modify pollination and fruit set. Treatments comprised zero, two, six, twelve, or all flowers, being left open to pollinators to determine if a threshold number of pollinated flowers alters the number of bulbils formed. Counts of the number of bulbils, flowers, fruits, seeds and unfertilized ovules were analyzed among treated plants. Using light and scanning electron microscopy techniques, fruit and bulbil development were also examined. Results suggest that bulbil formation is limited, but not restricted by seed production. Additionally, investment in sexual reproduction was observed to be larger at the Middleton location. There is anecdotal evidence of the presence of a specialist pollinator, Macropis nuda (Hymenoptera: Mellitidae), but none were observed. These findings provide new insight into the conditions influencing bulbil yield in L. terrestris and the ecological roles of both asexual and sexual reproduction.

Introduction

Lysimachia terrestris

Lysimachia terrestris (Myrsinaceae) (Swamp Candle, Earth Loosestrife) is a herbaceous, perennial plant which occupies marshy regions including riparian zones, bogs, wet ditches, and lake and pond margins (Cholewa, 2009). The species’ Canadian distribution comprises all provinces except Saskatchewan and Alberta. It is also present in regions of Kentucky, Arkansas, and Iowa (Sheffield, 2011) in the United States. Fruit production and seed set occurs in plants that have been visited by insect pollinators, primarily Macropis nuda (Hymenoptera: Melittidae; Sheffield, 2011). Vegetative propagation occurs through bulbil formation. Bulbil production is a defining reproductive character of L. terrestris and is particularly uncommon among other species within the genus, Lysimachia (Sheffield, 2011).

Figure 1A: Lysimachia terrestris Figure 1B: Bulbil formation at leaf axils inflorescence (Middleton, Nova Scotia) of Lysimachia terrestris

Plant stems in L. terrestris are glabrous, simple or branched and are 25-100 cm in length. Leaves are opposite, with absent or highly reduced petioles, and are without ciliation. Leaf blades are ovoid-lanceolate in shape, have a cuneate base and are smooth with entire margins. Leaves are 3-10 cm x 0.5-2 cm, and are tapered at the apex

(Cholewa, 2009). The inflorescence is terminal and forms a loose raceme (Cullen and

Alexander, 1984), 10-30 cm in length with flowering occurring from June to September

(Azan, 2011). The corolla is rotate and yellow with a reddish base. Like all other

Lysimachia species, L. terrestris has five longitudinally dehiscing anthers. The pistil comprises an inferior ovary, and a slender style which is crowned with a slightly enlarged stigma. The basal corolla and androecium are dotted by trichomatous elaiophores which actively secrete lipids. These structures are most numerous on the proximal upper surface of the petals of L. terrestris (Simpson, 1983). A basal cell, stalk cell and cap cell grouping comprise the trichome. The secretory cap consists of eight, largely cytoplasmic cells.

These are surrounded by an outer cuticle, under which the secretory products accumulate

(Simpson and Neff, 1983). Female M. nuda collect these lipid secretions from glandular regions of the androecium and corolla (Vogel, 1976). Lysimachia terrestris flowers are non-nectar producing but yield an abundance of pollen. If fertilized, fruiting is observed from August to October, during which ovoid seed capsules are produced. These are few seeded and are 3-4 mm in length.

Bulbils and Asexual vs Sexual reproduction

If conditions are not favourable for pollination and fertilization, such as in the absence of reliable insect-pollinators, L. terrestris may undergo vegetative reproduction.

It is also suggested that environmental abiotic conditions may affect bulbil yield (Allard,

1910). During bulbil formation, the development of secondary branches is arrested

(Ganong, 1901) and bulbil growth is initiated in the leaf axils (Smith and Gremaud,

2007). When fully developed, these are 1.5 cm long, red, cylindrical structures and comprise of 3-5 segments between nodes (Ganong, 1901). Morphologically, bulbils resemble suppressed branchlets (Allard, 1910). The vascular cylinder is in an underdeveloped condition and comprises only protoxylem and protophloem (MacDougal,

1908). Bulbils are shed from the plant, in the late season, when it is easily detached. In riparian zones, bulbil dispersion downstream is mediated by the flow of water (Basinger,

2000). Bulbils of L. terrestris are highly resilient to desiccation and freezing, thus readily overwintering in temperate climates (Ganong, 1901). In the following season, bulbil germination occurs, producing a root and leafy shoot. Bulbil development is completed when the bulbil becomes a rhizome, forming the primary axis of the plant (MacDougal,

1908).

Asexual breeding systems are favourable in disturbed environments including cleared woodland, and exposed or altered habitat (Lozano et. al, 2003). Rhizomatous growth is often observed among perennials in fluctuating environments, such as riparian habitats (Hoag, Melvin and Tilley, 2007). Richards (1997), suggests that agamospermous individuals are more adaptive to variances in physiological conditions than those which reproduce sexually. Fewer pollinators are often present in unstable environments, reducing the likelihood of pollination, and subsequent seed set. Disruption in either pollinator nesting sites or floral resources resulting from the conversion of wetland regions to urban development sites and agricultural land, is also attributed to the loss of pollinator communities (National Research Council, 2007). As vegetative reproduction occurs independently of external pollination, clonal colonies, or groupings of genetically

identical individuals arising from vegetative means, allow for population expansion under conditions wherein pollinators are limited (Barrett, 2015). Despite this apparent advantage, recombination in natural populations remains significant (Dagg, 2012).

