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2012 Floral Morphology and Development in Houstonia Procumbens (Rubiaceae), a Uniquely Distylous, Cleistogamous Species Eric (Eric Hunter) Jones

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COLLEGE OF ARTS AND SCIENCES

FLORAL MORPHOLOGY AND DEVELOPMENT IN HOUSTONIA PROCUMBENS

(RUBIACEAE), A UNIQUELY DISTYLOUS, CLEISTOGAMOUS SPECIES

By

ERIC JONES

A dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Degree Awarded: Summer Semester, 2012 Eric Jones defended this dissertation on June 11, 2012.

The members of the supervisory committee were:

Austin Mast Professor Directing Dissertation

Matthew Day University Representative

Hank W. Bass Committee Member

Wu-Min Deng Committee Member

Alice A. Winn Committee Member

The Graduate School has verified and approved the above-named committee members, and certifies that the dissertation has been approved in accordance with university requirements.

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I hereby dedicate this work and the effort it represents to my parents Leroy E. Jones and Helen M. Jones for their love and support throughout my entire life. I have had the pleasure of working with my father as a collaborator on this project and his support and help have been invaluable in that regard. Unfortunately my mother did not live to see me accomplish this goal and I can only hope that somehow she knows how grateful I am for all she’s done.

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ACKNOWLEDGEMENTS

I would like to acknowledge the members of my committee for their guidance and support, in particular Austin Mast for his patience and dedication to my success in this endeavor, Hank W. Bass for his help in acquiring reagents needed for lab work and. Alice Winn for her guidance with statistical analyses. I would also like to acknowledge my fellow graduate students who helped me learn the skills that I came to need in order to accomplish my research and for many engaging conversations and moral support. In particular I owe a debt of thanks to Abigail Pastore, Chris Buddenhagen, and Elise Gornish for help with statistical analyses. I would also like to acknowledge the undergraduate students who formed Team Houstonia as my research group for all their efforts and support. Last, but certainly not least I would like to acknowledge my family for their support and patience with this endeavor. In particular I would like to thank my father for all his help with my research and his support throughout this process and my wife, Penny, and our son, Thanial, for all their patience as I missed so much time with them through the course of this endeavor. I look forward to making it up to them in the years to come.

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TABLE OF CONTENTS

List of Tables ...... vi List of Figures ...... vii Abstract ...... viii 1. HOW UNIQUE IS THE CO-OCCURRENCE OF HETEROSTYLY AND CLEISTOGAMY AND WHAT IS THE EVOLUTIONARY HISTORY OF HETEROSTYLY IN THE COFFEE FAMILY (RUBIACEAE)? ...... 1 1.1 Introduction ...... 1 1.2 Methods ...... 6 1.2.1 Assessment of the co-occurrence of heterostyly and cleistogamy ...... 6 1.2.2 Phylogenetic inference and ancestral state reconstruction ...... 7 1.3 Results ...... 8 1.3.1 Taxonomic accounting of heterostylous and cleistogamous taxa ...... 8 1.3.2 Phylogenetic inference and ancestral state reconstruction ...... 14 1.4 Discussion ...... 24 1.4.1 Co-occurrence of heterostyly and cleistogamy ...... 24 1.4.2 The evolution of heterostyly in the Rubiaceae ...... 26 1.5 Conclusions ...... 27 2. RECIPROCAL HERKOGAMY IN HOUSTONIA PROCUMBENS (RUBIACEAE) . 29 2.1 Introduction ...... 29 2.2 Methods ...... 35 2.2.1 Specimen collection and imaging ...... 35 2.2.2 Morphometrics ...... 38 2.2.3 Pollination dynamics ...... 40 2.3 Results ...... 41 2.3.1 Organ reciprocity ...... 41 2.3.2 Floral morphology ...... 43 2.3.3 Pollination dynamics ...... 47 2.4 Discussion ...... 48 2.4.1 Reciprocal herkogamy in Houstonia ...... 48 2.5 Conclusions ...... 51 3. DISTYLOUS FLOWER DEVELOPMENT IN HOUSTONIA PROCUMBENS ...... 53 3.1 Introduction ...... 53 3.2 Methods ...... 57 3.3 Results ...... 59 3.4 Discussion ...... 71 3.5 Conclusions ...... 73 APPENDIX A – GENBANK ACCESSION NUMBERS FOR DNA SEQUENCES USED IN PHYLOGENETIC INFERENCE OF THE RUBIACEAE ...... 75 REFERENCES ...... 85 BIOGRAPHICAL SKETCH ...... 92

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LIST OF TABLES

1.1 Accounting of heterostylous species reported by order, family and genus ...... 9

1.2 Genera that contain both heterostylous and cleistogamous species ...... 13

1.3 Expected and observed co-occurrence of heterostyly and cleistogamy among angiosperms ...... 15

2.1 Summary of growth form and floral characteristics of Houstonia species used in this investigation ...... 33

2.2 Reciprocity measures for Houstonia ...... 43

2.3 Measurements of floral organs for Houstonia ...... 46

3.1 Metrics of distylous flower development ...... 61

3.2 Analysis of rates of flower development ...... 65

3.3 Cell sizes for floral organs of H. procumbens (Hpro) and H. caerulea (Hcae) ...... 70

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LIST OF FIGURES

1.1 Overview of the phylogeny of the Rubiaceae based on rbcL ...... 17

1.2 Details of the Rubiaceae phylogeny ...... 18 – 23

2.1 Phylogeny of Houstonia reproduced from Church and Taylor (2005) ...... 34

2.2 Maps of field sites and species distributions ...... 36

2.3 Image of dissected flowers used in this study ...... 37

2.4 Formulae employed in calculations of organ reciprocity for distylous taxa based on the method developed by Sanchez et al. (2008) ...... 39

2.5 Boxplots of reciprocity for Houstonia species ...... 42

2.6 Boxplots of bootstraps of reciprocity for species of Houstonia species investigated in this study ...... 45

2.7 Scatterplots of organ heights ...... 47

2.8 Plot of means of pollen diameters for Houstonia species ...... 49

3.1 Developing distylous flowers of H. procumbens ...... 62

3.2 Developing distylous flowers of H. caerulea ...... 63

3.3 Plot of anther stigma distance over the course of flower development ...... 66

3.4 Plot of anther height over the course of flower development ...... 67

3.5 Plot of stigma growth rates over the course of flower development ...... 68

3.6 Images of cleistogamous flowers of H. procumbens and H. pusilla ...... 69

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ABSTRACT

Among flowering plants, floral form is inherently linked to reproductive success and is therefore a key element in the evolution of angiosperm lineages. Variations in floral form and associated mating systems have demonstrated that flowers are well adapted in many cases to promote predominantly either outcrossing or self-fertilization among members of a given lineage. The focus of this work is to examine the developmental interactions and consequent effects on the morphology of two such flower types within individuals of Houstonia procumbens (J.F. Gmel.) Standl. (Rubiaceae). The two flower types in question are distylous and cleistogamous. Distyly is a form of heterostyly, wherein the reproductive organs of flowers in a given population are spatially separated (herkogamy) and arranged reciprocally to one another (reciprocal herkogamy) among individuals that represent two alternate morphotypes. Cleistogamy refers to the production of both flowers that open (chasmogamous) to interact with pollinators and flowers that self-pollinate precociously in bud (cleistogamous) on individuals of a given species. While both of these pollination syndromes, distyly and cleistogamy, appear to have arisen convergently, multiple times, across angiosperms both pollination syndromes apparently co-occur in only one species H. procumbens, an annual, herbaceous member of the coffee family (Rubiaceae). The goal of this work is to examine the interactions of these two pollination syndromes in H. procumbens by comparing the form and development of the various flower morphs (distylous morphs and cleistogamous flowers) of this species to those of closely related taxa that produce either distylous or cleistogamous flowers, but not both. The first chapter of my dissertation establishes the degree to which discoveries about the heterostylous, cleistogamous flowering plant species H. procumbens are applicable to other species. I use literature searches to establish that other species that are both heterostylous and cleistogamous are currently unknown, and I use phylogenetic inference and ancestral state reconstruction for the Rubiaceae to establish that heterostyly arose 27–36 independent times in the family and that the origin of heterostyly for H. procumbens occurred in an ancestor that produced at least 820 extant, descendent species. I also show that the joint probability of heterostyly and cleistogamy in angiosperm families, genera, and species is quite low, and the observed numbers of taxa in which the two co-occur at the ranks of family and genus (but not species) is

viii greater than expected, though not significantly so. The second chapter addresses the effect of cleistogamy on reproductive organ reciprocity for H. procumbens. I use comparisons among close relatives to show that the overall reciprocity of H. procumbens changed little with the evolution of cleistogamy but that reciprocity in a close distylous relative, H. caerulea, was sufficiently low to qualify it as “style dimorphic” by some standards—a situation that I suggest calls into question the use of the reciprocity index for such distinctions. I also demonstrate that H. procumbens does not have a pollen size or staining dimorphism, as is seen in other heterostylous members of Houstonia, making assessments of disassortative pollen transfer difficult. The third chapter examines the chasmogamous and cleistogamous flower development of H. procumbens and chasmogamous flower development of H. caerulea. I demonstrate that differences in the rate of elongation of styles and corollas lead to the difference in stigma and anther heights seen in the two chasmogamous morphs of H. procumbens and H. caerulea, and that this dimorphism in growth rates is established early in development. Additionally, I show that there is a relatively small anther-stigma distance in the early stages of floral development of H. caerulea, which is unexpected, since one might expect this to be a developmental pattern that makes cleistogamy (e.g., as in H. procumbens but not H. caerulea) evolutionarily simpler (by reaching sexual maturity in those earliest stages). Instead, I show that H. procumbens reduces the anther-stigma distance in its cleistogamous flowers by reaching sexual maturity when the corolla is short (and the anthers low) and by producing helical styles.

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CHAPTER ONE

HOW UNIQUE IS THE CO-OCCURRENCE OF HETEROSTYLY AND CLEISTOGAMY AND WHAT IS THE EVOLUTIONARY HISTORY OF HETEROSTYLY IN THE COFFEE FAMILY (RUBIACEAE)?

Introduction

This chapter establishes the degree to which discoveries about the heterostylous, cleistogamous flowering plant species Houstonia procumbens (Rubiaceae) are applicable to other species. Heterostyly and cleistogamy have broad taxonomic distributions across the angiosperms. They are found in 28 (Barrett and Shore, 2008) and 50 families (Lord, 1981; Culley and Klooster, 2007) of approximately 406 total families (The Plant List, 2010), respectively. Heterostyly and cleistogamy represent floral adaptations toward divergent mating strategies, and both pollination syndromes have received much research attention in the past (see for heterostyly: Darwin, 1877; Vuilleumier, 1967; Ganders, 1979; Barrett and Shore, 2008; Cohen, 2010; see for cleistogamy: Darwin, 1877; Lord, 1981; Culley and Klooster, 2007). The species-rich angiosperm family Rubiaceae contains more heterostylous taxa than any other. The goals of this chapter are (1) to determine how frequently heterostyly and cleistogamy co-occur in the flowering plants, (2) to determine the closest relatives of Houstonia, my focal genus, and (3) to determine whether heterostyly in the Rubiaceae represents many independent origins of the condition or a single origin with many subsequent losses. If heterostyly in Rubiaceae arose multiple times, an additional goal is to circumscribe the group of taxa that is heterostylous due to the origin of heterostyly shared with H. procumbens. Heterostyly is characterized by the reciprocal positioning of spatially separated reproductive organs (anthers and stigmas) among individuals of a population, a feature called reciprocal herkogamy (Webb and Lloyd, 1986). This organ reciprocity contributes to the female and male components of fitness in several ways. The spatial separation of anthers and stigmas (herkogamy) reduces sexual interference, defined as the interference of anthers with pollen deposition on stigmas or the interference of stigmas with pollen export from anthers (Barrett, 2002a). Additionally, herkogamy

1 serves to reduce pollen discounting (a reduction in the outcrossed siring success due to self-fertilization, Eckert et al., 2009), while an often-present biochemical self- and intramorph-incompatibility system contributes to the female component of fitness by promoting outcrossing among morphs of a given population (Barrett, 2002b). Further the reciprocal nature of herkogamy in heterostylous species serves to increase the precision of pollen transfer between heterostylous morphs providing a benefit to the male component of fitness. Heterostylous species may express any of a suite of ancillary polymorphisms in addition to reciprocal herkogamy and the self- and intramorph-incompatibility. These ancillary polymorphism may include pollen size and/or production dimorphisms and stigma size, shape, and/or stigmatic papillae dimorphisms (Dulberger, 1992). The most recent taxonomic review of heterostyly (Ganders, 1979) accounted for 24 families and 165 genera that contain heterostylous species of 406 families and 14,038 genera of flowering plants (The Plant List, 2010). While the development, genetics, and function of heterostyly have been examined in great detail (reviewed in Barrett, 1992) there is still some contention regarding the evolutionary origins of this mating system (Lloyd and Webb, 1992). The fundamental disagreement regards the order of appearance of the two principle aspects of heterostylous mating systems, the reciprocal herkogamy and the self- and intramorph-incompatibility. In his seminal work on heterostyly, Darwin (1877) proposed that the reciprocal herkogamy arose initially to improve the accuracy of pollen transfer between individuals of heterostylous species (disassortative pollination) and that the incompatibility arose secondarily to reinforce intermorph matings. What has followed has been a variety of hypotheses regarding the order of evolution of these two main components of heterostyly. Mather and deWinton (1941) hypothesized that both reciprocal herkogamy and self- and intramorph-incompatibility arose simultaneously. Charlesworth and Charlesworth (1979), using a genetic model, proposed that the self-incompatibility arose before reciprocal herkogamy. This result is based on an assumption that ancestors of heterostylous species were non-herkogamous (no spatial separation of stigmas and anthers) and that the self-incompatibility is more effective is promoting disassortative pollination than is reciprocal herkogamy. Then Lloyd and Webb (1992) proposed another hypothesis which contends that heterostylous species arose from an originally herkogamous ancestor. This they based on examination of the floral forms of genera and families that contain heterostylous taxa, in

2 which herkogamy is prevalent. They proposed that a stylar polymorphism could arise in a population that effects disassortative mating and that the self-incompatibility arose secondarily as a means to reinforce the morphological polymorphism. Cleistogamous (“closed marriage”) flowers do not achieve anthesis (opening of the flower), but self-pollinate in bud and are contrasted with chasmogamous (“open marriage”) flowers that achieve anthesis and are available for outcrossing. Cleistogamous species have been described as exhibiting dimorphic, induced, or complete cleistogamy (Culley and Klooster, 2007). Dimorphic cleistogamy refers to a species that produces two morphologically distinct types of flowers that differ developmentally from inception of the primordial buds. The timing of expression of chasmogamous and cleistogamous flowers may differ according to day length and/or temperature. Dimorphic cleistogamy is the condition for 77.3% of all cleistogamous species (Culley and Klooster, 2007) and is the type of cleistogamy exhibited by H. procumbens. Induced cleistogamy refers to the production of cleistogamous flowers that is induced by environmental changes in which developmental arrest of chasmogamous flowers results in the production of cleistogamous flowers. Complete cleistogamy refers to species that produce only cleistogamous flowers. These types of cleistogamy account for 8.3% and 10.4% of cleistogamous species, respectively, while 3.5% percent of cleistogamous species could not be assigned to one of these three categories (Culley and Klooster, 2007). In the most recent review of this pollination syndrome, Culley and Klooster (2007) examined the distribution of cleistogamy across angiosperms based on three family-level phylogenies (Soltis et al., 2000; Hilu et al., 2003; Soltis et al., 2005). At the family level it was estimated that cleistogamy has arisen 34–41 times. Cleistogamy may have arisen independently twice in the Eumagnoliids, six to eight times in the Monocots, and 24–31 times in the Eudicots (Culley and Klooster, 2007). These estimates are conservative, as cleistogamy may have arisen multiple times within some families (e.g., Caryophyllaceae and Nyctaginaceae). Rubiaceae (the coffee family) contains the greatest number of heterostylous species and genera of any angiosperm family (Darwin, 1877; Ganders, 1979). It is the fifth largest angiosperm family with 13,150 species distributed among 611 genera, and it is distributed worldwide, particularly in warm and tropical regions (Stevens, 2001 onwards). The names of major lineages within the Rubiaceae vary among taxonomists. Robbrecht and Mannen (2006) describe two subfamilies, Cinchonoideae and

3 Rubioideae, divided into two supertribes each (Cinchonidinae + Ixoridinae and Psychotriidinae + Rubiidinae, respectively), as well as a lineage treated as within Cinchonoideae composed of Luculia and Coptosapelta that is given a tribal designation (Coptosapeltae) but not a supertribal designation. Alternately, Stevens (2001 onwards) describes 3 subfamilies including Rubioideae, Cinchonoideae, and Ixoroideae, and the lineage [Luculia [Coptosapelta+Acranthera]] (see also Rydin et al., 2009, and Bremer, 2009). Heterostyly was used as a diagnostic character for describing taxonomic relationships in the family by some authors (e.g., Bremekamp, 1940 as cited in Bahadur, 1968), although Verdcourt (1958) remarked in his revision of the herbaceous Rubiaceae of Africa that the character is not consistent at the generic level and so might have arisen multiple times in the family. Verdcourt (1958) also noted that heterostyly is most common in the Rubioideae, an observation which is still true with current circumscriptions of the subfamily. Bahadur (1968), in the most comprehensive review of heterostyly in the family to date, reported 416 distylous species from 91 genera and 21 tribes; this equates to >3% of all species and 13–15% of all genera. While other families have a greater proportion of heterostylous taxa (species or genera; e.g., Primulaceae), they are at least an order of magnitude smaller in size relative to the number of species described in the Rubiaceae. Later, Ganders (1979) noted that 92 genera in the Rubiaceae contain distylous members, more genera than any other angiosperm family. The genus Houstonia is placed in tribe Spermacoceae of subfamily Rubiodeae (Church, 2003). Houstonia is comprised of 20 species with North American distributions. Species of the genus Houstonia display a variety of floral morphologies, including monomorphy, distyly and cleistogamy arrayed among both perennial and annual life histories (Terrell, 1996; Church, 2003; Church and Taylor, 2005). Within Houstonia 14 species (70%) are distylous and the remaining six species (30%) exhibit a monomorphic floral morphology. Two species are cleistogamous: H. procumbens, a distylous perennial, and H. pusilla, an annual species that bears monomorphic chasmogamous flowers. Both species are in H. section Houstonia. Although both heterostyly and cleistogamy have broad taxonomic distributions among angiosperms, the small proportions of each at the level of family (0.059 of the total number of families are heterostylous; 0.123 are cleistogamous) and genus (0.012 are heterostylous and 0.016 are cleistogamous) leads to low probabilities of the co- occurrence of the two at those taxonomic levels (0.007 and 0.00019, respectively).

