DORMANCY AND GERMINATION CHARACTERSITICS OF TWO FLORIDA NATIVE FORBS, ASCLEPIAS HUMISTRATA AND LUPINUS DIFFUSUS

By

GABRIEL CAMPBELL

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2016

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© 2016 Gabriel Campbell

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To Mother Earth, may we learn to appreciate your giving and to understand your mystery

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ACKNOWLEDGMENTS

I would like to thank all of my friends and family for their kind words of encouragement and wisdom. I could not have completed this document without the support of Dr. Mack Thetford, Dr. Debbie Miller, and Dr. Hector Perez. A shout out to the Moore family, Anna Dicks, Blakely Gill and the Perez Seed Lab for collecting seeds and graciously volunteering their time and expertise. Lastly, I would like to thank everyone involved in the Environmental Horticulture Department at the University of

Florida.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

ABSTRACT ...... 10

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ...... 12

Florida Panhandle and its Plants ...... 12 Florida Panhandle Sandhills and their Plants ...... 12 Florida Panhandle Barrier Islands and their Plants ...... 13 Are Florida Ecosystems in a State of Crisis? ...... 15 Restoration Ecology and Biodiversity...... 15 Seed Dormancy and Imbibition ...... 17 Experimental Preface...... 19

2 ASCLEPIAS HUMISTRATA SEED GERMINATION ...... 20

Introduction ...... 20 Asclepias ...... 20 Asclepias seed germination ...... 22 Asclepias humistrata ...... 23 Materials and Methods...... 24 Seed Collection and Storage ...... 24 Alachua County ...... 25 Citrus County ...... 25 Santa Rosa County ...... 26 Volusia County ...... 27 Seed Characteristics ...... 27 Seed Viability Tests ...... 27 Seed Imbibition Tests ...... 28 Germination Experiments ...... 28 Preliminary experiments ...... 29 Temperature gradient experiment ...... 30 Follicle maturity experiment ...... 30 Seed size experiment ...... 31 Illumination experiment ...... 31 Statistical Analyses ...... 31 Results ...... 32 Seed Characteristics ...... 32

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Seed Viability Tests ...... 32 Seed Imbibition Tests ...... 33 Germination Experiments ...... 33 Preliminary experiments ...... 33 Temperature gradient experiment ...... 34 Follicle maturity experiment ...... 35 Seed size experiment ...... 35 Illumination experiment ...... 35 Discussion ...... 36

3 LUPINUS DIFFUSUS SEED GERMINATION ...... 57

Introduction ...... 57 Lupinus ...... 57 Lupinus Seed Germination ...... 58 Lupinus diffusus ...... 59 Fire and Seed Germination ...... 60 Experimental Preface ...... 61 Materials and Methods...... 61 Seed Collection and Storage ...... 61 Seed Viability Tests ...... 62 Seed Imbibition Tests ...... 63 Germination Experiments ...... 63 Smoke-infused water experiment ...... 64 Rock polisher experiment...... 65 Freeze followed by hot bath experiment ...... 65 Scarification experiment ...... 66 Statistical Analyses ...... 67 Results ...... 67 Seed Viability Tests ...... 67 Seed Imbibition Tests ...... 67 Germination Experiments ...... 67 Preliminary experiments ...... 67 Scarification experiment ...... 68 Discussion ...... 68

4 CONCLUSIONS ...... 80

APPENDIX

A TETRAZOLIUM TESTING ...... 81

B SURVIVAL ANALYSIS...... 82

LIST OF REFERENCES ...... 85

BIOGRAPHICAL SKETCH ...... 96

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LIST OF TABLES

Table page

2-1 Summary of collection sites and seed characteristics for Ascelpias humistrata seedlots ...... 41

2-2 Viability percentages (n=100) using TZ staining of 13 Asclepias humistrata seedlots at different times after seed harvest...... 42

2-3 Effects of temperature and seedlot on germination probability for all - Preliminary Experiments on 2 seedlots of Asclepias humistrata...... 43

2-4 Effects of temperature and seedlot on germination probability for 7 seedlots of Asclepias humistrata in the Temperature Gradient Experiment ...... 44

2-5 Orthogonal contrasts of Temperature on germination probability across 7 seedlots of Asclepias humistrata in the Temperature Gradient Experiment ...... 45

2-6 Effects of follicle maturity and seedlot on germination probability for 3 populations of Asclepias humistrata...... 46

2-7 Effect of light on germination for 4 seedlots of Asclepias humistrata for the Illumination Experiment ...... 46

3-1 Viability (%) for 2 seedlots of Lupinus diffusus following 7 and 19 months of storage ...... 72

3-2 Summary of final germination percentages for all Lupinus diffusus Preliminary Experiments ...... 73

3-3 Effects of combinations of time in bleach and time scarifier on germination of 2 seedlots of Lupinus diffusus germination probability for the Scarification Experiment ...... 74

3-4 Summary of final diseases percentages pooled by time in bleach (n=1200), population (n=1900), and time in scarifier (n=600) for Lupinus diffusus in the Scarification Experiment ...... 75

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LIST OF FIGURES

Figure page

2-1 Seed imbibition curves for 2 Asclepias humistrata seedlots ...... 47

2-2 Effect of temperature and seedlot on relative percentages of germinated, viable, non-viable, and disease seeds for 2 seedlots of Asclepias humistrata exposed to seasonal temperatures in light and dark ...... 48

2-3 Effect of temperature and seedlot on relative percentages of germinated, viable, non-viable, and disease seeds for 2 seedlots of Asclepias humistrata exposed to seasonal temperatures in light and dark ...... 49

2-4 Effect of temperature and seedlot on relative percentages of germinated, viable, non-viable, and disease seeds for 7 seedlots of Asclepias humistrata in the Temperature Gradient Experiment ...... 50

2-5 Inverted Kaplan-Meier curves showing effect of temperature on germination probability for 4 seedlots of Asclepias humistrata used in the Temperature Gradient Experiment ...... 51

2-6 Inverted Kaplan-Meier curves showing effect of temperature on germination probability for 3 seedlots of Asclepias humistrata used in the Temperature Gradient Experiment ...... 52

2-7 Inverted Kaplan-Meier curves showing effect of Asclepias humistrata follicle maturity on germination probability representing 3 geographic locations in the Follicle Maturity Experiment...... 53

2-8. Effects of Asclepias humistrata follicle maturity on relative percentages of germinated, viable, non-viable, and diseased seeds representing 3 geographic locations in the Follicle Maturity Experiment ...... 54

2-9 Effects of seed size on relative percentages of germinated, viable, non- viable, and disease seeds for 2 seedlots of Asclepias humistrata in the seed Size Experiment ...... 55

2-10 Effect of light on relative percentages of germinated, viable, non-viable, and disease seeds for 4 seedlots of Asclepias humistrata in the Illumination Experiment ...... 56

3-1 Seed imbibition curves for nicked and unaltered seeds of 2 Lupinus diffusus seedlots ...... 76

3-2 Effects of combinations of time in bleach and time scarifier of 2 seedlots of Lupinus diffusus on relative percentages of germinated, viable, diseased, and non-viable seeds for the Scarification Experiment ...... 77

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3-3 Inverted Kaplan-Meier curves showing effects of a combination of time in bleach and scarifier on germination probability for the Santa Rosa County 2015 seedlot of Lupinus diffusus used in the Scarification Experiment ...... 78

3-4 Inverted Kaplan-Meier curves showing effects of a combination of time in bleach and scarifier on germination probability for the Walton County 2015 seedlot of Lupinus diffusus used in the Scarification Experiment ...... 79

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

DORMANCY AND GERMINATION CHARACTERSITICS OF TWO FLORIDA NATIVE FORBS, ASCLEPIAS HUMISTRATA AND LUPINUS DIFFUSUS By

Gabriel Campbell

December 2016

Chair: Mack Thetford Cochair: Debbie Miller Major: Horticulture Sciences

Asclepias humistrata (sandhill milkweed) and Lupinus diffusus (sky-blue lupine) are two Florida native forbs with potential uses in restoration projects and as ornamental landscape plants. No information exists on how to sexually propagate A. humistrata and the single paper on L. diffusus seed germination reported only 41% germination after chemical scarification. The objective of this study was to test for seed dormancy and describe germination requirements for A. humistrata and L. diffusus.

Thirteen Seedlots of A. humistrata were categorized by location, year, collection method, and seed characteristic, and were germinated in varying temperature and light exposures. Two seedlots based on location of L. diffusus were exposed to various dormancy alleviation techniques that included mechanical scarification, hot water baths with and without freezing, and exposure to smoke-infused water. A. humistrata had high

(greater than or equal to 70%) germination in light or dark with constant or fluctuating temperatures after 28 days. Orthogonal contrasts show optimal germination of 7 seedlots of A. humistarata exposed to a 12-hour photoperiod at constant temperatures was between 24 and 28 degrees Celsius after 28 days. However, survival analysis reveals

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seed source, seed size, and collection methods effect germination of A. humistrata.

Physical dormancy of L. diffusus seeds was eliminated and high germination percentage was achieved by 20-40 seconds of exposure in an electric seed scarifier followed by 15-

30 seconds in bleach. Bleached and scarified seeds were then incubated 23 days with a

12-hour photoperiod. Survival analysis indicates a significant three-way interaction (time in scarifier X time in bleach X seedlot), suggesting that a combination of bleach and scarification treatments differ across seedlots of L. diffusus.

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CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW

Florida Panhandle and its Plants

Almost a quarter of plant species found in the U.S can be found in Florida, including, 243 families, 1,402 genera, and 4,255 species (Wunderlin and Hansen 2011).

Of these 4,255 species, 2,857 are native, 229 are endemic, 500 are threatened or endangered, 1,398 are exotic, and 137 are invasive exotic (FNAI 2010; Wunderlin and

Hansen 2011). Florida’s plant biodiversity results from its unique geographic location and climate that ranges from subtropical in the extreme south to temperate in the northern part of the panhandle with a plant USDA hardiness zone of 10b and 8b, respectively. The panhandle of Florida constitutes the northwestern most portion of the state of Florida and is one of the most biologically diverse areas in North America

(Blaustein 2008). According to Clewell (1985), the Florida panhandle contains around

70% of the plant biodiversity found in all of Florida. Unfortunately, it is vulnerable to biodiversity loss due to climate change, sea level rise, and habitat loss (Cameron Devitt and Seavey 2012; Mulholland and others 1997; Reece and others 2013).

Florida Panhandle Sandhills and their Plants

Sandhills are xeric upland plant communities found on deep sandy ridges with well drained acid soils found throughout the panhandle of Florida (FNAI 2010). Sandhills are characterized by a tall scattered canopy of Pinus palustris (longleaf pine) and a midstory of Quercus laevis (turkey oak), Q. incana (bluejack oak), Q. margaretta (sand post oak), Q. geminata (sand live oak), and Crategus lacrimita (Pensacola haw) (FNAI

2010). Sandhill ground cover consists of Aristida beyrichiana (wiregrass), Sporobolus junceus (pineywoods dropseed), Schizachyrium scoparium (little bluestem), Licania

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michauxii (gopher apple), Serenoa repens (saw palmetto), and Gaylussacia dumosa

(dwarf huckleberry) (FNAI 2010). Sandhills are pyrogenic plant communities with low intensity ground fires that occur every 1-3 years (FNAI 2010).

Sandhills are part of a larger longleaf pine forest system that previously dominated the southeastern U. S., but logging practices and land conversion have destroyed all but 3% of longleaf pine habitat (Myers and Ewel 1990). Sandhills have been reduced by over 90% and in most cases the remaining sandhills are highly degraded and fragmented (Myers and Ewel 1990). Sandhill groundcover is more diverse than some other Florida panhandle vegetation types (Monk 1968), with the possibility of 50 or more herbaceous species within a several square meter area

(Clewell 1986).

Florida Panhandle Barrier Islands and their Plants

The coastline of the western half of the Florida panhandle is composed of narrow sandy linear barrier islands that parallel the mainland (Myers and Ewel 1990). Unlike south Florida’s coastal sand that is composed of calcium carbonate, north Florida coastal sand is composed of river deposited quartz from the granitic Appalachians, reworked by waves to create beaches and barrier islands during the Plestociene epoch

(Myers and Ewel 1990).

Barrier islands are highly sought out for residential development due to their proximity to beaches, water, and other tourist attractions. In the Gulf and Atlantic regions of the eastern United States, barrier island population densities are three times higher than coastal states and have increased 14% from 1990 to 2000, with 700,000 residents in Florida alone (Zhang and Leatherman 2011). Interestingly, barrier islands are not static and change constantly due to wind, waves, currents, tides and storms,

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posing problems for long-term sustainable inhabitance. These islands are especially vulnerable to sea level rise and hurricanes due to their location in hurricane prone areas and low elevations (Titus and others 1991).

For plants, Florida Panhandle Barrier Islands (FPBIs) pose unique environmental challenges, such as salt spray, desiccation, and stochastic large-storm events (Snyder and Boss 2002), as a result, uniquely adapted plant communities reside on FPBIs.

