Floral Biology of Alabama’s Spigelia species (Family Loganiaceae) By Gavin S. Shotts A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Master of Science Auburn, Alabama Date: May 1, 2021 Keywords: Conservation, pollination, seed production, reproductive biology Copyright 2021 by Gavin Shotts Approved by Robert S. Boyd, Chair, Professor of Biological Sciences James M. Affolter, Professor of Horticulture Matthew E. Wolak, Assistant Professor of Biological Sciences Abstract This thesis examines the pollination ecology of Alabama species of Spigelia. Alabama currently has two species that are under threat of extinction: S. gentianoides and S. alabamensis. The U.S. Fish and Wildlife Service has highlighted the need for studies of the reproductive biology of these species. I analyzed reproductive biology of the rare species S. gentianoides and S. alabamensis, as well as the common species S. marilandica. Determination of likely mating strategies of Spigelia was based on floral morphology and phenology, and evaluation of floral timing. Controlled pollination experiments were used to determine mating system (if flowers are self-compatible or self-incompatible, as well as the degree to which they can outcross). Additionally, floral visitors were observed to identify potential pollinators. Flowers were diurnal, with two-day to three-day longevity, with highly reduced herkogamy and dichogamy to the point where self-pollination is essentially assured. Controlled pollination experiments showed that Spigelia can set fruit via autopollination, selfing, or outcrossing. Floral visitor observations indicated low visitation rates to flowers and few potential pollinators. These results, in addition to prior genetic studies, suggest Spigelia species in Alabama are predominantly selfing. Survival of the remaining populations of the rare species, S. gentianoides and S. alabamensis, is dependent on effective conservation management. ii ACKNOWLEDGEMENTS I would like to thank my graduate advisor and committee chair, Dr. Robert S. Boyd for his support and encouragement throughout my graduate career. I also thank Dr. James Affolter and Dr. Matthew Wolak for serving on my committee and providing guidance when needed. I thank my former lab group Alan Jeon, Matthew Paek, Dr. Charles Ray and Bashira Chowdhury, for the countless hours in the field collecting data and who have been invaluable in numerous ways. I thank Jacob Botello for his help with some of my statistical analyses and offering advice whenever needed. I sincerely thank my family, my parents, Tammy and Stanley Shotts, for their love, support, encouragement, and belief in me, and my brothers Stan and Seth Shotts for always offering advice and support when needed. Lastly, I want to thank my wife Chelsea Shotts, who has showed endless love and support throughout this process, listening and assisting with even the most minor of problems every step of the way. Journal style used: Castanea iii TABLE OF CONTENTS ABSTRACT....................................................................................................................................ii ACKNOWLEDGEMENTS............................................................................................................iii LIST OF FIGURES.........................................................................................................................v LIST OF TABLES .........................................................................................................................vi I. INTRODUCTION ....................................................................................................................1-7 II. MATERIALS AND METHODS Study Species................................................................................................................................7-8 Study Populations.......................................................................................................................9-10 Floral Timing Study..................................................................................................................10-11 Floral Morphology and Phenology Study................................................................................11-13 Mating System Study...............................................................................................................13-15 Floral Visitation Observations.......................................................................................................16 III. RESULTS Floral Timing Study.................................................................................................................16-18 Floral Morphology and Phenology Study................................................................................19-20 Mating System Study...............................................................................................................21-23 Floral Visitation Observations..................................................................................................23-24 IV. DISCUSSION....................................................................................................................24-29 LITERATURE CITED ............................................................................................................30-39 iv LIST OF FIGURES Figure 1: Flowering period and peak bloom in 2018 and 2019 for S. marilandica populations..........................................................................................................................17 Figure 2: Flowering period and peak bloom in 2018-2019 for S. gentianoides and S. alabamensis........................................................................................................................18 Figure 3: Predicted probability of fruiting success from treatments in the Mating System Study……………………………………………………………………………………..22 v LIST OF TABLES Table 1: Comparison of phenotypic traits (Hershberger 2012) and conservation status (USFWS 2012) for Spigelia study species......................................................................................................8 Table 2: Herkogamous characteristics (mean+SD) in the three populations of Spigelia..............20 Table 3: Proportions of treated flowers that developed a fruit, and the estimated marginal mean probability of fruiting success (“emmeans”) by population and treatment (95% confidence intervals inside brackets) ...............................................................................................................21 Table 4. Mean seed set per fruit (+SD) for each treatment in the Spigelia populations. Different superscripts for the Alley Road S. marilandica population denote significantly different means by Tukey’s multiple comparison tests (P < 0.05) .........................................................................23 Table 5: Observed insect floral visitors to Spigelia flowers in 2019 and the numbers of each insect species visiting flowers during observations. (ND* for Not Determined) .........................24 vi Introduction The Earth is currently suffering a wave of anthropogenic biodiversity loss, with rates of extinction estimated to be 100–1,000 times the observed rate between previous mass extinctions in the fossil record (Barnosky et al. 2011, Pimm et al. 2014). For plants, which are the foundation of terrestrial food webs, an estimated 20% of all species are currently threatened with extinction (Brummitt et al. 2015). The International Union for the Conservation of Nature (IUCN) recently updated their Red List, concluding that there are 13,494 threatened species (47.7%) among 28,287 assessed species in major taxonomic plant groups (IUCN 2020). Havens (2014) argues that currently no country has an adequate conservation strategy for plants: they are becoming increasingly rare, their conservation is underfunded, they are not fully protected, and their value is underappreciated. Plant conservation efforts significantly increased with the passage of the U.S. Endangered Species Act of 1973 (USFWS, 1988). The Act established a legal mandate of unprecedented proportions to promote the collection, analysis, and exchange of biological information. The successful recovery of species requires a significant amount of scientific information, yet the present level of understanding and communication among researchers and resource managers is not sufficient (Shine and Doody 2011). Bridging this gap is a necessity for plant conservation moving forward. While many studies suggest management strategies for particular rare plants, few focus on reproductive biology, and even fewer involve the importance of vectors for pollen transfer (Boyd 1994, Kremen and Ricketts 2000). There is widespread consensus that documentation of the reproductive biology of endangered species may be useful for understanding why they are endangered (Schemske et al. 1994). Reproduction by seed is essential for plant species migration, promotes adaptation through production of genetic variation, and ultimately allows population persistence (Fenner and Thompson 2005). Two major components of plant reproductive biology are pollination and seed dispersal. These two factors play a critical role in determining genetic diversity and population size (Ellstrand and Elam 1993, Smith et al. 2015) and, despite their importance, they are often overlooked in conservation and management programs (Calviño-Cancela et al. 2012, Neuschulz et al. 2016,