Several species use both sexual and vegetative modes of propagation at an “evolutionary equilibrium”, indicating that both processes yield similar net returns (Abrahamson, 1980).

It is probable that asexual and sexual modes of reproduction have different ecological roles (Law, Cook, and Manlove, 1983). When population density is high, sexual propagation is most suitable (Abrahamson, 1980). In such regions, intensified competition, and the introduction of genotypes of greater fitness, may result in clone extinction. Sexual reproduction is evolutionarily advantageous in producing genetically variable and, therefore, adaptable populations (Abrahamson, 1980). Vegetative expansion may be favourable in facilitating the spread of successful genotypes in regions of low population density. During vegetative multiplication, an increase in the size of the population is observed, however genetic diversity is limited. Vegetative propagation will continue until environmental limitations restrict clonal growth. The clone must, therefore, distribute genetically identical propagules beyond its current region of occupation if it is to ensure its genetic survival. The greater the distance the propagule is dispersed, the greater the probability that it will encounter changed environmental conditions, and the greater the significance of recombination (Keddy, 2007). The two modes of sexual and asexual propagation constitute an inseparable system which are required for optimal plant fitness (Abrahamson, 1980).

Pollination ecology and Macropis nuda

The oligolectic bee species, M. nuda is thought to be reliant on L. terrestris for its pollen and floral oils (Sheffield, 2011). Gathered oil is used in constructing water- resistant cell linings and in developing larval provisions. Only females are known to participate in pollen and fluid collection (Buchmann, 1987). As oils are secreted in lieu of nectar in L. terrestris, foraging M. nuda must obtain nectar from alternate floral hosts

(Cane, Eickwort, Wesley and Speilholz, 1983). Nest sites of M. nuda readily occur within or adjacent to L. terrestris stands, often in sunlit regions with sandy soil and dense vegetative underbrush. Although M. nuda is solitary, nests are often aggregated on sloping banks (Cane et al., 1983).

Macropis nuda nests are parasitized by the cleptoparasitic bee, Epioloides pilosulus (Hymenoptera: Apidae). Historically, E. pilosulus was distributed from Eastern to Central North America but, as of 2011, has been designated as endangered under

COSEWIC. The species’ decline is likely attributed to the loss of wetland areas containing L. terrestris and the associated disappearance of M. nuda nesting sites

(Sheffield, 2011). Several marshes in Nova Scotia, for example, have been converted to cranberry fields or developed into urban centres. (Sheffield, 2011).

Although L. terrestris pollination is mediated largely by M. nuda, the mutualists appear to be unequally co-evolved. As the range of L. terrestris extends beyond that of M. nuda, the mutualism must be non-obligatory for the host plant (Buchmann, 1987). While

L. terrestris may not be wholly reliant on M. nuda, the bee species requires L. terrestris for the collection of larval provisions. Human disruption of M. nuda nesting sites is likely to increase the rarity of this bee genus. However, as L. terrestris is able to vegetatively reproduce in the absence of M. nuda, the species’ survival is highly probable.

Furthermore, the ability of L. terrestris to flourish in non-natural habitats (e.g.roadside ditches) favours its continued success (Simpson and Neff, 1983).

Objectives

Populations of L. terrestris were investigated in Middleton and Wolfville, Nova

Scotia to deduce the relative contributions of asexual and sexual modes of reproduction.

We propose that if fertilization and seed set occur, bulbil yield is correspondingly reduced. Furthermore, bulbil formation may be entirely prevented at an upper limit of pollinated flowers. Asexual and sexual reproductive modes were investigated to determine if there was a tradeoff between these alternate forms of reproduction. Variation in bulbil production was examined through manipulation of plant-pollinator interactions with exclusion bags that were applied to L. terrestris flowers prior to anthesis. Counts of number of bulbils, flowers, fruits, seeds and unfertilized ovules were analyzed among treated individuals. Floral and bulbil development were also examined through scanning electron and light microscopy to deduce specific, morphological stages relating to the ontogeny of these structures.

Materials and Methods

Collection Methods

Lysimachia terrestris samples were collected from the Harriet Irving Botanical

Gardens at Acadia University in Wolfville, Nova Scotia (20 T 392428 4993762

45.08914°N -64.36698°E). Specimens were harvested from a bog location, and a second courtyard site. Lysimachia terrestris samples were also collected from a natural site in

Middleton, Nova Scotia. This represented a larger natural population of L. terrestris, and

was positioned near exit 18A off Highway 101 (20 T 335340 4979837 44.95289°N -

65.08748°E). Plants were harvested upon initiation of observable inflorescence meristem activity, and inflorescences from two to three plants were collected weekly on June 8, 15,

23, 30 and July 7, 16 and 27 for examination of floral morphological development and subsequent bulbil formation. Specimens were stored in sterilized plastic vials containing formaldehyde acetic-acid alcohol (FAA: 90 parts 50% ethyl alcohol; 5 part formalin; 5 part glacial acetic acid). Prior to anthesis, exclusion bags were applied to L. terrestris, to prevent pollination. Among the treated plants, zero, two, six, twelve or all of the flowers were left open. Exclusion bags were prepared from a larger segment of breathable

Dacron, closed on three sides. Sections were cut from the outer corners of the bag and were sized to adequately cover the excluded portion of inflorescence. A fast-acting cyanoacrylate adhesive was used to fix the third, open side of the exclusion bags. The adhesive was applied sparingly to minimize the weight of the exclusion bag, and correspondingly, the influence of the bag on the physiological development of the treated plants. Upon drying, prepared exclusion bags were secured to the inflorescences using a twist- tie which was folded several times, loosely, about the inflorescence stem. Bags were then appropriately marked with the given treatment employed. If the treated portion of inflorescence became too large for the exclusion bag, such that curving of the inflorescence was observed within the bag, the exclusion bag was removed and, a larger bag reapplied.