Beach Dunes, Coastal Interdunal Swales, Coastal Grasslands, Marine Salt Marshes, and Coastal Scrub plant communities can all be found on FPBIs (FNAI 2010). Beach

Dunes are seaward, Coastal Scrubs occur at the highest elevations, and the rest of

FPBIs plant communities blend into an undulating matrix with embedded plant communities. Unfortunately, habitat destruction and habitat fragmentation from land development and recent hurricanes, create a need for restoration and conservation of

PFBIs plant communities and their plants.

Studies on FPBIs plants are diverse (Williams 2007) but have focused mainly on dune building and stabilizing plants which include Uniola paniculata (sea oats) (Barrios

2007; Gibson and others 1995; Hooton and others 2014; Stalter 1974; Sylvia 1989;

Williams 2007), Panicum amarum (bitter panicgrass) (Gibson and others 1995; Seneca and others 1977; Williams 2007), Cakile constricta (Gibson and others 1995) and Iva imbricata (Colosi and others 1978; Gibson and others 1995; Stalter 1974). However, perennial forbs found on PFBIs are not well studied despite their importance for PFBIs biodiversity and should be used in PFBIs plant restoration, xeriscaping, and native plant gardens within the state of Florida and beyond.

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Are Florida Ecosystems in a State of Crisis?

According to Myers and Ewell (1990), the arrival of Europeans to Florida marked a cataclysmic change for the health of ecosystems in Florida. Fire suppression, habitat fragmentation and conversion, and hydrology alteration permanently changed the land

(Myers and Ewell 1990). Land was deforested and soil was cleared for fuel and phosphate processing from the Tampa Bay area up the Suwanee River Basin cutting into the panhandle. The center of the state was converted to citrus plantations and in the southern part of the state wetlands were drained en masse for agriculture and development (Meyers and Ewell 1990), marking the end of many indigenous people’s relationship with their ancestral lands.

As Taylor (2013) stated, never in Florida’s history has the land been pillaged and raped as they are currently, with no community immune. Half of the pine flatwoods are gone, long leaf pine savannahs in Sandhill communities no longer prevail, hardwood communities are fragmented possibly beyond repair, 60% of upland coastal plant communities have disappeared, 95% of Pine Rocklands in the south have been destroyed by conversion or agriculture, and sand pine scrubs have been logged despite their endemism to Florida (Taylor 2013). Today, Florida has the third largest population in the United States with an estimated 20 million citizens and is the second largest producer of agricultural products attracting an undocumented amount of migrant farm workers, both point to further future ecosystem degradation.

Restoration Ecology and Biodiversity

Restoration ecology is a relatively new and exciting scientific field that holds promise in combining multiple disciplines (e.g., biology, botany, engineering, horticulture, and ecology), to improve the health of the planet by restoring valuable

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ecosystem services and functions, and biodiversity. The Society for Ecological

Restoration defines ecosystem restoration as “the process of assisting the recovery of an ecosystem that has been degraded, damaged, or destroyed” (SER 2002). An important component for any restoration project is deciding whether active revegetation is needed, and if so, what type of propagule to use and where to acquire plant material

(Johnson and others 2010; Jones and Monaco 2009; Mijnsbrugge and others 2010).

Conserving genetic diversity during revegetation projects is a widely recognized necessity for the long term survival of reintroduced plant populations (Johnson and others 2010; Jones and Monaco 2009; Kettering and others 2014; Sharma and others

2011), but questions still remain on how to restore populations that mimic the genetics and resilience of natural populations (Bischoff and others 2006; Dolan and others 2007;

Hufford and Mazer 2003; McKay and others 2005; Mijnsbrugge and others 2010;

Montalvo and others 1997; Sackville Hamilton 2001; Wilkinson 2001). Inbreeding depression, or reduced vigor and fitness due to inbreeding, and out breeding depression, or reduction of fitness of progeny due to distance of parents, are both important considerations for choosing plant materials that will be successful and representative of natural populations in any restoration project (Kaye 2010).

Regardless, there have been real world success stories that conserve the genetic integrity of plant populations during revegetation projects (Dolan and others 2008;

Pierson and others 2007).

Biodiversity is defined as the variety of species, genes within species, and ecosystems which sustain them. The economic, social, and spiritual value of biodiversity is transcended by our species’ dependence on it for survival (Gowdy 1997;

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Ghilarov 2000; Guo and others 2010). Current and predicted catastrophic habitat loss, habitat alteration, and climate change are well documented threats to global biodiversity

(Brooks and others 2002; Hoekstra and others 2005; Jantz and others 2015; Mantyka-

Pringle and others 2012). Predictions for future species extinction are as dire as they are convincing (Ceballos and others 2015; De Vos and others 2015; Ehrlich 1994;

Pimm and others 1995; Valiente and others 2015), yet uncertainty remains over the severity of species extinction and methods used to measure it (Dawson and others

2011; Fattorini and Borges 2012). Regardless, as reviewed in Bellard and others

(2012), the vast majority of models predict catastrophic biodiversity decline in the near future. A key step in preserving biodiversity and restoring terrestrial ecosystems is understanding the plants that sustain them.

Seed Dormancy and Imbibition

Angiosperms, or flowering plants, produce flowers that when pollinated form seeds that ultimately produce a new genetically unique plant. Seeds either germinate immediately or are dormant. Dormancy can be defined as a seed trait, present at shedding from the mother plant, that inhibits the completion of germination for intact, viable seeds exposed to favorable environmental conditions (Baskin and Baskin 2014;

Baskin and Baskin 2004). Seed dormancy has been separated into five classes: physiological (PD), morphological (MD), morpho-physiological (MPD), physical (PY), and combinational (PY+PD) (Baskin and Baskin 2004). Physiological dormancy manifests as a physiological inhibiting mechanism that limits the growth potential of embryos. Embryos with limited growth potential cannot rupture covering structures such as the testa to complete the germination process. A low embryo growth potential has been correlated with a high abscisic acid to gibberellic acid ratio. Physiological

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dormancy is further separated into non-deep PD, intermediate PD, and deep PD

(Baskin and Baskin 2014; Baskin and Baskin 2004). Non-deep PD dormancy describes seeds from which excised embryos produce normal seedlings, germinate in response to relatively low levels of gibberellins and cold or warm stratification (e.g. 4 weeks), and may germinate in response to scarification and after-ripening effects (Baskin and Baskin

2004). Intermediate PD dormancy describes seeds which excised embryos produce normal seedlings, may germinate in response to increasing concentrations of gibberellins, and germinate after 2-3 months of cold stratification (Baskin and Baskin

2004). Shorter periods of dry after-ripening treatments may also alleviate intermediate

PD (Baskin and Baskin 2004). Excised embryos from seeds with deep PD produce abnormal seedlings and application of gibberellins will not promote germination (Baskin and Baskin 2004). Seeds with deep PD require > 3-4 months of cold stratification for dormancy alleviation (Baskin and Baskin 2004). Morphological dormancy results when seeds are dispersed with an under-developed embryo (Baskin and Baskin 2004).

Morphological dormancy is alleviated when seeds are exposed to environmental conditions conducive for further embryo development (Baskin and Baskin 2004).

Application of gibberellins and stratification treatments may, in some species, substitute proper environmental conditions (Baskin and Baskin 2014; Baskin and Baskin 2004).

Morpho-physiological dormancy is a combination of morphological and physiological dormancy. Alleviation of dormancy in seeds with MPD is similar to treatments for seeds with PD or MD (Baskin and Baskin 2014; Baskin and Baskin 2004).

Physical dormancy results when the seed testa or other covering structures (e.g. endocarp of indehiscent fruits) is water impermeable. Physical dormancy is broken by

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actions, such as scarification treatments (e.g. mechanical, chemical) or repeated heating-cooling cycles, that render covering structures water permeable (Baskin and

Baskin 2014; Baskin and Baskin 2004). Seed imbibition is a passive process of water uptake by seed cells that marks the beginning of the germination process (Bewley and others 2013). Seed imbibition assays are a simple way to determine seed coat permeability and therefore test for physical dormancy. Seeds are first weighed and then exposed to water. Seeds are then re-weighed at defined increments of time. If seed mass does not increase with increasing exposure to water, then one can deduce a seed is water impermeable and that physical dormancy is present.

Experimental Preface

The objectives of this study are to explore the presence of dormancy and germination characteristics for Asclepias humistrata and Lupinus diffusus, two Florida panhandle native forbs. Seed collection techniques, storage of seeds, pre- and post- germination viability tests, seed surface sterilization techniques, and germination experiments will be used to accomplish the aforementioned objectives.

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CHAPTER 2 ASCLEPIAS HUMISTRATA SEED GERMINATION

Introduction

Asclepias

Asclepias (milkweed) is a genus formerly of the Asclepiadaceae which is now treated as the subfamily Asclepiadoidae within the Apocynaceae (Wunderlin and

Hansen 2011). The following morphological descriptions are found in Heywood and others (2007) and Wunderlin and Hansen (2011). Flowers are actinomorphic, mostly 5- merous (except for carpels) with fused petals varying from rotate or shallowly campanulate to tubular or funnel-shaped, and twisted lobes in the bud overlap to the left or right. Stamens are inserted in the corolla or on the gynostegium and the ovary is superior to subinferior and syncarpous. Fruits are generally ventrically dehiscent follicles. Asclepias has white but occasionally red or yellow sap. Leaves are simple, almost always entire, and are most commonly opposite, frequently whorled, and less often alternately arranged.

Clewell (1985) distinguishes Asclepias from other plants within Apocyanaceae as follows. Asclepias has entire mostly opposite leaves with a minute stipule. Sepals are distinct and basally united petals form an elaborate corona. Connate stamens are essentially adnate to the pistil just below the stigma. Two-carpel pistils are formed by adenation to stamens which compose the gynostegium. In addition, Asclepias transfer their pollen in large clumps called pollinia, resulting in fruits that have seeds from one male genotype (Wyatt and Broyles 1994).

Asclepias have been cultivated across continents and by multiple cultures for hundreds of years, mainly for stem (vascular tissue) and coma fibers (fiber attached to

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seed), and rubber derived from its latex sap (Whiting 1943). Coma fibers were also considered strategic war materials during World War II because of their use in warm clothing, life preservers, and life cushions (Berkman 1949). Currently, Asclepias is being studied for its use in conservation and restoration, effectiveness in the cleanup of oil spills using coma fibers (Choi and Cloud 1992; Rengasamy and others 2011), and use as native road side plants (Johnston and others 2015).

Asclepias play a complex roll in insect ecology (Abdala-Roberts and others

2012). Asclepias interact with aphids (Aphididae), ants (Formicidae), and bees

(Hymenoptera) (Borders and Lee-Mader 2014). Milkweed bugs (Oncpeltus) can be entirely dependent on Asclepias to complete their life cycle, and Asclepias can provide food for multiple insect species at a time (Root and Chaplin 1976).

However, Aslcepias are best known for their role as obligate larval host and food source for the charismatic monarch butterfly (Danaus plexippus). Monarch butterflies migrate from southern Canada to southern Mexico on both the east and west coasts of

North America, making them a spectacular insect that has received much attention by scientists, restoration practitioners, and the public alike. Monarch butterflies found in the eastern United States over winter in staggering numbers in the mountains of central

Mexico within Oyamel fir tree (Abies religiosa) forests (Anderson and Brower 1996;

Davis and Howard 2005). Upon the arrival of spring, reproductive diapause in monarchs ends (Herman 1981), and a single large mating event occurs before a mass migration northward begins (Van hook 1996). Pregnant females bound for the eastern U. S. deposit eggs on young Asclepias plants along the coast of the Gulf of Mexico (Brower

1995). As the eggs hatch and butterflies mature they migrate farther north where they

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form 2 or more breeding generations by the end of summer which migrate east over the

Appalachian Mountains until fall comes and they migrate back to Mexico (Brower 1996;

Brower 1995). Monarch butterflies breed as they pass through the pandhandle of

Florida during both spring and fall migration paths and some overwinter in southern

Florida (Luna and Dumroese, 2013), making the Asclepias population in the Florida panhandle critical for south Florida monarch populations. There are 18 naturally occurring Asclepias species in the panhandle of Florida, of which 2 are endemic, and are found in almost every plant community (Clewell 1985; Wunderlin and Hansen 2011).

Asclepias seed germination

Most studies of Asclepias seed germination describe pre-germination treatments which improve germination and are reviewed by Borders and Lee-Mäder (2014) in their conservation guide for milkweeds. Germination of Asclepias syrica (common milkweed) is overwhelmingly the most studied of all Asclepias species but studies are limited to geographic locations well north of Florida (Northeastern U.S. or southern Canada).

Asclepias syriaca seeds are innately dormant (Baskin and Baskin 1997; Omega and Fletcher 1972). Germination of 2, 3, and 9 week cold stratified seeds from A. syriaca germination was high (≥70% germination) compared to non-stratified seeds

(<10% germination) in light and dark when germinated at 35/20 or 30/15°C day/night temperature fluctuations but fell to less than 20% at 20/10°C for both stratified and non- stratified seeds (Baskin and Baskin 1997). Omega and Fletcher (1972) additionally found that exogenous hormones, particularly a combination of kinetin and gibberellic acid, removal of seed coat, and exposure to gases ball broke Canadian A. syriaca seed dormancy, with up to 100% germination for seeds with their seed coats removed.

