Exploring Reproduction in Wild Blue Lupine (Lupinus Perennis) in Comparison to L. Polyphyllus and L. Albus

Exploring reproduction in wild blue lupine (Lupinus perennis) in comparison to L. polyphyllus and L. albus

A thesis submitted to the committee of Graduate Studies in partial fulfillment of the requirements for the degree of Master of Science in the Faculty of Arts and Science

TRENT UNIVERSITY Peterborough, Ontario, Canada © Copyright by Heathyr E. Francis 2017 Environmental and Life Sciences M.Sc. Graduate Program September 2017

Abstract

Exploring reproduction in wild blue lupine (Lupinus perennis) in comparison to L. polyphyllus and L. albus Heathyr E. Francis

Wild lupine (Lupinus perennis) restoration efforts seek to increase and connect populations, using seeds, to facilitate the recovery of endangered butterflys in Ontario.

This study observed plant growth and phytohormone levels of L. albus, L. polyphyllus, and L. perennis through stages of seed development, each with varying strategies in growth and reproductive investment. L. polyphyllus is similar to L. perennis in morphology, acting as similar comparable with L. albus, a well-studied annual, as an outgroup comparator.

Wild lupines showed a lack of sexual reproductive effort as they did not put as much effort into above ground growth, and few in the population reproduces. They also showed cis-zeatin, a weaker cytokinin, throughout development and had higher amounts of abscisic acid at the end of seed maturity, impacting their ability to develop and germinate. These factors contribute to why wild lupines are difficult to restore using seeds, limiting expansion and challenging restoration.

Keywords: Wild blue lupine, L. perennis, L. albus, L. polyphyllus, physiology, cytokinins, abscisic acid, seed development, sexual reproduction, phenology, restoration, two-eyed seeing

Supervisor: Dr. R.J. Neil Emery

Committee: Dr. Eric Sager, Dr. Marcel Dorkin

Acknowledgements

g’chi miigwetch nii’kanaagnanaa

To express my deepest appreciation and gratitude for the opportunities within, surrounding, and extending from my thesis, I use these Anishinaabemowin words as they maintain their value and context beyond what I could explain. This language is a reflection and extension of living with the land, from the long-standing relationship with it and all of creation. It also serves to remind us whose land we are on and who holds that knowledge on all levels. From what I’ve come to understand, part of the (bio- cultural) restoration efforts is to maintain the language and the land (124, 69). I want to give acknowledgement of that and express my appreciation for the opportunities myself and my settler ancestors have been afforded on Turtle Island, and to specifically acknowledge that this research took place in Alderville First Nation within the traditional territory of the Mississauga Anishinaabe, locally within the Williams Treaty 20, 1923. I deeply appreciate the opportunity to come to know more about my place on this land through my lived, academic experience. Shawn Wilson, author of Research is Ceremony: Indigenous Research Methods says "If research doesn't change you as a person, then you aren't doing it right." (132). I am proud to say that it has for me. It has been through my work with TRACKS Youth Program and the Elders, traditional teachers, and those who have different perspectives and hold deep knowledge about all of creation and the land, that I have had the opportunity to explore this. From that, it reframes the research from being about the wild blue lupines to learning and knowing from the lupines and the context in which they grow (82). The journey has been challenging and I have strayed from the path, and am thankful for the many experiences and people that have brought me back to it. Within that challenge, I have come to know more about myself and question my thinking and understanding of things within, surrounding, and extending from this experience and research, which has been rewarding. And I hope that I will continue to be challenged for I have the experience, the family, and the friends, to support me in my path. thank you - all of my relations

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Table of Contents

ABSTRACT ...... II ACKNOWLEDGEMENTS ...... III TABLE OF CONTENTS ...... IV LIST OF FIGURES & TABLES ...... VII SUMMARY OF ABBREVIATIONS ...... VIII CHAPTER 1: INTRODUCTION TO WILD BLUE LUPINES (LUPINUS PERENNIS), L. POLYPHYLLUS, L. ALBUS AND PHYTOHORMONES, CYTOKININS AND ABSCISIC ACID...... 1 SAVANNA ECOSYSTEMS ...... 1 ALDERVILLE FIRST NATION’S BLACK OAK SAVANNA ...... 2 WILD LUPINE ECOLOGY AND RESEARCH ...... 5 LUPINE SPECIES COMPARISONS AS A MODEL TO UNDERSTAND PHYSIOLOGICAL LIMITATIONS ...... 10 LUPINE PHYTOHORMONES ...... 13 Cytokinins (CK) ...... 14 Abscisic Acid (ABA) ...... 15 FRAMING CURRENT RESEARCH ...... 16 CHAPTER 2: CONTRASTING INVESTMENTS IN VEGETATIVE GROWTH AND REPRODUCTION AMONG THREE LUPINE SPECIES WHEN PLANTED AT THE ALDERVILLE BLACK OAK SAVANNA. 20 INTRODUCTION...... 20 LUPINE SPECIES ...... 21 MEASURING GROWTH – HARVEST INDEX ALTERNATIVE ...... 22 MEASURING REPRODUCTIVE POTENTIAL AND OUTPUT ...... 23 RESEARCH FRAMEWORK ...... 24 METHODS ...... 27 FIELD PLOTS ...... 27 ALDERVILLE SITE ...... 28 PLANT GROWTH, VIGOR AND REPRODUCTION...... 29 DATA INTERPRETATION ...... 30 RESULTS...... 32 PLANT GROWTH: SHOOT HEIGHTS AND NUMBER OF LEAVES ...... 32 COMBINED GROWTH FACTOR ANALYSIS ...... 32 EXTENDED GROWTH OBSERVATIONS ...... 33 L. albus ...... 33 L. polyphyllus ...... 33 L. perennis ...... 33 REPRODUCTIVE GROWTH ...... 34 Rates of reproduction (population level fecundity) ...... 34 Number of flowers, pods, and seeds (plant level fecundity) ...... 34 SUMMARY OF PHENOLOGY RESULTS ...... 35

DISCUSSION ...... 38 VEGETATIVE GROWTH ...... 38 REPRODUCTIVE EFFORT ...... 40 RATES OF REPRODUCTION (POPULATION LEVEL FECUNDITY) ...... 41 NUMBER OF FLOWERS, PODS, AND SEEDS PER PLANT (PLANT LEVEL FECUNDITY) ...... 42 GROWTH AND REPRODUCTION ...... 47 CONCLUDING REMARKS ...... 49 CHAPTER 3: EXAMINING CYTOKININS AND ABSCISIC ACID DURING SEED DEVELOPMENT AMONG THREE LUPINE SPECIES WHEN PLANTED AT A ALDERVILLE BLACK OAK SAVANNA. .... 51 INTRODUCTION...... 51 PHYTOHORMONES ...... 51 SEED DEVELOPMENT ...... 52 ABSCISIC ACID ...... 53 CYTOKININS ...... 54 RESEARCH FRAMEWORK ...... 57 METHODS ...... 61 FIELD PLOTS ...... 61 SEED DEVELOPMENT STAGES ...... 62 HORMONE EXTRACTION ...... 62 HORMONE DETECTION AND ANALYSIS ...... 63 DATA INTERPRETATION...... 64 RESULTS...... 65 ABA ...... 65 TOTAL CK ...... 65 CK TYPES ...... 66 L. albus (CW) ...... 66 L. polyphullus (GL) ...... 67 L. perennis (WB) ...... 68 DISCUSSION ...... 73 ABA ...... 73 ABA in CW ...... 74 ABA in WB and GL ...... 74 OTHER ABA CONSIDERATIONS ...... 75 TOTAL CKS ...... 76 NT-CKs ...... 77 FB- CKs ...... 79 RS-CKs ...... 80 cis vs. trans-Zeatin ...... 81 OTHER CK CONSIDERATIONS ...... 82 Ratios of ABA: Total CK ...... 83 Ratio of ABA: FB-CKs...... 84 CONCLUSIONS ...... 84

CHAPTER 4 – CONCLUSIONS AND RESTORATION RECOMMENDATIONS ...... 86 CONCLUSION ...... 86 RESTORATION RECOMMENDATIONS ...... 87 REFERENCES ...... 91 APPENDIX: ADDITIONAL INFORMATION FOR EACH CHAPTER ...... 102 CHAPTER 2: ...... 102 EXTENDED METHODS ...... 103 CHAPTER 3: ...... 110 EXTENDED METHODS ...... 110 Hormone extraction ...... 110 HPLC-MS/MS ...... 112 Data interpretation ...... 113

List of Figures & Tables

Figure 1. Site map for the Alderville Black Oak Savanna 44°10'29.0"N 78°05'12.1"W __ 4 Figure 2. Map of lupine study areas at the Alderville Black Oak Savanna ______29 Table 1. Growth and seed data for determining harvest index alternative for each lupine species ______37 Table 2. Sample population reproductive data for each of the lupine species and locations and years. ______37 Figure 3. CK synthesis and metabolism scheme adapted from Kamada-Nobusada & Sakakibara (2009) showing CK biosynthesis MVA and MEP pathways CK-types and some key enzymes. ______56 Table 3. Five stages of growth in lupine seeds, based on Dracup & Kirby (1996) _____ 62 Figure 4. Abscisic acid measured in pmol(gFWt)-1 for L. albus (CW), L. polyphyllus (GL), L. perennis (WB) for five stages of seed development – described in Table 1 (Dracup and Kirby 1996). ______70 Figure 5. The total accumulation of cytokinins in measured in pmol(gFWt)-1 for L. albus (CW), L. polyphyllus (GL), L. perennis (WB) for five stages of seed development – described in Table 1 (Dracup and Kirby 1996). ______71 Figure 6. CK levels for iP, tZ, DHZ and cZ type CKs for seed development stages for L. albus (CW), L. polyphyllus (GL), L. perennis (WB) for five stages of seed development in pmol(gFWt)-1 – described in Table 1 (Dracup and Kirby 1996). ______72 Figure 7. Distances between Alderville and Harwood lupine populations and power corridor ______Error! Bookmark not defined.

Appendix: Figure 8. Illustration of field plot design. Plots are 1m2 with 0.5m spacing inbetween. ______102 Figure 9. A. The average number of leaves per plant for each of the lupine species, L. albus (CW), L. polyphyllus (GL), L. perennis (WB) in 2009. ______104 Figure 10. A. The number of flowers per plant L. albus (CW), L. polyphyllus (GL), L. perennis (WB) in 2009 and over five years 2007-2011. ______105 Figure 11 A. The number of seeds per plant for L. albus (CW), L. polyphyllus (GL), L. perennis (WB) in 2009. ______106 Figure 12A. The average number of leaves per L. perennis for each population of lupine in the Black Oak Savanna in 2009. ______107 Figure 13 A. The number of flowers per plant in the wild lupine quadrates at ABOS for over five years, 2007-2011. ______108 Figure 14. Total and average precipitation for the Alderville Black Oak Savanna from 2007-2011 ______109 Table 4. ABA, total CK, and CK FB amounts (average ±SE) for each stage and for each lupine species as well as the ration of ABA: Total CK and ABA: CK-FBs ______114

Table 5. Summary of hypothesis, predictions and supporting results ______114

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Summary of abbreviations

Lupine Species

• WB – wild blue lupine, L. perennis

• GL – garden lupine, L. polyphyllus

• CW – crop (white) lupine, L. albus

KBB – Karner Blue butterfly

ABOS – Alderville Black Oak Savanna

FP – field plot

Phytohormones

• ABA – Abscisic Acid

CK – Cytokinins iP types tZ types DHZ types cZ types

NTs – nucleotides iPRMP tZRMP DHZRMP cZRMP Isopentyladenosine trans-Zeatin Dihydrozeatin cis-Zeatin phosphate riboside riboside riboside phosphate phosphate phosphate

RSs – ribosides iPR tZR DHZR cZR Isopentyladenosine trans-Zeatin Dihydrozeatin cis-Zeatin riboside riboside riboside

FBs – freebases iP tZ DHZ cZ Isopentenyladenine trans-Zeatin Dihydrozeatin cis-Zeatin

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Chapter 1: Introduction to wild blue lupines (Lupinus perennis), L. polyphyllus, L. albus and phytohormones, cytokinins and abscisic acid.

Savanna ecosystems

Savanna ecosystems depend on fire to be maintained and thereby prevent them from succeeding into a woodland or forest habitats (86, 44). Savannas are characterized by 10-30% tree cover which subsequently controls species composition (8, 88). They will succeed into a forest or woodland habitat within 20-40 years without the presence of fire (86). Traditionally, Indigenous people facilitated wild fires (Russel 1983, Kay

1994, William 2000 in 19) which provided a variety of benefits to all the entities, biotic and abiotic (124, 69). With increasing settlement and development over the past few centuries (91), there is only 0.02% of this habitat left on Turtle Island (North America), for areas that were once savanna (and tallgrass prairie) (86). In Ontario, less than 3% of this habitat remains, and it is highly fragmented (8, 88).

Savannas are a biologically diverse and imperiled habitat (91), and are the host to several rare species, many of which have declined to the point of endangerment and expiration or extinction. The Karner Blue butterfly (KBB) (Lycaeides melissa samuelis) is a prime example of this. KBB is not only a flagship and umbrella species for savannas, but it is also an indicator species for high quality oak savannas with large populations of wild blue lupine, Lupinus perennis (WB) (18). KBB is dependent on WB to complete its two life cycles per year, where the eggs are laid primarily on full sun to partly shaded leaves (44). The caterpillars then feed and pupate on them, depending upon a specific

2 ant species to tend the chrysalis (19). KBB is non-migratory and can only fly approximately one kilometer, accessing nectar sources as adult butterflies. They overwinter on lupine leaves in the form of eggs (58). For more information about KBB in

Ontario, Bernard et al summarizes many of the current and relevant papers (13). Due to their limited flight ability and overwintering needs, patches of lupines need to be substantial in size to allow for adequate diversity in the KBB population, with patch connectivity that buffers against widespread ecological events such as fire, that could destroy all lupine plant material (19, 44). This need to increase lupine patch quality and quality has driven much research in Ontario and the upper Midwestern US, some of which occurs locally in the Rice Lake Plains.

Alderville First Nation’s Black Oak Savanna

Alderville First Nation’s Black Oak Savanna (ABOS) has the largest population of wild lupines in Ontario (58) and is the most northern aspect of savanna and potential

KBB distribution (19). As KBB habitat is impacted by climate change and retreats from its southern distribution, it may need to migrate northern limits of its range (47) where

ABOS and the Rice Lake Plains may need to support it (19). Currently, the site is approximately 60 hectares of which 35.6% is tallgrass prairie, 29.6% savanna, 19.6% woodlands and 15.2% other (Figure 1) (87). The Black Oak Savanna is located on First

Nation Reserve land, protected by Band Council since 2000 (2) but has long been known and tended to by Indigenous people (124, 69).

This area is known as Pemedashkotayang in Anishinaabemowin, which in fewer words, coarsely translates to ‘Lake of the Burning Plains’ (2). The language, which is a reflection

3 of/ originates from the land, describes the place, speaking to the understanding and relationships with all entities and of the people who live with this land. It tells of the use of fire and how essential that is to the entities that are connected to that place (92).

The tools of Eurocentric science would later help many of us understand this further in terms of fire’s effects on biotic and abiotic components of this system. This name was documented in about 1700 when the Mississauga Anishinaabe observed the

Haudenosaunee burning the area to clear land for crops and settlement (2). By 1853, a grouping of Mississauga Anishinaabe were settled on the south sides of Rice Lake, migrating from a Methodist mission on Lake Ontario. Around this time, the Rice Lake

Plains underwent agriculture conversion by European settlers, where they eliminated prairie and savanna habitat thinking that this area would be productive farm land.

Successful farming was a challenge as the soil is predominantly sand and low in available water and nutrients. This remnant of prairie persisted from that time due to the respect, care, and reciprocity of the Mississauga Anishinaabe as they continue to foster a relationship with this land and share their knowledge about it, allowing others to share in this as well. This can be viewed as an act of reciprocity for the land, taking responsibility for the care-giving and respect that it requires (69).

To achieve pre-colonial/settled landscapes, Indigenous perspectives must be considered. As the original knowledge holders and caretakers of this land, their perceptions and understanding of the landscape are valuable as they have a multigenerational relationship with the land which we are trying to restore (52, 124, 69).

Figure 1. Site map for the Alderville Black Oak Savanna 44°10'29.0"N 78°05'12.1"W

This can be challenging due to the systematic repression and elimination of Indigenous

Knowledge post-contact and the government policies and societal constructs that have supported this for hundreds of years (52, 1). To move past this, relationships must be repaired, treaties honoured, and mutual respect and understanding adopted, on an individual, community, governmental, and societal levels (1, 9, 52). One approach is using Two-Eyed Seeing, as described by Bartlett, Marshall & Marshall (9). Albert

Marshall, a Mi’kma Elder, explains that it is a gift of multiple perspectives, whereby strengths of Indigenous Knowledge and the gifts of western science are used together for the benefit of all beings and future generations (9). While Indigenous Knowledge is not always readily available or shared, it is important to maintain a space for it within research (132). In this context, it means honouring the land from which all knowledge stems from, acknowledging the spirit of what is studied, and reciprocity of life (69). For this research, it means acknowledging the Indigenous relationships with the land, specifically the Mississauga Anishinaabe of Alderville and surrounding territories and their understanding of savanna ecosystems. This consideration and placeholder for

Indigenous knowledge which is not shared in this research will be met with the best available scientific approaches and by using those tools to help understand why wild lupines struggle to be restored at the ABOS site.

