“Floral Morphology, Pollination Mechanisms, and Phylogenetics of Pleurothallis Subgenera Ancipitia and Scopula” A Senior Thesis Presented to The Faculty of the Department of Organismal Biology and Ecology, Colorado College BY Katharine Dupree Bachelor of Arts Degree in Biology 16th day of May, 2016 Abstract Pleurothallis is the largest myophilous (fly-pollinated) genus in subtribe Pleurothallidinae. Although many studies show highly specific relationships in pollination systems in the Orchidaceae, our understanding of these relationships in myophilous orchids is almost non-existent (Borba & Semir 2001). This study focuses on the floral micromorphology, specifically the lip and column, of species within subgenera Ancipitia and Scopula. Scanning electron microscopy of the micromorphology of floral structure shows a range of morphology and pollination mechanisms within the two studied subgenera. These include deceit pollination by pseudocopulation and reward pollination. In concert, phylogenetic analysis was performed to determine if a correlation existed between morphology or pollination mechanism and taxonomic groupings. Maximum parsimony trees were produced using ITS and matK sequences for subgenera Ancipitia, Scopula, and Pleurothallis, with species from the genera Laelia, Pabstiella, and Arpophyllum as outgroups. The ITS, matK and combined trees strongly support an Ancipitia/Scopula section within a monophyletic subgenus Pleurothallis. Within this section, both reward and deceit pollination mechanisms are found, meaning they are not restricted to the current taxonomic groupings. Morphological and genetic data therefore support the grouping of subgenera Ancipitia, Scopula, and Pleurothallis into one monophyletic subgenus. 2 Acknowledgements I would like to thank Dr. Mark Wilson, my advisor, for his guidance, inspiration, and encouragement. I would also like to thank former students for sequence contributions, Graham Frank for establishing protocols, and the Colorado College Organismal Biology and Ecology Department for funding. I thank Dr. Ron Hathaway for scanning electron microscopy instruction, Dr. Nick Brandley for editing assistance, Jaime Micciulla for providing supplies, and Donna Sison for her continued assistance. 3 Introduction Floral Morphology in Pleurothallidinae Within the family Orchidaceae, subtribe Pleurothallidinae comprises an estimated 4000 Neotropical species (Luer 1986). Species in the subtribe Pleurothallidinae exhibit a broad array of growth habits (epiphytic, terrestrial) and occupy many habitats (Higgins 2009). A typical pleurothallid is an epiphyte with a restricted distribution, frequently endemic, that lives in sympatry with other pleurothallids in extremely moist forests in the Andes (1800-2800 m) and is pollinated by flies (Borba & Semir 2001, Higgins 2009). In a typical Pleurothallis flower, the gynostemium, commonly referred to as the column, lies at the center, a result of the fusion of stigma and stamen (Claessens & Kleynen 2013). Within the stamen lies the pollinarium, a unit containing a variable number of pollinia. In some species, the pollinia are connected by the caudicle to the viscidium, a sticky structure. It is the viscidium that attaches to the pollinator. The entirety of the column is attached to the lip, a modified petal. The rest of the flower is comprised of two petals, a dorsal sepal, and a lateral sepal which results from the fusion of the two lower sepals (Figure 1). Figure 1: Illustration of typical Pleurothallis morphology (a) Petals, (b) lateral sepal, (c) column, (d) pollinarium and pollinia, (e) labellum or lip, and (f) synsepal. 4 In this study, I will be looking at the micromorphology, specifically the lip and column, of species within Pleurothallis subgenera Ancipitia and Scopula (Luer 1989). The micromorphology of these species can be very informative. From characteristics of the lip and column, we can speculate the size and type of pollinator, and the strategy employed to attract said pollinator. Pollination Mechanisms in Pleurothallidinae Pleurothallis is one of the two largest myophilous groups in the subtribe Pleurothallidinae (van der Pijl & Dodson 1966). Although there are many studies showing highly specific relationships in pollination systems in the Orchidaceae, our understanding of these relationships in myophilous (fly pollinated) orchids is almost non-existent (Borba & Semir 2001). This lack of attention is probably due in part to the reputation of Diptera as inefficient, irregular, unreliable and poor pollinators, characterized by lack of constancy and random, casual behavior (van der Pijl & Dodson 1966). Most knowledge about pollination of pleurothallids is inferred by study of morphology, as witnessing pollination events in the wild is