RELATIVE CONTRIBUTION OF SEXUAL AND ASEXUAL REPRODUCTION IN LYSIMACHIA TERRESTRIS by 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 © Copyright by Victoria C. Brown, 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 ii 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 iii 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. iv 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 v 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 vi 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 vii 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 viii 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 ix 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. x 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 1 Plant stems in L. terrestris are glabrous, simple or branched and are 25-100 cm in length.