4 Furthermore, the common presence of a self- and intramorph-incompatibility system in heterostylous lineages seems likely to preclude the evolution of cleistogamous flowers. However, cleistogamy could evolve as a means of reproductive assurance in lineages whose reproductive success depends on frequent and reliable pollinator service, if such services are unreliable. Testing whether the observed co-occurrence of heterostyly and cleistogamy is significantly more or less than expected by chance will allow me to infer whether self-incompatibility systems preclude the evolution of cleistogamy (lower than expected based on taxonomic distributions) or whether reproductive assurance, in response to unreliable pollinator service, promotes the evolution of cleistogamy in heterostylous lineages (greater than expected based on taxonomic distributions). As the presence of self-incompatibility mechanisms in heterostylous lineages is well documented (Barrett and Shore, 2008), I expect that heterostyly and cleistogamy co- occur less frequently than expected based solely on their taxonomic distributions. I will address the following hypotheses in this chapter: (1) based on the frequent occurrence of self-incompatibility in heterostylous species, I hypothesize that cleistogamy and heterostyly co-occur less frequently than expected by chance in angiosperms and that the single case in Houstonia procumbens is unique, and (2) based on the taxonomic distribution of heterostyly in the Rubiaceae, I hypothesize that there are multiple origins of distyly in the Rubiaceae but that heterostyly is plesiomorphic for the most recent common ancestor of Rubioideae because of the taxonomic breadth across which it is found in that subfamily. Subfamily Rubioideae contains Houstonia procumbens and the greatest number of other heterostylous taxa. I will address these hypotheses with parsimony reconstructions of ancestral states and by examining the proportional likelihoods of the data given the alternate states of distyly and monomorphy at the nodes of interest. Addressing these hypotheses will lead to an understanding of how broadly informative study of H. procumbens will be in our understanding of the interplay of heterostyly and cleistogamy and of heterostyly in the Rubiaceae.

5 Methods

Assessment of the co-occurrence of heterostyly and cleistogamy Heterostyly was recognized at the rank of genus by Ganders (1979) who described its occurrence in 166 genera from 28 families. Cleistogamy was accounted for to the rank of species by Culley and Klooster (2007) who described its occurrence in 693 species from 228 genera in 50 angiosperm families. An accounting of heterostyly at the rank of species was thus necessary, at least when genera contain both heterostylous and cleistogamous members. An accounting of heterostylous species was compiled from the primary literature based on reviews of heterostlyly (Vuilleumier, 1967; Bahadur, 1968; Ganders, 1979). Each of these reviews, with the exception of Bahadur (1968), lists heterostylous taxa by genus and the references therein were examined in order to accumulate a list of heterostylous species. I then used Web of Science and PubMed to search for more recent reports of heterostylous taxa using the following search terms: “heterosty*,” “disty*,” and “tristy*.” I cross referenced accounts of heterostylous species with those of cleistogamous species from the most recent review of the taxonomic distribution of cleistogamy, presented by Culley and Klooster (2007). I also conducted a search of Web of Science for other cleistogamous species using the search term “cleisto*.” I then searched Web of Science for any other taxa that are reported to be both heterostylous and cleistogamous using the following search terms: “cleisto* AND heterosty*,” “cleisto* AND disty*,” and “cleisto* AND tristy*.” I then examined these cross-referenced data sets to determine which lineages contained both cleistogamous and heterostylous taxa. I calculate the joint probability of these two pollination syndromes as the product of the probabilities of selecting the individual syndromes, given the proportion of the total number of families, genera, and species that contain heterostylous and cleistogamous taxa and assuming that the evolution of the two are independent of one another. It should be noted that my accounting of heterostylous taxa is not definitive due to some cases in which a species level accounting of heterostylous taxa was not successful. The result is that the proportion of heterostylous species reported here is certainly an underestimate. A binomial proportions test (prop.test) was conducted in R (R Development Core Team, 2011) comparing the proportions of expected and observed heterostylous and cleistogamous taxa at each taxonomic rank (family, genus, and species) in order to determine whether

6 differences between expected and observed co-occurrences of heterostyly and cleistogamy are statistically significant.

Phylogenetic inference and ancestral state reconstructions I generated a phylogeny of the Rubiaceae based on the rbcL sequences available from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) in January 2006. Data for the plastid gene rbcL represents the most complete taxonomic sampling for heterostylous species in the Rubiaceae to date (pers. obs.). The gene rbcL is ca. 1440 bases in length in Arabidopsis and encodes for the enzyme Ribulose-1,5-bisphosphate carboxylase (RuBisCO), large subunit (TAIR, accession no. 1009022826). The outgroups used were Cinchona laeve (Apocynaceae), a family closely related to Rubiaceae (Stevens, 2001 onwards) and also in order Gentianales (The Angiosperm Phylogeny Group, 2009), and Arabidopsis thaliana (Brassicaceae), a species from a different, but closely related order (Brassicales). The sequence of the rbcL gene for A. thaliana was taken from the complete plastid genome sequence for that species. The large number of taxa (over 350) precluded the use of currently available substitution model testing software (e.g., jModeltest; Posada, 2008) to assess which model is adequately parameter-rich using an information criterion such as the Akaike Information Criterion (Akaike, 1974). Instead, I used a moderately parameter-rich substitution model with separate parameters for transitions and transversions, HKY85 (Hasegawa et al., 1985). Phylogenies with branch lengths were generated using Bayesian methods in MrBayes (Ronquist and Huelsenbeck, 2003). In MrBayes, two independent Markov Chain Monte Carlo analyses (MCMCs) were run, each with one “cold” and three “heated” chains. These were run for 10,000,000 generations, sampling every 1,000th generation. The first quarter of the generations were discarded as the “burn in” for the purpose of assessing convergence using the potential scale reduction factor (uncorrected) of Gelman and Rubin (1992) and generating the consensus tree. I used parsimony and maximum likelihood methods in Mesquite version 2.75 build 566 (Maddison and Maddison, 2011) for the purpose of reconstructing ancestral states. I coded taxa as distylous if a member of the genus represented in the phylogeny is reported to be distylous. The parsimony inference used the consensus tree generated by MrBayes using the sumt command. Because likelihood analyses in Mesquite require

7 a fully bifurcating tree, the polytomies in that consensus tree were resolved randomly to 0-length branches and the 0-length branches were transformed to a very short length (0.0000001) in Mesquite prior to the likelihood calculations. Log likelihoods for a one- parameter and an asymmetric two-parameter model were evaluated for a statistically significant difference in their ability to describe the data using a likelihood ratio test (Goldman, 1993). Mesquite was used to report the proportional likelihoods for the data given the alternative states of distyly and monomorphy at nodes of interest (the most recent common ancestor (MRCA) of tribe Spermacoceae, subfamily Rubiodeae, and family Rubiaceae) with a threshold of significance set at 2. This is equivalent to the threshold of a log likelihood ratio of 7.4 : 1 advocated by Edwards (1972). It is a threshold used elsewhere for likelihood inference of ancestral states as a ‘rough minimum’ (Schluter et al., 1997).

Results

Taxonomic accounting of heterostylous and cleistogamous taxa Heterostyly was found to occur in 1,556 species from 160 genera representing 28 angiosperm families (Table 1.1). For several genera, accounts of heterostylous taxa at the level of species had not been found in the literature by the time of this reporting. In these cases the genus was counted as a single heterostylous species for the purposes of tabulating the number of heterostylous species. Unlike the distribution of cleistogamy, heterostyly is almost entirely restricted to the Eudicots. Three exceptions to this include the Amaryllidaceae, the Iridaceae, and the Pontideriaceae, and all but one species from those families is tristylous (only Narcissus albimarginatus is distylous). The five angiosperm families that contain the preponderance of heterostylous taxa are, in descending order by number of heterostylous species, Rubiaceae (418 spp.), Primulaceae (415 spp.), Boraginaceae (350 spp.), Turneraceae (178 spp.), and Erythroxylaceae (175 spp.).

8 Table 1.1 Accounting of heterostylous species reported by order, family and genus. Taxa are organized alphabetically by family. The type of heterostyly observed in a given genus is indicated with a “D” for distyly and a “T” for tristyly. The number of heterostylous species in a given genus is indicated (No. het. spp.). Genera for which an accounting of heterostylous species could not be found in the literature are indicated by a bold, italicized “1” in the “No. het. spp.” column.

Order Family Genus Distylous (D) / Genus No. het. tristylous (T) size spp. Lamiales Acanthaceae Oplonia D 19 3 Asparagales Amaryllidaceae Narcissus D/T 27 6 Unplaced Boraginaceae Amsinckia D 15 5 euasterid I Anchusa D 35 2 Arnebia D 25 1 Cordia D 320 319 Cryptantha D 100 1 Lithodora D 7 6 Lithospermum D 45 18 Mertensia D 45 1 Paracaryum D 9 1 Pulmonaria D 18 1 Oxalidales Connaraceae Agelaea T 50 1 Burttia D 1 1 Cnestis D 40 1 Connarus D 100 1 Ellipanthus D 10 1 Rourea D 55-60 1 Malpighiales Erythroxylaceae Erythroxylum D 250 1 Gentianales Gentianaceae Sebaea D 60 2 Gentianales Gelsemiaceae Gelsemium D 3 3 Mostuea D 8 8 Sebaea D 60 2 Malpighiales Hypericaceae Cratoxylum D 6 1 Eliea D 1 1 Hypericum D 370 3 Vismia D 35 1 Asparagales Iridaceae Nivenia D 5 3 Lamiales Lamiaceae Salvia D 900 1 Malpighiales Linaceae Hugonia D 32 2 Linum D 200 2 Gentianales Loganiaceae Gelsemium D 3 3

9 Table 1.1 continued.

Order Family Genus Distylous (D) / Genus No. het. tristylous (T) size spp. Myrtales Lythraceae Decodon D 1 1 Lythrum T 36 14 Nesaea D 56 8 Pemphis D 1 1 Rotala D 44 3 Malvales Malvaceae Hermannia D 100+ 1 Melochia D 54 5 Waltheria D 30-50 1 Asterales Menyanthaceae Menyanthes D 1 1 Nephrophyllidium D 1 1 Nymphoides D 38 38 Villarsia D 16 6 Santalales Olacaceae Schoepfia D 23 1 Santalales Oleaceae Forsythia D 7 7 Jasminum D 200 7 Schrebera D 10 10 Oxalidales Oxalidaceae Averrhoa D 2 1 Biophytum D 50 1 Dapania D 3 1 Oxalis D/T 500 48 Sarcotheca D 11 11 Caryophyllales Plumbaginaceae Acantholimon D 165 1 Armeria D 100 1 Ceratostigma D 8 2 Dyerophytum D 3 1 Goniolimon D 20 1 Limoniastrum D 9 1 Limonium D 350 1 – 6 Neslia D 1 1 Plumbago D 24 4 Ericales Polemoniaceae Gilia D 25 2 Caryophyllales Polygonaceae Fagopyrum D 8 8 Commelinales Pontederiaceae Eichhornia T 7 3 Pontederia T 5 3 Ericales Primulaceae Dionysia D 41 41 Hottonia D 2 1 Primula D 450 396 Vitaliana D 1 1 Gentianales Rubiaceae Agathisanthemum D 5-6 5 Amphiasma D 5-6 6 Anthospermum D 40 1 Aphaenandra D 2 1 Arcytophyllum D 15 2 Asperula D 90 1

10 Table 1.1 continued.

Order Family Genus Distylous (D) / Genus No. het. tristylous (T) size spp. Bouvardia D 20 2 Gentianales (cont) Rubiaceae (cont) Carphalea D 10 1 Carterella D 1 1 Chamaepentas D 1 1 Chasallia D 42 1 Cinchona D 23 5 Coccocypselum D 20 1 Craterispermum D 16 1 Cruckshanksia D 7 2 Danais D 40 1 Dibrachinostylus D 1 ? Diodia D 30 3 Ecpoma D 1 1 Gaertnera D 70 1 Gamotopea D 5 4 Geophila D 20 1 Gonzalagunia D 15 1 Guettarda D 80 1 Hedyotis D 250 11

Hedythyrsuys D 2 1 Heinsia D 4,5 1 Houstonia D 20 14 Hydnophytum D 52 1 Ixora D 300 1 Knoxia D 7 1 Lasianthus D 170 3 Lelya D 1 1 Leptodermis D 30 1 Luculia D 5 1 Machaonia D 30 1 Manettia D 80 1 Manostachya D 3 1 Mitchella D 1 1 Morinda D 80 3 Mussaenda D 100 49 Mycetia D 25 1 Mycetia D 25 1 Nertera D 15 1 Oldenlandia D 300 42 Ophiorrhiza D 150 4 Otomeria D 8 7 Paederia D 30 1 Palicourea D >200 1 Parapentas D 3,4 3 Paratriaina D 1 1 Pauridiantha D 25 1

11 Table 1.1 continued.

Order Family Genus Distylous (D) / Genus No. het. tristylous (T) size spp. Gentianales (cont) Rubiaceae (cont) Pentaloncha D 3 1 Pentanisia D 15 1 Pentas D 34 17 Pentodon D 2 2 Perama D 9 1 Pleiocraterium D 4 1 Pseudomussaenda D 4,5 2 Psychotria D 800,1500 91 Pyragra D 2 1 Rondeletia D 130 1 Rudgea D 150 4 Sabicea D 120 4 Sacosperma D 2 1 Schismatoclada D 20 3

Schwendenera D 1 1 Serissa D 2 1 Siderobombyx D 1 1 Sipania D 17 1 Spermacoce D 150 1 Spermadictylon D 1 1 Stenaria D 5 5 Stenotis D 9 1 Stephanococcus D 1 1 Temnopterynx D 1 1 Thridocalyx D 1 1 Trianolepis D 2 1 Tricalysia D 95 1 Wendlandia D 70 2 Santalales Santalaceae Arjona D 10 1 Saxifragales Saxifragaceae Jepsonia D 1-2 1 Santalales Schoepfiaceae Quinchamalium D 25 1 Malpighiales Turneraceae Hyalocalyx D 1 1 Loewia D 3 2 Piriqueta D 45 38 Stapfiella D 5 1 Streptopetalum D 6 5 Tricliceras D 12 12 Turnera D 128 109 TOTALS 28 160 1556

Culley and Klooster (2007) report that cleistogamy has been described for 693 species, from 228 genera in 50 angiosperm families. A search of the primary literature produced one putatively cleistogamous taxon not reported in this review, Lithospermum caroliniense (Boraginaceae; Kerster and Levin, 1968; Levin, 1968 and 1972). It was not

12 clear whether the cleistogamous flowers of L. caroliniense Levin (1968, p. 429) exhibit cleistogamy as it appears the putative cleistogamous flowers do eventually reach anthesis and as such exhibit what has been termed pre-anthesis selfing (Culley and Klooster, 2007). Additionally, Culley and Klooster’s reports of cleistogamy in distylous Houstonia caerulea (Rubiaceae) stemming from work by Ritzerow (1908) appears to be an error resulting from taxonomic revision which was not accounted for by Culley and Klooster (2007). Examination of that work revealed that the cleistogamous flowers that she examined, which she described as those of H. caerulea var. minor, were those of H. pusilla since H. caerulea var. minor is now considered a synonym of H. pusilla (Terrell, 1996). More recent treatments (Terrell, 1996; Church, 2003) record cleistogamy in H. procumbens and H. pusilla alone within the genus. Thus, the taxonomic accounting of cleistogamous species compiled by Culley and Klooster (2007) remains complete. Both cleistogamy and heterostyly were found to occur in six genera from five angiosperm families (Table 1.2). Those families that contain both heterostylous and cleistogamous members are all in the core Eudicots. In all cases where heterostyly and cleistogamy occur in the same genus, the type of heterostyly exhibited is distyly. Houstonia procumbens is the only species that produces both heterostylous and cleistogamous flowers.

Table 1.2 Genera that contain both heterostylous and cleistogamous species. The cleistogamous species reported for each genus are given (Cleist. spp.) with the source reference (Cleist. ref. from Culley and Klooster, 2007) and their status as heterostylous or not (Het.?).