Wild lupine ecology and research

Understanding WB ecology and savanna ecosystem characteristics to increase lupine density, patch connectivity and other supports for the KBB has been the focus of the body of lupine research in Ontario and the upper Midwestern United States. In

6 addition to this, presence and abundance of wild blue lupine is such that it can be considered as indicator species for high quality savannas (127). While there are no written records of WB existing at the ABOS site, seedlings were planted on the site starting in 2001, which Harwood (Fig. 7) was the source for seed, with 2348 being planted in 2002. When Chan studied the site in 2002, none had flowered and it was unclear if they would establish (19). Planting of plugs and monitoring of the plants continued at the site, with varying degrees of effort over the following ten years. In

2013 Jarvis assessed the site as Chan did, using different methodology and he found

19403 stalks, demonstrating that not only had they survived, but had spread and were denser, largely due to fire management and additional planting (58). While Jarvis counted a total of 19403 stalks on site, this is not representative of the number of plants, where personal observation in 2009 would estimate that there were ~450 plants on site. Their assessments showed that ABOS had the lupine density, variety of available light levels (sun, shade distribution), available nectar plants, and tending ant species to support KBB introduction. Both studies concluded that sites in Ontario (High

Park in Toronto, St. Williams Conservation in Norfolk County, Karner Blue Sanctuary &

Pinery Provincial Park in Grand Bend) need patch expansion and connection to be viable for KBB reintroduction. These studies also compared the Ontario populations to populations in the upper Midwest and found them comparable in morphology but population sizes and densities in Ontario were much smaller.

Chan and Jarvis both assessed other savannas in Ontario with WB for these factors, building off Boyonoski’s preliminary research that occurred in Pinery Provincial

Park in 1990. This was the first major research paper concerning wild blue lupine in both Canada and the US and thoroughly reviews all wild lupine research prior to 1990, including Dunn & Gillet 1966 and Harper 1977. In order to increase population size and quality for potential KBB reintroduction, Boyonoski conducted a thorough investigation of seed germination, patterns of emergence, growth and survival for both natural and manipulated variables in the field and greenhouse in order to understand how to increase establishment of wild lupines. Boyonoski’s contributions have been implemented in many subsequent restorations efforts in both Ontario and the upper

United States as the need for lupine patch maintenance, and expansion is needed throughout the lupine’s range (16, 91, 58).

Greenfield was next to contribute to the body of wild lupine knowledge, examining environmental variables that also impacted KBB distribution and assessing areas that had established population of both species. They assessed tree-moderated variables such as light and soil pH and landscape level forest characteristics, highlighting the work of Haack 1993 and Dirig 1988. Grundel, Pavlovic and Sluzman furthered the discussion with two papers, one exploring habitat use by KBB as influenced by canopy cover (44) and how shading impacts wild lupine chemistry, specifically nitrogen and their growth rates and senescence (45). They explored the tradeoff between reduced lupine size and density with canopy cover but KBB’s need to oviposit on shaded plants

(44). Oviposition was assessed for various phenological stages of WB as well as canopy cover, water stress soil type and presence of tending ants (45). These studies provided much of the foundation for an expansion of work that began in early 2000.

Halpern’s investigation into the potential for adaptation of WB to climate change, primarily drought, provided insight into seed response in juvenile and adult plants. By examining germination, leaf growth, and establishment (including root structure and the timing of reproduction), Halpern determined that lupines have the ability to avoid drought through seeds. Adults are well equipped to tolerate it, with some genetic variability (depending on population size) to adapt over a longer time frame (47). Halpern also initiated the exploration into maternal resource manipulation of lupines to determine their effect on seeds and their success (48). Halpern found that there were adaptive benefits to producing seeds of multiple sizes as the environment they encounter can be variable but non-adaptive environmental conditions also can influence seed size. This research provided a good baseline of what is known about WB seed morphology.

Bowling Green State University subsequently made a huge contribution to what is known about wild lupine reproduction with the work of several graduate students.

Shi explored the effects of inbreeding, population size, and selfing (47, 48, 115). Shenk initiated research into floral mechanisms in response to pollinators, expanding the knowledge about the flowers and how they work (114) and Bernhardt furthered this by extending this to looking at pollinator response at a population level (14). St. Mary investigated the trade-offs and effectiveness of increasing lupine populations through seeding and producing transplants, and monitoring survival, growth, and reproduction for two years (120). Plenzler focused on natural seed establishment, and determined what environmental factors can be manipulated, that will contribute to success (97, 96).

Both found that number of leaves is a predictor of survival, which is something that

Halpern also identified.

Grundel and Pavlovic contributed two papers confirming and extending on the work done in Bowling Green with one paper focused on KBB and the other on lupine reintroduction (91, 43). One of the main restoration conclusions for KBB is managing

WB for thermal conditions and lupine patch connectivity, as this determines what parts of the patch the plants can access and utilize (43). For the reintroduction of wild lupine to a site, they assessed the effects of canopy cover, surrounding vegetation and leaf litter over nine years. They explored how canopy cover and leaf litter determine how likely they are to persist (91). Pfitsh and Williams also published in this time frame, examining the Frosted Elfin, another imperiled butterfly, for which its life cycle also depends on wild lupines, and it thus shares many characteristics of the KBB including the need for open canopy for success (93).

More recently, Bowling Green State University students have examined the effects of predators on seeds, specifically mice and alydids, which appear to have some preference for seed coat colour (63, 64, 117). Seed coat colour may play a part in the fate of seeds as it is a trait that interacts with the light intensity it may be subject to water and degree of nodulation; but, this is impacted by other factors as well (121).

Beyond Bowling Green, Reinhardt explored adaptive management of oak savannas using observational, experimental, and focal species approaches, finding that environmental and climatic variables are important in defining suitable habitat and making predictions for future models (103). In Ontario, Nicol assessed canopy gaps in

Pinery Provincial Park, where it was found that species richness increased with the occurrence of open canopy created through the reintroduction of prescribed burns (82).

This body of research has outlined many of the environmental and habitat variables that impact lupine germination, establishment, growth, and reproduction as well as some of the phenological aspects of lupines that contribute to these observations. This has aided in the conservation and continued restoration efforts of

WB throughout its range and continues to be explored by researchers and restoration sites. However, beyond some genetic work done by Shi, very little is known about wild lupines internal limitations and controls. There is thus a complete gap in understanding the physiology of lupines, which is what this thesis will directly address within the field, as those are the growing conditions in which these plants are subject to. It is assumed that any factors limiting wild lupines at ABOS will also impact GL and CW when grown onsite. It is also assumed within this research that lupines are primarily spread (in a restoration context) through seed. This is congruent with other research and restoration practice although it can be observed in situ that wild lupine branch laterally underground. Many close stems, in clumps, are connected below ground via root structure though this is rarely documented in the literature. It is also assumed that the research conducted at other sites and with other lupine species is relevant and can be used for comparison to the lupines grown on the Alderville Black Oak Savanna site.

Lupine species comparisons as a model to understand physiological limitations

Other lupine systems have been studied for decades and this includes wild and cultivated situations. For example, in a west coast model, the relationship between L.

11 sulphureus ssp. Kincaidii and Fender’s Blue butterfly has been investigated (113).

Interestingly, there is very little cross-referencing in the research although the system is very similar to the WB and KBB of Severns’ work (113) and is congruent with the findings previously highlighted. Looking to other similar species may yield insight into the WB and KBB model and may provide additional insight into the challenges of propagating

WB by making comparisons with another lupine species that is more easily established like the Washington lupine, native to the west coast but present in Ontario as an escaped and potentially invasive garden perennial, L. polyphyllus (3).

L. polyphyllus, locally known as a garden lupine (GL) and the wild blue lupine have nearly identical flowers, whereby the functions of the flower and pollen loading system are the same (50, 114). They are both outcrossing species, serviced by bees, in sequence from the bottom to the top of the floral raceme resulting in about only 15% of the flowers per raceme being serviced (50). The greatest difference between GL and

WB is that the banner petal of WB changes once it has been successfully pollinated whereas GL does not (50). The timing of life history events is also parallel (15) but their growth patterns, (root structure and branching) are slightly different as are their leaf shapes, sizes, textures, and colours (personal obs.). Their seed size and shape are also similar and seed coat ornamentation are comparable, although all WB seeds at ABOS are white (personal obs). WB research, primarily Halpern’s work (48), has been used to support understanding of GL seed size versus seed number trade-offs (118). GL is well studied in Europe as it is an invasive species growing and spreading along roadways, and reducing species richness (51, 125, 55, 54, 59, 118, 75, 102). Mowing is recommended

12 for its control (125) and it has potential for use as a green manure crop to increase nutrients in depleted soils (75). Both positive impacts, where GL’s presence can increase pollination success in native species (54, 55), and negative impacts on pollination (competition between flowers for pollinator visits) within native species have also been explored (102). These factors make it a good comparator for WB in this research.

The meticulously well-researched crop species, L. albus, or the white lupine,

(CW) has had its physiology and phenology studied and reviewed for many decades with reviews of the knowledge as early as 1979, as identified by Van Staden &Davey (126) for this species. The many and varied physiological (90) and phenological (27) descriptions are a rich resource for the study of lupines. It acts as a physiological model lupine species bringing a strong background and perspective as a resource toward examining why wild blue lupines struggle to reproduce on a physiological level. The drawbacks are that the growth patterns and timing for life history events are much different for CW as compared to GL and WB. CW is an annual with a tendency for competitive traits such as high germination rates that establish a few leaves before initiating a few branches from the main stem along with its primary floral raceme (12). It has indeterminate branching which means additional branches, leaves and floral racemes may continue to develop after the primary has set pod if resources are available; although, this creates internal competition of limited resources (131). The flowers are self-compatible, but struggle to self-pollinate based on flower morphology (131). They have been subject to generations of breeding manipulations to create other traits that are beneficial to yield and crop

13 tending such as low alkaloid seeds (sweet lupine), and dwarfness (101, 130). In addition to this optimization of yields and management of field stressors, protein and nutritional content (122) has also been explored, and has progressed now to integrated approaches that combine previously distinct areas of study, such as ecophysiology (12). CW has also been a pivotal species in the identification and qualification of understanding the role, transport and biosynthesis of plant hormones, including cytokinins (23, 126, 123, 29, 32) and abscisic acid (104, 20), which is a major focus of this thesis.

Lupine phytohormones

Water, nutrients, and photosynthates, are all externally influenced growth elements that are transported from various parts of the plant (the sources), to areas of need during growth and development (the sinks). Examining the internal mechanisms of this would add perspective to understanding how external factors impact on wild lupine seed production as it is balanced with vegetative growth and clonal reproduction.

Plant hormones regulate cellular activity within the plant, and are involved in growth and reproductive development as well as stress responses as environmental factors impose limitations. They also play a role in the signaling in sink/source dynamics (106).

The five main classic families of phytohormones, which are produced within cells in minute quantities, are: cytokinins, abscisic acid, gibberellins, ethylene, and auxin. While the amounts of phytohormones present are important to function, the ratios in which these hormones occur together are important as well as they can be synergistic, antagonistic, or balancing in their resulting action. Function and receptivity of tissue is dependent on stage of development as well as location or organ in the plant (56). They

14 work at extremely low concentrations to signal within the plant and can be transported in the xylem or phloem (57, 106). Relatively higher concentrations of a hormone can signal, or cause, a growing organ to become a stronger sink for a needed nutrient, drawing more resources from the source organ where they are produced (147). This is observed during sexual reproduction where the developing seeds require support from the rest of the plant to develop, and the signal strength for nutrients, water and photosynthate is influenced directly and indirectly by hormone levels (106). The two phytohormones of interest in this study are cytokinins (CK) and abscisic acid (ABA) as they play a vital role in seed development and diversion coordination of source/sink dynamics.

Cytokinins (CK)

CK is an instrumental signaling and regulating factor for cell growth and division.

Cellular division and growth, especially in developing seeds, require support from the rest of the plant, and the establishment of a strong nutrient sink, drawing on large amounts of sugar metabolites manufactured and transported from distant sources like young leaves (32, 4, 77, 29, 46, 38, 106). There are over 30 different types of CKs and they range in their activity levels in the plant both in time and location (57). CK types and amounts are controlled by synthesis (and coordinated transport) and release of conjugate forms, and degradation by cytokinin oxidase (CKX) (56, 57, 38, 5, 110). CKs also exist in different configurations or types as illustrated in the CK pathway (Figure 3), where variations in the isopentenyl side chain are significant as they determine function

(7) and are only active within specific receptors (4). The pathways to the creation of

15 these CK types and their level of activity are explored further in chapter 3. Within this, there are two major pathways in which CKs are created, resulting in either a trans-zeatin

(tZ-CKs) or cis-zeatin (cZ-CKs) isomers as the active types ((Figure 3). Their role in reproduction, specifically in seed filling and development, has been studied in several crop species and CW is known to be tZ dominant system (32); whereas preliminary work with GL shows that it is cZ dominant system (29). This may also fluctuate based on stage of development as cZ tends to increase during times of growth limiting conditions such as drought and seed development (57). It is important to understand which types are present in maturing seeds as they can have slightly different effects and will help in understanding what is occurring in WB seed development that might be limiting their reproduction.

Abscisic Acid (ABA)

ABA is present and active during seed development and its roles are: to control dormancy, fruit ripening (119), and seed filling (starch and protein accumulation) (36), and to limit cell proliferation (68) as it is detected by various receptors (71, 22).

Normally during seed development there are often two peaks in ABA accumulation. The first is thought to be produced by the maternal plant and the second produced by the seeds themselves, thereby regulating seed maturity, dormancy and signaling for protein storage accumulation (111). Beyond seed development, and more broadly in the plant,

ABA signals for water stress responses (133) and stomatal control (129) as well as a broad up-regulation of gene activity (101). Amounts present in developing seeds tend to be lower compared to the rest of the plant but this can increase six-fold in times of

16 drought stress (36). ABA synthesis is controlled by 41 genes, 19 of which are impacted by drought stress (36). ABA is synthesized in the plastids and converted in the cytosol though it can also be transported in its conjugated forms (36). Monitoring ABA abundance and comparing it among species of lupines is worthwhile as ABA impacts dormancy and Boyonoski (16) identified it as potential source of strong wild lupine seed dormancy, which is difficult to break and may limit reproductive success, especially in a restoration context. ABA concentration is a better predictor of dormancy in a seed than

CK though ABA is not strongly correlated to germination (39). The interaction between these two phytohormones is also important as they perform different but connected roles within developing seeds and the ratios of their occurrence are important to observe (130, 129, 46, 30). Sampling seeds during key stages of development and monitoring overall plant growth will aid in understanding how all of these factors interact and impact the seeds produced by each plant species.

Framing current research The guiding question for this research is to explore sexual reproductive effort of wild blue lupine in comparison to other lupine species to help understand how they reproduce and continue work on expanding their range and density. This is primarily done by using collected seed at restoration sites such are the Alderville Black Oak

Savanna. More can be learned about this species by viewing it from multiple perspectives and taking both qualitative and quantitative approaches that honours

Indigenous, personal, and Eurocentric scientific ways of knowing (69). By comparing L. perennis to two other lupine species with differing phenological traits and physiological

17 characteristics, I hope to gain these perspectives through observation () and using the two eyed-seeing approach (9).

The scientific tools I planned to use was systematically establishing a plot-based study in the field but also maintaining observation of WB in the established long term monitoring plots at ABOS. This is necessary, as the wild lupine establishment in the field plot which followed a replicated block design, did not flower despite best efforts to support this and were monitored and sampled in the long-term monitoring plots established at ABOS. This modification in design allowed for observing the amount of effort put into growth and reproduction for each lupine species (L. perennis, L. polyphyllus, L. albus), as they are exposed to what is assumed to be substantially equivalent environmental variables found within the ABOS site. Growth was determined by measuring the heights and number of leaves, flowers, and pods. Rates of reproduction determined by the percent of plants that flower and set pod will also be calculated and examined to determine the degree of effort put forth into sexual reproduction and the success of that effort. The potential underlying controls will be further explored by looking into the types and amounts of CKs and ABA found throughout seeds development. Through this, it is hoped that the multiple ways of approaching understanding about wild blue lupine, some perspectives may be gained into why they difficult to restore, as compared to the other lupine species, with the goal of increasing their density and range.

The first hypothesis is that wild lupines put minimal effort into above ground growth and sexual reproduction as compared to CW and GL in the field. This will be

18 tested by measuring shoot height, and the number of leaves, flowers, and pods they produce per shoot as well as number of shoots within the research plots that put effort into reproduction. It was predicted that CW would be lower in vegetative effort and high in reproductive effort as it is an annual crop species; whereas GL would put more even amounts of effort into each. GL is a perennial that is bred to have a presence in a garden, both in number of flowers (but not number of pods and seeds per plant), and vegetative coverage. WB was predicted to put much less effort into both, with fewer flowers, pods, and less vegetative growth. The rational for this hypothesis and predictions are elaborated upon in chapter 2 and a summary of the hypotheses can be found on the following page

A second hypothesis states that CW, GL, and WB have different CK and ABA profiles that reflect their different reproductive strategies and output and this will negatively impact WB lupine seed production. CK levels were expected to be high during the early stages of development and taper towards maturity, which a shift away from active types, for all species. It was predicted that CW will have the most abundant

CK and ABA levels and would be confirmed as tZ dominant; whereas GL would have fewer active CK present and would be cZ dominant. WB was predicted to be similar to

GL, being cZ controlled, but with a less potent CK activity profile (lower quantity and fewer active types). Finally, WB was predicted to have higher levels of ABA as they have strong dormancy and are so difficult to germinate. The effects of this as well as the rational for this hypothesis and predictions are elaborated upon in chapter 3 and a summary of the hypotheses can be found on the following page.