Following fruit production, in late August, the exclusion bags were removed, and treated plants collected. Specimens were placed in sterilized plastic vials containing FAA.

After fixation of the plant tissue, all collected materials were analyzed in a petri dish containing 70% ethyl alcohol using the Nikon Stereoscopic Zoom Microscope SMZ1000.

Light and Scanning Electron Microscopy (SEM) Techniques

Upon completion of embedding in laboratory, Spurr resin blocks were cut into 0.5

µm sections using a Leica RM2065 ultramicrotome by Leica Biosystems (Concord,

Ontario) fitted with a diamond knife. Section were transferred to glass slides and were allowed to dry overnight. Spurr resin sections were subsequently stained in a solution of

0.5% Toluidine Blue (TBO) in 0.1% sodium carbonate using the procedure outlined by

Evans (2016). Embedded paraffin samples were sectioned at 10 µm thickness using a 820 rotary microtome by American Optical (Buffalo, New York) and were mounted onto glass slides and stained following Johansen’s Safranin and Fast Green Staining method.

Johansen’s Safranin and Fast Green Staining was carried out in accordance with the instructions provided in BIOL4173 Specialized Microscopy Techniques (Evans, 2016).

Red stained tissues indicated the presence of lignified, suberized or cutinized cells. Fast green appeared blue in cellulosic walls and in cytoplasmic regions.

Specimens to be examined under SEM were dehydrated in an ethanol series and critically point dried using carbon dioxide as the transitional medium. These were then mounted on stubs with double sided- conductive carbon tape, and sputter coated with gold-palladium in a sputter coater (Edwards S150A Sputter coating unit, Crawley,

England). The prepared specimens were viewed and photographed under the scanning electron microscope (JEOL JSL 6400, University of New Brunswick (UNB), Fredericton,

NB) at 5 kV in the Acadia Centre for Microstructrual Analysis (ACMA) in Wolfville,

Nova Scotia.

Statistical Analysis: Pollinator Exclusion Treatment

Counts of the number of bulbils, total number of flowers, fruits, seeds and unfertilized ovules were made using a Nikon Stereoscopic Zoom Microscope SMZ1000.

Relationships between variables were graphed using Numbers 3.5.3 (2150) by Apple Inc. at 1 Infinite Loop. Cupertino, CA 95014, Copyright ©2008-2011, which was used to generate a coefficient of determination value and an equation of the linear trend-line. The data analysis tool-pack in Microsoft Excel for Mac 2017 by Microsoft ©2017 (Redmond,

Washington), was used to conduct one-way ANOVAs comparing the number of fruits, number of bulbils, bulbil length and mean number of seeds per flower across treatment groups at the Highway 101 study site. Ordered difference reports (from the Tukey-

Kramer comparison (α = 0.05)) were developed to determine if a significant difference occurred between the mean values of treatment groups. A two-sample T-test for unequal variances (α = 0.05) was also conducted to determine if the average difference between groups of treated plants at the Harriet Irving Botanical Gardens was statistically significant for the independent variables studied.

Results

Floral and Bulbil Development

Distinctive stages of floral and bulbil development were observed among microscopic examinants. Young inflorescences are conical in shape and are surrounded by early bud primordia. Five early combined petal-stamen bundles emerge and grow toward the inflorescence axis. These are flanked by five, outer sepal primordia (Figures

2A-B). The gynoecium emerges from a centrally occurring ring primordia. Within the gynoecium a placenta forms, on which several ovule primordia emerge (Figure 2D).

Differentiation of the petals and stamens from the common-petal stamen then occur

(Figure 2D). Petal growth occurs exteriorly to the developing stamen primordia (Figure

2D). Stamen development is characterized by the formation of a longitudinal dehiscence on the interior surface of the anther. Eventually, the sepals grow large enough to cover floral apex (Figure 2E). An increase in filament length is observed in the developing flower (Figures 2F-J). The basal androecium becomes dotted with trichomatous elaiophores which consist of a rounded cap and cylindrical base (Figure 2I-J). Style elongation occurs and surpasses the length of the androecium (Figure 2F-J). Among mature flowers, petal growth occurs perpendicular to the floral axis (Figure 2J) and trichomes become apparent at the petal base (Figure 2J). Petal fusion occurs basally, forming the corolla tube (Figure 2J).