Order Family Genus Cleist. spp. Cleist. ref. Het.? Gentianales Rubiaceae Houstonia H. procumbens Terrell (1996) Y H. pusilla Terrell (1996) N Lamiales Lamiaceae Salvia S. cleistogama Uphof (1938) N Myrtales Lythraceae Lythrum L. portula von Marilaun N (1895) Oxalidales Oxalidaceae Oxalis O. acetosella Darwin (1877); N Gorczynski (1929) O. corniculata Uphof (1938) N O. micrantha Uphof (1938) N

13 Table 1.2 continued.

Order Family Genus Cleist. spp. Cleist. ref. Het.? O. montana Jasieniuk & N Lechowicz (1987) O. sensitiva Darwin (1877) N O. stricta Uphof (1938) N Unplaced Boraginaceae Cryptantha C. capituliflora Calvino and N Euasterid I Galetto (2003) Lithospermum L. calycosum Johnston (1952) N L. confine Johnston (1952) N L. incisum Gleason & N Cronquist (1991) L. incisuro Johnston (1952) N L. mirabile Johnston (1952) N L. parksii Johnston (1952) N

The joint probability of heterostyly and cleistogamy at the ranks of family, genus, and species is very low (Table 1.3). The observed amount of overlap in these two features is at least double that expected by chance at the rank of family and genus, but it is less than expected by chance at the rank of species (Table 1.3). The binomial proportions tests show that the expected and observed co-occurrences of heterostyly and cleistogamy are not significantly different however (Table 1.3).

Phylogenetic inference and ancestral state reconstruction The sequences represent 367 (Appendix A) operational taxonomic units (OTUs). The sample represents 236 (38.6%) of the 611 genera in the family (Stevens, 2001 onwards), 98 (72.1%) of the 136 genera in subfamily Rubioideae (Robbrecht and Mannen, 2006), and 26 (42.6%) of the 61 genera in tribe Spermacoceae (Groeninchkx et al., 2009). The sample represents 58 (72.5%) of the 80 heterostylous genera and 65 (18.7%) of the 348 confirmed heterostylous species in the family.

14 Table 1.3 Expected and observed co-occurrence of heterostyly and cleistogamy among angiosperms. Numbers (No.) of angiosperm families, genera and species taken from The Plant List (2010). Proportions of heterostylous (het) and cleistogamous (CL) taxa at each rank are indicated. P-values displayed are from the binomial proportions test of the expected and observed values of heterostyly and cleistogamy.

Group Families Genera Species No. Angiosperms (Plant list) 406 14038 273174 No. heterostylous 28 160 1556 Proportion het 0.06897 0.01140 0.00570 No. cleistogamous 50 228 693 Proportion CL 0.12315 0.01624 0.00254 Expected het and CL 3.448 2.599 3.955 Observed het and CL 5 6 1 p-value 0.849 0.413 0.380

The MCMC was run a sufficient length to get the potential scale reduction factor for all parameters to within 0.1 of 1.0. The 50% majority-rule consensus of trees sampled after the burn-in of the MCMC resolves three clades and a single species, Hintonia latiflora, in a polytomy at the base of Rubiaceae (Figures 1.1 and 1.2). The three clades correspond to supertribes Ixoridinae and Cinchonidinae (both of subfamily Cinchonoideae) and subfamily Rubioideae plus tribe Coptosapeltae (Robbrecht and Mannen, 2006). Within subfamily Rubioideae, supertribe Psychotriidinae is paraphyletic with respect to a monophyletic supertribe Rubiidinae. The relationships among members of Rubioideae are well resolved, with few polytomies, but the Cinchonoideae and Ixoroideae are less well resolved. Tribe Spermacoceae is resolved as monophyletic (Figure 1.2e). Alternative most parsimonious ancestral state reconstructions of distyly in Rubiaceae imply 27–36 origins and 8–18 losses (Figure 1.2). The MRCA of Rubiaceae and the MRCA of Rubioideae are each inferred to be monomorphic, whereas that of Spermacoceae is inferred to be either distylous or monomorphic under equally parsimonious alternatives. The distyly of Houstonia is inferred to have arisen on different branches depending on the equally parsimonious solution considered—as

15 early as the branch leading to the MRCA of Paederia and Houstonia (with at least 1640 descendant, extant species, based on the number of species in just the sampled genera; numbers from Groeninckx et al., 2009, and Mabberly, 2008) or as late as the branch leading to the MRCA of Debrachionostylus and Houstonia (with at least 820 descendant, extant species; numbers from Groeninckx et al., 2009). The log likelihood of the data given an asymmetrical two-parameter model (139.9786) was significantly better than given a one-parameter model (143.2829) at p=0.05 (df=1, 2 statistic=6.6085), and so the two-parameter model was used. Mesquite inferred a forward rate of 11.9857 and a reverse rate of 28.6701 resulting in a gain to loss ratio of 0.418:1 for distyly. The proportional likelihood of the data with the alternative states of monomorphy and distyly at the base of the family Rubiaceae, the subfamily Rubioideae, and the tribe Spermacoceae are 0.97:0.03, 0.86:0.14, and 0.02:0.98, respectively.

16 Figure 1.1 Overview of the phylogeny of the Rubiaceae based on rbcL. Black branches indicate distyly; white indicate monomorphy. Posterior probabilities for branches near the base are shown. Supertribes are indicated with colored bars; subfamilies are indicated with black bars; tribe Coptosapeltae corresponds to the yellow box. The magenta box in the Rubiidinae clade indicates the lineage that includes Houstonia. Details of the phylogeny are shown in Figure 1.2.

17

Figure 1.2 (following pages) Details of the Rubiaceae phylogeny. Higher taxa and branches are colored as in Figure 1.1. The posterior probability of each branch is given to the right of the branch. Supertribe Ixoridinae is on Figs. 1.2a–b, supertribe Cinchonidinae on Figs. 1.2b–d, tribe Coptosapeltae on Figure 1.2d, supertribe Psychotriidinae on Figs. 1.2d–e, and supertribe Rubiidinae on Figure 1.2e. In Figure 1.2e, tribe Spermacoceae is labeled and a magenta star indicates the position of Houstonia caerulea.

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23 Discussion

Co-occurrence of heterostyly and cleistogamy My review of the primary scientific literature found that both heterostyly and cleistogamy occur in six genera from five angiosperm families, but that they co-occur in only one species, Houstonia procumbens (Rubiaceae). The joint probabilities of these co- occurrences at each of the taxonomic ranks is quite low (p=0.0071 or less; Table 1.3), although the observed co-occurrence of these two pollination syndromes is not significantly more or less than expected by chance. This result does not provide support for the hypothesis that less than expected co-occurrences of heterostyly and cleistogamy would indicate self-incompatibility in heterostylous lineages hinders the evolution of cleistogamy in those lineages. Neither does it support the alternative hypothesis that a greater than expected co-occurrence of heterostyly and cleistogamy would indicate that cleistogamy arose in heterostylous lineages as a means of reproductive assurance. The greater than expected, albeit not significantly so, co- occurrence of heterostyly and cleistogamy at the level of family and genus is somewhat unexpected, due to the presence of self- and intramorph-incompatibility in many heterostylous species that should make the evolution of cleistogamy less likely. One could explain the apparent paradox in at least the large genera where one finds both heterostyly and cleistogamy (Salvia, 900 spp.; Oxalis, 500 spp.; and Cryptantha, 100 spp.; Table 1.2) by noting that the number of heterostylous taxa in each (1, 4, and 1, respectively; Table 1.1) is small and should not be taken as an indication of a widespread self-incompatibility system in the genus. In the smaller genera, where the number of heterostylous species is a larger fraction of the total (14 of 20 in Houstonia, 14 of 36 in Lythrum, and 18 of 45 in Lithospermum; Tables 1.1, 1.2) shifts from heterostyly to cleistogamy (or the reverse) is more likely to have occurred, and merit examination of the phylogenies for each. I will note that variation in the degree of expression of the incompatibility system in heterostylous species is known (Ferrero et al., 2012), and this variation could make more likely the evolution of cleistogamy. To test this hypothesis, one could examine whether the genera that contain both heterostylous and cleistogamous species exhibit a greater degree of variation in the expression of self- and intramorph-incompatibility than closely related, solely heterostylous genera. This

24 would not explain why heterostyly and cleistogamy co-occur, but it could explain the apparent paradox of them occurring in a small group of close relatives. Cleistogamy could evolve in lineages that contain heterostylous taxa that exhibit either a low degree of self- and intramorph-incompatibility or no incompatibility mechanisms altogether. Cleistogamy could evolve as a response to increased genetic load in these lineages as a means to purge this genetic load. Alternately, cleistogamy could evolve in such lineages as a means of reproductive assurance in response to reduced opportunities for outcrossing, likely due to poor or unreliable pollinator service (reviewed in Oakley et al., 2007). In lineages where the two do co-occur one would expect to find self- incompatibility mechanisms to be either labile in expression or absent. Inferring whether cleistogamy arose in those lineages that contain heterostylous taxa in response to inbreeding depression is possible if there is evidence to indicate that this is the case for those lineages. Inferring whether cleistogamy arose in lineages that contain heterostylous taxa as a means of reproductive assurance is more difficult to test. Given that this hypothesis is predicated on the abundance and action of pollinators, providing evidence for such historical factors would be difficult. An alternative possibility is that heterostyly arose in lineages that were already cleistogamous. It should be noted however that in all genera that contain both heterostylous and cleistogamous taxa, the heterostylous taxa represent a larger proportion of the genus than the cleistogamous taxa with the exception of Salvia, which contains one of each. These proportions are significantly different from one another (binomial proportions test) for Houstonia (p<0.0001), Lythrum (p=0.0004), and Lithospermum (0.009), all genera that contain fewer than 50 species. For Cryptantha and Oxalis, with 100 and 700 species, respectively, the proportions of heterostylous and cleistogamous taxa do not differ significantly (p=0.215 and 0.451, respectively). Notably, the co-occurrence of heterostyly and cleistogamy at the level of species appears to be unique to H. procumbens, a case of cleistogamy arising in a heterostylous lineage. There is another aspect of this research that bears examination, specifically that both cleistogamy and heterostyly are commonly associated with entomophilous pollination (Culley and Klooster, 2007, and Ganders, 1979, respectively). This in turn indicates that a more rigorous examination of the co-occurrence of these two pollination

25 syndromes would focus on those angiosperm lineages where entomophilous pollination is common rather than basing expectations on the proportions of all angiosperms represented by cleistogamous and heterostylous taxa.

The evolution of heterostyly in the Rubiaceae In the most complete sampling of Rubiaceae for the purpose of inferring the evolution of heterostyly to date (Figure 1.2), I found many origins of distyly (27–36), with over half (all but 12; Figure 1.2) occurring in the focal subfamily Rubioideae. With so many, one might consider the number of independent origins of heterostyly in Rubiaceae to be greater than any other single family and to begin to rival the number of independent origins in the other 27 families with heterostylous members combined. Thus, Rubiaceae represents a good lineage for understanding the repeated evolution of distyly. That is not to say that the origin of heterostyly for the focal species, Houstonia procumbens, did not lead to other descendants being heterostylous. Rather, the heterostyly of H. procumbens arose in either the most recent common ancestor of at least 820 descendant, extant species or perhaps at least 1640 descendent, extant species. Thus, knowledge about the heterostyly of H. procumbens could be applicable to the many instances of homologous heterostyly in its close relatives. However, a few caveats need to be recognized. My choice to code OTU's as heterostylous when any members of its genus are known to be heterostylous could lead to an underestimate (but not an overestimate) of the number of origins, since it assumes a single origin in a genus where there might be multiple. The absence of nearly one- third (27.9%) of the genera in the Rubioideae from the sampling could lead to difficult- to-predict changes. And also 27% of distylous genera are not represented in the rbcL data set. However, an exhaustive sampling of the family at the level of genera or species might be several years off, and this study gives an assessment of what we know with our current state of knowledge. The sister genus to Houstonia, among those sampled here, is inferred to be Stenaria (represented by S. nigricans). Stenaria is composed of 5 heterostylous spp. distributed in the Central and Eastern US, Mexico, and the Bahamas (Groeninckx et al., 2009). With multiple spp. of Houstonia sampled, Church (2003) found that Houstonia is paraphyletic with respect to Stenaria using sequences from the internal transcribed

26 spacers of the nuclear ribosomal DNA and the trnL intron from the plastid genome. Sister to these two genera, is Arcytophyllum, a genus of 17 spp. distributed from Mexico to western South America, none of which are heterostylous. This absence of heterostyly is here inferred to be due to a loss. The results of this analysis would indicate that distyly is plesiomorphic for the genus Houstonia, and as such any differences noted among distylous taxa in Houstonia represent modifications of the ancestral condition. There are only minor discrepancies between my results and that of the previous phylogeny inferred using rbcL (Bremer et al., 1995). Bremer et al. (1995), analyzing data from just 49 OTUs in Rubiaceae using parsimony, resolved a basal polytomy of Rubioideae, a clade composed of Cinchonoideae (here discussed as supertribe Cinchonidinae), Ixoroideae (here discussed as supertribe Ixoridinae), and Hintonia, and Luculia. The clade composed of Cinchonoideae, Ixoroideae, and Hintonia had little support (<60% bootstrap frequency), and there was a basal polytomy of those three in it. I resolved a basal polytomy comprised of Hintonia, supertribe Cinchonidinae, supertribe Ixoridinae, and a clade of subfamily Rubioideae and Luculia and Coptosapelta (provisionally the Coptosapelteae, Robbrecht and Mannen, 2006), but the last clade (the one including Coptosapelteae) was not strongly supported (posterior probability of 0.55). Given the lack of resolution at the base of the family in the two studies and even in recent multi-gene studies (e.g., Robbrecht and Manen, 2006), there is a clear need for intensive gene sampling of a few representatives of these major clades. The resolution of the position of Luculia within the family, for example, is of particular importance to descriptions of the evolution of distyly in this family because this genus potentially represents the earliest diverging distylous lineage in the Rubiaceae.

Conclusions

The results of this research show that heterostyly and cleistogamy co-occur in more angiosperm families and genera than expected based on the taxonomic frequency of these pollination syndromes, but not significantly so. These results suggest that research into the reasons why the two co-occur will be informative to descriptions of the evolution of both pollination syndromes. Further reasons why heterostyly and

27 cleistogamy might co-occur are posed as hypotheses that can be tested by future research efforts. It is also apparent that distyly has evolved more times in the Rubiaceae than in any other family of angiosperms. This makes the Rubiaceae an ideal system for examining the forces that lead to the evolution of this pollination syndrome. It is also clear from this research that more work needs to be done in accounting for heterostylous taxa at the species level. To this end I am collaborating with Dr. James I. Cohen (Texas A&M University at Laredo) to build a database of heterostylous species that will include measures of organ reciprocity, pollination dynamics, measures of self- incompatibility, and features of floral development. These factors will be anchored to populations and will include as much detail of the ecology of the populations as is feasible. It is our hope that this database will provide the scientific community with the information necessary for a better understanding of the evolutionary and ecological significance of this somewhat enigmatic pollination syndrome.

28 CHAPTER TWO

RECIPROCAL HERKOGAMY IN HOUSTONIA PROCUMBENS (RUBIACEAE)

Introduction

This study examines the interaction between heterostyly and cleistogamy on the morphology of flowers of Houstonia procumbens (Rubiaceae), a species that is uniquely heterostylous and cleistogamous (as shown in Chapter One). The species provides an opportunity to examine the degree to which cleistogamy, for which sexual interference results in 100% pollen discounting, interacts with reciprocal herkogamy, for which reduced sexual interference through herkogamy results in reduced pollen discounting. Reproductive assurance provided by cleistogamous flowers of H. procumbens could lead to a reduction in the degree of organ reciprocity of the distylous flowers as a reflection of relaxed selection for organ reciprocity in the distylous flowers of this species. Furthermore, the need of cleistogamous flowers to achieve pollination with anthers and stigma in close spatial proximity to one another could affect the degree of organ reciprocity in the chasmogamous, distylous flower of this species. The goals of this chapter are to (1) compare the reciprocal positioning of floral organs in H. procumbens with that in two non-cleistogamous, heterostylous relatives and (2) describe the effect of the degree of reciprocity on pollen flow in the species. The approach taken is to examine these features among H. procumbens and two close relatives chosen with their phylogenetic relationships to H. procumbens in mind in order to understand the evolutionary changes leading to differences seen among the three. Heterostyly is characterized by the reciprocal positioning of spatially separated reproductive organs (anthers and stigmas) among individuals of a population, a feature called reciprocal herkogamy (Webb and Lloyd, 1986). This organ reciprocity contributes to the male component of fitness by reducing pollen discounting (a reduction in the outcrossed siring success due to self-fertilization, Eckert et al., 2009), while an often present sporophytic self- and intramorph-incompatibility system contributes to the female component of fitness by promoting outcrossing among morphs of a given population (Barrett, 2002). Heterostylous species may express any of

29 a suite of ancillary polymorphisms in addition to reciprocal herkogamy and the self- and intramorph-incompatibility. These ancillary polymorphisms may include pollen size and/or production dimorphisms and stigma size, shape, and/or stigmatic papillae dimorphism (reviewed in Dulberger, 1992). The morphs of a heterostylous species are nominally designated based on the length of the style. In distylous species, such as those examined here, the long styled morph is called the “L-morph” or “pin,” and the short styled morph is called the “S-morph” or “thrum.” In tristylous species, where there are two sets of anthers at different heights in the flower, the morphs are designated as the L-morph, the S-morph, and the “M-morph” (where the stigma is at the mid-height and the anthers are above and below). One hypothesis forwarded to explain forces that have led to the evolution of heterostyly comes from Darwin (1877). His hypothesis is that reciprocal herkogamy promotes precision of pollen transfer between floral morphs and reduces loss of pollen to self-fertilization (pollen discounting) and imprecise pollen transfer. To some degree this hypothesis has received support from the empirical evidence (Ganders, 1974, 1975a, 1975b; Kohn and Barrett, 1992; Ree, 1997), but such studies can be complicated by other elements of the pollination biology of distylous species. The L-morph of distylous species tends to produce more pollen than the S-morph (Ganders, 1979), in which case the L-morph pollen is generally overrepresented in a given population in terms of pollen production. This has been documented to occur in Houstonia caerulea, with the L- morph producing 1.3 times more pollen per flower than the S-morph (Ornduff, 1980). However, Ornduff (1980) also showed that more pollen is left in the anthers of the L- morph—anthers more deeply positioned in the corolla tube—than the S-morph in that species. Similar disparities in pollen removal from thrum and pin anthers have been shown in other species (e.g., Primula vulgaris; Ornduff 1979). This can be further complicated if the morphs are not equally represented in a given population (anisoplethy, rather than isoplethy). To what degree reciprocal herkogamy produces dissasortative pollen flow is determined by the degree of precision of organ reciprocity exhibited by a given population (e.g., Ferrero et al., 2011), but it can also be affected by overall anther-stigma distances. For example, Ganders (1975b) found that populations of Amsinckia vernicosa var. furcata (Boraginaceae) exhibit greater inter-organ distance than A. spectabilis (7.4 mm average, n=1 population vs. 3.55 mm average, n=2 populations) and that A.