Summary of guiding question, hypothesis and predictions made in this research

Overarching • Why are wild lupines so difficult to spread by seed? question: What elements of their phenology and physiology contribute to this?

Phenology • Minimal effort into WB lupine reproduction are limit their sexual Hypothesis: reproduction, as compared to CW and GL in the field. Energy is not going into their growth either.

Methodology: • Measure growth using shoot height, the number of leaves. Measure reproduction effort by counting flowers, and pods. Count number of shoots that put effort into reproduction.

Predictions: CW: Minimal growth; high number of pods; all stems reproduce WB: Minimal growth; low number of pods; few stems reproduce

GL: Lots of vegetative growth; high number of pods; many stems reproduce

Physiology • WB CK and ABA abundance, types and timing are limiting their Hypothesis: sexual reproduction

Methodology: • Profile CK types and ABA through five stages of seed development

Predictions: CW: Low ABA at final stages; high amounts of CK, ABA; tZ-CKs WB: High ABA at final stages; low amounts of CK; cZ-CKs GL: Low ABA at final stages; low amounts of CK; cZ-CKs

Chapter 2: Contrasting investments in vegetative growth and reproduction among three lupine species when planted at the Alderville Black Oak Savanna. Introduction The Alderville Black Oak Savanna (ABOS), like many other restoration sites in

Ontario and the upper Midwestern United States, seek to increase wild blue lupine (WB) density and patch connectivity for Karner Blue Butterfly species conservation. Butterfly reintroduction can be considered by increasing the area that they might occupy (16, 19,

18, 58) and to establish populations in spatially separate but connected areas to guard against wild fires and other disturbances (42). Many of the environmental and habitat variables to support and understand how to expand wild lupine populations have been explored in Ontario and the upper Midwest (16, 18, 47, 48, 42, 93, 44, 45, 91) as well as factors affecting the species establishment (16) via seeds, including seed coat (121) and seedling recruitment (97, 96, 120). Particular attention has been paid to inbreeding

(115), population size (78), and self-pollination (116) including pollinator visitation (114,

14, 85) and seed predators like mice (63, 64) and alydids (121). Many of these studies include growth monitoring, (primarily tallying number of leaves) and reproduction assessment by counting flowers, pods, and seeds. No other study has compared wild lupine growth parameters to those of other lupine species with varying growth strategies as a means to assess how other lupines grow and reproduce in situ. This knowledge will be helpful in determining methods to assist in lupine patch maintenance and restoration.

Lupine species Lupinus polyphyllus, considered locally to be a garden lupine (GL), was chosen as

WB’s closest comparable species for its reproductive success, ease of growth and likelihood to thrive at this location. It is possible that local gardens could contain this species and there may be a possibility of interspecies cross pollination, (which, while not in the scope of this research, would be worthy of further study). The variety chosen was a dwarf garden cultivar, bred to be showy with its robust floral display and cut flower arrangement potential. Its hardy nature and quick growth were also considerations for planting onsite.

GL is native to the west coast North America although it can be found growing around the world (3, 54). It is a model species as there are many varieties that offer insight into breeding, pollination (55, 50, 54), acclimatization and invasive tendencies

(59, 125, 51, 102) and various other agricultural (75) and ecological points of study (3,

118, 35). It is a species of interest, with some research into the seed size, number, and recruitment as a means of understanding its abundance and interspecies dynamics

(Jakobsson & Erksson 2000 in 3, 118).

Lupinus albus, a crop lupine, often referred to as the white lupine (CW), was selected as an “outgroup” comparison, as it is an annual crop species, with lower alkaloid content and a different (indeterminate) growth pattern (53). The flowers are self-compatible (131) with large (100-1000mg) seeds (53) that can grow under a wide variety of environmental conditions, including frost, making it a suitable candidate, in some respects, for comparison. CW is also a well-studied crop species (i.e. since the

1970’s (126)), with research that examines its reproductive effort, nutrition and growth

22 and survivorship in the field (66, 53, 17, 11, 27, 122, 4, 12). While greenhouse germinating is not needed for these or the other two species, they were all started in the greenhouse to get them through the initial challenging steps of establishment while receiving supplemental water and nutrients (113). To compare the success of these two introduced species of lupine and their establishment on the site in a plot-based experiment along with WB, measures of their vegetative and reproductive effort were conducted. While there is no research into GL or CW’s optimal or in situ growing condition for Ontario or the upper Midwest, it is assumed that there is some robustness in its growing conditions given it is a common garden (GL) and crop (CW) species. Given the failure of the WB to flower in the replicated blocks during the monitoring year, they were monitored and sampled in the established plots within the savanna.

Measuring growth – harvest index alternative Harvest index is a common methodology for determining reproductive effort as a trade-off against vegetative growth in crops by measuring above ground biomass once seeds are harvested (49). Harvest indices can capture genetic, biotic, and abiotic factors, but it is hard to tease apart each of these effects (67). The destructive measurement of an actual harvest index is not ideal in a restoration setting, where minimal collection and disturbance is required by ABOS (16). Furthermore, WB leaves continue to be green and present in the savanna ecosystem until the late summer or fall, playing various ecological roles (44, 93, 43). Studies also show that the more leaves establishing plants have (91, 120, 16), the greater chance of survival and reproduction of the next year (97, 96, 48). Because of this, many studies use leaf tallies (97, 96) and leaf area (120) to determine size (16). This study used both leaf tallies and heights to

23 measure growth. Halpern has even used leaf number, as it is correlated to above ground biomass (r=0.86, p<0.0001, n=308), as a proxy for reproductive fitness for the next year (47, 48) and later work (97, 96, 120) supports this. Along with growth, reproductive potential is an important component of population health (16).

Measuring reproductive potential and output One of the most common measures of lupine reproduction is counting the number of flowers and pods per raceme produced by the plant. Counting racemes per plant is also important but challenging as WB branches underground, making it hard to determine how many racemes are produced by each genet. Because of this, within most WB studies, there is a reporting assumption that one raceme is counted as one plant even though many racemes may be connected to the same genet below ground

(25, 44), and one genet may actually have 60 racemes (116). This study will continue to report in this style but acknowledge that some racemes, were sampled in close proximity (the lupine monitoring quadrates at ABOS) may be from the same genetic individuals, through connected underground ramets. Where feasible, racemes from distinct clumps greater than 2m apart (47) were sampled to ensure as many individuals were sampled as possible.

Monitoring floral output is valuable as it shows which plants are attempting to potentially reproduce sexually and the amount of effort devoted to support this as they are pollinated, and the successfully fertilized flowers turn into pods. Flowering effort is also an indicator of current environmental conditions (16) and the genetic diversity, although the results of inbreeding depression manifest at high abortion rates within the pods (115). Population size and density contribute to this, where 125-800 flowering

24 plants is considered small, 1000-3000 large (14). While ABOS has a total of 19,403 stems (58), the individual plant number is in the 400-500 range (personal obs.). The size and duration of the floral display is also important in attracting bees to pollinate flowers

(55, 54) and once in the patch, they are directed visit specific WB flowers by the colour of the banner pedal (114). Since WB (and GL, 3) are largely outcrossing species (115), floral display is a worthy investment for lupines attracting pollinators (14, 55), and when the flowers become pods, successful pollination can be estimated with pod counts (16,

47). Counting pods is a non-invasive way to get a closer estimation of reproductive potential (97), with 5-6 ovules expected per WB pod (115). Seed counts can help refine this estimate even further but all per plant measures (like number of flowers, pods or seeds) should be examined in the context of the population as not every plant puts effort into reproduction. The percent of flowering plants can be used as an indicator for current environmental condition where low flowering rates indicate a regressive population with limited genetic flow (16).

Research framework It is challenging to tease apart ecological impacts when looking at reproduction on a per plant basis by exploring where plants are investing their effort into growth or reproduction (16). Annual lupines, like CW, tend to put far more of their available energy and resources into reproduction and seeds than roots (95). Herbaceous lupines put more energy and resources into root development and only a small portion into seed development (95, 14). Specifically, for wild lupine, micro habitat variations like shading by canopy cover (45) can impact reproduction, as can pollination (42) and growth (91) on a population scale. Plezler (97, 96) examined this as did Halpern (47, 48)

25 and Greenfield (42) where additional factors like fire, leaf litter (97, 96), changing weather patterns (water and temperature) (47, 48) as well as light and soil pH (42) varied throughout a WB lupine patch. Their work focused more on patch wide assessment, knowing that microclimates can vary, which is both beneficial to the WB lupine population as well as the other species it supports, with the goal of maintaining or increasing density and patch size. Rarely have any of these studies considered the physiology of the plants and, specifically, phytohormones have never been studied in this system. Some studies (16, 47, 48, 116, 94) mention energy or resource balance within the plants in supporting both growth, reproduction, and other various forms of stress. This supports taking a dual approach to assessing growth and reproduction balance within the plant via physiological assessment (chapter 3), at the individual plant level and also the population as a whole. Comparing the contrasting investments of vegetative and reproductive growth in this chapter will be built on by the physiological investigations in chapter 3, where hormonal measurements can elucidate the observed differences in growth and reproduction. By comparing this to other lupine species, examining the similarities and differences could yield insight about acclimatization of wild lupine populations in the savanna habitat that need continued restoration efforts.

The hypothesis tested was that wild lupines put minimal effort into sexual reproduction and growth as compared to the garden and crop lupines grown in the field.. This will be reflected in the height of the plants, and the number of leaves, flowers, and pods they produce as well as reproduction output.

It was expected that the L. polyphyllus, a dwarf variety of garden lupine, would be of moderate height, although other varieties of this species can be much more variable in height. It would also be bushy (highly branched) with many leaves and produce many flowering heads but relatively few pods (and therefore seeds). It was predicted that not all GL would flower, set pod or produce seed as there would be tradeoffs to balance reproductive output, vegetative growth, and root establishment.

It is expected that L. albus, a cultivated crop species, will have minimal leaves and produce many flowers that will consistently set pod and develop seed as it invests more of its available energy and resources into reproduction. The height of this species should be fairly standardized for tall cropping requirements, although this would also depend on environmental variables. It was expected that nearly all of the CW plants would attempt to reproduce, with many flowers, many pods and consistent number of seeds per plant. Reproductive output will be high and its vegetative growth lower than the others.

L. perennis was expected to be more variable in height as it is most often found growing amongst other savanna species, which all compete for the same limited resources. It was expected that, compared to the other two species, it would produce a moderate amount of leaves and a low number of flowers with fewer pods as it will likely invest much of its growth and resources into developing its large underground taproot system. Less than half of the WB plants observed were expected to flower, set pod or produce seed. It should have low reproductive output and persistent vegetative growth, with a fair portion of the latter being underground.

Methods Field plots In order to further examine the reproduction of WB at the Alderville Black Oak

Savanna site, two other lupine species were selected, planted on site, and exposed to similar environmental variables. The field plot location was in an abandoned field that was actively being restored to native vegetation conditions, close to a natural windbreak with some surrounding young oak trees and various tallgrass plants. The area was cleared of vegetation using herbicides, hand and mechanical removal with the use of a harrow, during the fall of 2008 to prepare for the 2009 field season (48). The plots consisted of 18, 1 m2 plots with 0.5m spacing in between, as illustrated in Figure 8 in the appendix.

Four WB and four GL plugs were planted in each plot in fall 2008 so that they would be established in 2009 for measurement and comparison with CW. Four CW plugs were planted in each plot in the late spring of 2009 as they are an annual species that could not have survived the winter at the site (3). In total, 72 individuals of each lupines species were planted, at a density of 12 per 1m2. All plugs were germinated and grown by Cathy Loukes, a local greenhouse operator who is very familiar with germinating lupines – which can be a difficult task as they have very low germination rates. This approach also leads to better survival following transplanting to the field

(120). Given the failure of the WB to flower in the replicated blocks during the monitoring year, they were monitored and sampled in the established plots within the savanna.

Alderville site Wild blue lupine were first established on the Alderville Black Oak Savanna in

2001. Thousands of plugs were planted within the Bowl and Hog’s Back at the site from seeds collected from a nearby natural population in Harwood, Ontario (19) (Figure 7).

Monitoring plots were established for each of the sub sections of the Bowl and the

Hog’s Back (Figure 2), each containing five 1m2 plots, totaling twenty quadrats (21). For more information about the plots and monitoring protocols used in these plots, see the appendix. These quadrats have been used in various research projects over the years that the lupine population has been re-establishing (although not Chan’s 2004 or Jarvis’s

2014 work at the site). Five year monitoring data helps frame the 2009 field season where GL and CW were planted on site in the field plots previously mentioned by establishing the context of the wild lupines on the site.

Figure 2. Map of lupine study areas at the Alderville Black Oak Savanna

Plant growth, vigor and reproduction All plants were monitored during the 2009 field season for establishment, growth, and reproduction using Dracup and Kirby’s guide to lupines (1996). Density was measured by counting stems (16 19), though it is known that one genet may produce multiple shoots, in close proximity to each other, via an underground root structures.

Keeping with the same protocols as the Alderville Black Oak Savanna (as opposed to the transect methods used by Chan (19) and Jarvis (58), two other wild lupine surveys done at ABOS), heights were measured during the flowering period, as no stem elongation occurs after flowering for GL and WB (27, 16). Lupine size as measured by diameter

(15), while used in the past at ABOS, was not used as the density of some clusters

30 overlaps and entirely fills each quadrat. St. Mary (120) and many others (96, 97) use the number of leaves as a measurement for size as it is non-destructive (better than measuring heights (97)). Potential fecundity was measured as the number of flowers and number of pods produced by each plant (16, 120, 14, 114). The number of plants that flowered and set pod were also noted and used to calculate reproduction rates (16,

11). These plants were sampled in parallel, throughout seed development, for hormone analysis, as described in chapter 3.

Seeds were collected and counted to further understand potential fecundity and seed development success (120, 11, 48, 112, 116, 115, 48, 14, 114). Collection from WB and GL required bagging of nearly developed pods so that they would not naturally dehisce and scatter (120). This was not needed for CW pods as they do not shatter, and remain attached to the plant. These seeds were counted and measured providing additional reproductive data and used for phytohormone sampling (germination experiments and restoration practices).

Once the 2009 field season was complete, the CW plants were removed from the plot. GL was allowed to over-winter in the plots and survivorship in the following field season (2010) was determined and then the plants were removed from the site. The

WB plants were left in the plots, the fence removed and the area was planted with additional species plug in an effort to restore the area to savanna.

Data Interpretation Measurements for growth and reproduction were taken several times over the summer and pooled at the end of the field season and the averages, standard deviation

(SD), standard error of the mean (SEM) calculated. The combined vegetative growth

31 factor multiplies the number of leaves per plant with its average height to create one measurement that can be compared among lupine species to evaluate the amount of effort put in to above ground vegetative growth (120, 16). The percent rate of pod set was calculated by dividing the number of flowers by the number of pods each plant produced. Reproduction effort was further explored by looking beyond individual racemes to determine the percent of flowers that flowered within the quadrats and field plots and how many of those plants that flowered continued to support pod development. This value was used to determine the species with the highest potential reproductive output (114, 120). An approximation to harvest index was calculated for each of the three lupine species to facilitate comparisons and understanding to how resources are allocated between vegetative and reproductive growth. Number of seeds per plant was divided by the above ground biomass where a proxy growth factor was generated by multiplying by number of leaves (Table 1).

Results Plant growth: shoot heights and number of leaves With respect to shoot heights and number of leaves for all lupine species observed, WB, on average was tallest and most variable, with the greatest range of heights (Table 1). Interestingly, the number of leaves on the plants that did not flower in the field plot also had the same number of leaves as those that did flower in the Bowl as illustrated in (Figure 9 in the appendix). The number of leaves per WB plant did not significantly vary across the four ABOS lupine monitoring quadrates but height did vary slightly (Figure 12 in the appendix). CW was taller than GL but with much smaller range of heights than observed in WB and GL and had a moderate amount of leaves (more than WB and fewer than GL). Overall the growth of this lupine species was strikingly different than other lupines studied, which is discussed in further detail below. GL, tended to be the shortest and had the fewest leaves (Table 1).

Combined growth factor analysis WB put the most effort into height when compared to the other species and GL put most effort into producing leaves, and both had a combined vegetative growth factor (height X leaves) of around 295 (Table 1). CW was balanced in height to leaf production and further differed from GL and WB by having a combined growth factor of

500 (Table 1). Based on observations in the field, GL and WB also put effort into establishing root structures that CW did not. While this measurement is helpful in comparing the growth of each lupine species, it needs to be considered in light of the energy each plant put forth into reproduction and how many of the plants within the sample reproduced.