Farmer and others (1986) tested 21 A. syriaca seedlots from 6 states in the

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Northeastern part of the U.S. and found that alternating day/night temperatures of

30/20°C was superior (94% germination) to a constant temperature of 30 and 25°C

(75% germination). Bhowmik (1978) found seed dormancy decreased as seed storage time increased when stored at -12, 5, and 21°C, and found that optimal germination temperature of A. syriaca was 27°C with almost 95% germination after 35 days.

Greene and Curtis (1950) compared germination percentages for cold-stratified

Wisconsin Ascleapis seeds (1-3 months at 4°C) against a non-stratified control for A. tuberosa and A. verticillata and found germination percentages increased (54 to 90%) and decreased (40 to 0%), respectively. A. ovalifolia and A. sullivantii had 77 and 89% germination after 4 and 5 months, respectively, when overwintered outdoors in

Wisconsin in germination flats (Greene and Curtis 1950).

Information for light-requirements of Asclepias seeds is scarce and conflicting

(Borders and Lee-Mäder, 2014). Light has been reported as necessary (Cullina, 2000;

Deno, 1993), unnecessary (Mitchell, 1926), and unnecessary but beneficial (Deno,

1993) for Asclepias seed germination. Existing literature does not consider pod maturity or seed size as possible factors in Asclepias seed germination.

Asclepias humistrata

Asclepias humistrata, is a slow-growing perennial that grows in Sandhills, ruderal areas, and locations landward of foredunes on barrier islands (Taylor 2013). A. humistrata has up to 3-foot-long erect to prostrate ascending stems with sessile, clasping, and glabrous dull green to yellow to purple leaves with pink veins (Taylor

2013). Flowers are terminal with pink to lavender umbels that have 5-lobed reflexed calyces and 5-lobed corollas (Taylor 2013). Fruits are erect narrow follicles that contain

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numerous seeds attached to a coma (floss-like fibers) that aids in wind dispersal (Taylor

2013), much like a dandelion.

Asclepias humistrata is a well-documented and consistent larval host for the monarch butterfly (Cohen and Brower 1982; Knight and others 1999; Malcolm and others 1987; Martin and others 1992). Monarch butterfly spring establishment is dependent on the health and growth stage of A. humistrata in the gulf coast of the USA, and on the timing upon which the monarchs migrate (Knight and others 1999).

Additionally, A. humistrata has been studied for a class of herbivory deterring steroids collectively known as cardenolides (Agrawal and others 2012). Cardenolides are found within A. humistrata leaf tissue which monarch butterflies ingest to deter predation

(Cohen and Brower 1983; Martin and others 1992; Nishio and Blum 1982).

A review of the literature reveals no information regarding A. humistrata germination requirements or propagation protocols. The overall objective of this work was to explore factors effecting germination of A. humistrata by addressing the following questions: 1) do seeds exhibit dormancy 2) how do constant or fluctuating temperatures influence germination 3) what role does follicle maturity and seed size play in germination and 4) does exposure to light inhibit germination?

Materials and Methods

Seed Collection and Storage

Dehisced follicles or follicles which dehisced upon the slight application of pressure were characterized as mature. Closed follicles which made an audible

“crunching” sound when squeezed but did not dehisce were characterized as immature.

Fully developed follicles of A. humistrata were cut from plants, immediately placed into paper bags, and placed in an opaque backpack for transport from the field to the lab. No

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more than half of the follicles from a single plant were collected as to reduce impacts of over-collection. Seeds were removed from their coma (floss fibers attached to seed) within 48 hours, allowed to air dry on paper towels for approximately 2 weeks, and ultimately stored in airtight jars at ambient laboratory conditions (≈24°C, 35% relative humidity.). A summary of seed collection site characteristics and dates of collection are provided in Table 2-1. Seedlots for the duration of this paper will refer to seeds with a similar geographic, temporal (year), collection method (immature versus immature), and seed characteristic (size).

Alachua County

Seeds from Alachua County in Hawthorne, FL were collected within a 10-mile radius of The Little Orange Creek Nature Park (29.621650, -82.068016) from late May to early June 2016 and stratified into mature and immature seedlots based on follicle maturity at the time of collection. The plants were on a recently restored Sandhill. The restoration effort was limited to mechanical understory Quercus (oak) tree removal that was mulched on site. No plants were found growing from under the mulch, they were strictly found in open sunny areas with bare mineral soil in Millhopper sands (USDA Soil

Survey). Controlled burns had been conducted within the last 2 years from collection date.

Citrus County

Seeds from Citrus County, FL (28.706603, -82.525908) were collected on the first week of June 2016 from the south side of US Route 98 in a 3-mile stretch between

The Suncoast Parkway and U.S. Route 19. Plants were located in a Scrub plant community with Candler fine sand and Astatula fine sand (USDA Soil Survey 2016).

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Santa Rosa County

Santa Rosa County collection sites included both coastal and inland locations.

Seeds from two A. humsitrata coastal populations were located 10-miles apart at

Pensacola Beach (30.349781, -87.051866) and Navarre Beach (30.378485, -

86.896179), in a highly fragmented barrier island (Santa Rosa Island, FL), and harvested from early June to late August 2015. Pensacola and Navarre beach collections were then combined as a single coastal seedlot. Seeds collected in

Pensacola Beach spanned a 2-mile-wide area of undeveloped land in Escambia County owned by the University of West Florida while seeds collected in Navarre Beach spanned a 0.5-mile-wide area of undeveloped county land. Both sites were North of state road FL-399. Populations were checked 1-2 times a month for the presence of mature follicles. Plants occurred in coastal grassland communities (FNAI 2010), often on the slopes of dunes on relatively high elevations. The Navarre beach population grew in a Newhan-Corolla complex soil while the Pensacola Beach population grew in a

Corolla-Duckston complex soil (USDA Soil Survey 2016).

The inland collection site was within or adjacent to Blackwater River State Forest,

FL, (30.704973, -86.874116), and was harvested twice a month from June to August

2015. This population occurred in Sandhill plant communities that had been burned within the last 2 years or on disturbed roadside areas on Lakeland sand (USDA Soil survey 2016).

Seeds from the same locations (coastal and inland) were harvested in 2016 from late May to early July at a frequency of twice a month. Follicles were stratified into mature and immature categories.

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Volusia County

Seeds from Volusia County in DeLeon Springs, FL (29.144228, -81.339911), were collected on May 22, 2016. This seedlot is located adjacent to a cow pasture on an unfertilized and unirrigated field mowed a few times a year (Hoblick, pers. comm).

Soil on this site was Astatula fine sand (USDA Soil Survey 2016).

Seed Characteristics

Average seed weight and area were determined for all A. humistrata seedlots.

Seed weight was determined by to the nearest 0.1 mg gravimetrically. Seed length and width were measured to the nearest millimeter using a ruler. Seeds, at their thickest part, were not more than 1mm. Therefore, seed volumes were not calculated.

Seed Viability Tests

All seedlots were subjected to a pre-germination trial viability test, hereafter referred to as TZ tests, and post germination trial TZ tests were conducted on non- germinated seed. Refer to Appendix A for more information on TZ tests. For all tests, seeds were nicked with a razor blade through the embryonic axis and part of the cotyledons, fully piercing half the seed. Once nicked, seeds were immediately immersed in a 1% solution consisting of 1 gram of 2,3,5-Triphenyltetrazolium chloride (≥95%) for every 100 mL of deionized distilled water. Seeds were placed in glass beakers or petri dishes sealed with Parafilm M® (Bemis NA, Neenah, WI) and were then immediately incubated in the dark at 32.5°C for approximately 24 hours. Pre-germination trial TZ tests consisted of 4 replications of 25 seeds (n=100) and seeds were incubated for 24 hours prior to visual assessment for staining. Seeds were either considered viable or non-viable with a caveat for post-experiment TZ tests where irregular staining (e.g. mosaic patterns of staining) were considered diseased.

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TZ tests were performed on seeds from Santa Rosa county 2015 and Santa

Rosa Island 2015 seedlots in January 2016, approximately 6 months after seed harvest.

Another TZ test was run on Santa Rosa county 2015 and Santa Rosa Island 2015 seedlots along with all other populations in July 2016, 1 year after seed harvesting for

2015 populations and 1-2 months after seed harvesting for 2016.

Seed Imbibition Tests

Santa Rosa County 2015 and Santa Rosa Island 2015 seedlots of A. humistrata were tested for water imbibition ability on January 17, 2016, approximately 6 months after seed harvest. Three replications of 7 seeds (n=21) for each population were placed in separate 60 X 15 mm glass petri dishes (Pyrex®, Germany) on top of one blotter paper immersed in 5 mL of distilled deionized (DI) water.

Seed weight was determined to the nearest 0.01 mg gravimetrically. Seed weights were measured at time 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 12, and 24 hours.

For each of the three replications, seeds were removed at each time interval, wrapped in a paper towel, and weighed individually prior to all seeds being returned to the moistened blotter in the petri dish. Water was added when a film of water around the seeds shrunk to <1mm thick.

Germination Experiments

For all germination experiments the following seed cleaning and germination trial procedures were used. Seeds were surface sterilized as follows in temporal sequence prior to the start of the experiment. Seeds were placed inside a plastic dish with cheesecloth held in place by a rubber band over the dish and placed under cold tap water rinse for 10 min. Water was decanted and seeds were immediately agitated with

10 mL of 50% ethanol for 1 minute inside a 100mL beaker. The ethanol was decanted

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and seeds remained in the beaker. Five milliliters of an 8.25% sodium hypochlorite solution diluted with 45 mL DI water was added to the decanted beaker along with 2 drops of Tween® 20 and placed on a gyroshaker at 60 RPM (Lab-line Inc. Junior

Orbitor shaker, Melrose Park, Illinois) for 10 minutes. Sodium hypochlorite was decanted and seeds were triple washed with DI water.

Seeds for the Preliminary and Temperature Gradient Experiments were put into

60 X 15 mm glass petri dishes on top of 1 blotter paper immersed in 5 mL of DI water.

Seeds from the Follicle Maturity, Seed Size, and Illumination Experiments were put into in 150 X 20 mm glass petri dishes (Pyrex®, Germany) on top of blotter paper immersed in 7 mL of DI water. All petri dishes were wrapped in Parafilm M® and placed in incubators (I-30-VL, Percival Scientific, Inc., Perry, IA, USA) with 57 ± 7 µmol m-2s-1 cool-white fluorescent light. Dark treatments were achieved by double wrapping petri dishes in aluminum foil. Distilled deionized water was added to petri dishes throughout experiments when blotter paper was no longer saturated.

Germination and disease events were recorded every 24 hours for 28 days.

Seeds were removed once germinated or considered diseased. Seeds were considered to have germinated upon the protrusion of the radicle at least 1mm out of the testa.

Seeds were considered diseased and removed from the experiment when over 10% of the seed coat had visible contamination.

Preliminary experiments

A preliminary round of experiments was conducted on Santa Rosa County 2015 and Santa Rosa Island 2015 seedlots of Asclepias humistrata beginning on April 15,

2016 to determine if seed dormancy was present and to see if seeds need constant or fluctuating temperatures and light or dark conditions to germinate. Three replications of

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10 seeds (n=30) were used for each of the 20 treatments. Seeds were exposed to 2 light (light or dark) and 2 temperature regimes (constant or fluctuating). Temperatures were either held constant at 15, 20, 25, 30, 35, or 40°C or fluctuated every 12 hours simulating Florida high/low seasonal temperatures in early spring/late fall, summer, early fall/late spring, and winter at 27/15, 33/24, 29/19, 22/11°C, respectively. For each temperature treatment both light and dark treatments occurred in the same incubator.

Seeds exposed to light were checked for germination events every 24 hours while seeds exposed to dark were unwrapped and checked once a week, never being exposed to more than 60 seconds of ambient white fluorescent light.

Temperature gradient experiment

A temperature gradient experiment was conducted to determine optimal germination temperatures for A. humistrata seedlots. Seeds were surface sterilized by immersion in sodium hypochlorite for 5 min without Tween® 20. On June 29, 2016 seeds from 7 seedlots were placed on a gradient table (Model 5010, Seed Processing

Holland, Enkhuizen, Holland) exposed to a 12-hour photoperiod of 43 µmol m-2s-1 cool- white fluorescent light. Petri dishes from each seedlot were spread equidistance on temperature lanes set at 18, 20, 22, 24, 26, 28, 30, 32 and 34°C. The surface area of the gradient table and size of the petri dish limited the experiment to two replicates of 25

(n=50) seeds.

Follicle maturity experiment

An experiment was conducted to determine the effects of follicle maturity at time of seed harvesting on A. humistrata germination probability and percentage among seedlots representing 3 locations. Alachua County 2016, Santa Rosa County 2016, and

Santa Rosa Island 2016 locations were each stratified into mature and immature

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seedlots (Table 2-1). Four replicates of 25 (n=100) seeds were germinated in light at

25°C.

Seed size experiment

A seed size experiment was conducted to determine if small seeds had different germination percentages for A. humistrata compared to large seeds. Seeds from Santa

Rosa County 2016 were divided into two categories – small and large. Seeds were measured longitudinally and considered small when ≤5 mm and large if >5 mm. Small seeds had a thinner testa, were green, and occurred on parts of the pod farthest away from location of pod dehiscence when compared to large seeds. Four replicates of 25

(n=100) seeds were germinated in the light at 25°C.