30 vernicosa var. furcata exhibits a greater degree of disassortative mating than A. spectabilis. Inter-population variation in reciprocity, as has been shown by studies such as Ferrero et al. (2011a), suggests that selection on the degree of organ reciprocity through interaction with pollinators may result in variation in the degree of organ reciprocity exhibited by populations of a given species. Previous research has demonstrated the importance of accounting for the fidelity and action of pollinators of heterostylous species. H. caerulea is known by Wyatt and Hellwig (1979) to be pollinated by long- tongued flies (Bombylius) and small native bees, the former of which Ornduff (1980) considered to be the primary pollinators responsible for intermorph pollination, given his observations of the species. Grimaldi (1988) considered the long proboscis of Bombylius to be “ideally suited for reciprocal pollen transfer” (p. 3) for H. caerulea. Grimaldi (1988) suggested that the flies are attracted both to nectar, which he observed at the base of some flowers, and pollen, which he found in the crop and could identify as solely from H. caerulea. The incompatibility system that makes intermorph crosses more successful than self and intramorph crosses can vary in its completeness, even within a group of close relatives (Ferrero et al., 2011b), leading to a greater or lesser contribution of intermorph crosses to the next generation, somewhat irrespective of the degree of disassortative pollination. Both Ornduff (1977) and Wyatt and Hellwig (1979) demonstrated that self- and intramorph pollinations in H. caurulea produce far fewer fruits and viable seeds than intermorph pollination. Wyatt and Hellwig (1979) showed that the former type of cross fruit only 2% of the time, whereas the latter produce fruit 95% of the time. Beliveau and Wyatt (1999) demonstrated that intermorph crosses of H. longifolia set significantly more fruits and seeds per fruit than intramorph crosses with up to 90% of fruits set in the case of the former vs. up to 15% (with their “thrip-corrected” result) in the case of the latter. Similarly, Levin (1974) found that no seeds were set with self and intramorph crosses in Stenaria nigricans (then called Hedyotis nigricans; a species nested in Houstonia; Church and Taylor 2005), whereas many were set in intermorph crosses. H. procumbens and its relatives in Houstonia offer a unique opportunity to explore the relationship between self-incompatibility and reciprocity further because the self- compatibility is required for the fertilization of the species’ cleistogamous flowers while the self-incompability of close relatives appears to be quite strong. Houstonia is a genus of 20 spp. native to the U.S., Canada, and Mexico. The potentially informative

31 comparison that I will make involves H. procumbens, the cleistogamous, distylous perennial, and H. caerulea and H. longifolia, two non-cleistogamous, distylous perennials (Table 2.1). While H. procumbens and H. caerulea share similar floral architecture and form, H. caerulea and H. longifolia share an erect growth habit. H. procumbens and H. caerulea both produce solitary flowers, while the infloresence of H. longifolia is a cyme, but the number of stems produced by all three species results in the production of many flowers by a given individual. H. procumbens is more closely related to H. caerulea than it is to H. longifolia (Figure 2.1), and so observations about H. longifolia can potentially be used to polarize the change inferred to produce differences in the reciprocity of H. procumbens and H. caerulea. That is to say that any similarities in organ reciprocity metrics between H. procumbens or H. caerulea with H. longifolia are assumed to describe the ancestral state of heterostyly for H. section Houstonia, the section to which H. procumbens and H. caerulea belong. Therefore, any differences in the same metrics between H. procumbens and H. caerulea can be interpreted as a difference from the ancestral condition for that section of the genus. Also, pollen dimorphisms between the two flower morphs of Houstonia are reported for the genus (Terrell, 1996) and specifically for H. caerulea (Wyatt and Hellwig, 1979; Ornduff 1980) and H. longifolia, which should facilitate assessments of disassortative pollen transfer. However, the flowers are extraordinarily small (Table 2.1; Figure 2.3) which offers challenges that would not be as significant with larger flowers. Selective forces that favor the evolution of cleistogamy may include reproductive assurance, the purging of inbreeding depression, lower relative cost and higher reliability of seed set for cleistogamous flowers, avoidance of disruption of locally adapted genotypes through meiotic recombination, and the automatic selection advantage of selfing (reviewed in Oakely et al., 2007). While research provides evidence for the maintenance of chasmogamous and cleistogamous flowers for cleistogamous species, the fitness benefits accrued through cleistogamous flowers appear to outweigh the purported benefits of chasmogamous flowering to such a degree that it is difficult to explain the maintenance of chasmogamy in cleistogamous species (reviewed in Oakley et al., 2007).

32

Jul)

– tableThe May ( May

–

May Jun ) Jan) – – – Phenology (Oct Mar Apr

species used in this investigation. investigation. this in used species Flower color Flower lavender) pale (rarely white sometimeswith yellow or center greenish blue, light blue, (rarely white occasionally lightpink or light violet) pinkish or purple to white purple

2.1) -

Houstonia 1.0 1.8 ( 1.6 – – –

(cm)

Flower form; length, lobes lobes length, form; Flower extended 0.45 Salverform; 0.58 Salverform; 0.35 Funnelform;

-

Inflorescence solitary solitary to one severalfew flowered cymes

Growth Growth form stems prostrate stems erect stems erect

Habit Perennial Perennial Perennial

adapted from Terrell (1996). Terrell from adapted

2.1 SummaryTable of growth form of floral and characteristics Species procumbens H. caerulea H. longifolia H.

was

33

Figure 2.1 Phylogeny of Houstonia reproduced from Church and Taylor (2005). A maximum likelihood phylogeny based on combined nuclear (ITS1 and ITS2 of the nuclear ribosomal DNA) and plastid data (the trnL intron and trnS-trnG intergenic spacer). Focal taxa for this study are indicated by blue boxes.

I will address the following hypotheses in this chapter. (1) H. procumbens will exhibit relaxed selection for reciprocal herkogamy, as evidenced by a lower degree of organ reciprocity, compared to its close relatives for two reasons: (a) it does not rely solely on outcrossing via heterostylous, chasmogamous flowers for reproductive success and (b) a developmental shift might have been necessary to reduce the stigma- anther distances in the cleistogamous flowers and this might also be expressed in the chasmogamous flowers (a possibility I will explore in Chapter Three). (2) Variation in organ reciprocity among the three species will be correlated to variation in disassortative pollen transfer, with greater disassortative pollen transfer where organ reciprocity is greatest.

34 Materials and Methods

Specimen collection and imaging Flowers of the following species were collected from the following locations: H. procumbens from Torreya State Park, FL (subsequently designated HproTSPFL; 30° 34’ 16” N 84° 56’ 24” W), Falling Waters State Park, FL (HproFWSPFL; 30° 43’ 45” N 85° 31’ 35” W), Tall Timbers Research Station, FL (HproTTRSFL; 30° 39’ 29” N 84° 13’ 10” W), and Birdsong Nature Center, GA (HproBNCGA; 30° 41’ 55” N 84° 11’ 58” W); H. caerulea from 6883 Charity Hwy Woolwine, VA (HcaeWWVA; 36° 49’ 52” N 80° 13’ 11.6” W), Fairy Stone Park, VA (HcaeFSPVA; 36° 47’ 33.5” N 80° 6’ 53” W), and Bowens Creek Park, VA (HcaeBCVA; 36° 45’ 58” N 80° 3’ 33.5” W); and H. longifolia from 6883 Charity Hwy Woolwine, VA (HlonWWVA), and DeHart Park, VA (HlonDHPVA; 36° 48’ 58” N 80° 15’ 09” W) (Figure 2.2). From one to five mature flowers were collected from each individual in a given population in FAA (ethanol, formaldehyde, acetic acid, and water 5:1:0.5:3.5) or 70% ethanol. Those collected in FAA were cleaned with serial dilutions of ethanol (30% X 1, 50% X 1, 70% X 2 for 10 min. each) and stored in 70% ethanol until dissection. From 1–5 flowers were collected from every flowering individual found in populations of H. procumbens from Falling Waters State Park and Torreya State Park throughout the flowering season (February through May). For all other species one to five flowers from 30 individuals of each morph were sampled with the exception of the population at Tall Timbers Research Station, which exhibited anisoplethy with 122 L-morph to 12 S-morph individuals counted in the area where collections were conducted (ca. 0.33 ha).

35

Figure 2.2 Maps of field sites and species distributions. Individuals of H. procumbens were collected from North Florida sites, and individuals of H. caerulea and H. longifolia were collected from Virginia sites. Inset panels show the distributions of (A) H. procumbens, (B) H. caerulea, and (C) H. longifolia taken from USDA Plants Database (http://plants.usda.gov/java/)

Specimens were dissected by slitting the corolla tube longitudinally, and the style and anthers were straightened parallel to the long axis of the flower. Dissected flowers were then photographed with a Nikon Coolpix 995 digital camera mounted on a Lecia WILD M3Z dissecting scope (Figure 2.3). Images included a metric ruler for scale. Measurements of stigma and anther heights were taken from the base of the corolla to the base of the stigma lobes and the mid-point of the two central anthers, respectively. Corolla lengths were measured from the base of the corolla to the tip of one of the two central lobes. Measurements were taken using ImageJ64 (http://rsbweb.nih.gov/ij/index.html).

36

Figure 2.3 Image of dissected flowers used in this study. (A) Dissected flowers of H. procumbens. (B) Dissected flowers of H. caerulea. (C) Dissected flowers of H. longifolia. Note the metric ruler for scale, hash marks indicate mm.

37 Morphometrics Reproductive organ reciprocity was measured using the method developed by Sanchez et al. (2008). This index calculates organ reciprocity as the absolute value of the average of the height differences (height gaps) of anthers and stigmas for a given organ level (long or short) weighted by the average of all organ heights in the study population. This is multiplied by the average of the standard deviation of the height gaps of anthers and stigmas for each level in order to account for variation of organ heights for each organ level (Figure 2.4). The reciprocity index describes perfect reciprocity of organ heights when the value of the index is equal to zero. A script for calculating organ reciprocity using the R platform (R Development Core Team, 2011) was generated by A. I. Pastore (Florida State University) and modified to perform bootstrap analyses by C. E. Buddenhagen (Florida State University). The reciprocity values generated in the R platform were checked against those derived from the version programmed into Microsoft Excel (2008) and provided by J. M. Sanchez. These R scripts are available from E. H. Jones upon request. In order to ensure that samples represent independent data points, one flower from each individual for which measurements were taken was chosen randomly for each individual represented by multiple flowers. Values of the reciprocity index for each population of each species were compared at the level of species using an analysis of variance (ANOVA) (Tukey corrected for multiple comparisons) using the R platform (R Development Core Team, 2011) to test for significant differences in organ reciprocity among species. It is assumed that the mean values of organ reciprocity for each species under investigation meet the criteria of normally distributed data required by the parametric ANOVA. This is assumed because according to the central limit theorem the means of the reciprocity values for each species should be approximately normally distributed. For each population bootstrap sampling was performed to produce a distribution of reciprocity values. Each bootstrap sampling comprised 10,000 replicates, sampling with replacement the total number of flowers for which organ heights were measured for each population. Along with calculating the overall reciprocity of reproductive organs for each population, the bootstrap analysis reported the relative reciprocity of organs at both the long and short levels. In order to test for differences between populations for each species a single-sample t-test was conducted on the distribution of

38 absolute values of the differences in organ reciprocity (overall reciprocity and relative reciprocity at the long and short organ levels) generated from the bootstrap samplings to determine if the absolute value of the differences in these metrics was significantly greater than 0.

n m |Ei − Sj| ra = /nm (1) ✓ x¯ ◆ Xi Xj

r = rd2 + rs2 (2) p

2 n m |Ei−Sj | v i j x¯ − ra (3) sdr = u h⇣ ⌘ i a uP P t nm sdr + sdr sdr = a s (4) 2

(5) R = r(sdr)

Figure 2.4 Formulae employed in calculations of organ reciprocity for distylous taxa based on the method developed by Sanchez et al. (2008). (1) Relative reciprocity at a given organ level (L or S). (2) Overall reciprocity for a given population. (3) Standard deviation of height gaps at a given organ level (L or S). (4) Overall standard deviation of height gaps for organs at both L- and S-levels. (5) Overall reciprocity for a given population, including variance in organ heights. Key: E=anther height at L-level, e=anther height at S-level, S=stigma height at L-level, s=stigma height at S-level, n=total number of single anthers (i) at L-level, p=total number of stigmas (k) at L-level, m=total number of anthers (j) at S-level, n=total number of stigmas (l) at S-level,

ra=relative reciprocity at the L-level, rs=relative reciprocity at the S-level, xbar=average heights of all organs (anthers and stigmas) in a population, r=overall reciprocity for a given population, sd=standard deviation, R=overall reciprocity for a given population including variance in organ heights.

39 Pollination dynamics Species were examined for pollen dimorphisms using pollen from mature anthers collected during emasculations from one individual of each morph for each species with the number of pollen grains examined equal to seven and ten for the L- and S-morphs of H. procumbens, 12 and 13 for the L- and S- morphs of H. caerulea, and nine and six for the L- and S-morphs of H. longifolia, respectively. Because H. procumbens did not exhibit a size or staining dimorphism as expected, pollen loads were only examined on stigmas of those populations of H. caerulea and H. longifolia. I emasculated flowers prior to anthesis and left them accessible to pollinators from 1–4 days before collection. Anthers from emasculations were collected separately in 70% ethanol and centrifuged at 13.2 X 1000 rpm for 2 minutes. The majority of the ethanol was then removed with a pipette. Uncapped tubes containing the anthers were then left overnight in a sterile flow hood to allow the remainder of the ethanol to evaporate. Staining pollen with iodine has been reported to reveal morph specific patterns in starch deposition in pollen of distylous morphs for Lythrum (Lythraceae), Hypericum (Hypericaceae), and Jepsonia (Saxifragaceae), with the pollen of the S-morph staining more darkly than that of the L-morph (reviewed in Dulberger, 1992). Accordingly, pollen was re-suspended in 10 uL of lugol solution and glycerol (1:1) and mounted on glass slides, heated to 55° C on a slide warmer and mounted under a coverslip. Pollen grains were photographed using a DP71 color digital camera (12.5 megapixles) mounted on an Olympus BX61 bright field microscope. Measurements of the diameter of the spherical pollen grains were used to verify significant differences in pollen size between pollen of L- and S-morphs through use of a t-test. The size of the spherical pollen grains was measured as the diameter using imageJ64 (http://rsbweb.nih.gov/ij/index.html). Stigmas from emasculations were centrifuged at 13.2 X 1000 rpm for 2 minutes. The majority of the ethanol was then removed with a pipette. Uncapped tubes containing the stigmas were then left overnight in a sterile flow hood to allow the remainder of the ethanol to evaporate. Stigmas were then re-suspended in 10 uL of glycerol:70% ethanol (1:1), mounted on a glass microscope slide with a coverslip. Images were taken of the stigmas and pollen grains using a DP71 color digital camera (12.5 megapixles) mounted on an Olympus BX61 bright field microscope (at 200X for pollen grains and 100X for stigmas). Pollen loads were scored by measuring all pollen

40 grains on a given stigma using imageJ64 and the resulting data analyzed with a chi- square test of the proportion of L-morph pollen to S-morph pollen on stigmas of L- morph and S-morph flowers.

Results

Organ reciprocity ANOVA analysis (Tukey corrected) of measures of reproductive organ reciprocity indicate that both H. procumbens and H. longifolia differ significantly from H. caerulea with respect to overall reciprocity (p=0.002 and 0.012, respectively), but not from one another in this respect (p=0.47) (Figure 2.5A and Table 2.2). While H. procumbens exhibits greater organ reciprocity at the long organ level than either of the other two species, H. longifolia exhibits greater organ reciprocity at the short level than either of the other two species (Figure 2.5B, C). The result of this is that overall reciprocity for H. procumbens and H. longifolia is more similar to one another than either is to that for H. caerulea. The reciprocity indices for H. procumbens and H. longifolia fall within the bounds described for distylous taxa (less than 0.035) from the previous research that used the same metric in other groups (Sanchez et al., 2008; Ferrero et al., 2009). However, H. caerulea exhibited a value of organ reciprocity more similar to those reported for style-dimorphic taxa (greater than 0.04; Ferrero et al., 2009; Table 2.2). Style-dimorphic flowers present stigmas at two different heights while anthers are presented at one height. A t-test of differences in organ reciprocity from bootstrapped reciprocity measures for all populations within species shows that the difference in overall reciprocity and relative reciprocity at the long and short organ levels are significantly greater than 0 (Figure 2.6; all p-values << 0.0001). This result is taken to indicate that the degrees of organ reciprocity for populations of the species under investigation differ significantly from one another.