Extended growth observations L. albus CW branched at the top of the stem as flowers bloomed, were pollinated and developed into pods. After the initial raceme began to develop pods, branching occurred and more tertiary floral racemes formed. The plant continued to put forth leaves and grow in overall height and bushiness (degree of branching) throughout seed growth and development. This appeared to continue until favorable growth conditions ended and the plant reached some environmental limit. These stalks were fairly woody throughout development, which may limit branching to upper stalks where the stem is still green. This type of growth was much different than the other lupine species studied as it is shows traits of indeterminate growth.

L. polyphyllus These plants had a thick tuberous root, just barely under the soil which is where most of the branching occurred and from which new shoots normally emerge in the spring. These lupines tended to be bushy, and develop branches that contained additional flowering racemes but more often contribute additional leaves without flowering. Stems remained fairly pliable throughout early pod development and continued to flower if the original raceme was un-pollinated or removed/lost.

L. perennis Stems remained fairly pliable throughout pod development but as the pods approached harvest maturity, the stalks became woody. Leaves were maintained throughout much of the summer but the upper stem that held the flowers and shattered pods dried out and became brittle. These plants rarely made a second attempt to flower even if the raceme was removed early in development. No attempt

34 to flower again was observed if pods in any stage of development were removed or destroyed.

Reproductive Growth Rates of reproduction (population level fecundity) None of the WB plants in the field plot flowered in 2009, so all floral measurements had to come from the established plots in the bowl at ABOS. In WB, only one quarter of the plants observed flowered and 47% of those plants that flowered, produced pods. In total, only 12% of the WB population of plants monitored produced pods which is comparable to the 14% observed in 116, which increased with additional resources and 13% found in 114 (Table 2). In CW, nearly all plants that established flowers (92%) also produced pods (89%) showing a 97% commitment of plants to maintaining and developing their pods (Table 2). In GL, approximately half (47%) of the plants studied produced flowers and 81% of those plants that flowered turned into pods. This shows a strong investment into sexual reproduction and developing fruits containing seeds, resulting in 34% of the plants having produced pods.

Number of flowers, pods, and seeds (plant level fecundity) Table 2 shows the average number and range of flowers produced and the number of pods produced by each lupine species studied. The WB Flowers in the field plot did not flower, flowering data came entirely from lupine monitoring quadrates at the ABOS. A key point to note from Table 2 (and explored further in Figure 10 in the appendix) was that the number of flowers observed in the 2009 field season is significantly (p<0.05) higher than the five-year average for WB although the number of pods produced per plant was similar. Overall, Figure 10 illustrates that each of the

35 lupine species produced many flowers but a minority developed into pods, especially

WB. In effect, approximately 30% of the flowers that CW produced were pollinated and grew into pods, which is quite similar to GL as approximately 32% of its flowers developed into pods. This was much different from WB, for which only 18% of its flowers developed into pods during the summer of 2009, compared to the 25% observed on average over five years of study (Table 1). This suggests that many of the flowers produced are meant to act as a floral display but WB continues to put less effort in to reproduction than the other lupine species monitored and grown at the Alderville

Black Oak Savanna.

Table 2 shows the average number and range of seeds produced per plant and the same measurement expressed as per pod for each the lupine species studied. CW was consistent in the number of seeds produced per plant and per pod although there were outliers. GL was highly varied in the number of seeds produced per plant and per pod. While it produced many pods, seed numbers on average were very low per pod.

WB had a comparable number of seeds per pod and per plant as did CW. In general, all lupines generated many flowers, a minority of which turned into pods and the number of seeds per pod was less than five, with consistently high abortion rates per pod.

Summary of phenology results With CW, an annual, each plant put forth great effort to produce seeds each year, as demonstrated by the increased number of flowers and pods and a moderate amount of large seeds produced as well as number of plants that put forth this effort.

They also put more effort into above ground vegetative growth than GL and WB. Many

GL and WB shoots do not flower or produce pods, though GL produced many more

36 flowers, pods, and seeds per plant than WB. Both of these plants put less into above ground vegetation than CW, but both species put effort into root structure, and likely

WB put much more effort into establishing deep tap roots to ensure seedling success.

Once a shoot invested in pods, WB showed comparable success in number of seeds per pod and number of seeds per plant to that of the CL.

Table 1. Growth and seed data for determining harvest index alternative for each lupine species Average Average # of Combined Seeds per Seeds per Seeds: Growth Seeds: Lupine type Height ± SE Leaves ± SE growth Seed n= plant ± SE pod ± SE combined n= leaves (range) (range) factor (range) (range) growth factor 23.4 ± 1.3 21.4 ± 2.07 13.63 ± 1.76 3.43 ± 0.33 L. albus, CW 38 (19-28) (13-28) 500.8 34 (4-19) (2.5-4) 0.6369 0.0272 6.9 ± 1.06 44.3 ± 3.66 58.08 ± 11.91 1.8 ± 0.2 L. polyphyllus, GL 60 (1-11) (19-59) 305.7 22 (13.5-77) (0.67-2.85) 1.3111 0.1902 L. perennis, WB 27.4 ± 0.44 10.5 ± 0.29 24.86 ± 2.29 2.9 ± 0.2 (2009, ABOS) 404 (23-31) (7-12) 287.7 42 (13.5-33.75) (2-3.6) 2.3676 0.0836 L. perennis (2009, 9.4 ± 0.53 FP) 64 N/A (6-11) N/A N/A N/A N/A N/A

Table 2. Sample population reproductive data for each of the lupine species and locations and years. # that % rate of pod Average % that % Number of Number of Number of Average % that Average % that set set set pod Lupine type n= Establishment plants that flowers pods flowered ± SE set pod ± SE pod (# of pods/ (pod n/ in FP flowered (n) (range) (range) (flower n/n) (pod n/n) (n) # of flowers) flower n) 24.0 ±3.1 7.4 ± 1.0 L. albus, CW 38 53% 35 (10-37) 34 (3-12) 31% 92% ± 4.5 89% ± 5.0 97% L. polyphyllus, 38.4 ±2.4 12.5 ±1.83 GL 60 83% 27 (27-46.5) 22 (4-20.5) 32% 47% ± 9.0 34% ± 7.8 81%

L. perennis, WB 30.2 ±0.6 5.4 ±0.2 (2009, ABOS) 404 N/A 111 (21-39) 52 (3-7) 18% 25% ± 5.3 12% ± 3.5 47% L. perennis, WB 18.4 ± 1.2 6.1 ±0.4 (2007-2011) 2226 NA 1519 (6.7-26.7) 1097 (4.5-7.3) 34% 56% ± 13 36% ± 10 72%

L. perennis, WB (2009 FP) 64 89% 0 0

Discussion The growth and reproductive results, show differences in investments for the three lupine species, supporting the main, overarching hypothesis that the differences would be evident in the number of vegetative and reproductive parts. A summary of hypothesis, predictions in light of results can be found in Table 5 of the appendix. In comparison to GL and CW, WB put the least amount of effort into above ground vegetative growth according to the combined vegetative growth factor. None of the WB plants in the field plots flowered in 2009 and all reproductive data came from the established quadrats at the Black Oak Savanna. When considering the population of plants, most CW plants produce pods and less than half of GL do and less than a quarter of WB produce pods. These results illustrate different investments in vegetative and reproductive effort, showing that wild lupines place a lower investment in growth and sexual reproduction, negatively impacting their ability to be restored. Exploring phytohormones during seed development will help understanding of these phenology results as they greatly impact the lupines physiological development (chapter 3). These perspectives combined allow for a deep understanding of why wild blue lupines struggle to reproduce from a restoration perspective, as compared to the other species in this study.

Vegetative Growth Both the number of leaves and heights of CW, GL, and WB varied between species, as predicted, showing different amounts of effort from each plant type into above ground vegetation. CW put in the most combined effort, followed by GL, and

39 then WB. These traits are congruent with the ecological function or role of each plant as initially explored in the hypotheses.

CW, a crop species with indeterminate branching, grew to be a consistent height with a limited number of leaves where number of leaves positively corresponds with height (53). In a farming operation, minimal effort for vegetative growth is ideal as increases the harvest index though partitioned resource availability for developing pods

(53). GL, bred to be showy, with disproportionate flowering effort, is a species that shows a wide variety of polymorphism (3) and is subject to selective breeding for a variety of traits. The variety of GL grown, in the field plot like most horticultural types, produced many leaves without growing very tall, which goes against the positive correlation between height and number of leaves (53). In a savanna ecosystem context,

WB must contend with intraspecific competition and showed more variety in measured heights. This is reflected in the slight differences in height between the lupine monitoring quadrats at ABOS (Figure 12 in the appendix). Interestingly, there was no significant difference in the number of leaves across locations including the field plot and among the lupine monitoring quadrats.

Of the two measures, number of leaves is a better predictor of vegetative effort than height and can be used as a non-destructive method to assess size (16, 120, 97, 96) and also a predictor for subsequent year’s survivorship and potential fitness (47).

Halpern reports that the above ground biomass is well correlated to total seed count

(r=0.86, p<0.001, n=1180) (4), making leaf counts an effective measurement for predicting seed generation for WB clumps in the field without disturbing the plants. It

40 can also be used to assess lupine plant health (113), as water stress (115) and nitrogen shortages impact leaf size and timing of emergence (76). Leaf health also influences herbivory rates as the more leaves present, the greater the likelihood of leaf predators locating them (37) and their palatability. For example, KBB prefer to utilize sun and shade leaves depending on sex, and leaf longevity is also important for larval feeing and ovipositioning (19, 44).

While both measurements can be used to determine effort put into vegetative growth (both independently and as ratios to one another), combining factors allows for an alternative perspective to this type of assessment that incorporates both measurements of the above ground biomass and growth effort. After multiplying height and number of leaves into the combined vegetative growth factor, GL and WB are closer in the effort they put into above ground vegetative growth whereas CW puts seemingly equal amounts of effort into height and number of leaves, putting more effort overall into vegetative growth. Based on observations made when removing GL from the field plot in the late fall 2009, GL put effort into developing below ground biomass, whereas

CW did not (personal obs.). This was true of WB as well, where some of the plugs planted in 2008 continue to survive, flower, and produce pods in 2016.

Reproductive effort Both the number of flowers and pods of CW, GL, and WB significantly varied among species in 2009, as predicted, showing different amounts of effort from each plant type into sexual reproduction. All lupines produced more flowers than pods, and in this study, ~30% of CW and GL flowers became pods but only 18% of WB flowers in

2009 set into pods. These measures are good predictors of seed production (15) making

41 them a worthwhile but non-invasive way to measure reproductive effort but population reproduction needs to be accounted for.

Rates of reproduction (population level fecundity) While determining the number of flowers, pods and seeds produced per plant can predict reproductive effort, this does not account for variations in the whole population, accounting for how many of them put effort into sexual reproduction.

When this is taken into consideration, CW puts far more effort in to reproducing, with

89% of the plants producing pods whereas only 35% of GL plants did and 12% of WB plants in 2009 (25% is the five-year average), which supports the hypothesis that the lupine species differ in their reproductive and growth strategies and aligns with the predictions.

When a measurement of the number of reproductive parts (either flowers, pods or seeds) is combined with the likelihood of that plant within the population flowering or producing pods, a more representative estimate of reproduction in a population can be estimated. By multiplying the number of flowers by the percent of plants that flower, it generates the following reproduction factors: CW 22.08, GL 18.05, WB 7.55 and 12.70 averaged for five years. This reframes reproductive effort, where CW now shows the greatest effort put into flowering followed by GL, whereas just looking at number of flowers produced would have GL as putting forth more effort. Examining the number of pods and the number of plants in the population that produce pods yielded similar results where CW has 6.59, GL 4.25, WB 0.65, and 2.44 over five years of monitoring which aligns strongly with the predictions, supporting the hypothesis.

Therefore, CW was most successful at producing seed, had a high reproduction rate

42 with a most pods set. GL also had a high number of pods and very high number of flowers but with a lower rate of plants that reproduce, and these factors balance out.

WB had a lower abundance of pods set from the number of flowers produced and the observed lupines showed a moderate rate of flowering and pod set where less than half of the number of plants observed that put forth this effort, which confirms the prediction that they differ in reproductive effort on a population level.

Number of flowers, pods, and seeds per plant (plant level fecundity) CW, as an annual, each plant must put forth effort to produce seeds each year in order to continue that genetic line (95), which it does through its indeterminate growth and continually initiating flowers until pod development is well underway (27). For CW,

30% of those flowers developed into pods. Both WB and GL have distinct floral racemes, initiated at one time, although a single GL plant will produce multiple racemes above ground. GL produces more flowers on average than the other lupines and with a

32% success rate of flowers developing into pods, produces many pods as well, with additional aboveground racemes per plant. WB produced many flowers from a single raceme but few of these developed into pods, as few as 18% in the 2009 study year and

25% over a five-year monitoring period. This is comparable to the reported 24% when open pollinated, 11% when only allowed to self-pollinate (116, 115). This illustrates that

WB puts some significant effort into sexual reproduction in 2009 but does not support the development of flowers into pods to the same extent as the other lupines do.

Extending the scope of study to five year (2007-2011), WB does show the same rate of pod setting, 34%, which will be explored further in this section. Therefore, looking at physiological processes that regulate the successful transition from flower to pod set

43 may shed insight into why wild lupines struggle to reproduce sexually and how phytophomones contribute to that (chapter 3).

Monitoring effort, timing and technique can contribute to difference in numbers of flowers reported. WB at the ABOS tend to produce fewer flowers (30.2 ± 0.6) than what has been recorded in Ohio, which reports 37.5 ± 13.97 by (114), ~35 by (14), and

30-50 reported by (115, 78, 116). In 2007 and 2011, the number of flowers were observed and recorded on a single day, which would result in the under reporting the number of flowers those years (Figure 13 in the appendix). This is because they open in synchrony (114) and the unopened flowers are difficult to count in the field. Pods however, are easier and more accurate to count and do not change over development and they were more consistently reported. What this demonstrates, for all species, is that lupines produce many more flowers than pods, but wild lupines in this study, produce even fewer than the others it was compared to. This supports the hypothesis that wild blue lupine reproduction is below that of the species it was compared to and that may contribute to its struggle to reproduce. Part of this may be the inability to support pod development and high abortion rates based on low self-compatibility (11) or even high temperatures (66) or division of assimilates to organs of higher investment priority. Large floral displays do aid in creating an attractive destination for pollinators where 20% of the flowers may be completely unviable (85).

Attracting pollinators and maintaining a floral display would aid in outcrossing and genetic diversity in the population (114, 14). CW is largely self-pollinating, with 5-

10% outcrossing by bees (53) while GL has a strong outcrossing rate (3, 54) similar to WB

44 which has an outcrossing rate of 0.85 (115). GL and WB flowers are serviced primarily by solitary bee species, where they are able to manipulate the flower to gain a dosed pollen reward as no nectar is provided by lupine species (14, 114, 85, 50). Both of these species are able to produce well beyond 30 flowers per raceme, but not all of them were open and viable at the same time (114). Each whorl of five flowers opens in synchronicity (114), extending the flowering period for several days to weeks (85). Bees only tend to visit 15% of the open flowers, where their attention is directed to viable flowers by the opening of the banner petal (27), starting at the bottom of the raceme and working up (50). The banner petal colour changes with quality and quantity of manipulation and resulting pollen tube growth (fertilization) (114, 85) signaling for the bees to stop visiting while maintaining the floral display from a distance. Cold wet weather slows this change and likely impacts pollinator visitation, as would the timing of rain (114, 15). The size of signal triggering pollinator visitation is determined by population size (14, 114) which validates looking at how much a population of lupines is flowering not just the individual plants.

The number of flowers available and how long they are viable along with their self-compatibility are key influences in the number of pods (53, 3, 115). Other environmental variables would also influence this such as pH range of soil, water stress

(drought or water logging) and nutrient availability (53, 76). Planting date would impact this (66) as well as available light (65, 82, 16). These variables have been well studied in

WB but may have been a factor in number of pods produced in all plants as the expected number of pods are lower than what was reported by others. CW produced 3-

12 pods per plant which is fewer than the reported number is 10-22 (66). WB has been reported to produce 6.7 pods per inflorescence (78, 116) and up to 9 with additional resources provided and only 3 when forced to self-pollinate (115). This demonstrates that all species may have been challenged to produce pods in the field plot, but across the site, WB was performing within its expected range, but still well below the two other lupine species (Figure 10). The number of pods for WB should be explored further within the five-year average data for the site.

While there was not much annual variation in the number of flowers and number of pods produced by WB over five years (Figure 13 in the appendix), the timing of rain may have impacted the ability of the flowers to be cross pollinated by bees (107).

Figure 14 in the appendix shows the total precipitation for the site over the five years as well as a breakdown of the average timing of precipitation and the annual variations of it. In 2009, the bulk of the annual precipitation fell in May, which corresponds with WB flowering and the need for bee pollination to allow for cross pollination. In June 2010, the bulk of the annual rain fell in June, where many flowers were produced but even fewer developed into pods. It would appear that the timing of rain for these two years impacted pod set, possibly due to lack of cross pollination by bees. While rainfall may impact flowers and pods, it also impacts herbivory and can impact the timing of life history events as well (14, 15, 107). Low water or water logging conditions would increase stress in the plant and may impact where resources in the plant are allocated

(above ground/belowground root structures, nodulation) (94, 45, 121, 53). The end

46 result of precipitation would impact seed production as both selfing and stress increase seed abortion rates (11, 14, 114, 85, 115, 78, 116).