Illumination experiment

An illumination experiment was conducted to test for differences of final germination percentage between seeds germinated in light and dark. On July 15, 2015 seeds from Alachua County 2016, Santa Rosa Island 2016, Santa Rosa County 2015, and Citrus County 2016 seedlots were either exposed to light or dark. For each seedlot, four replicates of 25 seeds (n=100) were grown at 25°C for 14 days. Seeds were checked once for germination and disease at the end of the experiment.

Statistical Analyses

Non- and semi-parametric time-to-event analyses, also referred to as survival analysis, were performed to evaluate temporal patterns of germination and build Cox regression models. Theoretical and applied aspects of time-to-event analysis are provided in Appendix B. Germination was the event of interest. Consequently, seeds that germinated during the course of any experiment were coded as 1. Seeds that did not germinate by the final day of the experiment and diseased seeds were right-

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censored and coded as 0. Kaplan-Meier estimates of survivor functions were generated for each covariate (e.g. illumination, population). The null hypothesis that survivor functions were the same within treatments was tested by calculating the log-rank statistic. R version 3.3.1 was used to analyze Kaplan-Meier estimates and the log-rank statistic.

Cox regression models were constructed using the so-called exact method to account for large proportions of tied event times. The proportional hazards assumption was evaluated using Schoenfeld residual analysis. A time-dependent covariate was included in final models if the proportionality assumption was not met (Allison 2010).

Orthogonal linear contrasts and custom hazard ratios were constructed to compare different levels of significant response variables. Cox regression analysis was conducted in SAS version 9.4.

Results

Seed Characteristics

Seed weight and area ranged from 5.53-9.40 mg and 17.0-50.1 mm2 (Table. 2-

1). Excluding immature and small seedlots, average seed weight and area was 6.92 mg and 43.17, mm2, respectively. On average, seeds weighed 28% more when immature yet were 22% smaller than their mature seedlot counterparts. Seeds from the Santa

Rosa Island locations were heavier than seeds from Santa Rosa County locations for

2015 and 2016 by 28% and 20%, respectively (Table 2-1).

Seed Viability Tests

All seedlots had high pre-germination test viability (≥70%) except for Santa Rosa

County 2016 Small, and Volusia County 2016 (Table 2-2). Seeds from Santa Rosa

County 2015 and Santa Rosa Island 2015 seedlots retained their high viability after 12-

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15 months in storage (Table 2-2). Small seeds (≤5mm long) from the Santa Rosa

County 2016 seedlot had low viability (≤50%) compared to very high viability (≥90%) of normal size (<5mm long) seeds from that location (Table 2-2). Seeds from Volusia

County 2016 had the highest rate of diseases (i.e., mosaic staining pattern) while seeds from the Santa Rosa County 2016 small seedlot had the highest rate of unstained embryos. Immature seedlots had similar viability compared to their mature seedlot counterparts for Santa Rosa County 2016 and Santa Rosa Island 2016 seedlots while immature seeds of Alachua County 2016 had higher viability than their mature seedlot counterparts (Table 2-1).

Seed Imbibition Tests

Seeds imbibed water rapidly until hour 6 when imbibition rates approached 0 for both populations (Figure 2-1). Seed coats appeared a deeper shade of brown when imbibed compared to when seeds were dry. Both populations approximately doubled in weight after 24 hours (Figure 2-1). Water imbibition was not altered by nicking the seed testa (data not shown). No visual signs of disease were present.

Germination Experiments

Preliminary experiments

Temperature and seedlot were significant factors for seeds treated with seasonal temperatures in light (Table 2-3). Temperature was the only significant factor for seeds treated with seasonal temperatures in dark and the constant temperatures in light

(Table 2-3). The interaction of temperature and seedlot was significant for seeds treated with constant temperatures in dark, therefore seedlots responded differently to temperature treatments.

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Asclepias humistrata germination was high (≥70%) in both constant and fluctuating temperatures regardless of the presence of light (Figure 2-2; Figure 2-3). For seasonal temperature experiments, winter (22/11°C) consistently had the lowest germination percentages (33-66%) regardless of light treatment and populations (Figure

2-2). For constant temperature experiments, 25 and 30°C consistently had the highest

(57-97%) germination percentages (when compared within experiments), regardless of light treatment and populations (Figure 2-3).

Temperature gradient experiment

The significant interaction of Temperature X Seedlot X Day suggests that the probability of germination differs based on temperatures but the response to temperature differs between seedlots (Table 2-4). Seeds germinated at all temperatures for all seedlots but differed drastically in final germination percentages (Figure 2-4) and probability of germinating across time and temperatures (Figure 2-5; Figure 2-6).

Orthogonal contrasts indicate optimal temperatures for Asclepias humistrata germination is 24-28°C (Table 2-5). Alachua County 2016 and Volusia county 2016 germination percentages and probability of germinating were low (≤50% and ≤0.5, respectively) across all temperatures (Figure 2-4; Figure 2-6), while all other seedlots had at least 1 temperature with high (≥70%) germination percentages and high (≥0.7) germination probability (Figure 2-4; Figure 2-6). Santa Rosa County 2016, Citrus

County 2016, and Santa Rosa County 2015 had similar germination probabilities (Table

2-4).

Disease occurred at least once for every temperature and in at least one temperature for every seedlot (Figure 2-4). Alachua and Volusia county seedlots had the highest amount of disease based on the presence of an odor that smelled like

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decomposing tissue which was overwhelming when petri dishes were opened.

However, no visual signs of contamination were present so it was impossible to cull diseased seeds.

Follicle maturity experiment

Follicle maturity and seedlot contributed independently to germination responses and there was no significant interaction of these two main effects (Table 2-6). Seeds from mature pods germinated faster and had a higher probability of germinating for

Santa Rosa County 2016 and Santa Rosa Island 2016 than seeds from immature pods

(Figure 2-7). The Alachua County 2016 seedlot had very poor germination probability

(≥0.3) regardless of pod maturity (Figure 2-7). No disease occurred in 67% of seedlots while 9 and 11% seeds were diseased by the end of the study for Alachua County 2016

Immature and Santa Rosa County 2016 Mature, respectively (Figure 2-8). Seeds from immature pods were not visibly different from seeds from mature pods pre or post- imbibition.

Seed size experiment

Small seeds which were less than 5mm at their longest point, had 0% germination while almost half of large seeds germinated (Figure 2-9). No disease occurred in this experiment (Figure 2-9).

Illumination experiment

Seeds from all A. humistrata seedlots germinated in light and dark (Figure 2-9).

Seeds from Santa Rosa County 2015 had improved germination response in the dark while all other seedlots showed no difference between light and dark treatments (Table

2-8). Disease occurred in Alachua County 2016 and Santa Rosa Island 2016 dark treatments at 1 and 11%, respectively (Figure 2-9).

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Discussion

Seed weights are highly variable in Aslcepias (Wilbur 1977). Average seed weights for mature and normal sized seeds of Asclepias humistrata were similar to A. syriaca seeds (Farmer and others 1986). Likewise, mature and normal (not small) A. humistrata seeds had a similar area to that of A. syriaca seeds (Morse and Schmitt

1985).

Physical dormancy is not present in A. humistrata because seeds readily imbibed water without being nicked unlike physically dormant seeds which have a water impermeable testa (Baskin and Baskin, 2014). Very high (≥90%) germination after 1-3 months of dry storage rules out the possibility of seed dormancy for Asclepias humistrata after 1-3 months in storage. These results run contrary to previous studies which indicate cold stratification (Baskin and Baskin 1977; Berkman 1949; Jeffery and

Robinson 1971), mechanical scarification (Evetts and Burnside 1972; Oegema and

Fletcher 1972), chemical scarification (Evetts and Burnside 1972), or hormone treatments (Evetts and Burnside 1972; Oegema and Fletcher 1972) are necessary for high (≥70%) germination percentages of wild-collected Asclepias seeds.

However, 2 and 8 days post-harvest seeds of Santa Rosa Island 2016 Asclepias humistrata collected in late summer show a significant reduction in germination rates and percentages compared to 1-3 month old seeds (data not shown). A period of after- ripening affects may be necessary to alleviate dormancy, if present, in A. humistrata seeds. Further studies are needed to determine whether 0-1 month seeds exhibit seed dormancy.

Similar to results found in the Preliminary Experiments for Asclepias humistrata

(Figure 2-2; Figure 2-3), A. syriaca was shown to have very high (≥90%) germination

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percentages with fluctuating temperatures (Baskin and Baskin 1977; Farmer and others

1986) and constant temperatures (Bhowmik 1978; Jeffery and Robinson 1971; Oegema and Fletcher 1972). Similarly, cold fluctuating temperatures, 15/6 and 22/11°C for A. syriaca and A. humistrata, respectively, limited germination to levels below high (≥70%) germination percentages (Baskin and Baskin 1977; Figure 2-2). Cold constant temperatures (15°C) resulted in low (≤50%) germination percentages for A. syriaca

(Bhowmik 1978) much like A. humistrata (Figure 2-3) in the Preliminary Experiments.

Highest germination percentages were achieved at 27°C (Bhowmik 1978) and

25°C (Jeffery and Robinson 1971) for Asclepias syriaca, both within the optimal range for A. humistrata (24-28°C) found in the Temperature Gradient Experiment (Table 2-6).

Seed germination percentages varied drastically among seedlots, from very low (≤30%) to very high (≥90%) for A. syriaca at optimal germination fluctuating temperatures of 30-

20°C for 16 and 8 hours, respectively (Farmer and others 1986), similar to our results with A. humistrata (Figure 2-4). Germination of A. humistrata remained high (≥70%) after one year in dry storage, as has been the case for A. syricaca (Bhowmik 1978;

Oegema and Fletcher 1972).

Mijnsburgge and others (2010) reviewed native seed collection for restoration projects but failed to address how to tell when fruits reach harvest maturity. Seeds from immature fruits often won’t germinate (Baskin and Baskin 2014) or have poor germination and low seedling survival (Bedane and others 2006). Baskin and Baskin

(1977) describe Asclepias syriaca seeds collected as mature while Berkman (1949),

Bhowmik (1978), Farmer and others (1986), Jeffery and Robinson (1971), and Oegema and Fletcher (1972) don’t discuss follicle or seed maturity when collecting A.syriaca for

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germination studies. Additionally, Wyatt (1996) describes collecting seeds of A. subulata for pollination experiments but also fails to describe follicle or seed characteristics at time of collection. Deno (1993) suggests collecting follicles immediately prior to dehiscence for ease of seed detachment from floss fibers but makes no other justification for this method.

Review of the literature found no study addressing how timing of seed collection

(i.e. follicle maturity) affects germination response of any Asclepias. However, Baskin and Baskin (2014) suggest only collecting mature, fully ripened seeds, because immature seeds tend to fail to germinate. Our study provides the basic information that collecting seeds from dehisced follicles, or follicles that dehisce upon slight application of pressure, yield more germinable seeds than immature follicles, or follicles which have an audible “crunching” sound when pressure is applied but fail to dehisce (Figure 2-6;

Table 2-7). Immature follicles can be further distinguished from other younger follicles that do not make an audible “crunching” sound upon the slight application of pressure. A phenological study comparing germination performance to days after pollination is needed to further refine collection methods which specifically target harvest maturity

(time at which seeds can be harvested without compromising viability) for A. humistrata.

Knowing time of harvest maturity may reduce seed loss to milkweed bugs (Oncopeltus fasciatus) because milkweed bugs devour seeds once follicles dehisce.

Germination response may vary among seed size classes. For example, large seeds tend to germinate faster and to higher proportions than smaller seeds, although this relationship does not necessarily hold for all species (Cideciyan and Malloch 1982).

Wyatt (1996) used the 10 largest seeds of Asclepias subulata with germination success

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for pollination studies but does not specify size of seeds. Farmer and others (1986) describe using only “large well-filled” seeds of A. syriaca in their germination experiments to reduce error caused by poor seed quality but failed to justify small seeds as poor performing. Seed area of A. syriaca was measured in Morse and Schmitt (1985) but not tested in relation to germination.

For Asclepias syriaca heavier seeds are correlated with increased germination performance (Morse and Schmitt 1985). In our experiments, small (≤5mm long) seeds did not germinate (Table 2-1). Therefore, anecdotal evidence corroborates heavier seeds, hence larger seeds, have increased germination performance for A. humistrata

(Figure 2-9).

Very high (≥90%) and high (≥70%) germination percentages have been reported for Asclepias syriaca with no light exposure (Baskin and Baskin 1977; Bhowmik 1978;

Farmer and others 1986; Oegema and Fletcher 1972). In addition, very high germination in A. syriaca is also possible in constant light as well as exposure to 14- hour photoperiod (Baskin and Baskin 1977). Light has no effect on A. tuberosa germination but is required by A. incaranata and A quadrifolia (Deno, 1993). A. phytolaccoides has very high germination in light but very low (≤ 30%) germination in dark. In the present work, very high and high germination percentages were found for A. humistrata in light and dark conditions (Figure 2-2; Figure 2-3). However, dark conditions improved germination of one of four seedlots tested in the Illumination

Experiment (Figure 2-10).