41

Figure 2.5 Boxplots of reciprocity for Houstonia species. Boxplots are derived from calculations of organ reciprocity for each population of each species under investigation. Lower values of the reciprocity index (Y-axis) indicate a higher degree of organ reciprocity. (A) Overall reciprocity for each species as the mean value of overall reciprocity for each population of the species in question. (B) Relative reciprocity at the long organ level. (C) Relative reciprocity at the short organ level.

42 Table 2.2 Reciprocity measures for Houstonia. Overall reciprocity (R) and relative reciprocity at the S- and L-levels (rs and rl, respectively) are indicated for each population (see text for population abbreviations). Relative reciprocity at the S- and L- levels (sdrs and sdrl, respectively) are indicated for each population. Sample sizes are indicated as the total number of individuals sampled from each population (n) as well as the numbers of L-morph (nL) or S-morph (nS) individuals examined for each population.

Species population R rs rl sdrs sdrl nL nS n H. caerulea HcaeFSPVA 0.0685 0.1215 0.3822 0.0963 0.2451 13 14 27 H. caerulea HcaeBCVA 0.0594 0.1983 0.3465 0.1144 0.1831 17 15 32 H. caerulea HcaeWWVA 0.0465 0.1390 0.3116 0.0955 0.1772 23 30 53 H. longifolia HlonDHPVA 0.0308 0.0852 0.2599 0.0588 0.1661 29 26 55 H. longifolia HlonWWVA 0.0265 0.0672 0.2732 0.0545 0.1343 18 11 29 H. procumbens HproFWSPFL 0.0198 0.1060 0.1531 0.0841 0.1281 94 81 175 H. procumbens HproTSPFL 0.0284 0.1373 0.1860 0.1041 0.1415 26 57 83 H. procumbens HproBNCGA 0.0181 0.1194 0.1429 0.0861 0.1079 29 33 62 H. procumbens HproTTRSFL 0.0160 0.1495 0.1189 0.0835 0.0838 38 12 50

Floral Morphology Measurements of floral morphology are provided in Table 2.3 and include all flowers measured for each individual from a given population (i.e., the complete data from which one flower for each individual was chosen for the analysis of organ reciprocity). Two-sample, unequal-variance t-tests indicate that stigma and anther heights within a given morph differ significantly from one another (all p-values << 0.001) within each population. Total corolla lengths (including corolla lobes) of H. procumbens only differed significantly between distylous morphs at Birdsong Nature Center (HproBNCGA, p < 0.05) with the corolla of S-morph flowers longer than those of the L-morph flowers. However, corolla lengths of H. caerulea differed significantly between morphs for all populations examined (p<<0.001 for HcaeBCVA and HcaeWWVA; p=0.0001 for HcaeFSPVA) with the corolla of the S-morph longer than that of the L-morph. This is consistent with Ornduff’s (1980) finding that the corolla tubes of L-morphs of this species, a feature not measured here, averaged 60% of the length of the S-morphs. A similar pattern is seen for both populations of H. longifolia (p=0.005 for HlonDHPVA; p<<0.001 for HproBNCGA), again with the corolla of the S- morph longer than that of the L-morph. For all populations of each species under investigation anther and stigma heights appeared to be reciprocally positioned (Figure 2.8). However, the average positions of

43 reciprocal organs within a given organ level differed within populations significantly (all p < 0.005; t-test, two-sample, unequal-variance) except for the S-level organs of H. caerulea (HcaeFSPVA) and H. longifolia (HlonDHPVA). Populations of H. procumbens have L-level stigmas that are on average higher than the L-level anthers, with the exception of HproBNCGA for which this is the reverse. On average the S-level stigmas of H. procumbens are shorter than the S-level anthers. Populations of H. caerulea have L- level stigmas that are on average shorter than the L-level anthers, while the S-level stigmas are on average higher than the S-level anthers, though in HcaeFSPVA this is reversed. The two populations of H. longifolia differed in this respect with the L-level stigmas on average higher than the L-level anthers and the S-level stigmas on average shorter than the S-level anthers for HlonWWVA, while the reverse was true for HlonDHPVA.

44

Figure 2.6 Boxplots of bootstraps of reciprocity for species of Houstonia investigated in this study. Panel A shows results for overall recipriocity (Rout), panel B shows results for relative reciprocity at S-level (rsout), and panel C shows results for relative reciprocity at L-level (rlout). Populations are depicted in the following order (from left to right) in all three panels: HcaeBCVA, HcaeFSPVA, HcaeWWVA, HlonDHPVA, HlonWWVA, HproBNCGA, HproFWSPFL, HproTSPFL, and HrpoTTRSFL.

45

6.658 (0.089) 6.569 (0.075) 11.757 (0.130) 11.757 14.590 (0.106) 12.580 (0.132) 13.655 (0.105) 12.105 (0.093) 12.784 (0.126) 12.997 (0.065)

corollaS-morph

6.324 (0.078) 6.455 (0.075) 11.584 (0.113) 11.584 (0.083) 11.611 11.120 (0.133) 11.120 (0.077) 11.165 12.845 (0.115)

10.814 (0.133) 12.859 (0.102) corollaL-morph

8.092 (0.104) 6.392 (0.151) 7.279 (0.122) 4.651 (0.092) 4.567 (0.068) 9.126 (0.082) 8.357 (0.132) 9.262 (0.137) 9.720 (0.072)

anther S-morph

2.903 (0.113) 2.823 (0.098) 2.617 (0.286) 2.206 (0.092) 2.228 (0.077) 5.988 (0.098) 6.539 (0.107) 7.272 (0.104) 5.885 (0.0873) L-morph anther L-morph . Measures of average with organ of heights the (lengths corolla) for . Measures 3.744 (0.146) 2.582 (0.157) 3.542 (0.141) 2.337 (0.122) 2.059 (0.082) 5.336 (0.142) 5.490 (0.122) 5.885 (0.144) 5.955 (0.0868) S-morph stigmaS-morph Houstonia 9.803 (0.111) 8.532 (0.112) 6.438 (0.090) 4.940 (0.173) 5.942 (0.098) 5.530 (0.084) 5.452 (0.081) 8.713 (0.101) 10.222 (0.101) L-morph stigmaL-morph population HcaeBCVA HcaeFSPVA HcaeWWVA HlonDHPVA HlonWWVA HproBNC HproFWSPFL HproTSPFL HproTTRS Species H. caerulea H. caerulea H. caerulea H. longifolia H. longifolia H. procumbens H. procumbens H. procumbens H. procumbens coefficient of variation in parentheses. Measurements are in mm. are in mm. of Measurements variation in parentheses. coefficient organs for of floral 2.3 Measurements Table

46

Figure 2.7 Scatterplots of organ heights. Plot of stigma (blue diamonds) and anthers (red squares) for all populations of Houstonia. The grey vertical line in each graph separates values taken from the L-morph individuals (left of the line) from those taken from S-morph individuals (right of the line). Population designations are as described in the text; species designations are: H. procumbens (Hpro), H.caerulea (Hcae), and H. longifolia (Hlon). Vertical scale is in mm, see Table 2.3 for average values of organ hieghts for each population.

Pollination dynamics In the end, relative pollen loads on stigmas of different morphs could not be determined for any of the populations under investigation. Pollen of H. procumbens did not exhibit the expected morph-specific staining pattern or the pollen-size dimorphism. Both H. caerulea and H. longifolia exhibited the pollen dimorphism typical of distylous taxa and documented previously (Wyatt and Hellwig, 1979; Ornduff, 1980), with S- morph pollen being significantly larger than L-morph pollen (p<<0.001; Figure 2.8). And morph-specific staining patterns were generally found to be the case for pollen of H. caerulea and H. longifolia with the pollen of the S-morph staining more darkly than

47 that of the L-morph, though this was not consistently the case. However, stigmas of H. caerulea were left mounted on slides long enough prior to imaging that distortion of pollen shape precluded the possibility of seeing size differences among the pollen of the two morphs. Further, staining pollen on these slides with lugol solution did not appear to provide the morph specific staining patter seen with freshly mounted pollen grains. Pollen loads on stigmas from emasculated flowers of H. longifolia from 7 S-morph and 7 L-morph individuals were examined from HlonWWVA, and pollen from 8 S-morph and 4 L-morph individuals were examined from HlonDHPVA (Table 2.4). While a clear pollen size dimorphism was apparent at 200X (Figure 2.8) and has been documented elsewhere (Beliveau and Wyatt, 1999), measurements of pollen on stigmas at 100X (as done for the pollen size measurements to make it easier to keep track of pollen already measured) did not produce as clear a size difference. This was likely due to a change of scale. As it was not possible to delineate L- and S-morph pollen grains among those seen on stigmas collected from emasculations, this analysis was not completed.

Discussion

Reciprocal herkogamy in Houstonia The results of this research does not indicate that there is relaxed selection for organ reciprocity in sampled populations of the uniquely cleistogamous and distylous species H. procumbens relative to the non-cleistogamous, distylous congeners H. caerulea and H. longifolia (Figure 2.5). This suggests that reproduction through the chasmogamous, distylous flowers of H. procumbens remains important for the reproductive success of this species despite the reproductive assurance provided by cleistogamous flowers. It also suggests that the developmental programs for distylous and cleistogamous flowers in this species may not interact appreciably with one another. The comparative developmental study of Chapter 3 will explore this further.

48

Morph

Figure 2.8 Plot of means of pollen diameters for Houstonia species. Hcae=H. caerulea, Hlon=H. longifolia, and Hpro=H. procumbens). Pollen was collected from anthers of one individual of each morph for each species. Pollen diameters indicated are in micrometers, error bars represent the standard error of the means. Mean values (with standard deviations in parentheses) and number of pollen grains measured are as follows: Hcae L=30.9 (2.3) n=12, S=38.8 (1.5) n=13; Hlon L=43.3 (1.6) n=9, S=50.1 (1.4) n=6; Hpro L=39.5 (1.1) n=7, S=37.8 (2.3) n=10.

Notably, the degrees of organ reciprocity for flowers of all populations under investigation differ significantly from one another within species (Figure 2.6, Table 2.2). These measures differ with regard to overall reciprocity and relative reciprocity at the long and short levels. Examination of organ heights at the long and short levels among populations within species indicates that there is variation among populations with regard to organ heights of mature flowers within species. This could be explained by selection imposed by interactions with pollinators that are likely to differ among populations.

49 One of the more interesting results of this research is that the reciprocity of populations of H. caerulea fall outside the bounds described for distylous taxa using this index in previous research and within the bounds of so-called “style-dimorphic” taxa that have anthers at a fixed position throughout the population. This calls into question the validity of setting boundaries for a distinction between distylous and style- dimorphic taxa based on the values of this index, since the anthers of H. caerulea vary reciprocally with the stigma heights (Figure 2.7). The lower reciprocity values for this species could be the result of relaxed selection for organ reciprocity due to the strong biochemical self- and intra-morph incompatibility system that complements it (Wyatt and Hellwig, 1979). Wyatt and Hellwig (1979) did not address reciprocity using the same measure as used here, but they did find that the distance separating stigmas and anthers differed significantly in L- and S-morphs in their study of H. caerulea, which is suggestive of variation in organ position at one of the levels or both, as I showed here (Figure 2.5). The difference in reciprocity among the three species can be interpreted as the evolution of a decreased reciprocity in H. caerulea, at least at the long level, using the phylogenetic contrast that I have designed. Given that the reciprocity of reproductive organs for H. procumbens and H. longifolia do not differ significantly from one another, and that the intended use of H. longifolia is to polarize the comparison of H. procumbens with H. caerulea, there appears to be relaxed selection for organ reciprocity in populations of H. caerulea compared to that of the other two species. It is also worth noting that the stigma lobes, and thus the receptive areas for pollen receipt, of H. procumbens and H. caerulea are longer than those of H. longifolia (pers. obs.), and variation in the degree of organ reciprocity for these species may be alleviated by the ability of the stigma lobes to collect pollen from a larger range of distances from the base of the corolla. Longer stigma lobes allow stigmas to capture more overall pollen, and this was seen in stigmas of H. caerulea compared to those of H. longifolia (pers. obs.). The composition of the genetic locus that confers heterostyly has been described in Primula (Primulaceae) as composed of three tightly linked genes: Gg, which controls gynoecium development and female incompatibility; Pp, which controls pollen development and male incompatibility; and Aa, which controls anther height (Ernst, 1925, 1936; Lewis, 1954; Dowrick, 1956). Together these three genes are commonly referred to as the S locus. Among the eight families where the dominance relationship

50 of the S locus has been determined for distylous taxa, six show the L-morph to be homozygous recessive (ss; gpa/gpa) for the S locus and the S-morph to be heterozygous (Ss; GPA/gpa) (Lewis and Jones, 1992). In two families this relationship is reversed (Hypericaceae (=Guttiferae) and Plumbaginaceae; Lewis and Jones, 1992). There have been no homozygous dominant genotypes of distylous species described in nature, and it is suspected that this is due to a recessive lethal allele linked to the dominant allele of the S locus (Mather and DeWinton, 1941; Baker, 1975). It is supposed that the dominant G allele suppresses style growth, resulting in short level stigmas while the dominant A allele results in the greater cell proliferation of the lower corolla resulting in L-level anthers (Webster and Gilmartin, 2006). Additionally, the dominant P allele of the S- morph is supposed to be responsible for the male component of the biochemical self- and intramorph-incompatibility system. For H. procumbens, the P gene, which controls the male incompatibility reaction, appears to be either non-functional or altered in its action downstream of its expression with a breakdown of the self- and intramorph incompatibility system in this species. Self-compatibility rendered through alteration of the G locus that confers female incompatibility would be accompanied by consequent changes in the position of stigmas and as that does not seem to be the case for H. procumbens, given the high degree of reciprocity for this species, it would not seem to be the case here. The differential starch deposition that leads to morph-specific staining in distylous taxa may be the result of the action of the P gene in the tapetum during pollen development in Houstonia (and perhaps its close relatives in Rubiaceae). In that case, it may be possible to predict the strength of the incompatibility by quantifying starch staining for those species, but testing this would require a different methodology than that employed here.

Conclusions The comparative phylogenetic approach used here suggests that the overall reciprocity of H. procumbens changed little with the evolution of cleistogamy (Figure 2.5a) and that the lesser overall reciprocity in H. caerulea evolved on a branch since its most recent common ancestor with H. procumbens. However, the absence of the pollen dimorphisms in H. procumbens that are seen in the other two species suggests some modification of the putative S-locus responsible for conferring reciprocal herkogamy

51 and self- and intramorph-incompatibility in many heterostylous species. Were the genes of the S-locus of Houstonia similar to those thought to occur in the S-locus of Primula, modification of a single gene (designated P in Primula) could alter both pollen dimorphism and the self- and intramorph incompatibility system, making the self- fertilization of cleistogamy possible. Alternately, it could indicate that the presence of cleistogamy in this species affects the expression of the S-locus such that the P gene is not expressed or the action of the P gene is inhibited downstream of its initial expression. Descriptions of the molecular genetics of the S-locus would provide the ability to test these hypotheses, among others. Overall, the results of this work indicate that any description of the degree of reciprocal herkogamy for a given species must take into account variation among populations before ascribing descriptions of the degree of reciprocity for a given species. This variation among populations could arise due to pollinator diversity, fidelity, and availability producing differences in reciprocal herkogamy over generations. It would be useful to examine correlations between pollinator assemblages, the associated pollination dynamics, and floral organ reciprocity for populations of distylous species.