GL was variable in its seed production (118), with a wide range of seeds per plant of slightly varying mass but a consistently low number of seeds per pod showing high abortion rates (112, 3). WB and CW were similar in their effort of producing consistently more seeds per pod, but lower number of seeds per plant, although the seed sizes are not at all comparable as CW seeds are much larger, approaching and sometimes exceeding 1000 mg per seed (53). The number of seeds per pod in WB are consistent with Shenk’s (114) 2.5 ±1.1 of the 5-6 expected ovules per pod reported by

Shi (115, 116) but far lower than the 99 seeds per plant observed by (48) who also found

0-7 seeds per pod.

Many genes control seeds size and seed development (67) just as they do for other components of reproduction like phytohormone signalling (especially ABA) and other internal plant functions. From a physiology perspective, both leaves and shoots and reproductive components such as flowers, pods and seeds, can be sinks for water and nutrients, where resources must be partitioned and can result in variable yields

(53). This would be most prevalent in CW which exhibits indeterminate growth, having new shoots, leaves, and flowers developing as pods are setting on the main stem (53,

27, 28). This will be explored further in chapter 3, where plant hormone types and abundance will help shed light on plant signaling dynamics. Harvest indexes have a weak response to variation within plants to stress and other environmental variables

(49) and the current and projected environmental conditions do not favorably support plant growth (67).

While collecting GL and CW seeds was required in the field trial, ABOS’s management policy mandated that only 10% of a populations seeds be collected to allow for natural regeneration (21) which limited monitoring efforts of WB seeds. More effort could have been put into monitoring seeds, as seed mass is often a reported measure but was not reported in this study as there was not a detectable trade-off between seed size and seed number (48). Parental resources (48, 115), population size

(78), and environmental factors like photoperiod and light (66) over the course of time

(3) can impact seed size. While the number of seeds per plant is valuable in understanding resource allocation within a single plant, it only offers a limited perspective. Adding an additional viewpoint of population dynamics offers another angle in which to assess why wild blue lupines struggle to restored. This study also stopped at the point of seed generation, it did not examine germination and establishment rates of seeds, which is an important component of reproductive success.

Many other studies do this and these perspectives will be assessed with the conclusions of this work in chapter 4.

Growth and Reproduction It is important to examine both vegetative growth and reproduction factors as they do impact each other. This is often examined with crops, through the use of a harvest index. Halpern (48) found that the number of leaves produced in WB correlated well with above ground biomass (r=0.86, p<0.0001, n=308) 2003. Furthermore, they found that above ground biomass was positively correlated to total seed count (r=0.86,

48 p<0.0001, n=1180). Since collection of above ground biomass was not possible in this study (16), using Halpern’s 2003 findings, a measure similar to a harvest index can be calculated by dividing the number of seeds per plant by the number of leaves resulting in seeds: leaves index of CW 0.6369, GL 1.311, WB 2.3676. This result contradicts the hypotheses which stated that WB would produce few leaves and few seeds. If all plants were taken into account, this ratio would be much lower as only 18% of WB in 2009 produced pods, which shows that the wild lupines that do produce seed, invest heavily into it. What this measure does illustrate is the need for additional perspectives as this only captures a per plant view of reproduction and many WB do not reproduce where are nearly every CW did. This helps frame the efforts of GL into this context, where they produced many leaves and many seeds per plant (though few per pod) and almost half of the population does not produce any pods and therefore seeds. WB’s above ground effort is even less, for which fewer plants reproduce and those that do, produce few seeds and leaves. This is to be expected though as seeds of wild relatives of crop species tend to be small and round (67) and that is observed (but not quantified) in the comparison of these species in this study.

Population size and reproductive effort are key factors in determining sexual reproductive success. Density of stems (14) and size of floral display impact pollinator visits, especially from a distance (114, 55, 54). Canopy cover (16, 93, 19), temperature

(66) and leaf litter (91) also influence flowering. This combined with the unpredictability of a fire managed landscape (96), population level fecundity is important for maintaining self-seeding and naturally spreading WB population while maintaining genetic variation

(115, 18). While natural seed dispersal is helpful in extending lupine distribution, it can only move seeds up to 3 m from the parent plant (18), although mice could carry them further, though this was not explored by (63, 64). Kappler focused on WB density’s impact on seed predation and the effects of prescribed burns and leaf litter, two factors that also impact lupine seed germination rates and seedling establishment (16, 96, 97,

120). Where seeds end up in the savanna is important as they have a 3-year viability in the seed bank (48), and require dormancy-breaking ques (120, 97, 96). This would be an interesting place for further study as would genetic studies to determine the impact of inbreeding (78) and patch relatedness across Ontario and the upper Midwest.

Concluding remarks In comparison to GL and CW, WB shows limited reproductive effort and pales in comparison for seed production when considering how many wild lupine produce pods and seeds. Flower production and pod set are promoted by cytokinins and that is a key variable worth exploring to elucidate physiological limitations in the weak reproductive output shown by WB in comparison to the other lupine species. This will be explored further in chapter 3 along with a contributor to WB dormancy, abscisic acid.

Given that ABOS is the most northern site of the WB and potential KBB distribution (18 58) and any potential reintroduction sites need to continue to work to extend patch connectivity and expanding WB lupine populations to support the KBB.

With the ABOS being the largest and most northern population (58), they need to continue to lead the way in this research, as explored further in chapter 4. There are also potential challenges on the horizon with climate change and Halpern suggests adaptation is possible within the genome, and innate tolerance to drought is present,

50 but will be slow and most responses will be shifts in life history (timing of germination, seasonal dormancy) (47). Examining phenological responses would benefit with concurrent physiological monitoring as ABA is a key player in water stress and would impact both leaves and seed development (20, 104, 15 and explored further in chapter

3). Considering that tallgrass prairies and savanna ecosystems are already water and nutrient challenged due to substrate type, WB will continue to be challenged with balancing water resources between vegetative and reproductive parts and may reduce reproductive effort, relying more heavily on vegetative reproduction (16). This could further exacerbate the issues with small, isolated populations and inbreeding depression (78, 116, 19). The additional ways in which these issues can be assessed by using various different scientific approaches and other ways of knowing, the better able we will be able to see the issue (52, 124, 1). By using multiple perspectives, it provides a holistic view of the issue and granting access to more tools from which to problem-solve with (9, 52, 1).

Chapter 3: Examining cytokinins and abscisic acid during seed development among three lupine species when planted at a Alderville Black Oak Savanna. Introduction Most research into L. perennis, the wild blue lupine (WB), investigates the ecological aspect of this plant’s growth, germination, reproduction, and preferred environmental variables in savanna ecosystems as explored in chapters 1 and 2. All of these approaches look at lupines on the macro scale, from whole plant to population level studies. There are very few studies that explore internal interactions in wild lupines such as genetic or plant metabolite investigations, including phytohormones.

These types of studies are important as genetics (10, 68) and source/sink dynamics (10,

40, 4, 106) are vital determinants of plant performance. This study attempts to address this gap, within the context of determining why, physiologically, wild blue lupines struggle to reproduce sexually as compared to the other species. By investigating the phytohormones present along with the previously investigated field observations of vegetative growth (chapter 2), and a creating space for indigenous ways of knowing as well (52, 1), generating a unique and holistic (9) approach to this question.

Phytohormones Cytokinins (CK) and Abscisic Acid (ABA) are phytohormones which have been identified as key influencers in seed development. CK is an influential signaling and regulating factor for cell growth and division. CK is a key factor for generating sink strength (ability to attract and use) for sugar and other metabolites to be sent to seeds for their growth and development (32, 4, 77, 29, 46, 56, 106, 6). Their role in

52 reproduction, specifically in seed filling and development has been studied in several crop species such as rice (72), legumes (100, 31), crop lupine (23, 126, 32), corn (80), barley (98), and others (56). ABA is also present and active during seed development and its role is to control dormancy (105), fruit ripening (119), as well as signaling for water stress responses (133) and triggers a broad up-regulation of gene activity (101). It also limits cell proliferation (68) and because of the varied roles it performs, there is often two peaks of ABA accumulation in developing seeds. One occurs early in development, ABA from the parent signaling for gene activity and limiting cell proliferation (101, 68) and one at the end of development, generated from the seed to enforce dormancy (81, 105). CK and ABA should be considered in concert as well since as they frequently act as antagonists (106). The study of ABA and CK concurrently, through samples taken throughout development, is important as it help illuminate what is happening internally at the time of sampling (33), which is often only monitored via external, phenological characteristics (60). There is also the ability to observe cross talk when monitoring for multiple phytohormones, as CK and ABA are likely not the only influential internal factors.

Seed development In addition to CKs and ABA, several other hormones like gibberellins (72), auxin

(33, 30), ethylene (110, 130, 119) perform key roles in seed development, particularly during pod set (as seeds are often aborted at this stage) and during later maturation

(105). The main interest in studying lupine seed growth and development pertains to the final stages of development, when the seed has progressed past its premature stages, to the ones where it is becoming viable. What happens in pod development on

53 parent plant, impacts the individual seed after dispersal wait for germination cues while undergoing dormancy breaking events or they persist in the seed bank until they die (39,

41). By capturing a succession of snapshots of the amounts and types of CK and ABA along the seed development scale, more can be learned about what is happening within

WB hormonally during seed development, which plays a key role in determining the fate of these developing seeds.

The lupine developmental scale used in this thesis was based on that developed by Dracup and Kirby 1996 (27). This modified scale attempts to categorize lupines into 5 stages, whereby, at stages one to three, the seeds are growing rapidly and are premature (27, 84) (Table 3). Stage four occurs when the seed is physiologically mature, the cotyledons are fully formed, and the seed is viable but still soft with a high moisture content. Stage five describes when the seed is drying out and can be stored as it has a lower moisture content, and in the agricultural sense is harvest mature (27). This scale serves as a developmental key to facilitate strategic study of the concentrations and types CKs and ABA during seed development at pivotal times like embryogenesis and later maturation stages (105, 56).

Abscisic Acid ABA performs various roles within a plant and the amount in seeds is generally lower than the rest of the plant but can increase under stress (111). Accumulation in seeds is important under ideal and stress conditions as it impacts many aspects of seed development and dormancy (111). Recent research is focused on its metabolism, receptors (71), biosynthesis, and active forms (22). Specific to this study, it is important to note that ABA accumulates in seeds especially just after anthesis, when the seed is a

54 major sink within the plant (105). It triggers various responses depending on concentration, cell development, and location as it has multiple receptors (101, 106) and is regulated by five major gene families (128). It plays a role in dormancy and germination (41), dry matter accumulation (111) and can delay cellular differentiation

(101) and proliferation (30) as well as delay the ripening of fruit (128). ABA performs a different role in premature and developing seeds and fruit, and the timing of exogenously applied ABA can either slow development (30) or hasten ripening (119).

ABA levels are often low during development increasing towards maturity, which generally correlates to the physiological maturity of the plant (101). This results in two peaks of ABA during seed development, the first is generated by the paternal plant, when there is rapid growth; whereas, the second is synthesized by the nearly developed seed itself as it matures and prepares for dormancy (111). It is also known that ABA concentration is a better predictor of dormancy in a seed than CK, although ABA is not strongly correlated to the opposite process of germination (39). The interaction between these two phytohormones is also important as they perform different but connected roles within developing seeds and the ratios of their occurrence can be important to observe (130, 129, 46, 30).

Cytokinins There are many different types of CKs and they range in their ‘active’ abilities and concentrations in the plant (for a current review, see 56). Different types of CKs can be found in developing seeds in changing abundances when there is rapid cell division and growth. This is supported by the rest of the plant, including organs like roots, or leaves which are the source of water and photosynthate that provide the seeds with

55 supplies to support this growth (the seed as a sink) (106). CK types and abundance are controlled by biosynthesis (isopentyltransferase, IPT), translocation (sink/source dynamics, 106), activation (lonely guy enzyme, LOG), inactivation creating conjugate forms (O-glucosyl transferase), re-activation (β-glucosidase) and degradation by CKX

(cytokinin oxidase/dehydrogenase) (56, 62, 36).

Using the current model for CK biosynthesis pathways by Kamada-Nobusada &

Sakakibara (2009), CKs can grouped into nucleotides (NTs), ribosides (RSs), and freebases (FBs) together for discussion (Figure 3). The most abundant, but least active types of CK are the NTs, as they are the first types created in the AMP, DMAPP pathway of synthesis (108, 100, 70, 7, 109, 27). Through a multistep activation (73), the monophosphate group is broken off the NTs, they can become RSs, which are more active in the cell (29, 7) but considered as having lower impact than the FBs. FBs are the most bioactive type of CKs although they are usually found in lower concentrations (33,

109, 73, 29, 62).

As illustrated in the CK pathway (Figure 3), NTs are converted into active FBs by

LOG enzyme (62). NTs can also be converted into RSs by 5’-ribonucleotide phosphohydrolase (or regain the phosphate group with adenosine kinase) which makes them more accessible to the receptors (33, 109, 73). This transfer is not always completely efficient, as many species will accumulate high amounts of NTs that are not converted into RSs, either because they are not needed or by a limiting factor, or plant deficiency in conversion (33, 109, 73). RS, once they have lost the riboside side chain through adenosine nucleosidase (or regained it through purine nucleoside

56 phosphorylase) become FBs. FBs are irreversibly degraded through CKX or converted to storage type CK (glycosides) as acted upon by O-glucosyl transferase. By examining the relative amounts of the NTs, RSs, and FBs in developing seeds, the CK profiles may demonstrate reasons how hormonal regulation may be limit WB so that it is not reproducing as well as other high yielding species like CW (chapter 2).

RSs

FBs

Figure 3. CK synthesis and metabolism scheme adapted from Kamada-Nobusada & Sakakibara (2009) showing CK biosynthesis MVA and MEP pathways CK-types and some key enzymes.

CKs also exists in different configurations or types as illustrated in the CK pathway, where variations in side chain are significant as they determine function (7) and are only active within specific receptors (4). This continues to be an area warranting further research (56, v148, 62). The four kinds of CK are, iP, DHZ, tZ and cZ, where cZ and tZ are sterioisometric but do not perform the same role in cell growth equally (31,

29, 6, 79, 33, 98, 38). Considerable research supports tZ-type as being the dominant and active CK type in many species such as barley (98), rice (72), lupine (32, 30), and many others but there is a growing body of research that suggests that cZ can be the active type in many legume species including chickpeas and lupines (29, 7, 79, 100, 31,

38). cZ is found various families throughout the plant kingdom, showing no evolutionary explanation (38). It is currently believed to be involved in energy conservation in growth limiting conditions (99, 38, 56). The research shows that CW is a tZ dominant species

(32) and unpublished data from Quesnelle and Emery reported in (29) suggests that GL is a cZR dominant species. There is no current research on which type of CK would be found in WB but it is likely to be cZ based on the preliminary findings in GL. Determining types of CKs present in developing seeds is important as cZ-CKs are currently understood as being less active and if present, would contribute to WB’s struggle to reproduce sexually, therefor limiting its ability to be spread through seed for restoration purposes.

Research framework By comparing the types and concentrations of CKs and ABA during reproduction of the wild lupine (WB) to CW, an annual crop species and GL, a common perennial garden species, more can be learned about WB’s seed development, which can help

58 guide restoration practices. It will help explore source/sink dynamics and the balance during the stages of seed development especially when considering other species with similar and/or desired traits. GL is used as a moderately similar comparator as shown in chapter 2. It is a perennial, but with slightly more ability to reproduce as demonstrated by its capacity to escape gardens and continue to colonize areas such as roadsides in eastern Canada. CW is the more extreme comparator/outlier as explored in chapter 2 since it has been modified and bred to produce many, large viable and nutritious seeds, and is farmed on large scales as a self-compatible, annual crop. This species was useful for comparison purposes as it exemplified the extreme case of high reproductive output balance by ability to be grown in a regulated and controlled manner. It demonstrates the traits of many years of selective breeding to achieve high yields and crop robustness and it has been well studied in the process (53, 27), providing much opportunity and abundance for quantifying CKs and to a lesser extent ABA (23, 32) and continues to be a crop of interest and research (122). Using these comparators and sampling during five stages of seed development and building on observations made in chapter 2 for these species by employing a physiological approach, the question of why wild blue lupine struggle to reproduce can be explored deeply. Using multiple approaches aids in the depth of understanding (52, 9) as it allows for dialogue between each method, aiding in new understandings. Together, it strengthens the conclusions generated in exploring why wild blue lupines struggle to reproduce in a restoration setting.

The primary hypothesis of this chapter is that the amounts and types of phytohormones (CK and ABA) would change through the five stages of seed

59 development, and that the course of change would be different in a way that reflected the varying levels of reproductive effort of each of the lupine species studied. It was predicted that ABA would shows a peak early in development that would be reflective of the sink strength of the seeds of the three lupine species (111) to help control and limit cell growth (68). Furthermore, it was predicted that the seed ABA concentrations would be different between annual and perennial lupines species as they approach maturity with varying levels of dormancy. CW was expected to have less ABA as it shows very little dormancy whereas WB dormancy is difficult to break; so WB ABA levels were expected to be high.

As for CK concentration, it was expected that CKs would occur in higher amounts early in seed development as that is a time of greatest cell division. They would also display lower overall amounts in later stages of seed development as growth and storage slow as the seed prepares for dormancy for all lupine species (32, 29). It was predicted that CW would contain greater amounts of CKs during development as the seeds produced are larger and would require increased sink strength for starches and possibly for the creation of additional cells. This will be especially high during the early stages of seed development resulting in a stronger sink signal, as compared to the other lupine species with much smaller seeds (32, 68). It was expected that GL and WB will also contain elevated concentrations of CKs in early seed development but these peaks would not be equal to the concentrations found in CW (29) and, therefore, sink signal will not be as great (106).