Disease rates were highest during the Temperature Gradient experiment (data not shown) compared to all other A. humistrata germination studies. Seeds from this

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experiment were sterilized in sodium hypochlorite without Tween® 20 for half the time when compared to all other A. humistrata experiments in this study. High infection rates for the Temperature Gradient Experiment when compared to all other experiments conducted suggests the need for 10 min exposure to sodium hypochlorite with Tween®

20 as an effective surface sterilization technique to reduce disease in germination studies.

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Table 2-1. Summary of collection sites and seed characteristics for 11 Ascelpias humistrata seedlots. Collection Location Location Coordinates Plant Community Soil Mature Collection Mean Mean Follicle Year Seed Seed Weight Area (mg) (mm2)

Yes 2016 8.10 44.2 The Little Orange Creek Nature Park Alachua County Sandhill Millhopper Sands (29.621650, -82.068016) No 2016 9.40 34.0

Suncoast Parkway and U.S. Route 19 Citrus County Scrub Astatula Fine Sand Yes 2016 6.73 40.3 (28.706603, -82.525908)

Yes 2015 6.40 37.6

Yes 2016 6.15 46.2 Blackwater River State Forest Santa Rosa County Sandhill Lakeland Sand (30.704973, -86.874116) Yes 2016 6.02 17.0 (Small)

No 2016 8.67 34.8

Yes 2015 8.14 50.1

Escambia County-Pensacola Beach (30.349781, -87.051866) Newhan-Corolla complex, Santa Rosa Island Coastal Grasslands, Beach Dunes Yes 2016 7.39 43.8 Santa Rosa County- Corolla-Duckston complex Navarre Beach-(30.378485, -86.896179)"

No 2016 9.31 42.9

DeLeon Springs, FL Volusia County Disturbed area Astatula Fine Sand Yes 2016 5.53 40.0 (29.144228, -81.339911) Immature seeds did not dehisce when pressure was applied and small seeds were ≤5mm long.

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Table 2-2. Viability percentages (n=100) using TZ staining of 13 Asclepias humistrata seedlots at different times after seed harvest. Seedlot Months in Storage % Viable Alachua County 2016 2-3 80 Alachua County 2016 Immature 2-3 92 Citrus County 2016 2 86 Santa Rosa County 2015 6-9 95 Santa Rosa County 2015 12-15 89 Santa Rosa County 2016 1-3 91 Santa Rosa County 2016 Immature 1-3 89 Santa Rosa County 2016 Small 1-3 37 Santa Rosa Island 2015 6-9 89 Santa Rosa Island 2015 12-15 89 Santa Rosa Island 2016 1-3 94 Santa Rosa Island 2016 Immature 1-3 93 Volusia County 2016 2 36 Immature seeds did not dehisce when pressure was applied to follicles and small seeds were ≤5mm long. Seeds were stored under ambient laboratory conditions (≈24°C, 35% relative humidity).

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Table 2-3. Effects of temperature and seedlot on germination probability of Asclepias humistrata for all 4 Preliminary Experiments. Treatment df Wald χ2 p Value Seasonal Temperature in Light Temperature 3 15.38 0.0015 Seedlot 1 5.44 0.0196 Temperature X Seedlot 1 1.73 0.1884 Seasonal Temperature in Dark Temperature 3 13.20 0.0042 Seedlot 1 2.34 0.1265 Temperature X Seedlot 1 0.00 0.9974 Constant Temperature in Light Temperature 3 66.46 <0.0001 Seedlot 1 0.00 0.9856 Temperature X Seedlot 1 0.06 0.8093 Constant Temperature in Dark Temperature 3 62.83 <0.0001 Seedlot 1 16.48 <0.0001 Temperature X Seedlot 1 15.43 <0.0001 Type 3 tests from cox regression models were used (Allison 2010). Temperature X Seedlot denotes interaction term. Fifteen and 40°C were removed from analysis due to no germination events. Seasonal temperatures fluctuate every 12 hours mimicking Florida’s early spring/late fall (27/15°C), summer (33/24°C), early fall/late spring (29/19°C), and winter (22/11°C) average high and low temperatures. Constant temperatures were 15, 20, 25, 30, 35, and 40°C. Light treatments had a 12-hour photoperiod during high temperatures (for seasonal temperatures only) while dark treatments had no photoperiod. Seeds were germinated for 28 days.

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Table 2-4. Effects of temperature and seedlot on germination probability for 7 seedlots of Asclepias humistrata in the Temperature Gradient Experiment. Treatment Coefficient SE of Wald p Value Hazard (βi) (βi) χ2 Ratio Temperature (v 34°C) 18°C -0.56 0.22 6.27 0.0123 0.57 20°C 0.39 0.19 4.24 0.0395 1.48 22°C 0.83 0.17 23.70 <0.0001 2.29 24°C 1.08 0.15 48.49 <0.0001 2.94 26°C 1.17 0.14 70.28 <0.0001 3.23 28°C 1.05 0.13 65.65 <0.0001 2.86 30°C 0.89 0.12 52.98 <0.0001 2.45 32°C 0.86 0.12 55.89 <0.0001 2.37 Seedlot (v Santa Rosa County 2016) Citrus County 2016 -0.05 0.17 0.09 0.7618 0.95 Volusia County 2016 -2.15 0.18 137.58 <0.0001 0.12 Santa Rosa County 2015 0.17 0.13 1.76 0.1846 1.19 Alachua County 2016 -2.14 0.15 213.31 <0.0001 0.12 Santa Rosa Island 2015 -0.75 0.10 54.45 <0.0001 0.47 Santa Rosa Island 2016 -0.93 0.09 104.46 <0.0001 0.39 Temperature X Seedlot 0.01 0.01 3.30 0.0694 1.01 Temperature X Seedlot X Day 0.00 0.00 3.98 0.0460 1.00 Hazard ratios generated using Cox regression (Allison 2010). Hazard ratios > or < 1 than with p value <0.05 indicate increased or decreased relative germination probability, respectively (Allison 2010). Row 2-9 represent comparisons of temperatures versus 34°C for each temperature tested. Row 11-16 represent comparisons of seedlots versus Santa Rosa County 2016. The second to last row represents interaction term. The bottom row represents interaction term with a time-dependent covariate to adjust for violation of the proportional hazards assumption (Allison 2010). Seeds were germinated with a 12-hour photoperiod for 28 days.

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Table 2-5. Orthogonal contrasts of temperature on germination probability across 7 seedlots of Asclepias humistrata in the Temperature Gradient Experiment. Comparison Hazard SE of Hazard Wald P value Ratio Ratio χ2 Temperature (°C) 18 v 20 0.39 0.06 43.31 <0.0001 18 v 22 0.25 0.04 95.02 <0.0001 18 v 24 0.19 0.03 118.61 <0.0001 18 v 26 0.18 0.03 120.30 <0.0001 18 v 28 0.20 0.03 89.27 <0.0001 18 v 30 0.23 0.04 60.73 <0.0001 18 v 32 0.24 0.05 49.91 <0.0001 18 v 34 0.57 0.13 6.27 0.0123 20 v 22 0.64 0.07 16.38 <0.0001 20 v 24 0.50 0.06 36.25 <0.0001 20 v 26 0.46 0.06 42.11 <0.0001 20 v 28 0.52 0.07 24.73 <0.0001 20 v 30 0.60 0.09 11.40 0.0007 20 v 32 0.62 0.10 8.30 0.0040 20 v 34 1.48 0.28 4.24 0.0395 22 v 24 0.78 0.08 5.95 0.0147 22 v 26 0.71 0.07 10.59 0.0011 22 v 28 0.80 0.09 3.63 0.0567 22 v 30 0.94 0.12 0.23 0.6280 22 v 32 0.97 0.14 0.05 0.8181 22 v 34 2.29 0.39 23.70 <0.0001 24 v 26 0.91 0.09 0.94 0.3312 24 v 28 1.03 0.11 0.06 0.8111 24 v 30 1.20 0.14 2.40 0.1214 24 v 32 1.24 0.16 2.67 0.1025 24 v 34 2.94 0.45 48.49 <0.0001 26 v 28 1.13 0.11 1.55 0.2135 26 v 30 1.32 0.14 6.85 0.0089 26 v 32 1.36 0.16 7.09 0.0077 26 v 34 3.23 0.45 70.28 <0.0001 28 v 30 1.17 0.12 2.41 0.1203 28 v 32 1.21 0.13 3.07 0.0797 28 v 34 2.86 0.37 65.65 <0.0001 30 v 32 1.03 0.11 0.09 0.7690 30 v 34 2.45 0.30 52.98 <0.0001 32 v 34 2.37 0.27 55.89 <0.0001 Hazard ratios generated using orthogonal contrasts from Cox regression models (Allison 2010). Hazard ratios > or < 1 than with a p value <0.05 indicate increased or decreased relative germination probability, respectively (Allison 2010). Seeds were germinated in 18, 20, 22, 24, 26, 28, 30, 32, and 34°C constant temperatures with a 12- hour photoperiod for 28 days.

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Table 2-6. Effects of follicle maturity and seedlot on germination probability for 3 populations of Asclepias humistrata in the Follicle Maturity Experiment. Treatment Coefficient SE of Wald p Hazard (βi) (βi) χ2 Value Ratio Pod Maturity (v Mmmature) Mature 2.10 1.03 4.18 0.0409 8.16 Seedlot (v Santa Rosa County 2016) Santa Rosa Island 2016 0.14 1.40 0.01 0.9231 1.15 Alachua County 2016 -1.76 0.74 5.56 0.0184 0.17 Pod Maturity X Seedlot -0.50 0.55 0.85 0.3559 0.60 Pod Maturity X Day 0.08 0.06 1.72 0.1896 1.08 Seedlot X Day 0.00 0.04 0.00 0.9596 1.00 Pod Maturity X Seedlot X Day 0.02 0.03 0.41 0.5221 1.02 Hazard ratios generated using Cox regression (Allison 2010). Hazard ratios > or < 1 than with p value <0.05 indicate increased or decreased relative germination probability, respectively (Allison 2010). The second row represents comparisons of mature versus immature follicles. The fourth and fifth rows represent comparisons of seedlots vs Santa Rosa County 2016. The sixth row represents the interaction term. The bottom 3 rows represent both treatments and the interaction term with a time-dependent covariate to adjust for violation of the proportional hazards assumption (Allison 2010). Seeds were germinated with a 12-hour photoperiod at 25°C for 28 days.

Table 2-7. Effect of light on germination percentages for 4 seedlots of Asclepias humistrata for the Illumination Experiment. Seedlot Pearson’s χ2 df p Value Alachua County 2016 0.13 1 0.7182 Santa Rosa Island 1.02 1 0.3123 2016 Santa Rosa County 34.94 1 <0.0001 2015 Citrus County 2016 2.24 1 0.1341 Pearson’s chi squared analysis were used to compare germination count data for each seedlot independently. A p value <0.05 indicates light had an effect on germination. Light treatments had a 12-hour photoperiod with while dark treatments had no photoperiod. Seeds were germinated at 25°C for 14 days.

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Asclepias humistrata Seed Imbibition Test 120

100

80

60 Santa Rosa Island 2015 40 Santa Rosa County 2015

% Increase of fresh weight freshofIncrease % 20

0 0 5 10 15 20 25 Time (hours)

Figure 2-1. Seed imbibition curves for 2 Asclepias humistrata seedlots. At time 0 seeds were weighed dry then immediately put into water. Seed weights were then measured at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 12, and 24 hour intervals.

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Asclepias humistrata Seasonal Temperatures 100% 90% 80% 70% 60% 50% 40%

30% Relative Percentage Relative 20% 10%

0%

Fall Fall Fall Fall

Spring Winter Spring Winter Spring Winter Spring Winter

Summer Summer Summer Summer Light Dark Light Dark Santa Rosa Island 2015 Santa Rosa County 2015 Germinated Viable Non-viable Disease

Figure 2-2. Effect of temperature and seedlot on relative percentages of germinated, viable, non-viable, and disease seeds for 2 seedlots of Asclepias humistrata exposed to seasonal temperatures in light and dark. Germinated seeds showed 1 mm protrusion, viable seeds stained regularly during post- experiment TZ tests, non-viable seeds stained irregularly during post- experiment TZ tests, and disease seeds showed presence of disease. Seasonal temperatures fluctuate every 12 hours mimicking Florida’s early spring/late fall (27/15°C), summer (33/24°C), early fall/late spring (29/19°C), and winter (22/11°C) average high and low temperatures. Light treatments had a 12-hour photoperiod during high temperatures while dark treatments had no photoperiod. Seeds were observed for 28 days.