Figure 2. Scanning electron microscopy image succession of morphological development of Lysimachia terrestris flower. A,B: Combined petal-stamen growth is initiated at common petal-stamen (cp), sepal primordia emerge (s) (week 1 of floral development) C: Sepal elongation (week 2) D: Anther progression and partitioning of stamens (st) and petals (p) from common petal-stamen (week 2-3 of floral development). E: Carpel comprises an inferior ovary, style (sy) and slightly enlarge stigma (sm). Five longitudinally dehiscing anthers definitively observed(week 2-3) F: Style length surpasses apex of androecium (week 3-4). G-J: Filament enlargement occurs, I-J: Numerous trichomes (t) on basal androecium and basal corolla, J: Petals grow perpendicular to the plane of the androecium (week 4). Scale bars: A= 50 µm, B=50 µm, 100 µm, C= 100 µm, D= 250 µm, E= 200 µm, F= 500 µm, G= 1mm, H= 1mm, I= 1mm, J= 1mm.

Fruit development was examined using SEM imaging. Seed capsule formation is marked by ovary wall thickening and the development of seeds from fertilized ovules.

The seed capsule is ovoid in shape (Figure 3). Both unfertilized ovules and seeds are embedded within spongy mesocarp within the ovary (Figure 4).

Figure 3. Scanning electron microscopy image of Lysimachia terrestris seed capsule. Capsule formation is characterized by drying and thickening of the pericarp (pc). The closed receptacle surrounds the developing seeds and placental tissue (p). The developed seed capsule is attached to only to the whorl of sepals (s) at its base and lacks adherence A B to the androecium and petals. Scale bar= 1mm C

Figure 4. A: Scanning electron microscopy images A. unfertilized ovules of Lysimachia terrestris (o), B: Developed seed (sd) and unfertilized ovules (o), C Spherical seed (sd) with ribbed sections. Scale bars: A= 200 µm, B= 100 mm, 200 µm.

Young bulbils have a small apical meristem and emerge from decussate, leaf-like projections. An increase in nodes is observed in the distal portion of the bulbil as the structure matures (Figure 5).

Figure 5. Scanning electron microscopy images of bulbil development. A. Bulbil apical meristem (am) emerges from intersecting protuberances (pr). Elongation occurs (B-J). Gradual narrowing of the primordium is subsequently observed. A node (n), or tapering of the bulbil occurs at its distal end. Foliar-like protuberances emerge at opposite ends of the node. These outgrowths enclose the newly developing internode (i) and occur perpendicular to the previously formed protuberance pair. Subsequent intermodal regions initiate in a similar sequence. Bulbil maturation is concluded, producing a propagule with three or more internodes. Scale bars: A= 500 µm, B= 1mm, 200 µm, C= 200 µm, D= 500 µm, E= 500 µm, F= 500 µm, G= 500 µm, H= 1mm, I= 1mm, J= 1mm.

The central cylinder comprises immature xylem and phloem tissue. Fully differentiated xylem occurs on the outer portion of the bulblike structure (Figure 6).

A B

Figure 6. Bulbil vascular cambium comprising proto-xylem (px) and proto-phloem (pp). Scanning electron microscopy images of 50X (Figure 6A) and 22X magnification (Figure 6B). Scale bars (Figure 6A) = 500 µm, (Figure 6B) = 1mm

The gynoecium chamber arises from a ring primordium at the floral axis (Figure

7C). Petal and stamen primordia are differentiated from the common petal-stamen

(Figure 7A). Petals occurs abaxially to the common primordum. Upward growth of the sepal and petal primordia occur and result in the eventual enclosure of interior floral structures (Figure 7I-J). As the petals and stamens move up, the ovary becomes semi- inferior (Figure 7G-J). Ovule development occurs on the conical placenta (Figure 7G).

Figure 7. Longitudinal paraffin sections illustrating development of L. terrestris flower. A-B: Emergence of sepal primordia (s) and common petal-stamen primordia(cp) (week 1 of bloom development) C-D: Emergence of ring of gynoecium (g) (week 2 of floral development) E-F: Style elongation (sy), (week 2-3 of floral development) F: Differentiation of petal (p) and stamen (st) from common petal-stamen (cp) (week 2-3 of floral development) G: Ovule formation (o) , petal and stamen elongation (week 3-4 of floral development), I-J: Petals enclose androecium and gynoecium (week 4) Scale bars: A= 50 µm, B= 50 µm, 100 µm, C= 100 µm, D= 250 µm, E= 200 µm, F= 500 µm, G= 500 µm, H= 500 µm, I= 1 mm, J= 1 mm.

Petal and stamen differentiation becomes more evident with increasing floral maturity (Figure 8). An increase in distance between petals and internal floral structures also occurs.

Figure 8. Floral ontogeny of L. terrestris in paraffin transverse sections (10 µm). Petals (p) and stamens (st) ascend relative to the gynoecium. Johansen’s Safranin and Fast Green Staining method. Red stain is present in lignified, suberized or cutinized cells and in nuclei-dense areas. Fast green appear blue in cellulosic walls and in cytoplasmic regions. Scale bars: A= 1 mm, B= 1mm, C= 1 mm.

Lysimachia terrestris seed capsule shows a central placenta which occurs freely from the inner gynoecium. The ovary, which lacks partitioning, is aseptate. Sections of placenta separate the developing ovules as the space within the gynoecium increases.

(Figure 9). At maturity, most of the floral parts wither and fall away from the gynoecium.

Only the calyx persists in its adherence to the semi-inferior ovary (Figure 9, Figure 10).