52 CHAPTER THREE

DISTYLOUS FLOWER DEVELOPMENT IN HOUSTONIA PROCUMBENS

Introduction

This study examines the interaction between heterostyly and cleistogamy on the development of both flower types of Houstonia procumbens (Rubiaceae), a species that is uniquely heterostylous and cleistogamous (Chapter One). My approach is to compare the development of its heterostylous flowers to those of H. caerulea, a close relative that is also heterostylous but is not cleistogamous. The species provide an opportunity to examine the degree to which constraints of cleistogamy influence the development of flowers that are reciprocally herkogamous and vice versa. The goals of this chapter are to (1) determine the developmental basis for reciprocal herkogamy for H. procumbens and H. caerulea and (2) determine whether the cleistogamous and chasmogamous flowers of each morph of H. procumbens share similarities that differ from the morphs of H. caerulea. Heterostyly is characterized primarily by the reciprocal positioning of stigmas and anthers among individuals in a given population (Webb and Lloyd, 1986), although suites of ancillary polymorphisms are also often exhibited (e.g., pollen size as shown in Chapter Two). The developmental basis for this organ reciprocity has been examined in a diverse array of heterostylous taxa (e.g., as reviewed in Hernandez and Ornelas, 2007). Some features of floral form are common across heterostylous taxa, including sympetalous, tubular corollas, relatively few stamens that are adnate to the corolla, syncarpous ovaries with few carpels, and flowers between five and thirty mm in length (Richards and Barrett, 1992). Tubular corollas restrict the ways in which pollinators can interact with the flowers, and limits on flower size may reflect co-adaptation between heterostylous species and their pollinators (e.g., Grimaldi, 1988). If flowers are too small, the reciprocal positioning of reproductive organs may not facilitate segregation of pollen of the different morphs on discreet portions of the bodies of their pollinators (Richards and Barrett, 1992). Alternately, upper limits on heterostylous flower size may be imposed by developmental constraints because minor variation in organ growth in

53 early development may be magnified through a prolonged process of floral maturation and result in greater variation in organ heights at maturity (Richards and Barrett, 1992). The anthers and stigmas represent the terminal ends of the male and female reproductive organs, respectively, and the positions of these floral features depend on the development of filaments and the lower corolla for anthers (the latter when the filaments are adnate to it) and the style for stigmas. Although it is commonly the case that length of either the filament or the lower corolla, and in some cases both, is responsible for anther position, in other cases bending of the filaments is ultimately responsible for anther position (reviewed in Richards and Barrett, 1992). Likewise, growth of the style is commonly responsible for stigma position, but the position of the stigma can also be the result of bending of the style (reviewed in Richards and Barrett, 1992). Since Darwin’s pioneering work on heterostyly, members of the genus Primula (Primulaceae) have served as models for examining floral form and development of distylous flowers. Webster and Gilmartin (2003, 2006) describe heteromorphic flower development in Primula vulgaris var. Blue Jeans. They found that there were no discernable differences in the earliest development of the long-styled morph (L-morph) and short-styled morph (S-morph) of this variety (Webster and Gilmartin, 2006), but that the differences arise later (Webster and Gilmartin, 2006). At those later stages, the difference in stigma height between morphs arises due to greater elongation in the style cells of the L-morph than in the S-morph, rather than differences in cell number. However, difference in anther height arises due to differences in cell number in the lower corolla (to which the filaments are adnate for some of their length), with divisions starting earlier in the S-morph (which ultimately has anthers at the high position) than in the L-morph. Given that the composition of the S-locus that confers distyly has been described in Primula to be composed of three linked genes, G, P, and A (Dowrick, 1956), where the S-morph genotype is GPA/gpa and the L-morph genotype is gpa/gpa (Bateson and Gregory, 1905 as cited in Lewis and Jones, 1992), the authors infer from these results that the action of the dominant G allele is to inhibit style growth while the action of the dominant A allele is to promote cell division in the lower corolla (Webster and Gilmartin, 2006). Cohen et al. (2012) examined whether species that represent three independent origins of heterostyly have similar floral development in Lithospermum (Boraginaceae).

54 They found that the dimorphism in reproductive organs for these species is due to differences in growth rates of the corollas and the styles. In the L-morph of all species the growth rate of the style was greater than that of the corolla, with the stigma extending beyond the height of the anthers by the point at which the corolla is approximately a third to a half of its final length. In the S-morph of all species, the initial growth rates of the style and corolla are similar, but at approximately half the final corolla length style growth rates slowed dramatically while corollas continued to grow at the same rate. Additionally, as reported for Primula (Webster and Gilmartin, 2006) both dimorphisms in style length and anther height is due to analogous anatomical elements of development. Dimorphism in style length is due to increased length of cells of the styles of the L-morph relative to the S-morph. Dimorphism in anther height is due to a greater number of cells of the lower corolla, below the point of anther insertion, of the S-morph relative that that of the L-morph. Comparisons of developmental studies in Rubiaceae from species representing independent origins of heterostyly (as determined based on the results of Chapter One) demonstrate greater variety in the means by which species attain the alternative floral morphologies. There have been studies of distylous flower development in members of supertribes Conchonidinae (Richards and Koptur, 1993), Psychotriidinae (Hernandez and Ornelas, 2007; Faivre, 2000), and Rubiidinae (Faivre, 2000; Riveros et al., 1995). These studies suggest four developmental pathways to achieving differences in stigma height and two for achieving differences in anther height (Hernandez and Ornelas, 2007). Since perhaps just two of these focal species (Bouvardia ternifolia and Oldenlandia salzmanii) share a common origin of distyly with Houstonia (Fig. 1.2E), I will focus on the pattern seen for each of them. Faivre (2000) found in Bouvardia ternifolia that anther position differs between morphs due to differences in the relative growth rates of the corolla, to which the filaments are adnate, and an earlier cessation of corolla elongation in the L-morph, resulting in a shorter corolla in that morph. Faivre (2000) found that differences in stylar growth rates throughout bud elongation produce the dimorphism in stigma heights for the morphs. Riveros et al. (1995) found similar results in Oldenlandia salzmanii (which they discussed as the synonym Hedyotis salzmanii) where relative growth rates of both the style and corolla differed between morphs and the corolla of the L-morph ceased elongation earlier, resulting in a shorter corolla in that morph.

55 The production of cleistogamous flowers by a heterostylous species is unique to H. procumbens (as shown in Chapter One), so the examination of cleistogamous and chasmogamous flower development in a heterostylous species is entirely novel. Previous work describing cleistogamous flower development and form has been limited to a relatively few species. In their study of the development of chasmogamous and cleistogamous flowers of Viola odorata, Mayers and Lord (1983, 1984) found that size differentiation between the two flower types began to occur at the stage of pollen mother cell development and that the anthers of chasmogamous flowers were two-fold greater in size at maturity than those of cleistogamous flowers. In V. odorata, divergence in the gynoecium was evidenced by significantly greater ovule production in chasmogamous flowers and the style of the cleistogamous flowers being recurved 180°, remaining in close proximity to the anthers. Precocious development of anthers and pollen in the cleistogamous flowers, which is the mechanism of pollination in this species, occurs by growth of pollen tubes through the anther wall, which lacks a well- developed endothecium. In Lamium amplexicaule (Lamiaceae), the stage at which chasmogamous and cleistogamous flowers diverge anatomically is pollen mother cell production, when cleistogamous flowers produce fewer pollen mother cells than do chasmogamous flowers (Lord, 1982). The morphology of the two flower types in this species is the same across ontogeny, although the rate of growth for cleistogamous flowers is accelerated relative to that of chasmogamous flowers. In Collomia grandiflora (Polemoniaceae) precocious anther development is also seen, and the anthers of the chasmogamous flowers are twice the size of anthers of cleistogamous flowers (Minter and Lord, 1983). Additionally, the anthers of the chasmogamous flowers developed four locules whereas the anthers of cleistogamous flowers developed only two locules. Mature pollen grains of the chasmogamous flowers were also significantly larger and more numerous than those of cleistogamous flowers. Most pertinent to my research, Ritzerow (1908, as cited in Lord, 1981) described cleistogamous flowers of Houstonia pusilla (using the synonym, H. minor), the only other species in the genus besides H. procumbens with cleistogamous flowers. Ritzerow (1908) was said by Lord (1981) to have found pollination proceeding by the growth of pollen tubes through undehisced anthers, which exhibited a rudimentary endothecium. She also reported a reduction in the size of the ovaries of cleistogamous flowers relative to those of chasmogamous flowers.

56 I will address the following hypotheses in this chapter. (1) Differences in the rate of elongation of styles and corollas lead to the difference in stigma and anther heights, respectively, seen in the morphs of the heterostylous species H. procumbens and H. caerulea, as seen in the development of closely related heterostylous species (Faivre, 2000; Riveros et al., 1995). My expectation is that style elongation will occur at a higher rate for the L-morphs than for the S-morphs as described by slope parameters of regression models of flower organ height over developmental time. Likewise, I expect anther height to occur at a higher rate for the S-morphs than the L-morphs if rates of organ development are the means by which floral organ dimorphisms are achieved in these species. (2) However, if one of these species is expected to vary from this developmental pattern, it is H. procumbens, which might be expected to maintain an anther-stigma distance close to zero (and similar rate of elongation for the style and corolla) for much of its development to make the evolution of cleistogamy simpler (by means of precocious sexual development, as in Viola odorata (Mayers and Lord, 1983, 1984) and Collomia grandiflora (Minter and Lord, 1983). (3) If this is not the case, I expect that the cleistogamous flowers of each morph of H. procumbens will have solved the problem of ending development with the anthers and stigma in close proximity in a way somewhat independent of style and corolla length (e.g., with curvature of the style, as seen in Viola odorata; Mayers and Lord (1983, 1984)). This is based on the apparent absence of an effect on reciprocity in chasmogamous flowers of the need by cleistogamous flowers to end development in this way (Chapter Two). And (4) greater cell proliferation (rather than cell elongation) in the lower corolla of S-morph flowers relative to L-morph flowers will be responsible for the greater height of anthers in these species, and greater cell elongation of the styles of L-morph flowers relative to S-morph flowers will be responsible for the difference in stigma height for these species, as seen in Primula and Lithospermum by Webster and Gilmartin (2003) and Cohen et al. (2012), respectively.

Materials and Methods Flowers intended to represent the full range of distylous development of the following species were collected from the following locations: H. procumbens from Torreya State Park, FL (HproTSPFL; 30° 34’ 16” N 84° 56’ 24” W), Falling Waters State Park, FL (FWSPFL; 30° 43’ 45” N 85° 31’ 35” W), and H. caerulea from 6883 Charity Hwy

57 Woolwine, VA (HcaeWWVA; 36° 49’ 52” N 80° 13’ 11.6” W (Figure 2.2). Cleistogamous flowers of H. procumbens were collected from HproTSPFL, HproFWSPFL, and Birdsong Nature Center (HproBNCGA; 30° 41’ 55” N 84° 11’ 58” W; Figure 2.2). Flowers were collected in FAA (ethanol, formaldehyde, acetic acid, and water 5:1:0.5:3.5) or 70% ethanol. Those collected in FAA were cleaned with serial dilutions of ethanol (30% X 1, 50% X 1, 70% X 2 for 10 min. each) and stored in 70% ethanol until dissection. A portion of the flower buds representing early distylous developmental stages and cleistogamous flowers were dissected using a Lecia WILD M3Z dissecting scope and photographed with a Nikon Coolpix 995 digital camera including a metric ruler for scale (when details of floral anatomy were visible). Flower buds were then dehydrated with a graded ethanol series (80%, 85%, 90%, 95% X2, 100% X2) then critical point dried and sputter coated with Iridium at approximately 5 nm thickness and examined with a Nova 400 Nano field emission electron microscope at 3 KV. Measurements of corolla length, stigma height, and anther height (all from the base of the corolla) for both sets of images were made using ImageJ64 (http://rsbweb.nih.gov/ij/index.html). Cell lengths from tissue of lower corollas and styles from mature flowers were examined using several methods. In some cases cell lengths were measured from whole flowers using a dissecting light microscope with an ocular micrometer. In other cases lower corolla tissue and styles were cleared, mounted on glass microscope slides, and imaged with a DP71 color digital camera (12.5 megapixles) mounted on an Olympus BX61 bright field microscope. Tissue clearing was accomplished by heating in 0.24 molar HCl plus 20% methanol at 57° C for 15 minutes, transferred to a solution of 7% NaOH in 60% ethanol at room temperature for 15 minutes. The tissue was then re- hydrated with a graded ethanol series (40%, 20%, and 10%), infiltrated with 5% ethanol plus 25% glycerol and mounted in 50% glycerol on glass slides (protocol adapted from Malamy and Benfey, 1997). From 10 – 29 cells representing 1– 3 individuals of each morph for each species were examined. Cell lengths from digital images were measured using ImageJ64 (http://rsbweb.nih.gov/ij/index.html). Flower developmental stages were described as the length of the corolla relative to the average length of mature corollas for the species and population in question (relative corolla size). This was done in order to determine the ranges of floral development represented by the samples analyzed. Organ developmental rate was described by plotting the height of anthers and stigmas, measured from the base of the

58 corolla, against corolla length. Anther-stigma distance (ASD) was plotted against corolla length to determine the rate at which the distylous morphs of each species reach a herkogamous state. Developmental rates were determined by fitting a linear regression to the plots of organ heights versus corolla lengths and the slope parameters of the models compared. The use of a linear regression model was compared to a regression model that included a second order term using analysis of variance in order to determine whether a linear model adequately described the data. Distribution of the data (non-transformed and log transformed) were checked for normality using a Kolmogorov-Smirnov test (ks.test) and the constancy of variance and normality of errors were checked by plotting residuals vs. fitted values of the model and residuals vs. theoretical quantiles (normal Q-Q plot), respectively. The R2 values of the raw data and log-transformed data were compared using analysis of variance to determine if log transformation significantly improved the fit of the regression model to the data. I compared the slope parameters of the regression models with an analysis of covariance (ANCOVA) using organ height as the response variable, corolla length as the explanatory variable, and distylous morph as the fixed effect. The interaction term (morph by corolla length) from the ANCOVA was used to detect significant differences in the slope parameters of the two models (i.e., models for the L- and S-morphs for a given species). Significantly greater slope parameters are taken to indicate a higher rate of organ growth in all comparisons of organ height to corolla length. In order to determine whether rates of style growth were different from that of the corolla, the slope parameters of the regression models describing growth of these organs were compared to one. For each regression model (i.e., for each morph of each species) the 95% confidence interval was calculated for the slope parameters to determine whether it overlapped the value of one indicating an isometric relationship between style growth and corolla growth. Differences in cell lengths of lower corolla tissue and styles were compared using a t-test (two-tailed, two-sample, unequal-variances).

Results I examined flowers of H. procumbens that ranged from 0.114–0.697 of the final corolla length for the L-morph and from 0.078–0.501 for the S-morph (Figure 3.1), and I examined flowers of H. caerulea that represented a range from 0.105–0.526 for the L- morph and from 0.085–0.607 for the S-morph (Table 3.1, Figure 3.2). Consequently all

59 analyses are valid only in relation to descriptions of early flower development. Inclusion of a second order term in the regression models did not significantly improve the ability of the models to describe the data. Consequently, linear models were used in the analyses. Distributions of the data did not differ significantly from normality whether or not they were log transformed (all p-values were greater than 0.2). Additionally, the constancy of variation and normality of the error terms were supported by the residuals vs. fitted values plot and normal Q-Q plot, respectively, regardless of whether the data were log transformed. Log transforming the data did not significantly improve the ability of the regression models to describe the data and yielded lower R2 values in as many cases as it yielded higher R2 values. Consequently, the untransformed data were used in the analyses. Plots of ASD versus corolla length (Figure 3.3) show that all the heights of reproductive organs begin diverging from one another (ASD is > 0) early in development with ASD taking on a significantly different rate of change (slope) for both morphs of each species examined (Table 3.2). The regression models describing organ height as a function of corolla length showed significant differences in their slope parameters between both morphs for each species (Table 3.2). The slope parameter of the regression models for stigma height as a function of corolla length was greater for the L-morph than the S-morph for both H. procumbens (1.04 vs. 0.58, respectively) and H. caerulea (0.90 vs. 0.50, respectively). Likewise, the slope parameter of the regression models describing anther height relative to corolla length was greater for the S-morph than for the L-morph for H. procumbens (0.80 vs. 0.62, respectively) and for H. caerulea (0.59 vs. 0.23, respectively). For both H. procumbens and H. caerulea anther stigma distance increased at a greater rate for the L-morph than the S-morph. The predicted values of organ heights for stigmas and anthers of both morphs of each species were greater than the average values of heights for those organs at maturity except for anther height of the L-morph of H. caerulea. The 95% confidence intervals for the slope parameters of all models were less than one, except for that describing stigma height of the L-morph of H. procumbens, which showed an isometric relationship with respect to corolla length. Cell size in the style and lower corolla did not differ significantly between morphs in either species (Table 3.2). However, cell size in filaments does in H. procumbens, with those of the L-morph longer than those of the S-morph (Table 3.3; p<0.001); the filament of H. caerulea was very short and could not by easily measured.

60

Table 3.1 Metrics of distylous flower development. Flower buds sampled for this work have a species (H. procumbens = Hpro, H. caerulea = Hcae) and morph (S-morph = S, L-morph = L) designation. Bolded rows present averages for the respective morph and species determined in Chapter Two. Relative (Rel.) metrics represent the proportion of the average value for that metric from mature flowers (the value in bold for the respective morph and species). Anther stigma distance (ASD) is the calculated absolute value of the difference between the midpoint of the anther and the base of the stigma lobes, as employed in reciprocity measures for Chapter Two.

Sp. morph Anther Rel. Stigma Rel. Corolla ASD Rel. designation height anther height stigma length corolla height height length Hpro_S 0.571 0.068 0.349 0.064 0.921 0.222 0.078 Hpro_S 0.563 0.067 0.370 0.067 1.171 0.193 0.100 Hpro_S 1.151 0.138 1.071 0.195 2.393 0.080 0.204 Hpro_S 1.650 0.197 1.107 0.202 2.522 0.543 0.215 Hpro_S 2.492 0.298 1.973 0.359 3.507 0.519 0.298 Hpro_S 3.192 0.382 2.393 0.436 4.836 0.799 0.411 Hpro_S 4.640 0.555 3.259 0.594 5.890 1.381 0.501 Hpro_L 0.544 0.091 0.622 0.071 1.325 0.078 0.114 Hpro_L 0.663 0.111 0.6 0.069 1.516 0.063 0.131 Hpro_L 1.284 0.214 2.249 0.258 3.166 0.965 0.273 Hpro_L 1.285 0.215 1.492 0.171 3.282 0.207 0.283 Hpro_L 1.682 0.281 3.003 0.345 3.939 1.321 0.339 Hpro_L 2.219 0.371 2.805 0.322 4.442 0.586 0.383 Hpro_L 2.885 0.482 4.737 0.544 5.525 1.852 0.476 Hpro_L 4.764 0.796 7.566 0.868 8.098 2.802 0.697 Hcae_S 0.514 0.071 0.5 0.141 1.163 0.014 0.085 Hcae_S 0.599 0.082 0.584 0.165 1.182 0.015 0.087 Hcae_S 1.121 0.154 1.075 0.303 2.630 0.046 0.193 Hcae_S 1.472 0.202 1.427 0.403 2.753 0.045 0.202 Hcae_S 1.423 0.195 1.544 0.436 2.766 0.121 0.203 Hcae_S 4.778 0.656 4.116 1.162 8.290 0.662 0.607 Hcae_L 0.440 0.152 0.382 0.064 1.173 0.058 0.105 Hcae_L 0.618 0.213 0.518 0.087 1.495 0.100 0.134 Hcae_L 0.717 0.247 1.726 0.290 2.553 1.009 0.230 Hcae_L 0.843 0.290 2.587 0.435 3.445 1.744 0.310 Hcae_L 1.605 0.553 4.207 0.708 5.728 2.602 0.515 Hcae_L 1.490 0.513 4.803 0.808 5.849 3.313 0.526

61

Figure 3.1 Developing distylous flowers of H. procumbens. The three panels on the left show the early developmental stages for flowers of the L-morph and panels on the right show early developmental stages for flowers of the S-morph.