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Asclepias humistrata Constant Temperatures 100% 90% 80% 70% 60% 50% 40%

30% Relative Percentage Relative 20% 10%

0%

20°C 35°C 35°C 30°C 25°C 30°C 25°C 20°C 35°C 30°C 25°C 20°C 35°C 30°C 25°C 20°C Light Dark Light Dark Santa Rosa Island 2015 Santa Rosa County 2015 Germinated Viable Non-viable Disease

Figure 2-3. Effect of temperature and seedlot on relative percentages of germinated, viable, non-viable, and disease seeds for 2 seedlots of Asclepias humistrata exposed to constant temperatures in light and dark. Germinated seeds showed 1 mm protrusion, viable seeds stained regularly during post- experiment TZ tests, non-viable seeds stained irregularly during post- experiment TZ tests, and disease seeds showed presence of disease. 15, 20, 25, 30, 35, and 40°C were grown in light with a 12-hour photoperiod while dark treatments had no photoperiod. 15 and 40°C are excluded due to 0% germination. Seeds were observed for 28 days.

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Asclepias humistrata Temperature Gradient Experiment 100% 90% 80% 70% 60% 50% 40% 30%

Relative Percentage Relative 20% 10%

0%

18 22 26 30 34 20 24 28 32 18 22 26 30 34 20 24 28 32 18 22 26 30 34 20 24 28 32 18 22 26 30 34 Alachua County Citrus County 2016 Santa Rosa County Santa Rosa County Santa Rosa Island Santa Rosa Island Volusia County 2016 2016 2015 2016 2015 2016

Germinated Viable Non-viable Disease

Figure 2-4. Effect of temperature and seedlot on relative percentages of germinated, viable, non-viable, and disease seeds for 7 seedlots of Asclepias humistrata in the Temperature Gradient Experiment. Germinated seeds showed 1 mm protrusion, viable seeds stained regularly during post-experiment TZ tests, non-viable seeds stained irregularly during post-experiment TZ tests, and disease seeds showed presence of disease. Seeds exposed to 18, 20, 22, 24, 26, 28, 30, 32, and 34°C constant temperatures with a 12-hour photoperiod for 28 days.

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Figure 2-5. Inverted Kaplan-Meier curves showing effect of temperature on germination probability for 4 seedlots of Asclepias humistrata used in the Temperature Gradient Experiment. Numbers in legend denote degrees Celsius. Ninety-five percent confidence intervals are excluded for clarity. Seeds were germinated with a 12-hour photoperiod for 28 days.

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Figure 2-6. Inverted Kaplan-Meier curves showing effect of temperature on germination probability for 3 seedlots of Asclepias humistrata used in the Temperature Gradient Experiment. Numbers in legend denote degrees Celsius. Ninety-five percent confidence intervals are excluded for clarity. Seeds were germinated with a 12-hour photoperiod for 28 days.

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Figure 2-7. Inverted Kaplan-Meier curves showing effect of Asclepias humistrata follicle maturity on germination probability representing 3 geographic locations in the Follicle Maturity Experiment. Ninety-Five percent confidence intervals are shown bracketing the germination probability for both mature (solid line) and immature (dashed line) seeds. Seeds were germinated with a 12-hour photoperiod at 25°C for 28 days.

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Asclepias humistrata Follicle Maturity Experiment 100% 90% 80% 70% 60% 50% 40% 30% 20% Relative Percentage Relative 10% 0% Mature Immature Mature Immature Mature Immature Alachua County 2016 Santa Rosa County Santa Rosa Island 2016 2016 Germinated Viable Non-viable Disease

Figure 2-8. Effects of Asclepias humistrata follicle maturity on relative percentages of germinated, viable, non-viable, and diseased seeds representing 3 geographic locations in the Follicle Maturity Experiment. Germinated seeds showed 1 mm protrusion, viable seeds stained regularly during post- experiment TZ tests, non-viable seeds stained irregularly during post- experiment TZ tests, and disease seeds showed presence of disease. Seeds were germinated with a 12-hour photoperiod at 25°C for 28 days.

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Asclepias humistrata Seed Size Experiment 100% 90% 80% 70% 60% 50% 40%

30% Relative Percentage Relative 20% 10% 0% Large Small Germinated Viable Non-viable Disease

Figure 2-9. Effects of seed size on relative percentages of germinated, viable, non- viable, and disease seeds for 2 seedlots of Asclepias humistrata in the seed Size Experiment. Germinated seeds showed 1 mm protrusion, viable seeds stained regularly during post-experiment TZ tests, non-viable seeds stained irregularly during post-experiment TZ tests, and disease seeds showed presence of disease. Small seeds were ≤to 5 mm long and large seeds were over 5 mm long. Seeds were germinated with a 12-hour photoperiod at 25°C for 28 days.

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Asclepias humistrata Illumination Experiment 100% 90% 80% 70% 60% 50% 40% 30%

Relative Percentage Relative 20% 10% 0% Light Dark Light Dark Light Dark Light Dark Alachua County Citrus County Santa Rosa Santa Rosa 2016 2016 County 2015 Island 2016 Germinated Viable Non-viable Disease

Figure 2-10. Effect of light on relative percentages of germinated, viable, non-viable, and disease seeds for 4 seedlots of Asclepias humistrata in the Illumination Experiment. Germinated seeds showed 1 mm protrusion, viable seeds stained regularly during post-experiment TZ tests, non-viable seeds stained irregularly during post-experiment TZ tests, and disease seeds showed presence of disease. Light treated seeds had a 12-hour photoperiod while dark treated seeds had no photoperiod. Seeds were germinated at 25°C for 28 days.

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CHAPTER 3 LUPINUS DIFFUSUS SEED GERMINATION

Introduction

Lupinus

Lupinus is a genus within the cosmopolitan Fabaceae (legume) plant family which is the third most diverse family of flowering plants, the second most economically important plant family, and unique in their ability to enrich nutrient-poor soils via a nitrogen-fixing symbiosis with the Rhizobium bacteria (Heywood and others 2007).

Fabaceae can be characterized usually by compound leaves that are usually stipulate

(Heywood and others 2007). Flowers are hermaphroditic or unisexual, bilaterally or radially symmetrical, terminal or axillary, racemes, heads or spikes, with 5 joined sepals,

5 petals, and 2 to 5 stamens in whorls that are monadelphous or diadelphous (Heywood and others 2007). Fruits are dry or fleshy dehiscent or indehiscent legumes with seeds that generally have a strong testa with little endosperm and 2 large cotyledons

(Heywood and others 2007). Seeds within the Fabaceae family commonly have physical dormancy (Baskin 2003; Quinlivan 1971; Rolston 1978).

Lupinus can be identified by, 1-foliate or palmate glandular leaves, several- seeded dehiscent fruit, and monadelphous stamens (Wunderlin and Hansen2011). The

Florida panhandle contains 6 Lupinus species, 4 which are native and can be found in xeric plant communities (Clewell 1985).

The economic value of Lupinus includes the use of L. polyphyllus for feedstock and soil nutrient enrichment (Heywood and others 2007), the use of L. texensis

(bluebonnets) for native wildflower roadside plantings in Texas, and the prevalence of

Lupinus within the horticulture industry as an ornamental plant. Lupinus can also play

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critical roles in ecosystem biodiversity, for example, the threatened L. sulphreus ssp. kincaidii (Kincaid’s lupine) is a host plant for the endangered Icaricia icarioides fender

(Fender’s blue butterfly) (Kaye and Kuykendall 2001). Most importantly, Lupinus forms nodulating symbioses with nitrogen-fixing bacteria (Rhizobium) that enhance soil fertility by bringing Nitrogen from the air into the soil (Simms and others 2006).

Lupinus Seed Germination

Water impermeable seeds have been found in Lupinus. angustifolius, L. leteus, and L. varius (Quinlivan 1970), L. diffusus (Dehgan and others 2003), L. angustifolius

(Valenti and others 1989), and L. digitataus (Gladstones 1958). Lupinus seed physical dormancy alleviation mechanisms include mechanical scarification (electric seed scarifier, sandpaper abrasion, cutting with scissors, and nicking with a blade) chemical scarification (sulfuric acid), hot water baths with and without pre-freezing, cold stratification, seed burial, and salt spray (Davis and others 1991; Dehgan and others

2003; Jones and others 2016; Karaguzel and others 2004; Kaye and Kuykendall 2001;

Tiryaki and Topu 2014).

Kaye and Kuykendall (2001) found two wild populations of L. sulphureus ssp. kincaidii had different highest germination percentages (95 and 55%) after mechanical scarification (sandpaper abrasion) and cold stratification (4°C for 8 weeks) compared to a control which had less than 10% germination for both populations. Davis and others

(1991) found cutting, filing, and piercing seed testa and exposing seeds to 30-60 minutes of an 18M Sulfuric acid bath equally improved germination rates to 85-95% over a control treatment (0-50% germination) by improving imbibition for some but not all of the 4 commercial seedlots of L. texensis tested. L. varius showed improved germination rates over control treatments (30% germination) when mechanically

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scarified (100% germination), immersed in 18 M sulfuric acid for 8-24 hours (93-100% germination), and when boiled for 6 minutes germination improved to 80% (Karaguzel and others 2004).

Jones and others (2016) treated 4 Great Basin lupines (Lupinus arbustus, L. argenteus, L. prunophilus, and L. sericeus) with mechanical scarification (removal of seedcoat), a constant temperature hot water bath (95°C) for 15, 20, 25, 30, 40, 45, 50,

55, and 60 seconds, or treated seeds for a constant time (60 seconds) with a 50, 55, 60,

65, 70, 75, 80, 85, 90, or 95°C hot water bath. L. argenteus seeds treated with hot water bath for 60 seconds improved seed germination to 92% compared to a control (52%).

Mechanical scarification improved germination for L. argenteus (52 to 85%), L. prunophilus (32 to 46%), and L. sericeus (22 to 66%) but reduced germination for L. arbustus (68 to 18%).

Tiryaki and Topu (2014) found freezing seeds for 1, 2, 4, or 7 days at -80°C followed by hot a 5 second hot water bath (90°C) broke physical dormancy. Highest germination was 88% for seeds frozen for 1 day. Untreated seeds only had 3% germination and seeds only frozen or treated with hot water had less than 10% germination.

Lupinus diffusus

Lupinus diffusus (sky-blue lupine) is characterized by its perennial life cycle, hairy

1-foliate alternate leaves with a prominent midvein, and hairy seed pods (Taylor 2013).

Inflorescences are identified by terminal racemes with blue pea shaped flowers that have a white to cream colored spot on its upper petals and its two lipped calyx which appear in late winter to early spring (Taylor 2013). L. diffusus shows promise for use in roadside plantings due to its showy flowers, restoration of barren sites due to its ruderal

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nature, and soil remediation due to its ability to fix Nitrogen in soils. However, due to its physical dormancy, mechanisms must be constructed to prime seeds pre-broadcasting to obtain quick and uniform germination (Dehgan and others 2003).

Only one existing study on Lupinus diffusus germination requirements has been conducted by Dehgan and others (2003). Non-scarified seeds had 5% germination percentage due to physical dormancy. Only chemical scarification (24 hours in 18M sulfuric acid) improved germination to 41% while gibberellic acid (GA3), mechanical scarification (sandpaper abrasion) and hot water immersion made no difference or reduced germination when compared to non-scarified seeds. Seeds put in hot water

(90°C) and left to cool down at room temperature for 24 hours had 0% germination.

Fire and Seed Germination

One unique and newly discovered way to break seed dormancy in fire-prone ecosystems is replicating environmental conditions that occur during fires (e.g. heat and smoke). A large amount of literature has been amassed concerning the efficacy of using smoke to promote seed germination across plant taxa and geographic origin since 1990

(Amin 2011; Baxter and others 1994; Brown and others 1993; Dixon and others 1995;

Fornwalt 2015; Jäger and others 1996; Norman and others 2006) the year which the first smoke stimulated germination study was published (De Lange and Boucher 1990).

Increased seed germination in response to fire for species within the Fabaceae family is well documented (Moreira and others 2010) and includes Lupinus luteus (Kopyra and

Gwozdz 2003). Landis, 2000 reviews the techniques used to replicate fire for horticulture purposes, pointing to the success of using plant derived smoke infused in water to increase germination rates, though it should be noted that perhaps not all types of smoke promote germination for a particular species (Baxter and others 1995),

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specific temperatures are needed to volatize germination promoting compounds (Jäger and others 1995), and smoke can inhibit germination in certain species and at certain concentrations (Adkins and Peters 2001; Fornwalt 2015).

Tieu and others (1999) described a method for making a smoke-infused water concentrate by burning local flora and forcing smoke through water. The smoke-infused concentrate, and a series of dilutions, were then tested for improved germination of dormant Australian native plants that grow in fire-prone communities. They found various smoke-infused water concentrations improved germination across families. A similar method is used in this study to test if smoke-infused water promotes germination of Lupinus diffusus.

Experimental Preface

The objective of the following experiments was to find effective and practical treatments to alleviate physical dormancy and facilitate germination in 2 different

Lupinus diffusus seedlots collected in the panhandle of Florida. Seeds were treated with smoke-infused water, scarification by time in a rock tumbler, dry freezing, hot water baths, a combination of dry freezing and hot water baths, and a combination of times in an electric seed scarifier immediately followed by immersion in sodium hypochlorite.

Materials and Methods

Seed Collection and Storage

Seeds from two locations (Walton and Santa Rosa County, FL) were harvested by hand in early May 2015. Whole inflorescences with intact legumes were harvested when legumes were fully developed and no green tissue was visible on the inflorescences. Inflorescences were placed into paper bags and allowed to air dry at

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ambient laboratory conditions (≈24°C, 35% relative humidity) for 2 months. Seeds were stored in airtight jars under ambient laboratory conditions.