Figure 9: Paraffin sections of Lysimachia terrestris seed capsule (10 µm). A, B: Longitudinal sections, B: Unfertilized ovules (u) embedded in porous tissue within seed capsule (c) C:Transverse section: Seed capsule encloses placenta (p) and is flanked by five sepals (s). Johansen’s Safranin and Fast Green Staining method used. Lignified, suberized or cutinized cells are stained a brilliant red. Fast green appears blue in cellulosic walls and in cytoplasmic regions. Scale bars: A= 500 µm, B=500 µm, 250 µm

Figure 10. Paraffin longitudinal section (10 µm) of seed capsule of Lysimachia terrestris (style detached) stained using Johansen’s Safranin and Fast Green Staining method (10 µm). Outermost layer (epicarp) (e) encases mesocarp (m) and inside layer of pericarp (endocarp) (en) which surrounds the seeds (sd). Highly lignified, suberized or cutinized regions are stained red by Johansen’s Safranin. Cellulosic walls and cytoplasmic cells appear blue. Scale bar= 500 µm

Bulbils resemble suppressed branchlets and are comprised primarily of underdeveloped vascular tissue. The structures are jointed and form a pointed apex

(Figure 11, Figure 12). Bulbil primordia have an apical meristem at the tip that is enclosed by two foliar- like projections (Figure 12).

Figure 11. Ontogenic development of Lysimachia terrestris bulbil (paraffin transverse sections) (10 µm) stained using Johansen’s Safranin and Fast Green Staining method A: Central region of bulbil flanked by protuberance pair (pr) resembling foliar organs. B: Second protuberance pair (pr2) envelop newly developing internode. Pr2 occur perpendicular to the plane of the pr. Proto-xylem (px) and porto-phloem (pp) comprise the bulbil interior. C: An increase in intermodal diameter is observed among bulbils at maturity. A-C: Proto-xylem (px) and proto-phloem (pp) comprise the bulbil interior. Scale bars= A= 500 µm, B= 1mm, C= 1mm.

Figure 12. Progressive development of early bulbil primordia (Spurr resin longitudinal sections) (5 µm) stained in a solution of 0.5% Toluidine Blue (TBO) in 0.1% sodium carbonate. Polysaccharides appear purple. A: Growth of the bulbil primary axis is initiated at the apical meristem (am). The apical meristem is encircled by two, laterally occurring protuberances (pr) which shield the region of rapidly dividing cells. B-C: Upward growth of the bulbil occurs at is primary axis. C: Growth of the primary body occurs occurs at an accelerated rate relative to the protuberances and the bulbil apex (a) approaches the interior surface of these projections. Scale bars= A=250 µm, B=250 µm, C=500 µm.

Figure 13: Progressive morphological development of Lysimachia terrestris bulbils. Bulbil adherence to leaf axils occurs at the base (b). Bulbil growth is initiated at the apical meristem (am) located at the apex of the vegetative structure. Nodes (n) and internodes (i) are present in greater number among mature bulbils. Scale bars: A= 750 µm, B=1 mm, C= 1mm, D= 1mm, E= 1 mm.

Pollinator Exclusion

Mean bulbil number decreased with increasing number of open flowers at the

Highway 101 and K.C.I.C locations, and generally decreased with increasing mean number of seeds per flower. Though a tradeoff does exist between these alternate forms of reproduction, results suggest that bulbil formation is limited, but not restricted by seed production (Table 1).

Table 1. Mean number of fruits, number of bulbils, bulbil length and number of seeds per flower for treated L. terrestris at Highway 101 in Middleton Nova Scotia and at the Harriett Irving Botanical Gardens.

Highway 101 Harriett Irving Botanical Gardens

Number x̄ x̄ x̄ Bulbil x̄ x̄ x̄ x̄ Bulbil x̄ of Numbe Number Length Number Number Number Length Number Unbagged r of of of Seeds of Fruits of of Seeds Flowers Fruits Bulbils per Bulbils per Flower Flower

0 0.000 13.750 0.609 0.000 0.000 34.330 1.286 0.000

2 1.750 11.500 1.118 0.169 — — — —

6 5.000 8.500 1.084 0.935 — — — —

12 10.000 8.250 1.019 0.442 — — — —

All 28.250 7.000 0.422 3.237 11.000 20.250 1.397 0.932

Comparison between treatments with zero, two, six, twelve or all flowers unbagged indicated significant differences between number of fruits ( F4,15= 47, p <

0.001; Table 2). A Tukey-Kramer post hoc-comparison (Appendix C), indicated significant differences between treatments: zero versus all flowers uncovered, two versus all flowers uncovered, six versus all flowers uncovered and 12 versus all flowers uncovered for the number of fruits at Highway 101. A significant difference in means also

exists between the groups 12 flowers uncovered and 0 flowers uncovered (p < .01), and between the groups, 12 flowers uncovered and 2 flowers uncovered (p < .05). The differences in mean values between each of the other groups are not statistically significant.

Table 2. Summary: Mean number of fruits (Highway 101) (+/- SEM) (n= 4 for all treatments). Means followed by different letters were significantly different from one another (Tukey-Kramer, p < 0.05).