An extended physiological hypothesis is that the types of CKs present in development will also vary among lupine species. It is known that CW’s predominant active CK types are tZ (32) whereas for GL it is cZ (29). It was predicted that WB would have similar CKs to those of GL, as those two species have many of the same morphological traits and patterns of growth.

It was expected that WB would be very similar to GL in terms of CK and ABA concentrations and forms during seed development as they are both perennial lupines and share many similar life history traits. WB will show a higher accumulation of ABA than the other lupine species as it approaches maturation because it has a greater dormancy than the other species. Other differences will include cZ as the most active

CK form during seed development and changes in CK forms over development will tend towards cZ types, as based on previous research with GL.

Methods Field plots In order to physiologically examine the reproduction of WB at the Alderville

Black Oak Savanna site, L. polyphyllus (GL) a common garden lupine, and L. albus (CW) a crop lupine, were selected, planted on site, and exposed to similar environmental variables. The field plot was located in an abandoned field that was actively being restored to native vegetation conditions, close to a natural windbreak with some surrounding young oak trees and various tallgrass plants. The area was cleared of vegetation using herbicides, hand pulling, and mechanical removal with the use of a harrow, during the fall of 2008 to prepare for the 2009 field season. The plots consisted of 18, 1 m2 plots with 0.5 m spacing in between, as illustrated in Figure 8 in the appendix.

Four WB and four GL were planted in each plot in fall 2008 so that they would be established in 2009 for measurement and comparison with CW. Four CW plugs were planted into each plot in the late spring of 2009 as they are an annual species that could not have survived the winter at the site. In total, 72 lupines of each species were planted, 12 per 1m2. All plugs were germinated and grown by Cathy Loukes, a local greenhouse operator who is very familiar with germinating lupines – which can be a difficult task as they have very low germination rates. Given the failure of the WB to flower in the replicated blocks during the monitoring year, they were monitored and sampled in the established plots within the savanna.

Seed development stages Dracup and Kirby’s lupine development guide was modified to develop the five stages of seed development for hormone monitoring in this experiment (1996).

Table 3. Five stages of growth in lupine seeds, based on Dracup & Kirby (1996)

Stage Description

Stage one – premature young green pod dark green seeds, water contents Stage two – premature seeds filling space, seed bulges little to no water in seeds, green and soft Stage three – premature greed pod, septa split seed coat changing, green to yellow cotyledons Stage four - physiologically mature pods drying, khaki coloured soft, pale seed coat, yellow to gold seed coat Stage five – harvest mature pods dry, dehisce, hard pale seeds

These stages were used for selecting tissue for hormone sampling of seeds for

CKs and ABA, as described below. Samples were collected in the field and quickly frozen on dry ice (-78.5°C) until processed in the lab where pods removed, seeds weighed, and stored -80°C until extraction.

Hormone extraction Extraction of CK and ABA from homogenized plant tissue followed the procedure established in Emery et al 2006, (100), and also described by Powel et al 2013. Internal

2 standards were added and consisted of 50 ng of each of the following: [ H6]iP

2 2 (isopentenyl adenine), [ H6][9R]iP (isopenteyl adenosine), [ H6][9RMP]iP (isopentenyl

2 2 adenosine 5' monophosphate), trans-[ H5]Z (zeatin), trans-[ H5[9R]Z (zeatin riboside),

2 2 2 [ H3]DHZ (dihydrozeatin), [ H3][9R]DHZ (dihydrozeatin riboside), [ H6][9RMP]DHZ

2 dihydrozeatin 5' monophosphate, and [ H4]ABA (abscisic acid) (OlChemIm Ltd.,

Olomouc, Czech Republic). After homogenization, extraction, purification, and reconstitution steps further elaborated on in the appendix, ABA and the CK types were separated into different fractions using Reverse-Phase-Ion exchange with cation exchange and reverse phase characteristics of MCX columns (6 cc, with 150mg of sulfonated sorbent). NTs underwent a further process that cleaves phosphate groups and the resultant CK-ribosides were further purified using C18 solid phase extraction columns (500 mg, Accubond ODS; Fisher Scientific, Mississauga, Ontario). After further drying, reconstituting and transferring, the samples were loaded into vial inserts and analyzed by HPLC-ESI-MSMS (26, 33, 98). ABA samples were kept in amber glass and wrapped in tinfoil to reduce light degradation (41, 74).

Hormone detection and analysis HPLC-ESI-MS/MS methods follow those described by Ferguson et al 2005.

Specifications of the instrumentation used as well as particulars about run times, limits of quantification, and detection limits can be found in Powell et al (2013) and Farrow and Emery (2012). Both CK and ABA fractions were loaded using the Agilent 1100 binary

HPLC system (Mississauga, Ontario) and separated by a Finesse Genesis C18 reversed- phase column (4µm, 150 x 2.1 mm; Jones Chromatography, Foster City, California). All

CK samples were analyzed in positive-ion mode (turbo V-spray ionization source) by the

API 4000 triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex,

Concord, Ontario).

Data interpretation The peaks generated in each run were analyzed and integrated using Analyst software (version 4.2.1). Using the three biological replicates, average abundances and their corresponding standard errors were calculated for each of the CK types then aggregated to find total CK amounts (89, 70). This was also done for the ABA samples.

Statistical analysis was performed in Graphpad Prism 5 where one-way ANOVAS with

Tukey’s post hoc were used to test for significant differences among values (p<0.05) between ABA. One-way ANOVAs were used to test for differences among values for total CK values for each of the lupine species between replicates, Bartlett’s test for equal variances was used along with the Tukey post hoc.

No statistical tests were used in the snapshot of each CK type for each of the three lupine species for seed development stages in Figure 6 as the overall picture captures the general differences between species.

Results ABA The patterns and magnitude of ABA throughout development appear quite different for each of the lupine species. Overall, ABA was observed in greater amounts early in development, while the seed were premature and rapidly growing, both in size and number of cells, and lower concentrations in the final stages. The highest amounts of ABA were found in stage three for all species: CW 30243±2141 pmol/gFWT, WB

3577±212 pmol/gFWT, GL 3300 ±267 pmol/gFWt (Figure 4). The lowest amounts of ABA were found in stage four for all species: CW 137± 5 pmol/gFWT, WB 1018 ±199 pmol/gFWT, GL 634±39 pmol/gFWT. This change in ABA concentration between stages three and four was significant and corresponded with the seeds reaching physiological maturity. In the final stage of development, ABA concentrations were very low in CW

(137 ±5 pmol/gFWT), which shows little to no dormancy. Both GL and WB had an increase in ABA levels from stage four to stage five, (GL 947±92 pmol/gFWT. WB

1899±304 pmol/gFWT), which may contribute to the seeds dormancy after leaving the parent plant.

Total CK Trends in total CK included higher relative concentration of CKs earlier in development (stages 1-3) for each of the lupine species. It was also important to note that the concentration of CKs in CW was on average, over all stages, several magnitudes greater than GL and WB. Total CK averaged over all stages was 144000 pmol/gFWt, where GL and WB was 700 pmol/gFWt, approximately 200 times more abundant!

Both CW and GL showed elevated CK concentration in the early development stages one to three, with their highest’s peaks in stage two, as observed in Figure 5.

This was especially profound in CW, stage two, where the greatest abundance of CKs in all species for all stages was found at 460848 ± 62690 pmol/gFWT. GL also had a high accumulation of CKs at stage two as well 1719 ± 247 pmol/gFWT, but not the same magnitude as CW. WB’s greatest amount of CKs was observed in stage one, 1062 ±77 pmol/gFWT. WB maintained a more consistent concentration of CK throughout development (stages one to four then it drastically decreases) in stage five. CW and WB showed very little accumulation, 21 ±3 and 79±7 pmol/gFWT respectively of total CK while GL still had 245±26 pmol/gFWT in stage five.

CK Types Looking into the larger groups of CKs illustrated in Figure 6. NTs were the most abundant, followed by RSs, and relatively low levels of FBs found through all stages for all lupine species. Interestingly, on average, all lupine species had the same total concentration of FBs, through all five stages of development. It is also clear that CW is dominated by tZ with large amounts of DHZ, which is rarely observed. WB and GL showed no occurrence of tZ but were clearly cZ dominated, with some accumulation of

DHZR and iPR. This illustrates that bulk of WB and GL CKs were being generated via the

MVA pathway (Figure 3) whereas CW’s CKs were being generated via the MEP pathway, with the CYP735A enzymes active.

L. albus (CW) Most CKs found in seed development in CW were found in the premature stages and were tZ and DHZ types. It is clear that tZ CK forms were the active type with most

CKs being generated via the MEP pathway and with IPT and CYP735A enzymes active.

While there was some presence of cisZ and iP types throughout development, it was never more than tZ.

CK-NTs tended to accumulate throughout development, and DHZRMP had the largest concentrations of all CK types in stages two and three. ZRMP was also abundant, with 78000 ±20000 and 57000 ± 34000 pmol/gFWt in stages two and three (which was significantly greater than all other stages for ZRMP). All other CK types occurred in low amounts (<500 pmol/gFWt) compared to these peaks with the exception of cZRMP in stage three which had 1800 ±800 pmol/gFWt.

Similar trends were found in CK-FBs, where DHZ was clearly the most present at earlier stages of seed development with 60-270 pmol/gFWt in stages one to three. This was similar to t or cZ, which occurred at ~125 ± 30 pmol/gFWt through stages two and three. iP and cZ levels were detectable but low in stages two and three. There was little to no accumulation of any CK FBs in stages four and five.

With CK-RSs forms, DHZR was abundant in stages one to three and ZR in stage two and three. Even cZR had a distinct peak in stage two, as did iPR in stages two and three, as illustrated in Figure 6.

L. polyphullus (GL) In contrast to CW, GL showed very little to zero presence of tZ and clearly CKs are synthesized via the MVA pathway and heavily cZ dominant. It also showed some presence of cZ-CKs until the seed reached harvest maturity as displayed in Figure 6.

CK NTs accumulated throughout out seed development, and cZRMP was the most abundant form with some in every development stage. Stages two and three had the greatest amount, with 553 ±89 pmol/gFWt and 4303 ±52 pmol/gFWt. Stage one had 132 ±39 pmol/gFWt, which was very similar to stage five with 156 ±42 pmol/gFWt.

Stage four had the lowest presence with 48 ±16 pmol/gFWt. Only DHZRMP occurred in any accumulation (>25 pmol/gFWt) for CK-NTs, with a peak of 154 ± 6 pmol/gFWt.

CK-FBs were found in low concentrations in GL, and only cZ is present in an amount greater than 15 pmol/gFWt, greater concentration found during the premature growth stages. Stage one had 55 ± 39 pmol/gFWt, which was similar to stage three 54 ±

26pmol/gFWt. Stage two had of 546 ± 479pmol/gFWt. Stage four had the lowest amount, with 7 ±2 pmol/gFWt and stage five had 15± 8 pmol/gFWt.

For CK-RS, cZR followed the same general trend of more CKs in the premature stages than later stages. There was a clear peak in stages one to three, with a larger peak in stage two. iPR and DHZR was the only other CK-RS with a presence greater of

~50 pmol/gFWt

L. perennis (WB) Similar to GL, WB contained very low or no presence of tZ throughout development and was clearly cZ dominant, with CKs being synthesized via the MVA pathway. Even though total CK (Figure 5) showed that CKs levels were fairly consistent throughout early development, by looking at cZ, and cZR, clear peaks are shown in

Figure 6. WB also maintained some level of CK throughout the entirety of seed development, primarily cZ.

Again, CK-NTs were the most represented form of CK found throughout seed development. CK-NT persisted at consistent levels throughout stages one to four. Stage one had 292 ± 76 pmol/gFWt. Stages two, three, and four each contained more than

500 pmol/gFWt with concentrations of 505 ± 165 pmol/gFWt, 702 ±307 pmol/gFWt, and 600 ± 172 pmol/gFWt, which was comparable to the concentrations found in GL for stages two and three. Stage five of cZRMP concentration in WB is 37 ±10 pmol/gFWt.

Like the other lupine species studied, CK-FBs were the least abundant CK form recovered. Only cZ was found in concentrations greater than 15 pmol/gFWt, although iP was also detected in stage one. cZ showed intermittent levels of CK accumulation between stages of seed development, which is different than both GL and WB and CK-

RSs and CK-FBs for WB. Stage one had 36 ±7 pmol/gFWt, which was very similar to stage four 43 ±10 pmol/gFWt and stage three with 18 ±2 pmol/gFWt. Stage two had the greatest accumulation of cZ, with 210 ±117pmol/gFWt. Stage five had the lowest amount, with 1 ±0.5 pmol/gFWt.

Like GL, CK-RS in WB showed the most telling trends, which consistently followed the development of greater occurrence of CK in earlier seed development stages. cZR was clearly present throughout seed development and decreased over time, although this was not statistically significant. Other CK types were present in small amount at the

RS level, mainly iPR and DHZR in stage one and DHZR in stage four. All other CK-RS occurred at amounts less than 30 pmol/gFWt.

Figure 4. Abscisic acid measured in pmol(gFWt)-1 for L. albus (CW), L. polyphyllus (GL), L. perennis (WB) for five stages of seed development – described in Table 3 (Dracup and Kirby 1996). Letters indicate significate difference, as tested by one way ANOVA, Tukey multiple comparison test, P< 0.05.

Figure 5. The total accumulation of cytokinins in measured in pmol(gFWt)-1 for L. albus (CW), L. polyphyllus (GL), L. perennis (WB) for five stages of seed development – described in Table 3 (Dracup and Kirby 1996).

Figure 6. CK levels for iP, tZ, DHZ and cZ type CKs for seed development stages for L. albus (CW), L. polyphyllus (GL), L. perennis (WB) for five stages of seed development in pmol(gFWt)-1 – described in Table 3 (Dracup and Kirby 1996).

Discussion In comparing CKs and ABA of the wild lupine to other lupine species during reproduction, differences in amounts and types were observed, while general patterns of presence were similar between species. WB utilized the MVA pathway and showed cZ-CKs present through-out development, in a pattern and abundance similar to GL.

This was in contrast to CW which was tZ type dominate (MEP pathway) and at a magnitude far exceeding GL and WB, which would impact signal strength within source/sink dynamics. WB also showed an accumulation of ABA at the end of development which the other species did not. This illustrates source/sink dynamics differences of that of CW, a high yielding crop species with little dormancy, and GL as showy garden species with invasive qualities, both of which reproduce better than WB.

By exploring these results further and in light of other research, wild blue lupine reproduction, can be further examined. A summary of hypotheses, predictions and if they were confirmed in this research can be found in Table 5 of the appendix.

ABA ABA early in development is important as it reduces seed abortion and promotes embryo growth, and all lupine species showed some amount in stage one, which is in line with the hypothesis and predictions made that two peaks of ABA would appear in seed development (81). For all species, levels of ABA were highest during stage three of seed development when the seeds are at a pivotal spot in development, as predicted and supports the hypothesis. This is a time of transition from cell proliferation (68) and rapid growth (30) where ABA is highest, to reaching physiological maturity (62) where

74 the ABA signal decreases to the lowest amounts observed in stage four (Figure 4). ABA is needed at these end stages, for fruit ripening (119) and triggering colour change (128) which would be occurring beyond the physiological maturity stage (62). ABA is also produced within the seed to control and enforce its own dormancy (111, 81). At stage five, harvest maturity, the patterns in ABA accumulation vary between species and must be examined within in the context of each lupine’s growth patterns individually for further understanding.

ABA in CW ABA levels exist in trace to low amount in stages four and five of maturing CW seeds. This may explain why these newly formed seeds lack dormancy, which is a desired trait in an annual crop species. In this situation, the desired trait would be for these seed to have high germination rates (with the lowest input of any dormancy breaking strategies) as well as high establishment rates (40, 41, 39). This is also a desirable trait in many annual species, and crops bred to be dependent on human selection of appropriate growth conditions and not have persistence in the seedbank

(27).

ABA in WB and GL GL and WB have ABA within a comparable magnitude of each other and generally follow the pattern of accumulation of more ABA during early stages, as predicted, with a peak early in development to control growth. WB had the most ABA in stage one than any other species, which would impact early signal sink strength (along with CKs) and abortion rates (81). There is also ABA accumulation at the end of development which is different from CW and supports the hypothesis and confirms the

75 predictions that WB accumulate ABA at the end of development, which would make germination difficult and this would contribute to the difficulty for seeds to be used in restoration. ABA levels are low in stage four and then increased slightly in stage five, not to levels seen in premature seeds but higher than that of stage four. This effect is more often observed in seeds (101), where ABA levels generally correlate to physiological maturity of seeds and preparation of the seed for dormancy (81). This effect was more pronounced in WB than GL and should be compared to CK amounts to understand hormone cross talk and source/sink dynamics.