Walton County seeds were collected from a private residence in DeFuniak

Springs, FL (30.741024, -86.150908). Plants were growing within Lakeland sand and

Chipley sand soil types (USDA Soil Survey). Santa Rosa County seeds were collected from a private residence in Milton, FL (30.605615, -86.920558). Plants were growing in a Lakeland sand soil type (USDA Soil Survey). The collection site was a treeless disturbed abandoned field within a residential development. The landowner is an amateur apiarist with several dozen manmade beehives adjacent to plants from which were collected. At the time of collection, bees from the beehives were seen visiting flowers.

Seed Viability Tests

For all TZ tests (Appendix A) seeds were nicked with a razor blade on the distal end of the seed. Once nicked, seeds were immediately immersed in a 1% TZ solution consisting of 1 gram of 2,3,5-Triphenyltetrazolium chloride (≥95%) for every 100 mL of deionized distilled water. Seeds were then immediately incubated at ambient laboratory temperature (≈24°C).

TZ tests were performed on seeds from Walton County 2015 and Santa Rosa

County 2015 approximately 7 months after seed harvest. Four replicates of 25 seeds

(n=100) were put into 4 separate 15 mL glass beakers containing 4 mL of TZ solution covered with Parafilm M®

Another TZ test was run on Walton County 2015 and Santa Rosa County 2015 seedlots in September 2016, 1.5 years after seed harvesting. For this round of TZ tests,

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100 seeds from each seedlot were immersed in 4 mL of TZ solution in 50mL glass beakers that were covered with Parafilm M®.

TZ tests were also run on post-experiment non-germinated seeds. Seed numbers were variable given the fact that seed germination percentages were not uniform across seedlots and treatments. These seeds were immersed in 4 mL of TZ solution in 50mL glass beakers that were covered with Parafilm M®.

Seed Imbibition Tests

A seed imbibition test was conducted to test for physical dormancy (Baskin and

Baskin 2014). Walton County 2016 and Santa Rosa County 2016 seedlots were tested independently for water imbibition ability on February 26, 2016, approximately 9 months after seed harvest. Seeds were either nicked with a razor blade on the distal end or were left unaltered. Three replications of 7 seeds (n=21) for each seedlot and treatment were placed in separate 60 X 15 mm glass petri dishes on top of one blotter paper immersed in 5 mL of distilled deionized (DI) water.

Seed weight was determined to the nearest 0.01 mg gravimetrically. Seed weights were measured at time 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 12, and 24 hours.

For each of the three replications all seeds were removed at each time interval, wrapped in a paper towel and weighed individually prior to all seeds being returned to the moistened blotter in the petri dish. Water was added when a film of water around the seeds shrunk to <1mm thick.

Germination Experiments

A preliminary round of experiments was conducted on the Walton County 2015 seedlot of Lupinus diffusus. Four experiments to test efficacy of scarification techniques

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on improving germination percentages compared to a control for L. diffusus were conducted on June 6, 2016 for 10 days.

Seeds were surface sterilized as follows in temporal sequence prior to the start of the experiment. Seeds were placed inside a plastic dish with cheesecloth held in place by a rubber band over the dish and placed under running cold tap water for 10 min.

Water was decanted and seeds were immediately agitated with 10 mL of 50% ethanol for 1 min inside a 100mL beaker, ethanol was decanted and seeds remained in the beaker. Five mL of 8.25% sodium hypochlorite solution diluted with 45 mL of distilled deionized (DI) water was added to the decanted beaker along with 2 drops of Tween®

20 and placed on a gyroshaker at 60 RPM for 10 min. Sodium hypochlorite water was decanted and seeds were triple washed with DI water. Seeds were put on top of 1 blotter paper immersed in 5 mL of DI water within 60 X 15 mm glass petri dishes wrapped in one layer of Parafilm M®. Seeds were exposed to ambient lab conditions

(≈24°C) under cool white fluorescent lights for approximately 10 hours a day. Seeds were either unaltered (control), nicked with a razor blade, exposed to various concentrations of smoke-infused water, put in a rock polisher for various lengths of time, or given a hot bath for various amounts of time with or without freezing beforehand. Two replicates of 10 seeds (n=20) were used for each treatment. Nicked seeds had their testa ruptured by razor blades on the distal end. Seed testas were considered ruptured when the seed would stick to the razor blade, overcoming the force of gravity.

Smoke-infused water experiment

Nicked seeds were exposed to smoke infused water to determine if smoke promotes germination in L. diffusus. Infused smoke water stock solution was made by combusting equal amounts of leaf litter and small branches of Florida native fire-

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adapted trees (Pinus palustris, Quercus geminata, and Q. laevis) in a grill modified with a hose attachment and pulled through 500 mL of deionized water using a vacuum pump for 1 hour. Seeds were exposed to 5 mL of 0.5, 1, or 3% dilutions of liquid-smoke stock solution.

Rock polisher experiment

Luinus diffusus seeds were mechanically scarified using a hobby-sized rock tumbler to test for efficacy of physical dormancy alleviation. Seeds were mixed with silicon carbide 46/70 grit (Sisters Rocks inc. Westminster, CO) and mechanically scarified for various lengths of time using a rock polisher (Rotary Tumbler Model 3A,

Lortone inc, Mukilteo WA). Approximately 200 seeds and 100 cm3 of grit were placed within the barrel and seeds were taken out after 1.5, 3, 4.5, 16.5, 24, 48, 72, 96, and

122 hours.

Freeze followed by hot bath experiment

Seeds were subjected to differing intervals of freezing at -10°C prior to submersion at various lengths in a hot water bath (97-99°C) to determine the efficacy of alleviating physical dormancy. An additional interval of submersion in the hot water bath was conducted for a subset of treatments following a 30 second ice water bath with treatments denoted with a (2X). Seeds with a (2X) therefore received the stated time twice with a cold water bath between treatments. Non-frozen seeds (0 day) were in a hot water bath for 1(2X), 1.5(2X)1, 2(X), 1.5, or 2 minutes ,1 day frozen seeds were in a hot water bath for 1(2X), 1.5(2X), 2(2X), 5(2x), 7.5(2X), 10(2X), 30(X), 5, 7.5, 10, 15, or

30 minutes, and 7 day frozen seeds were in a hot water bath for 1(2X), 1, 1.5, or 2 minutes.

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Scarification experiment

Seeds were mechanically scarified using an electric seed scarifier and subsequently surface sterilized with sodium hypochlorite to determine optimal scarification and sterilization techniques to alleviate physical dormancy of Lupinus diffusus. Walton County 2015 and Santa Rosa County 2015 seedlots were mechanically scarified using a Forsberg seed scarifier (Seedburo Equipment, Des Plaines, Illinois) for

0, 10, 20, 30, 40, 50, or 60 seconds. Seeds were surface sterilized immediately post- scarification by submersing seed for 0, 15, or 30 seconds in 5 mL of 8.25% sodium hypochlorite solution diluted with 45 mL of deionized water. Four replicates of 25 seeds

(n=100) were used for a total of 20 treatment combinations (6 levels of scarification X 3 levels of sterilization) with the caveat that seeds in scarifier for 0 seconds were not bleached post-scarification. Seeds were placed on 3 sheets of filter paper immersed in

3.5 mL of deionized water within each petri dish and placed in a growth chamber at

25°C with a 12-hour photoperiod. Germinated and diseased seeds were recorded every

24 hours for 23 days. Germinated seeds were seeds with a >1mm radicle protruding from the seed testa while diseased seeds were either seeds with visible hyphal growth or seeds which tissue appeared to liquefy and stain blotter paper.

Seeds were surface sterilized before the experiment as follows in temporal sequence. Seeds were triple washed with cold tap water for 10 minutes and then immersed in 10 mL of 50% ethanol for 1 min inside a 100mL beaker while being agitated. After, seeds were immersed in 5 mL of 8.25% sodium hypochlorite solution diluted with 45 mL of deionized water in the same beaker for 10 minutes while being hand agitated. Lastly, seeds were triple washed with deionized water before being put into petri dishes.

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Statistical Analyses

Survival analysis (Cox regression and Kaplan-Meir curves) was conducted as described in Chapter 2 and Appendix B for the Scarification experiment. For all other experiments only final germination percentages were compared.

Results

Seed Viability Tests

Seeds retained high viability (≥70%) after 7 and 17 months in storage under ambient laboratory conditions (Table 3-1). No visible contamination was present.

Seed Imbibition Tests

Water imbibition rates for both seedlots of Lupinus diffusus were severely restricted for non-nicked seeds when compared to nicked seeds (Figure 3-1). Only two seeds from the Santa Rosa County 2015 seedlot and no seeds from the Walton County

2015 seedlot imbibed water after 6 hours while all seeds from nicked treatments were imbibed after 6 hours. No visible contamination was present.

Germination Experiments

Preliminary experiments

Preliminary tests exhibited low germination (≤ 30%) for all treatments except for seeds that were nicked with a razor blade, which had 75% germination (Table 3-2).

Non-nicked seeds had 10% imbibition and no germination. All seeds from the Smoke water experiment imbibed but germination was inhibited to 20, 10, and 15% for 0.5, 1, and 3% liquid smoke concentrations, respectively (Table 3-2). The rock polisher experiment’s highest seed imbibition rate was 30% with only 5% germination (Table 3-

2). Even though imbibition rates reached 65% with hot water/cold water treatments the highest germination rate was a meager 5% (Table 3-2).

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Scarification experiment

The highest order interaction term (Bleach X Seedlot X Scarification) was significant in the Scarification Experiment (Table 3-3), indicating germination probability is not equally affected by time in scarifier and bleach across seedlots. Therefore, no specific prescription based on survival analysis for time in scarifier and bleach can be given to promote germination in Lupinus. diffusus. Both seedlots had very low (≤ 30%) germination percentages and probabilities and percentages for seeds which weren’t scarified (Figure 3-2; Figure 3-3; Figure 3-4). High germination percentages (≥70%) were found with 20 seconds in scarifier followed by 15 seconds in bleach and for 30 seconds in scarifier followed by 30 seconds in bleach for the Santa Rosa County 2015 seedlot (Figure 3-2). High germination percentages (≥70%) were found with 40 seconds in scarifier followed by 15 seconds in bleach for the Walton County 2015 seedlot (Figure

3-2).

Disease incidence was ubiquitous across all treatments (Figure 3-2; Table 3-4) and occurred in every petri dish. Equal disease percentages were found across seedlots (Table 3-4). Time in bleach post-scarification did not reduce disease (Table 3-

4). Disease percentages were variable for time in scarifier (Table 3-4).

Discussion

As was the case in Dehgan and others (2003), Lupinus diffusus seeds exhibited physical dormancy in this study due to a water impermeable testa (Baskin and Baskin

2014). Very low (≤30%) germination percentages (in part due to dormancy) for untreated Lupinus has been reported for L. arboreus (Davidson and Barbour 1977), L. diffusus (Dehgan and others 2003; Figure 3-2), L. sericeus (Jones and others 2016), L.

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sulphureus ssp. Kincadii (Kaye and Kuykendall 2001), L. texensis (Davis and others

1991), and L. varius (Karaguzel and others 2004).

Landis (2000) and Brown and Staden (1997) both review the use of smoke to promote germination in seeds which grow in fire-prone communities. Dehgan and others (2003) posed the possibility of smoke promoting germination of Lupinus diffusus.

However, preliminary results in this study show all concentrations of native plant smoke infused water inhibits L. diffusus germination (Table 3-2).

In Dumroese’s and others (2009) guide for tribal nurseries they discuss the successful use of small rock tumblers to alleviate physical dormancy for different native plant species. Lupinus diffusus appears to have too hard of a seed coat for this type of scarification to work (Table 3-2). Although, Dreseen (2004) describes a method of wet tumbling to relive physical dormancy. Future studies on L. diffusus should investigate wet tumbling as a way to break physical dormancy.

Freezing followed by a hot bath did not increase germination in Lupinus diffusus

(Table 3-2) but did increase germination in L. albus (Tiryaki and Topu 2014). However,

Tiryaki and Topu (2014) used a -80°C freezer while seeds in this study were frozen in a

-10°C freezer. A hot bath didn’t increase germination in L. diffusus (Dehgan and others

2003; Table 3-2), L. albus (Tiryaki and Topu 2014), L. prunophilus and L. sericeus

(Jones and others 2016) but did increase germination for L. arbustus and L. argenteus

(Jones and others 2016), L. texensis (Davis and others 1991), and L. varius (Karaguzel and others 2004).

Chemical (Sulfuric acid) scarification has led to high (≥70%) germination percentages in Lupinus argenteus and L. sericeus (Jones and others 2016), and L.

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varius (Karaguzel and others 2004). The most effective known treatment to alleviate L. diffusus dormancy is a sulfuric acid bath yet only achieves 41% (Dehgan and others

2003). Even though acids can be an effective treatment to break physical dormancy in

Lupinus, they are hazardous to human health and the environment, and require infrastructure for proper disposal that complicates practical use in nurseries.

