Plant Biology ISSN 1435-8603 RESEARCH PAPER Pollination ecology of two species of Elleanthus (Orchidaceae): novel mechanisms and underlying adaptations to hummingbird pollination C. E. P. Nunes1, F. W. Amorim2, J. L. S. Mayer3 & M. Sazima3 1 Programa de Pos-Graduac ßao~ em Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Sao~ Paulo, Brazil 2 Departamento de Botanica,^ Instituto de Biociencias,^ Universidade Estadual Paulista “Julio de Mesquita Filho”, Botucatu, Sao~ Paulo, Brazil 3 Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Sao~ Paulo, Brazil Keywords ABSTRACT Epidendroideae; micromorphology; nectar secretion; ornithophily; protandry; Sobraliae; Relationships among floral biology, floral micromorphology and pollinator behaviour Trochilidae. in bird-pollinated orchids are important issues to understand the evolution of the huge flower diversity within Orchidaceae. We aimed to investigate floral mechanisms Correspondence underlying the interaction with pollinators in two hummingbird-pollinated orchids C. E. P. Nunes, Departamento de Biologia occurring in the Atlantic forest. We assessed floral biology, nectar traits, nectary and Vegetal, Instituto de Biologia, Universidade column micromorphologies, breeding systems and pollinators. In both species, nectar Estadual de Campinas, Caixa Postal 6109, CEP is secreted by lip calli through spaces between the medial lamellar surfaces of epider- 13083-970 Campinas, Sao~ Paulo, Brazil. mal cells. Such a form of floral nectar secretion has not been previously described. E-mail: [email protected] Both species present functional protandry and are self-compatible yet pollinator- dependent. Fruit set in hand-pollination experiments was more than twice that under Editor natural conditions, evidencing pollen limitation. The absence of fruit set in interspe- A. Dafni cific crosses suggests the existence of post-pollination barriers between these sympatric co-flowering species. In Elleanthus brasiliensis, fruits resulting from Received: 2 October 2014; Accepted: 5 cross-pollination and natural conditions were heavier than those resulting from self- February 2015 pollination, suggesting advantages to cross-pollination. Hummingbirds pollinated both species, which share at least one pollinator species. Species differences in floral doi:10.1111/plb.12312 morphologies led to distinct pollination mechanisms. In E. brasiliensis, attachment of pollinarium to the hummingbird bill occurs through a lever apparatus formed by an appendage in the column, another novelty to our knowledge of orchid pollination. In E. crinipes, pollinarium attachment occurs by simple contact with the bill during insertion into the flower tube, which fits tightly around it. The novelties described here illustrate the overlooked richness in ecology and morphophysiology in Orchidaceae. 1969), which can be produced in different structures such as INTRODUCTION sepals, lip and column (Stpiczynska et al. 2003; Stpiczynska Most orchid species are insect-pollinated, whereas pollination et al. 2005, 2011). In most cases, the mechanism of pollinaria by vertebrates such as birds is less common (van der Pijl & deposition on birds is simple contact of the viscidium with the Dodson 1969; Schiestl & Schluter€ 2009). Although birds are bird’s bill (Johnson 1996; Singer & Sazima 2000; Liu et al. not among the most frequent pollinators in the Orchidaceae, 2013). However, less common mechanisms are also observed, bird pollination occurs in many species in the tropics, and such as pollinaria deposition on the birds’ feet or head feathers hummingbirds are the main bird pollinator of orchids in the (Johnson & Brown 2004; Micheneau et al. 2006; respectively). Neotropics (van der Pijl & Dodson 1969; Rodrıguez-Robles The genus Elleanthus consists of approximately 111 species et al. 1992; Singer & Sazima 2000; Machado & Carvalho 2006). (Govaerts et al. 2010) distributed throughout tropical America Hummingbirds invaded and diversified in South America and concentrated in the Andean mountain chain (Dressler approximately 22 million years ago (McGuire et al. 2014), driv- 2006). Nine species occur in Brazil (Barros et al. 2010) and ing a ‘pollination revolution’ in the diversification of several three in the Atlantic forest (Stehman et al. 2009). Elleanthus plant taxa, including orchids, by selecting floral traits favour- brasiliensis (Lindl.) Rchb.f. occurs from French Guiana to able to their nectar foraging habits, leading to adaptations in southern Brazil (Govaerts et al. 2010). In the Brazilian Atlantic flower morphology and adaptive radiation of some plant taxa forest, this species occurs at altitudes ranging from sea level to (Whittall & Hodges 2007). 