There is a increase of ABA within the seeds as they approached harvest maturity and the pod dehiscing in WB (81, 111). These higher levels of ABA would indicate that the seeds are more dormant (although ABA is not always a good predictor of dormancy

(41)) is an advantageous trait for native species in North America, with a shorter growing season (40). This would ensure that some seeds would be contributed to the seed bank – a desirable trait for a species that typically grows in disturbed areas such as roadsides or fire maintained oak savannas (39, 41). This finding has great impacts on restoration practices – which will be discussed in further detail in chapter 4. It could be speculated that ABA loading at harvest mature in GL has been limited as it is a garden species that tends to self-seed and spread along roadsides easily, without as much trouble trying to germinating WB seeds (personal obs.).

Other ABA Considerations ABA accumulation could also be a result of stress induction (133) through low water conditions (104), which is a strong possibility for the 2009 growing season at the

Black Oak Savanna as discussed in chapter 2 and the timing of rain (Figure 14). The

76 sandy, nitrogen poor soil that these lupines were grown in does not hold water well and both water stress, soil drying, and low nitrogen can trigger ABA transport from the roots, to the developing seeds (133, 130, 40, 129). Further tests could be done to explore this variable and quantify its potential impact by sampling phloem to determine if ABA is being transported. Supplemental watering could have also been done to determine if water stress was a factor.

Overall - ABA is perceived in a complex manner as it has multiple receptors (71,

22) where the response is influenced by cell type and development (101) and the pathways to synthesis (105) and its many responses are still being explored both in seed development and seed germination test (39, 130, 71). Further work is needed to explore the connection and potential for cross-talk between ABA sources and sinks (111) and other phytohormones like GA (105), IAA, and CK and the ratios that these exist in

(101, 30, 39).

Total CKs The results confirm that there is a difference in accumulation in the stages of seed development for total cytokinins and there are differences in pathways and dominant CK types among the species. CW produces drastically more CK (nmol/gFWT) than GL and WB (pmol/gFWT), making it obvious that the differences either derived from being an annual or crop put CW in a category by itself. CW is also tZ-CK dominate, with CKs being biosynthesized using the MEP pathway whereas cZ is predominate in GL and WB and cZ’s are generated using the MVA pathway, which fits the hypothesis that

WB utilizes the less robust pathway that generates cZ-type CKs and this contributes to its struggle to reproduce through reduced reproductive effort and success. The

77 bioactivity between tZ and cZ differs in many plant systems so this is an important result

(38) and may be attributed to the larger, faster growing, non-dormant seeds for crop which are much different from the smaller seeds of wild types (68).

In terms of an overall developmental pattern, the trend for total CK, is similar to that of ABA for which there are higher concentrations in the early stages of development, and less in stages four and five, as predicted. GL and CW show an increase of total CK during the earlier stages of premature growth, with peaks in total

CKs in stage two and three respectfully. WB appears to maintain steady and low CK production throughout seed development, only showing a slightly reduced production in stage five. This pattern of CL and GL is to be expected as CKs are required during seed development, especially during the yearly stages when the cells are rapidly dividing and growing (68, 31, 100). CKs would contribute to developing seed signal strength through growth and demand for photosynthate and other nutrients from the rest of this plant, resulting in a strong sink signal (56, 106). It would be expected that CK levels, especially active types of CKs like FBs (and to a much lesser extent, RSs), would be found in low concentrations as the seed approached maturity and ready for dormancy (65). While this supports the hypothesis that inactive/storage CK types accumulate at the end of seed development, further investigation into the types of CK is will help shed light on what contributions this has to reproduction and the bigger question of why wild lupines struggle to reproduce sexually.

NT-CKs NTs are the most abundantly observed type of CKs found in each of the lupine species, but they are also not considered active (100, 98, 109). NTs are highly abundant

78 for CW (which has been well documented in the literature at similar levels to the present study (29, 32)), with mostly t-ZRMP and DHZRMP in the early stages of seed development. This supports the body of work that suggests the MEP pathway (Figure 3) is the primary active contributor of CKs that results in the accumulation of t-ZRMP (with the action of CYP735A) (109, 33, 56, 62, 81). DHZRMP, results, in great amounts for CW, when ZRMP is acted upon by zeatin reductase (109, 33). CW’s excessive accumulation of NTs may be an indicator of a bottle neck in the conversion of CKs to more active type, particularly LOG enzyme to directly convert NTS into bioactive FBs (62, 81), but also shows the vast ability to synthesis CKs. This would increase signaling capacity for sugars, aid in cell division and growth to support these huge (by comparison to WB and GL) seeds (10, 29, 32).

ZRMP and DHZRMP are not the only NTs observed though, as CW also shows some accumulation of cZRMP during stages three and four, and WB and GL only show cZRMP through stages one to four. This supports the idea that the MVA pathways is active in each of the lupine species, where tRNA-IPT generate cZ type CKs (Figure 3), as indicated by the primary presence of cZRMP in WB and GL as well as CW (33, 109, 81,

62, 56, 106). WB and GL do not show an accumulation of any other CK-NTs during any stage of development except for GL in stage four. At this stage, there is a prominent drop in cZRMP, but suddenly there appears to be a significant peak at the same time, in

DHZRMP. This may be an indication of some cellular switching or activation occurring during the physiological maturing of the seed (79, 33, 29, 38).

FB- CKs While LOG enzymes (Figure 3) are able to convert NTs into FBs (140, 149), the accumulation of active FBs (29) is lower in all stages and each of the three lupine species have comparable levels, despite highly variable amounts of NTs and RS between species throughout growth. CW continues to show active amounts of Z, cZ, and DHZ in the early stages of seed development, which the timing and concentration of these three types of

CK likely contribute to the large seed size, filling and growth of these seeds during stages one to three (98, 79, 6). This suggests that CW is likely overproducing CKs and may cause it to use the MEP pathway, which appears to be more effective in producing large seeds. The larger seeds, lack of dormancy, and higher yields for CW make it far more appealing as a crop than the small seeded, perennial GL and the wild perennial lupine that is difficult to cultivate. CK content would most certainly impact pod set, seeds set, seed filling and size, cell growth and proliferation, and dormancy of these three species

(29, 100, 77, 4, 10).

WB and GL show only an accumulation of cZ, with more at the earlier stages of development (100), decreasing towards the end of stages four and five. This would be expected as the seeds are reaching maturity and are not growing (98). As the exception to this, WB shows a peak of cZ at stage four not seen in the other species. This seems a bit peculiar, as one would not expect a peak of an active CK so close to the final stages of development. There is very little accumulation of CKs for WB in stage five and this is likely impacting WB seeds after they are developed and the dormant seeds wait their fate beyond the parent plant (40).

RS-CKs At the RSs level, more CK types appear and these types, especially tZR, cZR, and to some extent DHRZ, likely have some level of bioactivity and the amounts of each observed in the three species, would help support this (29, 7). All three species show some iPR which may be evidence of the AMP+DMAPP pathway (At the RSs level, more

CK types appear and these types, especially tZR, cZR, and to some extent DHRZ, likely have some level of bioactivity and the amounts of each observed in the three species, would help support this (29, 7). All three species show some iPR which may be evidence of the AMP+DMAPP pathway (Figure 3) contributing CKs to aiding in seed signaling and development (108, 109). CW continues to show an accumulation of tZR during the early stages of development but there a larger accumulation of DHZR, and some presence within WB and GL. This is an interesting result as DHZR is not often observed in other species and has been suggested to be a spillover pathway for Z type CKs (but in this case, possibly cZ types as well) (33, 29, 100).

These RS peaks appear after high NT peaks in previous stages, for example CW and GL in stages one and two. This shows good conversion of NTs to the putatively active RS types of CKs via 5’ribonucleotide phosphorylase (109, 33, 108). This conversion of NT to RS is what contributes to the high peak of CK found in both CW and

GL in stage two, high levels of NTs in Stage one are converted, over time to more active types (29, 7). This trend does not appear in WB, as there is a consistent decreasing presence of cZR over the stages of development, which could be contributing to it struggle to reproduce. There is a significant peak of DHZR at stage four for WB, which may indicate some significant intercellular switching of CK types at a critical point in

81 development, similar to that observed in GL at stage four for NT-CKs (79, 33, 29). These

RS trends are worth noting as RS do contribute to growth and development, although their roles are not totally understood, they are still active, just not to the extent of FBs

(29). cis vs. trans-Zeatin While cZ is present in the early stages of seed development in all lupine species,

CW is the only species to also have an accumulation of tZ at the early stages of development. It is common for lupines to have both cZ and tZ during development as they perform similar but different roles (38) and it is likely that the critical timing in transitioning between the two play a pivotal role in development (36, 98, 79). This is what contributes to CW’s reproductive success, as Z functions better at signaling and influencing growth in developing seeds (79, 31) and found during seed filling (98).

The timing, amount, and types of CKs present in the cell (36) are critical for signaling for the needs and amplifying sink strength (10), for sugar loading (77), seed filling (98), cell division (29), and overall growth and development. The growth characteristics and the reliance of CW on the MEP pathway which results in increased iP and Z type CKs, further confirms that CW has been and continues to be a good candidate for domestication. These characteristics seem to support its increased reproduction, producing many large seeds, as internally signaled and controlled through phytohormones, like ABA and CK, and demonstrate an ability to put more effort into sexual reproduction than GL and WB (98, 29, 100, 10, 68). GL and WB seem to be reliant on the activity of cZ and CK’s generated out of the MVA pathway as there are only limited amount of other CK kinds (29, 109), which appears to signal for controlled

82 growth, smaller seeds and greater dormancy. While there are many other factors in this, both internally and external abiotic factors, it allows for some understanding as to why wild lupines struggle to be spread in a restoration context.

Other CK Considerations There are a few internal and external abiotic factors that could impact CK as well as some sampling, extraction, and analysis sources of variance. Within the seeds – CK content can change rapidly, even within an hour (33) and when collecting tissue, from multiple plants, this could impact results. Pod position (29, 32) and seed selection can also contribute to variance that could potentially be measured individually given the sensitivity of mass spectrometers currently (33, 89, 72, 83) but that could have been avoided by pooling a few very similar biological replicates and sampling from them. This technology, along with column, and extraction methodology improvements could also be used to further investigate CK and ABA concentrations as well as other phytohormones like IAA and gibberellins to get a more holistic view of what is happening in the seeds as well (89, 33, 72). This would help capture some the cross-talk that happens between hormones and how they are perceive in different tissues, resulting in divergent growth and development patterns (83).

Lupines offer a unique approach to monitoring transport of phytohormones as phloem can be collected from them to monitor whole plant signaling and sink strength

(6, 10, 36). This would aid in the exploration of abiotic factors like nutrient (nitrogen phosphorus, sulphur, iron) and water availability (77, 5, 110, 4) as well as cold and light stress (5, 6). Biotic factors may also be explored where sugars are being created in leaves and transported (61, 77, 6). Another unique feature to studying lupines is their

83 symbiotic relationship with rhizobia to form root nodulations, which in the nitrogen poor soils of the Black Oak Savanna, would provide a growth advantage (70, 31, 5). The role of bacteria and fungi in creating and excreting CKs is still an area to be explored and how this might impact and be transported in the plant could be explored further in lupines (39, 80, 5). Connecting these kinds of in-depth biochemical surveys to field trials and life trait monitoring should also be encouraged. It unites knowledges among plant physiologists and community ecologists and the types of research each field tends to rely on. This aids in the dynamic ability to understand complex systems that allow plants to react to biotic and abiotic factors in the environment (60).

Ratios of ABA: Total CK While examining the accumulation of CKs and ABA both valid individually, assessing them in comparison, using ratios can help determine cross-talk, balance and complex cellular processes controlled by various gene families (46, 33, 89). When comparing the concentrations of ABA to the total amount of CK observed at the same stage, CW consistently has more total CK than ABA in stages one to three and has similar amount in stages four, but in stage five, ABA does exceed the amount of CK by 100 pmol/gFWT. The magnitude of difference in concentration of CKs when compared to

ABA that is makes comparison difficult for CW, though it must be contributing to the observed difference in seed size, dormancy, and potential for germination (68, 10, 39).

The presence of ABA, triggers broad up-regulation of gene activity, and extreme levels of total CK, primarily tZ (and DHZ) type CKs, could be having synergistic impact on cell division, differentiation, and seed filling, encouraging seeds to grow very large (68).

GL and WB, on the other hand, had more ABA present than CK, 2.7-3 times as much on average. The only time when these ratios approach evenness for any of the species, is WB at stage four. Interestingly, the greatest amount of ABA to total CK is observed in WB at stage five, where there is 24 times as much ABA as there is total CKs.

While this has not been discussed frequently in the literature, it would seem in this survey of three species across five stages of development, this may be biologically significant. ABA concentration is a better predictor of dormancy than CK (39, 40), but perhaps the ratio of the two is also important (46), as well as the way these concentrations change through time in the dormant seed (39, 101). This would be an area for further study with these three species and will be discussed in chapter 4.

Ratio of ABA: FB-CKs Looking deeper into this and assessing the amount of ABA to the amounts of CK-

FBs, ABA always exceeds the amount of CK-FBs present, for all stages of each lupine species. This is most pronounced in stage five for WB, where ABA levels exceed CK-FBs

(primarily only cZ) by 475 times! This dramatic difference is emphasized in earlier stages where the low ratio of ABA:CK-FBs in stages one and two for all species. It then jumps considerably in stage three, due to a large increase in ABA and a decrease in CK-FBs for all species but nowhere near the difference in magnitude for WB in stage five. This likely contributes to the difficulty in germinating WB seeds, which would contribute to

WB difficulty reproducing.

Conclusions Each of these lupine species has distinct characteristics while they do share many similarities as lupines. For example, CW is an annual, large seed producing crop with

85 indeterminate branching, and highly viable seeds that have huge amounts of CK (mainly tZ and DHZ types) and ABA that are high during the earlier stages of seed development.

Comparing these traits to GL and WB, has yielded some key insights into some of the intercellular processes as to why WB struggle to reproduce and are harder to grow.

GL and WB are perennials that have smaller seeds (but greater pod set (29), longer and controlled seed development (38), exhibit limited branching, and produce seeds with some dormancy and persistence in the seed bank (39). While each of these characteristics may be influenced by the ratios of ABA: total CK (or CK-FBs) and the other phytohormones, and internal and whole plant processes, it is also subject to much broader abiotic (nutrients, water, light), and biotic (inter and intra specific plant competition) influences (56). While controlled studies are often done to minimize or eliminate these types of variables, in situ studies are important as these factors do impact plants and allowing these aspects to play their role can enrich the depth of knowledge in understanding of the challenges they face (60).

Chapter 4 – Conclusions and restoration recommendations Conclusion Using multiple tools and perspectives, some reasons why wild blue lupines populations are difficult to expand were explored for the northern aspect of this plant’s range. From a phenology perspective, WB does not put as much effort into above ground growth, leaves and height, as GL and CW do. Less than half of the WB lupines in a population reproduce, and few of the abundant flowers develop into pods as compared to GL, which illustrates that it does not put effort into sexual reproduction from that respect either, which is observed within its range of Ontario and the upper

Midwest (116, 114). From a physiological perspective, WB and GL were different from

CW as they showed cZ-CKs throughout development, generated by the MVA pathway and WB had higher amounts of ABA at the end of seed development (79, 62, 38). This would impact the ability of WB seeds to germinate (39), as compared to CW which showed no ABA at the time of dispersal and GL which had some, but far less than WB

(22). When ABA and CK-FBs (or even total CKs) are considered in relationship to each other, WB truly stands out as having far more ABA to CKs present at the time of dispersal (46, 39, 101). Each of these factors contribute to why wild lupines struggle to reproduce sexually, illustrating some challenges to its expansion of population using seeds with the extended restoration goal of having greater patch connectivity for the recovery of the Karner Blue Butterfly and other imperiled butterfly species.

Restoration recommendations Using the knowledge gained from the different scientific tools used within this research and examining a wide variety of others work, some restoration recommendations and areas of further study can be brought forward. In the interest of continuing to spread and connect lupine populations, a variety of methods should be employed. This provides a bet hedging approach, and embraces the idea that multiple methods are valid and using the best and most relevant aspects of each of them, a dynamic restoration plan can be implemented (52). Previous research described in chapter 1 and the results from this research confirm that steps must be taken to maximize viable seed by knowing how, where and when to use various methods for lupine expansion (120). Established lupine patches will continue to expand by naturally dispersed seed and vegetatively through underground colonization, also increasing plant density (43, 18).

Seed collection should take a dualistic approach, where seeds continue to be collected after harvest maturity and stored, stratified, and germinated the next year when they can be planted and tended, to ensure that they establish. These plugs would be best used when establishing more plants at sites where a few lupines already exist as there is higher costs associated with this method. But the cost is justifiable as there is potential for greater success given the effort provided in tending to the seedlings until they establish 5-7 leaves before planting, which increases success and likelihood of flowering in the following year (97, 19, 47). Seeds should also be collected after physiological maturity and before harvest maturity (Table 3) to avoid having to employ dormancy breaking techniques, based on the ABA levels and CK amounts and types

88 present found in this study (chapter 3). These seeds can be used in two ways – germinating and growing into plugs for transplant in the field in the fall if time and resources allow, or for a lower investment of time, directly sowing them into the field shortly after collection (97, 120). This method would be advisable when trying to establish them in areas where it is unknown if they are likely to establish as there is lower cost to this method. Once they are established in an area, then plugs can be used to supplement the patch until the population is large enough to avoid the effects of inbreeding depression.