Mechanical scarification has been used to successfully break Lupinus physical dormancy. Sandpaper abrasion relived physical dormancy for L. sulphureus ssp.

Kincaidii (Kaye and Kuykendall 2001) and cutting L. varius seeds with a scissor broke physical dormancy (Karaguzel and others 2004). Nicking seed coats with a razor, metal file abrasion, and seed coat piercing with a hammer and nail improved germination in L. texensis (Davis and others 1991). While the previously mentioned mechanical scarification techniques improve germination of Lupinus, they are labor intensive.

For Lupinus diffusus, mechanical scarification via time in a Forsberg electric seed scarifier increased germination probability from near 0 to 0.8 (Figure 3-3; Figure 3-4) unlike decreased germination in response to mechanical scarification via sandpaper abrasion in Dehgan and others (2003). The Great Basin Lupinus species L. argenteus and L. sericeus showed increased germination while L. arbustus and L. prunophilus germination was not increased in response to the same seed scarifier used in this study

(Jones and others 2016). However, in Jones and others (2016) the longest seed scarification treatment was 10 seconds while in this study the shortest treatment was 10 seconds, suggesting L. diffusus has a relatively thick testa.

Germination percentages of non-diseased seeds were 100% for 36% of scarified seeds. Disease was the major issue limiting germination percentages of Lupinus

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diffusus to below very high (≥90%) percentages (Table 3-4). Future studies on L. diffusus should use fungicides and antibacterials to reduce failed germination due to disease.

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Table 3-1. Viability (%) for 2 seedlots of Lupinus diffusus following 7 and 19 months of storage. Months After Collection 7 19 Seedlot Viability Percentage Santa Rosa County 2015 89 82 Walton County 2015 96 90 Seeds were stored under ambient laboratory conditions (≈24°C, 35% relative humidity).

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Table 3-2. Summary of final germination percentages for all Lupinus diffusus preliminary experiments. Treatment % % Treatment % % Imbibed Germinated Imbibed Germinated Control 10 0 0 Day freeze Control 100 75 1 min (2X) 50 0 nicked Liquid 1.5 min (2X) 25 0 smoke 0.5% 100 20 2 min (2X) 25 0 1% 100 10 1 min 40 5 3% 100 15 1.5 min 30 0 Rock 2 min 35 0 polisher 1.5 hours* 20 0 1 Day freeze 3 hours* 20 5 1 minute (2X) 25 0 4.5 hours 10 0 1.5 minutes (2X) 15 0 16.5 hours 0 0 2 minutes (2X) 35 0 24 hours 10 5 5 minutes (2X) 65 0 48 hours 30 5 7.5 minutes (2X) 50 0 72 hours 5 0 10 minutes (2X) 45 0 96 hours 10 0 5 minutes 30 0 122 hours 15 0 7.5 minutes 30 0 10 minutes 15 0 15 minutes 40 0 30 minutes 55 0 7 Day Freeze 1 minute (2X) 10 0 1 minute 5 0 1.5 minutes 45 0 2 minutes 10 0 On the left treatment column, the control treatment was unaltered and the control nicked treatment was nicked with a razor blade on the distal end. The liquid smoke treatment percentages (0.5, 1, and 3%) denote dilution strengths from stock solution. The rock polisher treatment times describe continuous time in rock polisher. On the right treatment column 3 different freeze times (0, 1, and 7 days) with subsequent hot baths. Minutes denote length submerged in hot water (97-99°C) and 2X represents treatments which were exposed to times twice with a 30 second ice water bath between hot baths. The symbol (*) denotes n=10. Seeds were germinated under ambient laboratory conditions (≈24°C) for 10 days.

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Table 3-3. Effects of combinations of time in bleach and scarifier on germination probability for 2 seedlots of Lupinus diffusus for the Scarification Experiment. Treatment Parameter SE of Wald P Hazard Estimate (βi) (βi) χ2 Ratio Time in bleach (seconds) v 30 0 -1.42 0.42 11.35 0.0008 0.24 15 -0.64 0.21 9.01 0.0027 0.53 Time in scarifier (seconds) v 60 0 -2.75 0.55 25.14 <0.0001 0.06 10 -0.05 0.35 0.02 0.8753 0.95 20 0.38 0.28 1.83 0.1766 1.47 30 0.43 0.22 3.77 0.0522 1.53 40 0.47 0.16 8.83 0.0030 1.59 50 0.14 0.11 1.68 0.1946 1.15 Walton County 2016 v Santa 0.37 0.17 4.88 0.0272 1.45 Rosa County 2016 Bleach X seedlot -0.04 0.01 17.27 <0.0001 0.96 Bleach X scarification 0.00 0.00 7.03 0.0080 1.00 Seedlot X scarification 0.01 0.00 3.84 0.0500 1.01 Bleach X seedlot X 0.00 0.00 12.44 0.0004 1.00 scarification Hazard ratios > or < 1 than with p value <0.05 indicate increased or decreased relative germination probability, respectively (Allison 2010). Times in bleach are contrasted with 30 seconds in bleach (row 2-3) and times in scarifier with 60 seconds in scarifier (row 5- 10). The bottom four rows are interaction terms. Seeds were germinated with a 12-hour photoperiod at 25°C for 23 days.

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Table 3-4. Summary of final diseases percentages pooled by time in bleach (n=1200), population (n=1900), and time in scarifier (n=600) for Lupinus diffusus in the Scarification Experiment. Comparison Percent Diseased Population Santa Rosa County2015 41 Walton County2015 41 Time in bleach (seconds) 0 40 30 39 60 45 Time in scarifier 0 32 10 36 20 33 30 42 40 37 50 53 60 47

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Lupinus diffusus Seed Imbibition Test 140

120

100 Walton County 2015 nicked

80 Walton County 2015

60 Santa Rosa County 2015 nicked

40 Santa Rosa County 2015 % Increase of fresh weight weight freshofIncrease % 20

0 0 1 2 3 4 5 6 Time (hours)

Figure 3-1. Seed imbibition curves for nicked and unaltered seeds of 2 Lupinus diffusus seedlots. Nicked seeds had their testa pierced with a razor blade on the distal end. At time 0 seeds were weighed dry then immediately put into water. Seed weights were then measured at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 12, and 24 hours. Hour 12 and 24 data are not shown due to highly reduced imbibition at these times.

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Lupinus diffusus Scarification Experiment 100% 90% 80% 70% 60% 50% 40%

30% Relative Percentage Relative 20% 10%

0%

0,0 0,0

0,10 0,20 0,30 0,40 0,50 0,60 0,10 0,20 0,30 0,40 0,50 0,60

30,30 50,30 60,30 10,15 20,15 30,15 40,15 50,15 60,15 10,30 20,30 40,30 50,30 60,30 10,15 20,15 30,15 40,15 50,15 60,15 10,30 20,30 30,30 40,30 Santa Rosa County 2015 Walton County 2015 Germinated Viable Non-Viable Disease

Figure 3-2. Effects of combinations of time in bleach and time scarifier of 2 seedlots of Lupinus diffusus on relative percentages of germinated, viable, non-viable, and disease seeds for the Scarification Experiment. Numbers on X-axis denote time in scarifier and time in bleach, respectively. Germinated seeds showed 1 mm protrusion, viable seeds stained regularly during post-experiment TZ tests, non-viable seeds stained irregularly during post- experiment TZ tests, and disease seeds showed presence of disease.

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Figure 3-3. Inverted Kaplan-Meier curves showing effects of a combination of time in bleach and scarifier on germination probability for the Santa Rosa County 2015 seedlot of Lupinus diffusus used in the Scarification Experiment. Numbers in legend denote time in scarifier. Ninety-five percent confidence intervals are excluded for clarity. Seeds were germinated with a 12-hour photoperiod at 25°C for 23 days.

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Figure 3-4. Inverted Kaplan-Meier curves showing effects of a combination of time in bleach and scarifier on germination probability for the Walton County 2015 seedlot of Lupinus diffusus used in the Scarification Experiment. Numbers in legend denote time in scarifier. Ninety-five percent confidence intervals are excluded for clarity. Seeds were germinated with a 12-hour photoperiod at 25°C for 23 days.

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CHAPTER 4 CONCLUSIONS

The Florida panhandle native forbs, Asclepias humistrata (sandhill milkweed) and

Lupinus diffusus (sky-blue lupine), have potential restoration and ornamental use but propagation protocols are lacking for both species. This study pioneered A. humistrata seed germination understanding by describing optimal germination temperatures (24-

28°C), size (≥5mm long), and follicle maturity (dehisced or dehisced after slight application of pressure). Additionally, it was found that seed germination can be improved in dark depending on seedlot. L. diffusus germination was improved by over

30% from existing germination studies by using time in an electric seed scarifier followed by time in sodium hypochlorite rather than time in a sulfuric acid bath. Future greenhouse and outplanting studies, and additional germination studies, should build on this study to make propagation protocols for A. humistrata and L. diffusus complete and accessible to restorationists and native plant growers alike.

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APPENDIX A TETRAZOLIUM TESTING

Tetrazolium (TZ) tests are useful in the native seed industry for quickly identifying the viability of populations by taking subsamples of populations and extrapolating TZ viability data to predict seed viability for the sampled population. TZ tests should be conducted on subsamples of a population before any large-scale native seed collection projects to estimate number of seeds to collect. Seed viability data can be used to interpret intrapopulation variability over time and interpopulation variability.

The TZ test is named for the colorless water-soluble bioactive chemical, 2,3,5- triphenyltetrazolium chloride, which is reduced to the nondiffusible red-colored compound formazan by enzyme dehydrogenases present in respiring seed tissue (Patil and Dadlani 2009). Therefore, only seeds which are stained red to pink are considered viable. However, seeds infected with pathogens or other organisms also stain red and give false viability data. Therefore, it is important to understand typical staining patterns, which are described in some detail in the TZ Testing Handbook, when analyzing stained seeds during a TZ test.

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APPENDIX B SURVIVAL ANALYSIS

Traditional statistical methods such as analysis of variance and curve-fitting have been the typical means of analyzing germination data. However, germination data present several unique challenges that render traditional methods inappropriate mainly due to assumption violations. For example, germination data are binary. A seed either germinates or it does not. Methods such as ANOVA and curve-fitting cannot account for the nature of this data. Perhaps, logistic regression could be used to analyze binary data. But, logistic regression does not consider the presence of right-censored data

(see below). Next, germination data is collected over some temporal scale (e.g. days, weeks or months) dictated by experimental design. It is rare that all seeds will germinate prior to the end of the experimental period. This is especially true for seeds of non- domesticated plants. Moreover, observations are repeatedly made on the same cohort of seeds throughout the experimental period. The temporal spread of germination events means that data are not normally distributed. Assumptions of independence are violated because repeated observations on the same cohort of seeds results in serial auto-correlation of residuals. Also, the presence of non-germinated seeds at the end of an experiment underestimate means and variances. Standard statistical methods are not capable of handling such right-censored data (McNair and others 2013)

Alternatively, survival analysis, known also as time-to-event analysis, failure-time analysis and reliability analysis, is a powerful group of statistical methods used to study the timing and occurrence of events (e.g. seed germination) on individuals (e.g. seeds) across numerous scientific disciplines (Allison 2010; McNair and others 2013). Non- and semi-parametric survival analyses offer several advantages over traditional

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statistical techniques including, but not limited to: 1) accommodation of censored observation, non-normal data, non-independent samples and heterogeneous variances;

2) analysis based on the distribution of event times for individual experimental units rather than cumulative results; 3) evaluation of multiple quantitative and qualitative covariates and 4) the ability to incorporate time-dependent covariates.

Non-parametric (i.e., Kaplan Meier) and semi-parametric (i.e., Cox proportional hazards) survival analysis statistical methods are based on the survivor and hazard functions, respectively (McNair and others 2013). Non-parametric statistics do not assume any specific probability distributions while semi-parametric statistics include non-parametric and parametric (defined probability distributions) assumptions. The

Kaplan-Meier estimator is a non-parametric statistical model that estimates the survivor function using exact data and continuous observations (McNair and others 2013).

Germination experiments use interval data with periodic simultaneous observation, unless a very dedicated researcher observes seeds non-stop for the duration of an experiment, which is impractical. However, McNair and others (2013) justify that it is statistically safe and appropriate to use statistical models which assume exact data with continuous observations when conducting germination experiment survival analyses.

The Kaplan-Meier estimator is easily visualized via a step-function that shows temporal patterns of germination events while simultaneously providing an estimate for the probability of germination.

Cox-regression is a semi-parametric survival analysis statistical method, based on the hazard function, that allows for the assessment of multiple covariate effects which can change over time (e.g. time-dependent covariates). The hazard function

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quantifies the instantaneous risk of an event occurring (i.e., seed germination) at an infinitesimal time, given that the individual (seed) in question has not already experienced the event (Allison 2010; McNair and others 2013).

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BIOGRAPHICAL SKETCH

Gabriel graduated from the University of Oklahoma in 2011 with a Bachelor of

Science degree in Botany with a minor in Chemistry. He then took horticulture classes while working in nurseries from 2013 to 2014. Most recently he obtained a Master of

Science degree in December of 2016 from the Environmental Horticulture department.

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