1000 m. Previous studies have provided preliminary informa- Bird-pollinated orchids, as purported for many Elleanthus C. tion on pollination of E. brasiliensis. Singer & Sazima (2000) Presl. species, usually fit the bird pollination syndrome (Faegri and Singer (2003) recorded the hummingbird Phaethornis pre- & van der Pijl 1979). For orchids, these traits include narrow trei visiting the flowers, Wolowski et al. (2013a) recorded Phae- tubes formed by the lip and/or sepals, pinkish, orange or red- thornis squalidus visiting flowers and Wolowski et al. (2013b) dish colours and nectar secretion (van der Pijl & Dodson studied the natural fruit set of this species. Miller et al. (1996) Plant Biology 18 (2016) 15–25 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands 15 Nectar and ornithophily in Elleanthus Nunes, Amorim, Mayer & Sazima observed butterflies and bees as visitors. However, there are no opening, flowers are exposed above it (Figs 1C arrow and 2C). detailed data on the importance of these visitors for pollination Resupination of the flowers in the pendant inflorescence pro- biology of E. brasiliensis. duces the lip on the upper side of the flower. Flowers are 18- Elleanthus crinipes Rchb.f. is endemic to the montane (high- mm long and have reddish short floral bracts, purplish-red land) Atlantic forest (Stehman et al. 2009). It is restricted to sepals, white petals and two purplish-red spots on the lip highland forest areas and occurs primarily at altitudes above (Figs 1C and 3A). 800 m, where it can occur in sympatry with E. brasiliensis.As Elleanthus crinipes has erect stems with spiky, lax and elon- in most Elleanthus species, there are no detailed studies or gated inflorescences without visible mucilage. Flowers are not other sources of information on visitors to or pollinators of resupinate and the lips form tubes parallel to the inflorescence this endemic species. Even though these two co-occurring axis (Figs 1D and 2D–E). Flowers are approximately 12 mm in orchid species are allocated to different sections of Elleanthus length and have light pink floral bracts surpassing the length of genus, they are useful subjects for testing the hypothesis for the the flowers. The sepals, petals, lip and eight pollinia are white existence of pre- and post-zygotic barriers isolating orchid spe- or cream and the anther cap is violet. cies (Sun et al. 2011). This work focuses on the pollination ecology of E. brasilien- Floral biology and nectar sis and E. crinipes, with emphasis on floral biology, breeding system and pollination mechanisms. To better understand the Flowering phenological pattern was classified following New- floral features and pollination mechanisms involved in bird strom et al. (1994). Time, sequence and duration of anthesis pollination in these species, we addressed the following ques- (hereinafter, the period in which the corolla opened and pre- tions: (i) what are the floral and morphological traits that cor- sented male or female functional organs) were assessed in the relate with pollinator morphology; (ii) what are the nectar field. Flower scent production was assessed in situ by smelling traits (sugar composition, secretion pattern) and micromor- the blossoms. Stigma receptivity was also assessed in situ on phology of the nectary; (iii) what is the breeding system, and five open flowers and five flower buds, taken randomly from are the species dependent on pollinators for sexual reproduc- different individuals of the two species, following Dafni et al. tion; (iv) what are the main pollinators and how efficiently do (2005). In order to verify the function as floral resource of the they transfer pollen among plants; (v) is there hybridisation mucilaginous secretions of the inflorescences of E. brasiliensis, between these sympatric congeneric species related to the this secretion was tested for the presence of water-soluble sug- breaking of pre- or post-pollination barriers to interspecific ars in five inflorescences from different individuals using a crossing; and (vi) how are the micromorphology of the column pocket refractometer (N-1a; Atago, Tokyo, Japan). In order to and its appendage involved in the attachment of pollen onto understand the mechanisms of pollinaria deposition, we exam- the pollinator’s body? ined both fresh and fixed flowers and inserted tweezers into the corolla tube, simulating pollinator mouthparts. Total nectar volume was quantified in flowers, previously MATERIAL AND METHODS bagged at the bud stage, using microliter syringes (Hamilton, Reno, NV, USA; E. brasiliensis:n= 72 flowers from seven indi- Study sites and species characterisation viduals; E. crinipes:n= 10 flowers from two individuals). Sugar The study was carried out between 2007 and 2010 at two sites: concentration (%