Treatment Mean (+/-) SEM

0 0.00a 0.000 2 1.75a 0.2500

6 5.00ab 0.4082 12 10.0b 0.9129

All 28.3c 3.568

Mean number of bulbils was significantly different between only the zero and all treatments at the Highway 101 site (Fdf1= 4, df2= 15= 4.50, p < 0.05 (Table 3). No other treatments exhibited significant difference in bulbil number.

Table 3. Summary: Mean number of bulbils (Highway 101) (+/- SEM) (n=4 for all treatments) Means followed by different letters were significantly different from one another (Tukey-Kramer, p < 0.05).

Treatment Mean (+/-) SEM 0 13.8a 2.323 2 11.5a 0.6455 6 8.5ab 0.6455 12 8.25b 0.7500 All 7.00c 1.291

Mean bulbil length was significantly different between the following treatment groups: two and versus flowers uncovered (p < 0.001), six versus all flowers uncovered

(p < 0.001), twelve versus all flowers uncovered (p < 0.001), and between two versus zero flowers uncovered (p < 0. 001), six and zero flowers uncovered (p < 0.001), and twelve and zero flowers uncovered (p < 0.001) (Tukey-Kramer, Appendix E) (Fdf1=4, df2=191= 4.50, p < 0.001) (Table 4)

Table 4. Summary: Mean bulbil length (cm) +/- SEM (Highway 101) Means followed by different letters were significantly different from one another (Tukey-Kramer, p < 0.05).

Treatment Count Mean (+/-) SEM 0 55 0.609a 0.05682 2 47 1.12a 0.07746 6 33 1.08ab 0.07659 12 33 1.02c 0.08180 All 28 0.422d 0.04052

Mean seed set was significantly different between treatments ((F=90.8 (df1=4, df2=610) ; p < 0.10)) (Table 5). Specific differences were found between treatment groups of all versus zero, all versus two, all versus six, all versus 12, 12 versus zero, 12 versus 2, and six versus zero flowers (p < 0.001) (Appendix E). A significant difference was found between the treatment groups having six versus two flowers uncovered

(p < 0.01).

Table 5. Summary: Mean number of seeds per flower (+/- SEM) (Highway 101). Means followed by different letters were significantly different from one another (Tukey- Kramer, p < 0.05).

Treatment Count Mean (+/-) SEM 0 124 0a 0.1415 2 124 0.169a 0.1415 6 108 0.935ab 0.1516 12 120 1.44c 0.1439 All 139 3.24d 0.1337

Mean number of bulbils was significantly different between zero (34.3) and all

(20.3 +/- SEM) treatments at the HIBG site (t (3) =5.67; p < 0.01). Mean bulbil length was not significantly different between zero (1.29) and all treatments (1.40 +/- SEM) at

HIBG (t(3)= -1.28; p= 0.204). A significant difference, however, was found to occur between zero and all treatment groups for mean number of seeds per flower. At the HIBG site, the mean values for number of seeds per flower were 0 and 0.972(+/- SEM), for zero and all treatments, respectively (t (3) =-6.95; p < 0.001).

Mean bulbil length was generally lower among L. terrestris at the Highway 101 site (Figure 15). Mean number of bulbils was negatively correlated with fruit production at both study sites (rHIBG= -0.7026) (rHighway 101= -0.6406) (Figure 18). A weak negative linear trend occurred between mean bulbil length and number of bulbils at the HIBG

(rHIBG= -0.3576). At the Highway 101 location, the relationship between bulbil length and number was weakly positive ( rHighway 101= 0.0990)(Figure 16). As fruit production was absent among plants with zero unbagged flowers, self- fertilization was not exhibited among L. terrestris. An increase in fruit production also occurred among plants with greater number of exposed flowers (Figure 17).

◊= Highway 101 ◊= Harriet Irving Botanical Gardens

Figure 14. Mean length of bulbils amongst treated Lysimachia terrestris at Middleton, NS, and the Harriet Irving Botanical Gardens in Wolfville, NS.

◊= Highway 101 ◊= Harriet Irving Botanical Gardens

Figure 15. Mean length of bulbils amongst Lysimachia terrestris having varied number of bulbils at the Highway 101 and the Harriet Irving Botanical Gardens (Error bars represent standard error of the mean).

◊= Highway 101 ◊= Harriet Irving Botanical Gardens

Figure 16. Total number of fruits from Lysimachia terrestris treated with pollinator exclusion bags (Error bars represent standard error of the mean).

◊= Highway 101 ◊= Harriet Irving Botanical Gardens

Figure 17. Number of bulbils developed by Lysimachia terrestris having varied number of fruits.