When patches are small, signal strength can be boosted by placing potted lupines or other bee pollinated species. Potted GL would be ideal once it is verified that they cannot cross pollinate and hybridize, which is an area for further research. Placing the potted plants in close proximity to help increase bee traffic will aid in the outcrossing and inbreeding depression of isolated lupine patches (14). This is something that could be tested locally in the Harwood lupine patches1 , to help increase local lupine densities and population sustainability.

As suggested by (34), working to establish and connect populations via power- line corridors would be advisable. The Alderville site is 2 km away from a power-line corridor and the Hardwood population is 6 km away from both ABOS and 0.65 km the corridor, as seen in Figure 7. By connecting these two sites, much would be done to

1 Harwood was the natural occurring, parent population for the ABOS lupines planted on the site in 2002 and in 2009 consisted of approximately five distinct clumps southwest of ABOS in the area indicated on the map in Fig. 7 (personal obs.)

89 buffer against any chance of the entire lupine population being burned, making KBB reestablishment more feasible in this area.

Figure 7. Distances between Alderville and Harwood lupine populations and power corridor

Alderville First Nation’s Black Oak Savanna has been site of active engagement in understanding, restoration practices, and a place where individuals can demonstrate a renewed relationship with the land. They provide opportunity for (decolonized/

Indigenised) research and allow those not indigenous to Turtle Island to become indigenous to place by promoting and hosting opportunities to reclaim responsibility for

90 sustaining the land that sustains us (52, 69). This site has provided opportunity for relationships with the land to be repaired, treaties honoured, and mutual respect and understanding adopted, on an individual, governmental, and societal levels (1, 9, 52). It also sustains the opportunity for bio-cultural restoration, a term used by Kimmerer which describes the restoration of both the ecosystem as well as cultural practices, which are a reflection of their relationship and knowledge of the land (69). Innovation in restoration at the ABOS site comes from the unique combination of the scientific tools used within and beyond restoration ecology, and integration of indigenous knowledges and practices (1). This exemplifies what Mi’kma Elder Albert Marshall describes as a journey of co-learning for long term and sustained change where:

“Two-Eyed Seeing adamantly, respectfully, and passionately asks that we bring together our different ways of knowing to motivate people, Aboriginal and non- Aboriginal alike, to use all our understandings so we can leave the world a better place and not compromise the opportunities for our youth (in the sense of Seven Generations) through our own inaction” (9) This allows many people to see themselves in the restorative relationship of the land, calls them to action and provides space for the multiple perspectives that we hold in doing this work. Engaging in this kind of understanding, there can be reconciliation with our relationship with each other and the land (69).

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131 Williams, I. H., Martin, A. P., Ferguson, A. W. & Clark, S. J. 1990. Effect of pollination on flower, pod and seed production in white lupin (Lupinus albus). The Journal of Agricultural Science 115: 67–73. 132 Wilson, S. 2008. Research is ceremony: indigenous research methods. Black Point, N.S., Fernwood Pub. 133 Xing, X., Zhou, Q., Xing, H., Jiang, H. & Wang, S. 2016. Early Abscisic Acid Accumulation Regulates Ascorbate and Glutathione Metabolism in Soybean Leaves Under Progressive Water Stress. Journal of Plant Growth Regulation 35: 865–876.

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Appendix: Additional information for each chapter Chapter 2:

Figure 8. Illustration of field plot design. Plots are 1m2 with 0.5m spacing inbetween.

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Extended methods I started monitoring the Alderville lupines in 2007 and began to adjust the standard monitoring protocol to better gauge the success of the lupines on site. Height monitoring continued, as did floral stem counts, along with new measures (number of flowers and number of pods per plant) to assess potential per plant reproductive effort and population fecundity. It is important to note that Bowl Central quadrat 1 was unintentionally removed in 2009 and further monitoring was not possible, as the exact location of the original plot could not be re-established.

To gain some understanding of how many seeds were produced by these plants without interrupting the normal drying and dispersal of the seed pods, panty hose and/or very thin nylon disposable socks were used to enclose the seed heads before pod shatter and seed scattering. This allowed the seeds to be collected and counted (120).

This was also beneficial for further restoration effort on the site as seeds were easily collected at harvest maturity and stored until the next season for the germination and creation of plugs for further lupine restoration.

While this method of collection is efficient, it cannot be applied to more than

10% of the plants, as restricted by standard restoration seed collection protocols (21). It was also noted on several occasions that seeds would occasionally germinate in the collection device shortly after dispersal (personal obs.). Further investigation in the field after seed dispersal revealed that the bags were not necessarily the cause of this, as many other seemed to be germinating on the savanna floor and establishing into the late summer and early fall, as weather conditions and site availability permitted.

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A. B.

A B A B A C C

Figure 9. A. The average number of leaves per plant for each of the lupine species, L. albus (CW), L. polyphyllus (GL), L. perennis (WB) in 2009. B. The average height of each lupine species, L. albus (CW), L. polyphyllus (GL), L. perennis (WB) in 2009. Note that L. perennis in the field plot did not set flower, therefore height measurements are taken from the established plots in the Black Oak Savanna.L. albus n=27 (field plot), L. polyphyllus n=63 (field plot), L. perennis n=64 (field plot), L perennis n=283 (ABOS). Box plot shows 75% percentile, 25% percentile and median with the min and max values for number of leaves and heights (b) per plant measured. Letters indicate significate difference, as tested by one way ANOVA, with Bartlett’s test for equal variance and Tukey multiple comparison test, P< 0.05.

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A. B. B A B C

AC A A C

Figure 10. A. The number of flowers per plant L. albus (CW), L. polyphyllus (GL), L. perennis (WB) in 2009 and over five years 2007-2011. B. The number of pods per plant L. albus (CW), L. polyphyllus (GL), L. perennis (WB) in 2009 and over five years 2007-2011. L. albus n=35 (field plot), L. polyphyllus n=35 (field plot), L. perennis 2009 n=404 (ABOS), L perennis 2007-2011 n=375 (ABOS). Note that L. perennis in the field plot did not set flower, therefore reproductive measurements are taken from the established plots in the Black Oak Savanna.

Box plot shows 75% percentile, 25% percentile and median with the min and max values for number of flowers (a) and pods (b) per plant measured. Letters indicate significate difference, as tested by one way ANOVA, with Bartlett’s test for equal variance and Tukey multiple comparison test, P< 0.05.

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A. B. B A

A A B A

Figure 11 A. The number of seeds per plant for L. albus (CW), L. polyphyllus (GL), L. perennis (WB) in 2009. B. The number of seeds per pods per pod for L. albus (CW), L. polyphyllus (GL), L. perennis (WB) in 2009. L. albus n=43(field plot), L. polyphyllus n=35 (field plot), L. perennis 2009 n=42 (ABOS). Note that L. perennis in the field plot did not set flower, therefore reproductive measurements are taken from the established plots in the Black Oak Savanna.

Box plot shows 75% percentile, 25% percentile and median with the min and max values for number of seeds per plant (a) and number of seeds per pod (b) per plant measured. Letters indicate significate difference, as tested by one way ANOVA, with Bartlett’s test for equal variance and Tukey multiple comparison test, P< 0.05.

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A. B. B AC ABC AC

Figure 12A. The average number of leaves per L. perennis for each population of lupine in the Black Oak Savanna in 2009. B. The average height of L. perennis for each population of lupine in the Black Oak Savanna in 2009. Bowl East n= 78, Bowl Central n=63, Bowl West n=55, Hog’s Back n=33.

Box plot shows 75% percentile, 25% percentile and median with the min and max values for number of leaves and heights (b) per plant measured. Letters indicate significate difference, as tested by one way ANOVA, with Bartlett’s test for equal variance and Tukey multiple comparison test, P< 0.05.

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A. B.

Figure 13 A. The number of flowers per plant in the wild lupine quadrates at ABOS for over five years, 2007-2011. B. The number of pods per plant in the wild lupine quadrates at ABOS for over five years, 2007-2011. Box plot shows 75% percentile, 25% percentile and median with the min and max values for number of flowers (a) and pods (b) per plant measured. 5 yr. average 18.4 ± 1.2 (6.7-26.7) flowers and 6.1 ±0.4 (4.5-7.3) pods per plant (n=2226)

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Figure 14. Total and average precipitation for the Alderville Black Oak Savanna from 2007-2011

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Chapter 3: Extended methods

Hormone extraction Extraction of CK from homogenized plant tissue followed the procedure established in Emery et al 2006, and also described by Powel et al 2013. Frozen tissue was homogenized using the MM300 Ball Mill (Retsch, Switzerland) at a speed of 25 rad/s for 5 minutes in pairs of steel containers, with a steel ball in a small amount of

-1 cold Bieleski #2 Fixative ([CH3OH/H2O/HCOOH] 15/4/1, v/v/v) along with 10 ng μL of CK and 50 ng μL-1 of ABA deuterated internal standards. The CK internal standard consisted

2 2 of 50 ng of each of the following: [ H6]iP (isopentenyl adenine), [ H6][9R]iP (isopenteyl

2 2 adenosine), [ H6][9RMP]iP (isopentenyl adenosine 5' monophosphate), trans-[ H5]Z

2 2 2 (zeatin), trans-[ H5[9R]Z (zeatin riboside), [ H3]DHZ (dihydrozeatin), [ H3][9R]DHZ

2 (dihydrozeatin riboside), [ H6][9RMP]DHZ dihydrozeatin 5' monophosphate, and

2 [ H4]ABA (abscisic acid) (OlChemIm Ltd., Olomouc, Czech Republic). The pulverized plant material was collected into 15 ml tubes. Additional Bieleski extraction buffer was used to wash any remaining pulverized seed tissue and stored over night at -20ºC (47).

The solution was centrifuged for 15 minutes until a distinct pellet formed of plant tissue at the bottom of the 15 ml tube. The supernatant was collected and the pellet re-suspended by adding additional cold Bieleski’s solution and vortexing and sonicating the pellet and storing at -20ºC before re-extracting the pellet (26). The combined supernatant was dried in either in a 15 mL tube with a speed vacuum concentrator (SPD11V-115 Thermo Electron Corporation, Mississauga, Ontario) or in a

111 round-bottomed flask with a Büchi Rotavapor R-200 (Postfach, Flawil, CH), both of which operated in vacuo at ~40 °C.

The dried samples were reconstituted in 5 mL of 1M formic acid (HCOOH), and the pH verified to be between 1.4 and 2.0 to ensure CKs remained protinated for further purification and separation using Reverse-Phase-Ion exchange. MCX columns, (6 cc, with 150mg of sulfonated sorbent) were used to separate ABA from CK using cation exchange and reverse phase characteristics (26). Any plant material remaining in the samples was spun down during centrifuging as the columns were activated using analytical grade methanol (MeOH) and 1M formic acid (HCOOH). The samples were loaded in the columns and washed with 1M formic acid (HCOOH). ABA was the first to be eluted and collected using analytical grade methanol (MeOH). Collection tubes were changed and CK-NTs were eluted using 0.35 M ammonium hydroxide (NH4OH). Once again, collection tubes were changed and FBs and RSs were eluted with 0.35 M ammonium hydroxide (NH4OH) in 60% (v/v) methanol (MeOH). Collected under slight vacuum pressure, at a flow rate not exceeding 1.0 mL/min. All samples were dried using the SpeedVac Concentrator.

NTs were further processed by reconstituted in 1 mL of 0.1 M ethanolamine (pH

10.4) and adding three units of a bacterial phosphatase enzyme. Samples were placed in a 37°C water bath overnight then dried to ensure compete bacterial action cleaving the phosphates, transforming the NTs into RSs that can be detected by the HPLC-

MS/MS. NTs were further purified by running them through C18 solid phase extraction columns (500 mg, Accubond ODS; Fisher Scientific, Mississauga, Ontario). The columns

112 were conditioned using 10 mL of methanol (MeOH) and then 20 mL of Milli-Q water while the samples were brought up in 4 mL of Milli-Q water. The samples were then loaded into the columns then washed with 20 mL of Milli-Q water. The RSs (previously

NTs) were eluted using 15 mL of an 80:20 (v/v) methanol (MeOH) and Milli-Q water solution then dried.

All samples were reconstituted in 500μL of methanol (MeOH), sonicated and vortexed then transferred into a 1.5 mL tube. This was done three times, then the

1.5mL sample dried. Samples were loaded into vial inserts and analyzed by the HPLC-

ESI-MSMS (26, 16v 2). ABA samples were kept in amber glass and wrapped in tinfoil to reduce light degradation (15, 121)

HPLC-MS/MS HPLC-ESI-MS/MS methods follow those described by Ferguson et al 2005 (47).

Specifications of the instrumentation used as well as particulars about run times, limits of quantification, detection limits can be found in Powell et al 2013 and Farrow and

Emery 2012. Both CK and ABA fractions were assessed using the Agilent 1100 binary

HPLC system (Mississauga, Ontario), utilizing as turbo V-spray ionization source. All CK samples were analyzed in positive-ion mode by the ABI 4000 triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Concord, Ontario).

A sample volume of 20 µL was run through a Finesse Genesis C18 reversed-phase column (4µm, 150 x 2.1 mm; Jones Chromatography, Foster City, California). The CKs were eluted from the column with a gradient of MeOH (A) and 0.08% acetic acid (B)as described in Farrow and Emery 2013. At a flow rate of 400 µL/min. Initial conditions were 1% A and 99% B, increasing linearly to 50% A and 50% B over ten minutes, then

113 immediately changing to 99% A and 1% B after 10.1 minutes. This was maintained until the conditions were immediately switched back to initial conditions 15 minutes after commencement of the run. The column was then allowed to re-equilibrate to 19 minutes, at which time the run ended.

Data interpretation The peaks generated in each run were subsequently analyzed and integrated using Analyst (version 4.2.1) software. Using the three biological replicates, average abundances and their corresponding standard errors were calculated for each of the CK types then aggregated to find total CK amounts (42, 91). This was also done for the ABA samples. Statistical analysis was performed in Graphpad Prism 5 where two-way

ANOVAS were used to test for significate differences among values. One-way ANOVAs with Tukey’s post hoc were used to test for difference among replicates for each lupine species between stages of development. One-way ANOVAS were used to test for differences among values for total CK values for each of the lupine species between replicates, Bartlett’s test for equal variances was used along with the Tukey post hoc.

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Table 4. ABA, total CK, and CK FB amounts (average ±SE) for each stage and for each lupine species as well as the ration of ABA: Total CK and ABA: CK-FBs

ABA Total CK Ratio ABA: CK FBs Ratio ABA: mean SE mean SE Total CK mean SE CK FBs 1 190.6 19.6 16829.7 6606.5 0.011 153.6 6 1.2 2 4446.3 165.6 460848.7 62690.5 0.01 487.7 34.1 9.1

3 30243.1 2141 239685 25582.8 0.126 317.6 49.6 95.2

CW 4 335.7 23.7 496.8 249.5 0.676 3.7 0.6 91.7 5 136.9 5.1 20.9 2.8 6.549 2.2 0 62.7 AVE 7070.5 823.6 143576.2 13621.2 0.049 192.9 13.7 36.6 1 2972.4 503 1062.2 77.1 2.798 58.3 3.4 51 2 2354.2 85.7 641.4 247.6 3.67 215 143.6 11 3 3577.2 211.9 934.6 191.2 3.827 27 2.1 132.4 WB 4 1017.7 198.7 956 117.8 1.064 47.8 5.9 21.3 5 1898.8 303.6 79 7.5 24.039 4 0.8 476.9 AVE 2364.8 81.5 734.6 35.7 3.219 60.1 7 39.3 1 1476.8 111.2 611.9 55.1 2.413 83.5 21.8 17.7 2 3262 699.7 1718.6 246.8 1.898 566.9 277.2 5.8

3 3346.1 473.8 778.6 46.4 4.297 65.6 13.7 51 GL 4 633.8 39.3 242.4 17 2.614 15.7 3.8 40.3 5 946.9 92.4 245.2 25.7 3.861 25 4.7 37.8 AVE 1933.1 102.6 719.4 42.2 2.687 151.3 25.5 12.8

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Table 5. Summary of hypothesis, predictions and supporting results

Overarching Why are wild lupines so difficult to spread by seed? question What elements of their phenology and physiology contribute to this? Phenology Minimal effort into WB lupine reproduction are limit their sexual Supported by partially confirmed predictions; Hypothesis reproduction, as compared to CW and GL in the field. Energy is modified hypothesis is that effort is going to not going into their growth either. roots and vegetative reproductions in WB Methodology Measure growth using shoot height, the number of leaves. Measure reproduction effort by counting flowers, and pods. Count number of shoots that put effort into reproduction. Predictions CW Minimal growth; high number of pods; all stems High growth, lowest seeds, most reproduced (as reproduce predicted), showed highest effort (as predicted) WB Minimal growth; low number of pods; few stems Lowest growth (as predicted), highest seeds, reproduce very few stems reproduced (as predicted) GL Lots of vegetative growth; high number of pods; many Lower growth, moderate seeds, few stems stems reproduce reproduced Physiology WB CK and ABA abundance, types and timing are limiting their Supported by confirmed predictions Hypothesis sexual reproduction Methodology Profile CK types and ABA through five stages of seed development Predictions CW Low ABA at final stages; high amounts of CK, ABA; tZ-CKs Observed as predicted WB High ABA at final stages; lower amounts of CK; cZ-CKs Observed as predicted GL Low ABA at final stages; lower amounts of CK; cZ-CKs Observed as predicted