62

Figure 3.2 Developing distylous flowers of H. caerulea. The two panels on the left show the early developmental stages for flowers of the L-morph and panels on the right show early developmental stages for flowers of the S-morph.

Examination of cleistogamous flowers of H. procumbens did not show any morph- specific differences in morphology. The corolla of the one mature cleistogamous flower of this species for which an SEM image was obtained reached an overall length of 0.83

63 mm within which was contained a style with a length of 1.2 mm (Figure 3.6 panel C). This is accomplished through the coiling of styles in a helical fashion within the corolla of mature flowers for this species (Figure 3.6). Cleistogamous flowers of H. pusilla proved to be too small to effectively dissect for examination either with light or scanning electron microscopy (Figure 3.6 panel A), being on the order of 0.5 mm long at maturity, and so I could not collect morphological or developmental information from them.

64

AVG Actual maturity at value 5.98 8.35 8.71 5.49 2.90 7.28 5.94 3.54

Predicted Predicted at value maturity 6.71335 9.082896 10.90867 6.62143 2.749696 7.947103 9.378178 6.820839

C.I. 97.5% 0.72076459 0.9647166 1.2407900 0.664379397 0.2936913 0.66147241 1.0338565 0.5724709

C.I. 2.5% 0.5256284 0.6502563 0.8439155 0.5087709 0.17041676 0.5355495 0.7754714 0.4313644

Df 11 11 11 8 8 8

06 05 06 - - - and slope parameter values for each linear regression model model linear regression each for values parameter slope and

2 value R P- 0.02 0.0009 0.06 3.77E 4.74E 7.81E

Slope 0.62 0.81 1.04 0.98 0.41 0.22 0.23 0.59 0.90 0.50 0.64 0.09

2 R 0.97 0.97 0.96 0.58 0.86 0.81 0.96 0.99 0.99 0.98 0.96 0.95

Organ Organ height Anther Stigma ASD Anther Stigma ASD

Morph L S L S L S L S L S L S

describing flower development by morph and species. P-values and degrees of freedom (df) from ANCOVA are from (df) freedom of P-values degrees and species. and by morph development flower describing The models. the of of regression parameters the (C.I.) intervals slope confidence along withpresented 97.5% the and 2.5% the average alongside are presented model the on regression based organ stigma) (anther each and for values predicted organ reciprocity of (chapter calculation for two). measured flowers organ from each for values Table 3.2 Analysis of of rates 3.2 Analysis Table flower development. Species procumbens H. caerulea H.

65

3.0 2.5 2.0 1.5 AntherStigma Distance 1.0 0.5 0.0

2 4 6 8

corolla length

Figure 3.3 Plot of anther stigma distance over the course of flower development for both morphs of H. procumbens and H. caerulea. Species by morph combinations are depicted by the following color schemes: Hpro L-morph (blue) slope=0.41 R2=0.86, Hpro S-morph (black) slope=0.22 R2=0.81, Hcae L-morph (red) slope=0.64 R2=0.96, and Hcae S-morph (green) slope=0.09 R2=0.95. Measurements indicated are in mm.

66 4 3 antherheight 2 1

2 4 6 8

corolla length

Figure 3.4 Plot of anther height over the course of flower development by morphs for H. procumbens and H. caerulea. Species by morph combinations are depicted by the following color schemes: Hpro L-morph (blue) slope=0.62 R2=0.97, Hpro S-morph (black) slope=0.80 R2=0.97, Hcae L-morph (red) slope=0.23 R2=0.96, and Hcae S-morph (green) slope=0.59 R2=0.99. Measurements indicated are in mm.

67 6 4 stigmaheight 2

2 4 6 8

corolla length

Figure 3.5 Plot of style growth rates over the course of flower development. Species by morph combinations are depicted by the following color schemes: Hpro L-morph (blue) slope=1.04 R2=0.96, Hpro S-morph (black) slope=0.58 R2=0.98, Hcae L-morph (red) slope=0.90 R2=0.99, and Hcae S-morph (green) slope=0.50 R2=0.98. Measurements indicated are in mm.

68

Figure 3.6 Images of cleistogamous flowers of H. procumbens and H. pusilla. Panel A shows a cleistogamous flower of H. pusilla after an attempted, unsuccessful dissection. Panels B and D show cleistogamous flowers of an S-morph individuals of H procumbens while panels C and E show cleistogamous flowers of an L-morph of H procumbens.

69

P- value 0.89 0.799 0.276 0.065 0.00015 - were of

S cells No. morph 20 15 29 20 29

- H. procumbensH.

S ind. No. morph 2 1 2 1 2 Data represent average (AVG)

-

(Hcae). SD S SD morph (um) 8.62 5.99 9.41 9.41 12.40

-

H. caerulea H. AVG S AVG (um) morph 41.37 30.866 37.601 41.872 36.860 -

(Hpro) and (Hpro) and

No. cells L cells No. morph 28 10 20 10 20 -

No. ind. L ind. No. morph 3 1 2 1 2 H. procumbensH. of -

SD L SD 6.3 7.861 11.22 8.089 3.62 morph morph (um)

-

AVG L AVG (um) morph 40.841 30.107 34.218 35.42 26.439

Species Hpro Hcae Hpro Hcae Hpro

for 3.3 CellTable lengths floral organs Cell lengths Style Lower corolla Filament and standard deviations (SD) of cell lengths of the corresponding tissues for the morphs indicated. Numbers (No.) of (No.) of Numbers indicated. the for lengths morphs of cell of tissues (SD) the corresponding deviations standard and of Only filaments species. and morph each for are indicated measured cells and (ind.) individuals sufficient length to measure cell lengths. The p-values indicated are from two-sample, unequal-variance t-tests. unequal-variance two-sample, p-values lengths. are from indicated The cell lengthsufficient to measure

70

Discussion As seen in the development of closely related heterostylous species (Faivre, 2000; Riveros et al., 1995), differences in the rate of elongation of styles and corollas lead to the difference in stigma and anther heights seen in the morphs of the heterostylous species H. procumbens and H. caerulea (Figures 3.4 and 3.5). Slope parameters of the regression models describing height of the long organs were significantly greater for each morph (i.e., stigmas of the L-morph and anthers of the S-morph). Given that all developing flowers examined represent early stages of development, this indicates that the basis for this floral heteromorphism is established early in development. Additionally, discrepancies between predicted and average values of mature organ heights for anthers and stigmas of both morphs for each species seem to indicate that late floral development should exhibit lower rates organ growth than that seen early in development. It is therefore important to include data describing late flower development, in addition to those data presented here, in future work with this system. The relationship between style growth and that of the corolla is one of isometry for the L-morph of H. procumbens and one of negative allometry (slope<1) for styles of the S-morph of H. procumbens and styles of both morphs of H. caerulea. This result describes the means by which stigmas of the L-morph of H. procumbens end development extended beyond the corolla tube whereas that is not the case for stigmas of the L-morph of H. caerulea, which are retained within the corolla tube at maturity (pers. obs.) Since cell length in the lower corolla does not differ significantly between morphs, one could infer that relative length differences in the lower corolla that arise between morphs of each species are due to differences in cell number, as seen in Primula by Webster and Gilmartin (2003) and in Lithospermum by Cohen et al. (2012), rather than cell length. Contrary to the pattern seen in Primula (Webster and Gilmartin, 2003) and Lithospermum (Cohen et al., 2012), differences in cell proliferation, rather than cell elongation, appear to be responsible for differences in style length in these species, inferred from the absence of significant differences in the length of cells of the style among morphs. Future research should aim to verify empirically, rather than inferentially, that cell proliferation is responsible for the dimorphism seen here and strive to take into account features of late flower development for these species.

71 The anther-stigma distance grows from early in development in both morphs of H. procumbens and the L-morph of H. caerulea, but for the S-morph of H. caerulea the anthers and stigmas are at the same height until perhaps half of the corolla’s elongation, after which it increases until it is finally greater than that for the L-morph (on average; Chapter 2). This is paradoxical, since one might expect this to be a developmental pattern that makes cleistogamy (e.g., as in H. procumbens) evolutionarily simpler (i.e., requiring fewer modifications to developmental programming). I would also expect to see delay in organ growth over developmental time for both morphs of H. procumbens rather than solely in the S-morph of H. caerulea. In that case, flowers early in development that reach precocious sexual maturity could take advantage of the close proximity of the stigma and anthers, as seen in Viola odorata (Mayers and Lord, 1983, 1984) and Collomia grandiflora (Minter and Lord, 1983). Instead of modifying style length to produce stigmas and anthers at the same height in cleistogamous flowers, H. procumbens is doing something similar to the bending of the style in Viola odorata (Mayers and Lord 1983, 1984). It is producing shorter corollas and helical styles in both morphs that do not appear to show morphological similarities to the chasmogamous flowers of those morphs (Fig. 3.6). It is possible that the cleistogamous flowers of this species reach maturity before differences in the distylous morph on which they are born arise. Given that cleistogamous flowers are notably precocious in their development this seems to be a likely scenario and one worth investigating. The morphology of cleistogamous flowers raises an interesting question regarding investment in biomass. One clear advantage of cleistogamous flowers over chasmogamous flowers is the relatively low cost in terms of biomass for their production (e.g., Winn and Moriuchi, 2009). It is therefore notable that the cleistogamous flowers of H. procumbens produce styles longer than their corollas have capacity to enclose, leading to the helical coiling of the styles of these flowers. While the extra style growth exhibited by these flowers may not seem at first glance to be substantial in terms of biomass, it is possibile that some element of the overall floral developmental program for the chasmogamous flowers of this species is the reason for this particular arrangement of the style in the cleistogamous flowers. This may be the case for other cleistogamous flowers that have been described (i.e., Mayers and Lord, 1983 and 1984) as well.

72 Certainly the greatest weakness of this chapter’s research is related to sample sizes. In particular it is regrettable that cleistogamous flowers of H. pusilla and H. procumbens were so difficult to dissect since more data from those two flower types would fill gaps in the sampling of these flowers toward the goals of this research. Additionally, more samples of developing distylous flowers of H. procumbens and H. caerulea would provide a more complete picture of the development of these flowers, particularly with regard to later stages of floral development.

Conclusions In summary, it appears that rates of organ growth are not only responsible for the dimorphism seen in distylous flowers of the species examined, but that these differential rates of organ growth are established early in development. The taxonomic distribution of heterostyly across angiosperms seems to indicate that the genes responsible are relatively easily co-adapted. The variability in expression of distyly as reflected in degrees of organ reciprocity would seem to indicate that the genes responsible for this heteromorphism are at least somewhat removed from their downstream molecular targets, allowing for selection through interaction with pollinators to modify the ultimate expression of genes that confer the floral heteromorphism. It has been shown that genes that are differentially expressed among morphs of Primula vulgaris (Primulaceae) include stress-related genes, proteins that govern cell wall development and expansion, and RNA binding proteins (McCubbin et al., 2006). These results indicate that indeed the S-locus genes that govern distylous flower development do so by regulating suites of genes, which in turn produce the dimorphism seen among floral organs of distylous species. With regard to the form of cleistogamous flowers of H. procumbens it appears that either the S-locus that confers distyly in the chasmogamous flowers of this species does not interact with the developmental program that produces cleistogamous flowers or the cleistogamous flowers reach sexual maturity before any expression of the S-locus has an opportunity to have such an effect. It is noteworthy that modifications to the style of cleistogamous flowers for this species seem to reflect an accommodation of long styles within a short corolla. I suspect that the genes that confer cleistogamy interact with the general floral developmental program rather than supplanting it entirely,

73 leaving the cleistogamous flowers with a need to adjust to the default floral morphology for a given species. Molecular characterization of the heterostyly S-locus will obviously be necessary to examine these hypotheses in rigorous detail. Although, it is unfortunate that only H. procumbens produces both heterostylous and cleistogamous flowers, or these hypotheses might be testable without knowledge of the molecular basis for the S-locus by comparing the development of heterostylous and cleistogamous flowers of several species including perhaps manipulated lines that are mutated in the genetic regions responsible for flower development.

74 APPENDIX A

GENBANK ACCESSION NUMBERS FOR DNA SEQUENCES USED IN PHYLOGENETIC INFERENCE OF THE RUBIACEAE

Species GenBank accession no. Adina pilulifera AJ346964.1 Adina rubella AJ346965.1 Adinauclea fagifolia AJ346966.1 Agathisanthemum bojeri Z68787.1 Aidia micrantha Z68844.1 Aidia racemosa AJ286693.1 Alberta magna Y18708.1 Alibertia edulis Z68843.1 Alseis lugonis Y18709.1 Amaracarpus sp. AJ002176.1 Amphiasma benguellense AJ616209.1 Amphiasma luzuloides AJ288594.1 Amphidasya ambigua Y11844.1 Anthospermum herbaceum X83623.1 Antirhea lucida X83624.1 Aoranthe penduliflora Y11845.1 Appunia guatemalensis AJ288593.1 Arabidopsis thaliana NC 000932.1 Arcytophyllum arisatum AJ288595.1 Argostemma hookeri Z68788.1 Batopedina pulvinellata AJ288596.1 Bertiera breviflora X83625.1 Bertiera guianensis AJ224845.1 Borojoa sp. AJ286694.1 Bouvardia glaberrima X83626.1 Breonadia salicina AJ346967.1 Breonia chinensis AJ346968.1 Breonia decaryana AJ346969.1 Breonia havilandiana AJ346970.1 Breonia macrocarpa AJ346971.1 Breonia perrieri AJ346972.1 Burchellia bubalina Z68833.1

75 Species GenBank accession no. Burttdavya nyasica AJ346973.1 Caelospermum balansanum AJ288598.1 Caelospermum monticolum AF331644.1 Calochone redingii Z68845.1 Calycophyllum candidissimum X83627.1 Canthium coromandelicum Z68851.1 Capirona decorticans Y18710.1 Carpacoce sp. AJ288597.1 Carphalea glaucescens Z68789.1 Casasia clusiifolia Z68831.1 Catesbaea fuertesii AY205347.1 Catesbaea spinosa X83628.1 Cephalanthus glabratus AJ346974.1 Cephalanthus natalensis Y18711.1 Cephalanthus occidentalis CAA58608.1 Cephalanthus salicifolius AJ346975.1 Chassalia parviflora Z68790.1 Chazaliella abrupta Z68791.1 Chimarrhis hookeri Y18712.1 Chiococca alba L14394.1 Chomelia sp. Y11846.1 Cinchona calisaya AY538478.1 Cinchona macrocalyx AY538479.1 Cinchona officinalis AY538480.1 Cinchona pitayensis AY538481.1 Cinchona pubescens X83630.1 Cinchonopsis amazonica AY538482.1 Coccocypselum hirsutum X87145.1 Coddia rudis AJ286695.1 Coffea arabica X83631.1 Coffea sp. AJ286691.1 Condaminea corymbosa Y18713.1 Conostomium longitubum AJ616210.1 Conostomium quadrangulare Z68792.1 Coprosma pumila X87146.1 Coptosapelta flavescens Y18714.1 Corynanthe mayumbensis AJ346976.1 Corynanthe pachyceras AJ346977.1 Corynanthe paniculata AJ346978.1 Cosmibuena grandiflora AY538483.1

76 Species GenBank accession no. Coussarea macrophylla Y11847.1 Craterispermum brachynematum AJ288629.1 Craterispermum laurinum AF331646.1 Cremaspora triflora Z68856.1 Crossopteryx febrifuga AJ347009.1 Cruckshanksia hymenodon AJ288599.1 Cubanola domingensis X83632.1 Cynanchum laeve DQ006054.1 Damnacanthus indicus Z68793.1 Danais xanthorrhoea Z68794.1 Declieuxia fruticosa AJ002177.2 Deppea grandiflora X83633.1 Dialypetalanthus fuscescens AJ251366.1 Dibrachionostylus kaessneri AJ616211.1 Dictyandra arborescens AJ286708.1 Didymaea alsinoides Z68795.1 Didymosalpinx norae Z68834.1 Diodia sarmentosa AJ288600.1 Diplospora polysperma AJ286703.1 Dolicholobium oxylobum AJ318445.1 Duperrea pavettifolia AJ286709.1 Duroia hirsuta AJ286696.1 Emmenopterys henryi Y18715.1 Enterospermum coriaceum X83634.1 Erithalis fruticosa X83635.1 Erithalis vacciniifolia AY205349.1 Ernodea littoralis AJ288601.1 Euclinia longiflora Z68835.1 Exostema acuminatum AY205359.1 Exostema caribaeum AY205358.1 Exostema ellipticum AY205358.1 Exostema ixoroides AY205355.1 Exostema lineatum AY205353.1 Exostema longiflorum AY205352.1 Exostema mexicanum AY205357.1 Exostema myrtifolium AY205356.1 Exostema nitens AY205361.1 Exostema parviflorum AY205354.1 Exostema salicifolium AY205360.1 Exostema spinosum AY205350.1