Discussions

Stages of Floral Morphological Development

The stages of floral morphological development in L. terrestris are similar to those of Maesa species. Like L. terrestris, species of the Maesa genus, belong to Myrsinaceae, and are distinguishable from other genera of by the development of a semi-inferior ovary, and the growth of many-seeded fruits (Caris et al., 2000). Though L. terrrestris seed set is limited, the species also bears an ovary which is semi-inferior. In the species, Maesa argentea, initiation of floral growth is characterized by the emergence of five outer sepal primordia, which fuse basally to form the calyx (Caris et al., 2000). This developmental stage is similarly observed in L. terrestris. In M. argentea, five common petal-stamen primordia are subsequently formed in the remaining portion of the floral receptacle. As in

L. terrestris, these common primordia later differentiate into the petals and stamens (Caris et al., 2000). Petal growth occurs an accelerated rate relative to the stamen primordia and results in the eventual enclosure of the interior floral parts. At this time, the anthers of M. argentea become progressively definitive in structure. A longitudinal dehiscence develops on each of the anthers (Caris et al., 2000). L. terrestris anther progression occurs in a similar manner. In a subsequent stage of M. argentea development, the gynoecium develops from a ring primordia, positioned centrally. Similar to L. terrestris, a conical placenta develops within the cavity of the gynoecium. Style elongation, also, similarly, occurs at the gynoecium apex and ends in a lobed stigma. Ovule primordia emerge from the placenta and become separated by outgrowth of placental tissue (Caris et al., 2000).

As the ovary of M. argentea matures, all floral parts other than the calyx, wither and fall.

Like L. terrestris, M. argentea flowers develop trichomes. However, trichome

development is limited to the pedicel, and is not present on the basal androecium or basal corolla as in L. terrestris (Caris et al., 2000).

Bulbil Developmental Stages

The ontogenic development of L.terrestris bulbils occurs in definitive stages. These bear some resemblance to the stage of growth observed in the bulberifous species

Remusatia vivipara (Pflanzenreich). Like, L. terrestris, R. vivipara can produce both sexual flowers and asexual bulbils (Huang and Hsieh, 2014). Bulbils of R. vivipara have hooked scales which develop similarly to L. terrestris bulbil propagules. In stage one of

R. vivipara bulbil growth, primordium elongation and thinning occurs. This bears resemblance to L. terrestris development in which gradual narrowing of the bulbil propagule occurs at its distal end. Subsequent scales are initiated at the inside of the first scale primordium and develop at an angle of 120 degrees from the previous scale primordium (Huang and Hsieh, 2014). L. terrestris bulbil protuberances resemble the scales of R. vivipara. Protuberances, which appear as foliar organs, also initiate in a sequential manner in which newly developing protuberance pairs occur at the bulbil node and develop perpendicular to the previously formed protuberance pair. Subsequent intermodal regions initiate in a similar sequence.

Pollinator Exclusion

Among the L. terrestris populations studied, it is evident that bulbil formation and seed production are alternate processes, and as investment in fruit production increases, there is a corresponding decline in bulbil yield. The allocation of resources to bulbil production was quantified through measurement of bulbil length and number of bulbils per plant. A

moderately-strong negative correlation occurred between number of bulbils and number of fruits at both the Harriet Irving Botanical Gardens and Highway 101 study sites. Fruit production was absent among plants with zero unbagged flowers, This indicates that self- fertilization was not exhibited among L. terrestris. An increase in fruit production also occurred among plants with greater number of exposed flowers (Figure 15).

Treated plants having “0” or “All” flowers uncovered had substantially reduced bulbil length relative to treated plants in the “2”, “6”, and “12” experimental groups. While it was hypothesized that leaving all flowers uncovered would decrease the apportionment of resources to the bulbil pathway through reduced bulbil length, a definitive explanation regarding the decrease in bulbil length under the “0 flowers unbagged” condition is unclear. It is possible that application of the pollinator exclusion bag resulted in plant physiological effects that were most apparent in the “0” condition. Damage caused to the stem of the inflorescence, sagging of the pollination bag and the propensity of the bag to collect or retain water may have contributed to reduced plant fitness. A reduction in available resources would result in a decrease in bulbil length.

Wang and Cronk (2003) indicate that inflorescence growth of other bulberifous species is regulated by the interaction of genetic and environmental cues. It is probable that investment in bulbil formation and asexual reproduction is less at the Middleton location as a result of the presence of specialist pollinator, M nuda. A lack of reliable pollinators at the Harriet Irving Botanical Gardens location may reduce the likelihood of pollination and subsequent seed set, thus favouring clonal propagation.

The forces that predominate in the maintenance of sexual reproduction are highly disagreed upon amongst biologists (Law, Cook and Manlove, 1983). The cause of variation between bulbil formation and flower number has yet to be rigorously identified.

One possibility for variation in these characters is that bulbil propagule and seed formation have differing ecological roles (Law et al, 1983). Abrahamson (1980) theorized that clonal expansion is favourable at low population densities. When population density is high, he suggested sexual propagation is most suitable for distribution to new sites (Abrahamson, 1980). This theory assumes that sexual propagules are more readily dispersed than those produced asexually (Law et. al, 1983). However, as

L. terrestris bulbils are highly buoyant, their ability to be distributed in wetland zones exceeds that of L. terrestris produced seeds. Genetic factors which contribute to the preservation of sexual reproduction among L. terrestris must, therefore, be considered.

Occasional seed set might be necessary for the maintenance of genetic variability (Wang and Cronk, 2003). As clonality reduces genetic variation among populations, some seed set and gene migration between and within populations is necessary for sustaining viable populations (Wang and Cronk, 2003).