rare (Borba & Semir 2001). The variety of lip morphology in the species belonging to subgenera Ancipitia and Scopula suggests many different pollination strategies are present (Diaz-Morales & Karremans 2015). Using scanning electron microscopy (SEM), I will examine morphology and use observations to speculate on pollination mechanisms. Deceit and reward strategies are known to occur, and the use of sexual and aggregation pheromones have been reported (Karremans et al. 2015). Reward pollination refers to when the flower offers a reward to solicit pollination. The “small pan” on the lip of some species (Vogel 1990) is termed the glenion in Pleurothallidinae and its presence is probably indicative of reward pollination (Frank 5 2015). Deceit pollination refers to when the lip of the flower appears to offer a reward, but does not. This deception can take the form of either resembling the scent/color scheme of a rewarding flower or resembling the female of the pollinator species. This elicits pseudocopulation by the male of the pollinator species, during which the pollinia are attached. The absence of a glenion, the overall structure of the lip, and the presence of an osmophore may be taken to indicate that a species utilizes pseudocopulation as a pollination mechanism (Frank 2015). Though osmophores are also present on some reward-pollinated flowers, and are therefore not a definitive indicator independent of other characteristics, they are critical to the efficacy of pseudocopulation (Frank 2015). Due to the highly variable lip and column morphology present in species of subgenera Ancipitia and Scopula (Luer 1989), I hypothesize that several pollination strategies will be present within and between each subgenera; there may be a distinction along the taxonomic division in the form of pollination mechanism or morphological feature(s). Phylogenetics Genera within the subtribe Pleurothallidinae have been traditionally circumscribed on the basis of morphological characters and although this classification has been useful, several problems arise at the generic level (Higgins 2009). These problems are particularly apparent in the genus Pleurothallis, the largest genus of the subtribe (Dressler 1993, Luer 2002). Luer (1986) described a Pleurothallis “as any pleurothallid that does not fit into any of the other genera.” As described by Luer (1999), this misfit mega-genus has 6 historically included some 2000 species grouped artificially into 32 subgenera with numerous sections, subsections, and series. Several molecular studies (Pridgeon et al. 2001; Wilson et al. 2011; 2013) have confirmed the occurrence of polyphyly and paraphyly in the genus circumscribed on the basis of morphology, necessitating the re-circumscription of the genus in the light of phylogenies based on molecular data (Wilson et al. 2015). In Genera Orchidacearum Vol. 4, Pridgeon (2005) proposed that a monophyletic genus Pleurothallis would include subgenera Ancipitia, Mirandia, Pleurothallis, Restrepioidia, Rhynchopera, Scopula and Talpinaria. However, Luer suggested elevating the subgenera Ancipitia and Scopula to the generic level as Ancipitia (Luer) and Colombiana for the species of subgenus Scopula (Luer 2004). It is these two subgenera that are of interest in this study. The subgenus Ancipitia was first described in 1986; the species within the subgenus being recognized by “the laterally compressed, two-edged ramicauls” and the “solitary flowers produced from the apex of the ramicaul in a fascicle of usually elongated peduncles,” (Luer 1989). Ancipitia currently consists of 28 species, all originating from Central and South America (Luer 1989). The subgenus Scopula was also first described in 1986; the species within the subgenus being recognized by “the tuft of single-flowered peduncles emerging near the apex of the leaf from the median sulcus,” (Ospina & Dressler 1974). Scopula currently consists of six species, all restricted to South America. Aside from the emergence position of the flowers, plant and flower morphology are very similar between species of Ancipitia and Scopula (Luer 1989). Luer (1989) noted these similarities, but maintained a distinction between the two subgenera. Due to the conflicting philosophies on the generic status of Ancipitia and Scopula, further analysis is needed on the genetic level. Following the studies of 7 Wilson et al. (2011; 2013), analysis using nuclear internal transcribed spacer (ITS) region and the plastid maturase K (matK) gene will provide a more complete phylogenetic history. The internal transcribed spacer region, ITS, is commonly used for phylogenetic analysis due to the highly variable ITS1 and ITS2 sequences, which are separated by conserved coding regions (Salvolainen & Chase 2003). The matK gene is