77 Species GenBank accession no. Faramea multiflora Z68796.1 Feretia aeruginescens Z68857.1 Fernelia buxifolia AJ286704.1 Gaertnera sp. Z68797.1 Gaillonia yemenensis AJ288630.1 Galium aparine DQ006124.1 Galium mollugo AY395538.1 Galopina circaeoides AJ288602.1 Gardenia angusta AJ286697.1 Gardenia hansemannii AJ318446.1 Gardenia thunbergia X83637.1 Genipa americana Z68839.1 Geophila repens Z68798.1 Glossostipula concinna Z68846.1 Gonzalagunia affinis Y11848.1 Guettarda speciosa AY538485.1 Guettarda uruguensis X83638.1 Gynochthodes coriacea AJ288603.1 Gynochthodes epiphytica AF331649.1 Gyrostipula comoriensis AJ346979.1 Gyrostipula foveolata AJ346980.1 Haldina cordifolia X83639.1 Hallea stipulosa AJ346981.1 Hamelia cuprea X83641.1 Hamelia papillosa AY538487.1 Hedyotis fruticosa Z68799.1 Heinsenia diervilleoides CAC16586.1 Heinsia crinita Y11849.1 Hekistocarpa minutiflora AF332366.1 Hemidiodia ocimifolia AJ288607.1 Hillia triflora X83642.1 Hillia valerii AJ288608.1 Hintonia latiflora X83643.1 Hippotis sp. Y11850.1 Houstonia caerulea AJ288604.1 Hydnophytum formicarum X83645.1 Hydrophylax maritima AJ616212.1 Hymenocoleus hirsutus AJ002178.1 Hymenodictyon decaryi AJ347013.1

78 Species GenBank accession no. Hymenodictyon flaccidum AJ347014.1 Hymenodictyon floribundum CAC87381.1 Hymenodictyon parvifolium CAC87382.1 Hyperacanthus amoenus AJ286698.1 Hypobathrum racemosum AJ286705.1 Isertia coccinea AY538489.1 Isertia laevis AY538490.1 Isertia pittieri Y11851.1 Isidorea cf brachyantha AY205348.1 Ixora biflora Z68866.1 Ixora coccinea X83646.1 Ixora hookeri Z68864.1 Ixora parviflora AJ224844.1 Janotia macrostipula AJ346982.1 Joosia aequatoria AY538491.1 Joosia umbellifera AY538492.1 Kailarsenia ochreata Z68847.1 Keetia zanzibarica X83647.1 Kelloggia chinensis AY570776.1 Kelloggia galioides AY570777.1 Kerianthera preclara AY538493.1 Knoxia platycarpa AJ288631.1 Kohautia caespitosa Z68800.1 Kraussia floribunda Z68858.1 Kraussia socotrana AJ286706.1 Ladenbergia amazonensis AY538494.1 Ladenbergia carua AY538495.1 Ladenbergia macrocarpa AY538496.1 Ladenbergia oblongifolia AY538497.1 Ladenbergia pavonii Z68801.1 Ladenbergia sp. AY538498.1 Lasianthus batangensis AY538499.1 Lasianthus pedunculatus Z68802.1 Lelya prostrata AJ288609.1 Leptactina platyphylla Z68867.1 Lerchea bracteata AJ288610.1 Luculia grandifolia X83648.1 Luculia pinceana AJ347012.1 Ludekia borneensis AJ346983.1 Manettia bicolor Z68803.1

79

Species GenBank accession no. Manostachya ternifolia AJ616213.1 Mapouria umbrosa Z68804.1 Maschalocorymbus corymbosus AJ288611.1 Massularia acuminata Z68841.1 Metadina trichotoma AJ346984.1 Meyna tetraphylla X83649.1 Mitchella repens AF190440.1 Mitchella undulata AF190445.1 Mitracarpus villosus AJ288632.1 Mitragyna diversifolia AJ346985.1 Mitragyna inermis AJ346986.1 Mitragyna rotundifolia AJ346987.1 Mitragyna rubrostipulata AY538486.1 Mitragyna speciosa AJ346988.1 Mitrasacmopsis quadrivalvis AJ616214.1 Mitriostigma axillare X83650.1 Morinda citrifolia AJ318448.1 Mussaenda arcuata Y11854.1 Mussaenda erythrophylla X83652.1 Mussaenda raiateensis AY538500.1 Mussaenda scratchleyi AJ318447.1 Mussaendopsis beccariana AJ347010.1 Mycetia malayana Z68806.1 Myonima violacea AJ286710.1 Myrmecodia platyrea X87147.1 Myrmeconauclea strigosa AJ346989.1 Nauclea diderrichii AJ346994.1 Nauclea orientalis X83653.1 Nauclea orientalis AY538501.1 Nauclea subdita AJ346996.1 Nauclea xanthoxylon AJ346995.1 Neolamarckia cadamba AJ346990.1 Neonauclea brassii AJ346991.1 Neonauclea clemensiae AJ318450.1 Neonauclea forsteri AJ346992.1 Neonauclea longipedunculata AJ346993.1 Nertera granadensis X83654.1 Neurocalyx zeylanicus Z68807.1 Ochreinauclea maingayi AJ346997.1 Oldenlandia cf corymbosa X83655.1

80

Species GenBank accession no. Oldenlandia goreensis Z68808.1 Opercularia vaginata Z68809.1 Ophiorrhiza mungos X83656.1 Oreopolus glacialis AJ288612.1 Otiophora cupheoidea AJ288613.1 Otomeria oculata AJ288614.1 Oxyanthus cf zanguebaricus Z68838.1 Oxyanthus pyriformis Z68836.1 Oxyceros sp. AJ286699.1 Paederia foetida AF332373.1 Paederia scandens AY654280.1 Palicourea lasiorrachis AJ002179.1 Palicourea sp. Z68810.1 Paracoffea melanocarpa Z68853.1 Paracorynanthe antankarana AJ347017.1 Paragenipa lancifolia AJ286707.1 Parapentas silvatica X83657.1 Paratriaina xerophila AJ288633.1 Pauridiantha paucinervis Z68811.1 Pauridiantha sp. AY538502.1 Pausinystalia johimbe AJ346998.1 Pausinystalia lane-poolei AJ346999.1 Pausinystalia macroceras AJ347000.1 Pavetta abyssinica Z68863.1 Pavetta lanceolata Z68865.1 Pavetta platyclada AJ318451.1 Pentagonia macrophylla X83658.1 Pentanisia longituba Z68812.1 Pentanopsis fragrans Z68813.1 Pentanopsis gracilicaulis AJ616208.1 Pentas lanceolata L13931.1 Pentodon pentandrus X83660.1 Pertusadina eurhyncha AJ347001.1 Pertusadina hainanensis AJ347002.1 Pertusadina malaccensis AJ347003.1 Phyllis nobla Z68814.1 Phylohydrax carnosa AJ288615.1 Pinckneya pubens X83661.1 Placopoda virgata Z68815.1 Plocama pendula Z68816.1

81

Species GenBank accession no. Pogonopus speciosus X83662.1 Porterandia crosby Z68840.1 Posoqueria latifolia Z68850.1 Pouchetia gilletii Z68859.1 Praravinia suberosa AJ288616.1 Pravinaria leucocarpa AJ288617.1 Prismatomeris beccariana AJ288618.1 Pseudomussaenda arborea Y11856.1 Pseudomussaenda flava Y11855.1 Psilanthus ebracteolatus CAC16594.1 Psilanthus mannii Z68852.1 Psychotria borucana AJ002180.1 Psychotria bremekampiana AJ002181.1 Psychotria dukei AJ002182.1 Psychotria graciliflora AJ002183.1 Psychotria ipecacuanha AJ002184.1 Psychotria kirkii X83663.1 Psychotria leptothyrsa AJ318452.1 Psychotria mariniana AJ002185.1 Psychotria micralabastra AJ318453.1 Psychotria micrococca AJ318454.1 Psychotria nervosa AJ002186.1 Psychotria peteri Z68817.1 Psychotria poeppigiana Z68818.1 Psychotria ramuensis AJ318455.1 Psychotria sp. AJ002188.1 Psyllocarpus laricoides AJ288619.1 Putoria calabrica AJ288620.1 Ramosmania rodriguesii Z68860.1 Randia aculeata Z68832.1 Randia decemcostata AJ286700.1 Randia fitzalani Z68848.1 Randia moorei Z68849.1 Randia ruiziana Z68830.1 Randia schumanniana AJ318456.1 Randia truncata AJ286701.1 Relbunium hypocarpium AJ288621.1 Remijia chelomaphylla AY538503.1 Remijia macrocnemia AY538504.1 Remijia pacimonica AY538505.1

82

Species GenBank accession no. Remijia pedunculata AY538506.1 Remijia ulei AY538507.1 Retiniphyllum pilosum AF331654.1 Rhachicallis americana X83664.1 Richardia pilosa Z68820.1 Rogiera suffrutescens X83665.1 Rondeletia odorata Y11857.1 Rothmannia longiflora Z68837.1 Rubia tinctorum X83666.1 Rudgea lorentensis Z68821.1 Rudgea sessiliflora AJ002187.1 Rustia splendens Y18716.1 Rutidea orientalis Z68862.1 Sabicea aspera AY538508.1 Sabicea villosa Y11858.1 Sarcocephalus latifolius X83667.1 Sarcocephalus pobeguinii AJ347005.1 Schenckia blumenaviensis AJ288622.1 Schradera subandina Y11859.1 Schumanniophyton magnificum AJ286702.1 Scyphiphora hydrophyllacea Y18717.1 Serissa foetida Z68822.1 Simira viridiflora Y18718.1 Sinoadina racemosa AJ347004.1 Sipanea biflora AY538509.1 Spermacoce hispida AJ288623.1 Spermacoce tenuior Z68823.1 Spermadictyon suaveolens Z68824.1 Stenaria nigricans AJ288606.1 Stilpnophyllum grandifolium AY538510.1 Stilpnophyllum oellgaardii AY538511.1 Strumpfia maritima Y18719.1 Sukunia longipes Z68842.1 Tamridaea capsulifera Y11860.1 Tarenna buruensis AJ318457.1 Tarenna drummondii AJ286712.1 Tarenna neurophylla Z68861.1 Tarenna supra-axillaris AJ286711.1 Theligonum cynocrambe X83668.1 Timonius sp. Y18721.1

83

Species GenBank accession no. Timonius timon AJ318458.1 Triainolepis hildebrandtii AJ288625.1 Tricalysia cryptocalyx Z68854.1 Tricalysia ovalifolia Z68855.1 Trichostachys sp. AJ288626.1 Uncaria africana AJ347006.1 Uncaria guianensis AJ347007.1 Uncaria rhynchophylla X83669.1 Uncaria sp. AJ347008.1 Urophyllum ellipticum AJ288627.1 Vangueria madagascariensis X83670.1 Versteegia cauliflora AJ318459.1 Versteegia grandifolia AJ286713.1 Virectaria major Y11861.1 Warszewiczia cordata Y18722.1 Xanthophytum capitellatum AJ288628.1

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91 BIOGRAPHICAL SKETCH

Eric Hunter Jones

Professional preparation Tallahassee Community College, Tallahassee, FL; Science Ed.; A.A. (Cum Laude) 2000 Florida State University, Tallahassee, FL; Biological Science; B.S. 2003

Appointments 2005 & 2012 Teaching assistant for Botany laboratory (BOT 3015L), supervisors: Dr. Austin R. Mast and Dr. Hongchang Cui, Florida State University, Department of Biological Science 2011 Teaching assistant for Botany (BOT 3015), supervisor: Dr. Austin R. Mast, Florida State University, Department of Biological Science 2009 – 2011 Teaching assistant for Field Botany (BOT 3143), supervisor: Dr. Austin R. Mast, Florida State University, Department of Biological Science 2010 Botanical Society of America, teaching section, committee for determining criteria for awarding the teaching section’s Samuel Postlethwait award 2010 Teaching assistant for Evolution (PCB 4674), supervisor: Dr. Darin R. Rokyta, Florida State University, Department of Biological Science 2008 – 2012 Instructor for non-majors biology (BSC 1005), supervisors: Dr. Ann S. Lumsden and Caroyln Schultz, Department of Biological Science, Florida State University 2008 Fa Ecology and Evolution student representative for the Department of Biological Science Integrating Genotype and Phenotype search committee 2004 – 2012 Laboratory manager for Dr. Austin R. Mast, Florida State University, Department of Biological Science 2002 – 2003 Non-majors biology laboratory instructor (BSC 1005L), supervisor: Dr. Ann S. Lumsden, Florida State University, Department of Biological Science 2001 – 2003 National Science Foundation Young Scholars Program teaching assistant (Biochemistry of the gene), supervisor: Dr. Lloyd Epstein, Florida State University, Department of Biological Science

92 2001 – 2002 Sea to See instructor, K – 12 outreach program, supervisor: Robert Lutz, Florida State University, Department of Biological Science 1999 – 2001 College preparatory mathematics instructor, supervisor: Dr. Sally Search, Tallahassee Community College, Academic Support Department

Peer Reviewed Publications Mast, A. R., E. F. Milton, E. H. Jones, R. M. Barker, W. R. Barker and P. H. Weston. In review. Does the time-calibrated phylogeny of the woody Australian genus Hakea (Proteaceae) support the origin of bird pollination among insect-pollinated ancestors? American Journal of Botany 99: 472-487.

Mast, A. R., C. L. Willis, E. H. Jones, K. M. Downs, and P. H. Weston. 2008. A smaller Macadamia from a more vagile tribe: inference of phylogenetic relationships and divergence times in Macadamia and relatives (tribe Macadamieae; Proteaceae). American Journal of Botany 95: 843-870.

Mast, A. R., Jones, E. H., and Havery, S. P. 2005. An assessment of old and new DNA sequence evidence for the paraphyly of Banksia with respect to Dryandra (Proteaceae). Australian Systematic Botany 18: 75-88.

Honors/Awards 2012 Recipient of the Outstanding Teaching Assistant Award from the Program for Instructional Excellence at Florida State University 2008 Co-recipient of the Integrative Training in Biology and Society Fellowship sponsored by the National Science Foundation (Supervisors: Dr. Tom Miller, Dr. Brian Inouye, Dr. Nora Underwood, Dr. Fredrick Davis) 2007 & 2009 Robert K. Godfrey Fellowship recipient for study in Botany ($500.00; Department of Biological Science, FSU) 2002 Fa National Science Foundation Research Education for Undergraduates (Department of Biological Science, FSU) Supervisor: Dr. Hank W. Bass 2002 Fa Varina Vaughn/Wynonna Jordan Botany Scholarship ($1,500; Department of Biological Science, FSU) 2002 Su James R. Fischer American Cancer Society Fisher Research

93 Fellowship ($2,400; Department of Biological Science, FSU); Supervisor: Dr. Hank W. Bass 2001 Fa Francenia E. Fisher Botany Scholarship ($2,500; Department of Biological Science, FSU) 2000 Fa Phi Theta Kappa International Honor Society (for two year colleges)

Outreach/Public Service 2008 – 2011 PlantingScience Master Plant Science Mentor; sponsored by the Botanical Society of America; alpha and beta testing of new teaching modules, working with gifted and ESOL students 2004 – 2010 Florida State University Department of Biological Science Teaching Workshop - invited lecture “Engaging your audience” 2010 Botanical Society of America, Botany Conference (Brown University, Providence, RI), ½ day workshop - PlantingScience Fostering Student Scientific Thinking Through Mentoring; presented by: Eric H. Jones (Florida State University), Laura Lagomarsino (Harvard University Herbaria), Laura Super (University of British Columbia), Lindsey K. Tuominen (University of Georgia) 2008 Southeastern Ecology and Evolution Conference, conference co- administrator (5th annual; served 171 participants from 46 academic institutions), Florida State University, Department of Biological Science 2007 – 2012 PlantingScience mentor, sponsored by the Botanical Society of America and the American Society of Plant Biologists 2006 – 2007 President of the Ecology and Evolution Research Discussion Group (EERDG), Department of Biological Science, Florida State University

Scientific Presentations 2010 Botanical Society of America - Reciprocal Herkogamy in Houstonia procumbens, a Heterostylous, Cleistogamous Species (poster presentation) 2010 Plant Science Seminar (Florida State University) - Floral development and evolution in Houstonia procumbens (Rubiaceae) a uniquely heterostylous, cleistogamous species 2010 Southeastern Ecology and Evolution Conference (Georgia Institute of Technology)

94 Old dogs learning new tricks. Asking philosophical questions using biological systems, an integrative approach (oral presentation) 2009 European Society for Evolutionary Biology Congress (Torino, Italy) - Searching for a causal explanation of a fitness difference in the model system, X174 Bacteriophage; Eric Jones, S. Brian Caudle, Donald V. Griffin, and Darin R. Rokyta (poster presentation by Donald V. Griffin) 2007 Southeastern Ecology and Evolution Conference (University of Central Florida) - Convergent Evolution of Heterostyly - two family scale investigations (oral presentation) 2007 Ecology and Evolution Seminar Series Department of Biological Science FSU - The evolution of heterostyly across angiosperms (oral presentation) 2006 Botanical Society of America (California State University, Chico) - How many times, and in what evolutionary contexts, did heterostyly arise in the angiosperms? (oral presentation)

Professional Society Memberships Botanical Society of America American Society of Plant Taxonomists American Society of Plant Biologists American Association for the Advancement of Science National Education Association

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