An understanding of the reproductive development, conditions influencing bulbil yield, and the ecological roles of both asexual and sexual colonization in L. terrestris is significant in the maintenance of this plant species. The action of preserving and restoring L. terrestris strands is of importance, provided the loss of wetland regions to natural and anthropogenic- induced causes (Sheffield, 2011). A gradual diminishing of populations of L. terrestris has contributed to local diminution of E. pilosilus and its host bee, M. nuda, which requires L. terrestris floral oils for the development of nest provisions (Sheffield, 2011). This study provides insight regarding how asexual and sexual reproductive pathways might facilitate L. terrestris expansion under variable environmental conditions. Future directions for research may include an investigation of floral and bulbil development in other plants. Ontogenic observations may contribute to an improved understanding of asexual and sexual

reproductive strategies and the relationships between bulberifous species.

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Appendix A: Mean, standard deviation, standard error and raw data: Number of fruits and number of bulbils

Appendix B: Mean, standard deviation, standard error and raw data: Bulbil length and number of seeds per flower

Appendix C: Comparisons for all pairs using Tukey- Kramer HSD: Ordered Difference Report: Number of Fruits (Highway 101)

Level - Level Difference Std Err Dif Lower CL Upper CL p-Value All 0 28.3 2.35 21.0 35.5 <.001 All 2 26.5 2.35 19.2 33.8 <.001 All 6 23.3 2.35 16.0 30.5 <.001 All 12 18.3 2.35 11.0 25.5 <.001 12 0 10.0 2.35 2.75 17.3 <.01 12 2 8.25 2.35 0.997 15.5 <.05 12 6 5.00 2.35 -2.25 12.3 0.259 6 0 5.00 2.35 -2.25 12.3 0.259 6 2 3.25 2.35 -4.00 10.5 0.647 2 0 1.75 2.35 -5.50 9.00 0.942

Appendix D: Comparisons for all pairs using Tukey- Kramer HSD: Ordered Difference Report: Number of Bulbils (Highway 101)

Level -Level Difference Std Err Dif Lower CL Upper CL p-Value 0 All 6.75 1.84 1.07 12.43 p<0.05

0 12 5.50 1.84 -0.180 11.2 0.0600 0 6 5.25 1.84 -0.430 10.9 0.0768

2 All 4.50 1.84 -1.18 10.2 0.156

2 12 3.25 1.84 -2.43 8.93 0.426 2 6 3.00 1.84 -2.68 8.68 0.502

0 2 2.25 1.84 -3.43 7.93 0.739 6 All 1.50 1.84 -4.18 7.18 0.922 12 All 1.25 1.84 -4.43 6.93 0.958

6 12 0.250 1.84 -5.43 5.93 0.100

Appendix E: Comparisons for all pairs using Tukey- Kramer HSD: Ordered Difference Report: Bulbil length (Highway 101)

Level - Level Difference Std Err Dif Lower CL Upper CL p-Value 2 All 0.696 0.105 0.407 0.989 <.001 6 All 0.662 0.113 0.350 0.974 <.001 12 All 0.597 0.113 0.286 0.909 <.001 2 0 0.509 0.088 0.268 0.750 <.001 6 0 0.475 0.0970 0.208 0.742 <.001 12 0 0.410 0.0970 0.143 0.677 <.001

0 All 0.187 0.102 -0.0946 0.469 0.361 2 12 0.0991 0.100 -0.176 0.375 0.859 6 12 0.0648 0.108 -0.234 0.364 0.975 2 6 0.0343 0.100 -0.241 0.310 0.997

Appendix F: Comparisons for all pairs using Tukey- Kramer HSD: Ordered Difference Report: Mean number of seeds per flower (Highway 101)

Level - Level Difference Std Err Lower Upper CL p-Value Dif CL All 0 3.24 0.195 2.70 3.77 <.001 All 2 3.07 0.195 2.54 3.60 <.001 All 6 2.30 0.202 1.74 2.86 <.001 All 12 1.80 0.196 1.26 2.33 <.001 12 0 1.44 0.202 0.890 1.99 <.001 12 6 1.27 0.202 0.720 1.82 <.001 6 0 0.935 0.207 0.368 1.50 <.001 6 2 0.766 0.207 0.198 1.33 <.01 12 6 0.506 0.209 -0.0654 1.08 0.111 2 0 0.169 0.200 -0.378 0.717 0.916

Appendix G: ANOVA single factor analysis for Levene’s test

ANOVA single factor analysis for Levene’s test using group means: Number of fruits (Harriet Irving Botanical Gardens)

Source of Variation SS df MS F P-value F crit Between Groups 198.1 1 198.1 208.5 <0.001 6.608 Within Groups 4.75 5 0.95

Total 202.9 6

ANOVA single factor analysis for Levene’s test using group means: Number of bulbils (Harriet Irving Botanical Gardens)

Source of Variation SS df MS F P-value F crit Between Groups 268.9 1 268.86 23.12 <0.01 6.608 Within Groups 58.15 5 11.63

Total 327.0 6

ANOVA single factor analysis for Levene’s test using group means: Mean bulbil length (Harriet Irving Botanical Gardens)

Source of Variation SS df MS F P-value F crit Between Groups 0.7147 1 0.7147 6.536 <0.05 3.893 Within Groups 19.90 182 0.1093

Total 20.62 183

ANOVA single factor analysis for Levene’s test using group means: Mean number of seeds per flower (Harriet Irving Botanical Gardens)

Source of Variation SS df MS F P-value F crit Between Groups 7.771 1 7.771 249.62 <0.001 3.887 Within Groups 6.351 204 0.031

Total 14.12 205