See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/336776678 FLORAL ANATOMY AND EVOLUTION OF POLLINATION SYNDROMES IN LEPANTHES AND CLOSE RELATIVES Conference Paper · July 2019 CITATIONS READS 0 329 6 authors, including: Diego Bogarín Melania Fernández University of Costa Rica University of Costa Rica 143 PUBLICATIONS   3,714 CITATIONS    35 PUBLICATIONS   138 CITATIONS    SEE PROFILE SEE PROFILE A. P. Karremans Franco Pupulin University of Costa Rica University of Costa Rica 113 PUBLICATIONS   735 CITATIONS    201 PUBLICATIONS   1,707 CITATIONS    SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Systematics of Trichosalpinx View project Pleurothallidinae from Colombia View project All content following this page was uploaded by Diego Bogarín on 24 October 2019. The user has requested enhancement of the downloaded file. CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. FLORAL ANATOMY AND EVOLUTION OF POLLINATION SYNDROMES IN LEPANTHES AND CLOSE RELATIVES Diego Bogarín*1,2,3,7, Melania Fernández1,3, Adam P. Karremans1,2, Franco Pupulin1, Erik Smets2,7,8, and Barbara Gravendeel2,5,7 1Jardín Botánico Lankester, Universidad de Costa Rica, Cartago, Costa Rica; 2Naturalis Biodiversity Center, Endless Forms group, Leiden, The Netherlands; 3Herbarium UCH, Universidad Autónoma de Chiriquí, Chiriquí, Panama; 4Re- search Associate, Royal British Columbia Museum and, American Museum of Natural History; 5University of Applied Sciences Leiden, Science and Technology Faculty, Leiden, The Netherlands; 7Institute Biology Leiden, Leiden University, Leiden, The Netherlands; 8KU Leuven, Ecology, Evolution and Biodiversity Con- servation, Leuven, Belgium *Corresponding author: diego.bogarin@ucr.ac.cr Pleurothallidinae is the largest Neotropical orchid floral convergence in plants pollinated by biting midges subtribe encompassing >5100 species that are mainly and also which other pollination strategies can be in- dipteran-pollinated. Various pollination syndromes, ferred from flower anatomy in the sister genera. Based targeting hump-backed flies (Phoridae), fungal gnats on histochemistry, LM, and SEM we found similar (Sciaridae), and vinegar flies (Drosophilidae) have been floral secretions such as carbohydrates, proteins, and documented in detail for Acianthera, Dracula, Lepanthes, lipids in different organs of Anathallis, Lankesteriana, Octomeria, Pleurothallis, and Specklinia, all belonging to and Trichosalpinx, supporting the hypothesis of floral different clades. Among them, the highly diverse genus parallelism in these genera. Lepanthopsis with a pa- Lepanthes, including about 25% of the species of the pillose lip and secretory glenion and Zootrophion with Pleurothallidinae, is most closely related to Anathallis, closing flowers and verrucose-papillose inner surface of Draconanthes, Frondaria, Lankesteriana, Lepanthopsis, sepals might employ different pollination systems. This Trichosalpinx, and Zootrophion. Members of this “Lepan- study provides additional micromorphological and his- thes clade” display high floral divergence and are likely tochemical data to support future pollination studies of adapted to different pollinators . However, only two pol- other members of the Lepanthes clade. lination studies have been published for the group: one for Lepanthes, pollinated by Bradysia (fungal gnats) and Keywords: Bulbophyllum, histochemistry, glenion, Lep- another for Trichosalpinx, pollinated by Forcipomyia (bit- anthes, micromorphology, myophily, Pleurothallidinae, ing midges). Floral traits present in Trichosalpinx, such as Trichosalpinx purple flowers and a mobile, pubescent lip, evolved to accommodate pollination by biting midges. These traits With more than 5100 species, Pleurothallid- are also found in other plant species pollinated by bit- inae (Epidendreae) is the largest Neotropical ing midges such as Bulbophyllum, Ceropegia spp. (Ascle- orchid subtribe (Pridgeon et al. 2005). The piadaceae), and Theobroma cacao (Malvaceae). Because some members of the Lepanthes clade exhibit similar species diversity of the group is concentrated floral traits, we hypothesize that pollination by biting in few genera. One of these is Lepanthes Sw., midges evolved in these orchids as well. In this study, containing over 1200 species (about 25% of we discuss the micromorphological and histochemical the species of Pleurothallidinae). The genus is features of the flowers among some of the members of phylogenetically most closely related to Ana- the Lepanthes clade in order to test the hypothesis on thallis Barb.Rodr., Draconanthes (Luer) Luer, 389 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. Frondaria Luer, Lankesteriana Karremans, Lep- anthopsis (Cogn.) Ames, Trichosalpinx Luer and Zootrophion Luer, all considered members of the Lepanthes clade (Chase et al. 2015; Kar- remans 2016; Fig. 1). These genera are much less diverse than Lepanthes and account for only 1% of the species of the clade (Bogarín et al. 2016). Studies of the evolution of the Pleu- rothallidinae showed that the most speciose lineages of the subtribe diversified recently (within the last 15 Mya), and the Lepanthes clade underwent the highest rate of species di- versification (Pérez-Escobar et al. 2017). How- ever, the factors that have shaped this incred- ible species diversity in the Pleurothallidinae such as the role of plant-pollinator interac- tions are still largely unknown because of the lack of knowledge of pollination strategies. Shifts in pollination strategies or adaptations to new pollinators exert evolutionary forces that enhance rapid speciation in angiosperms (Johnson 2010; Kay and Schemske 2008; Smith 2010). In other plant groups, however, species radiations without changes in pollina- Fig. 1. Phylogeny summary of the Lepanthes clade showing the tor specialization have been documented (Ol- main lineages/taxa, the number of species in each taxa, and pollinator information. Phylogeny based on our unpublished lerton et al. 2009), and other studies pointed data using nrITS and matK. out that certain pollination systems may in- crease species diversification independently of its different floral traits compared to Lepan- the pollination shift (Valente et al. 2012). thes and consequently a different pollination mechanism. Trichosalpinx targets exclusively fe- In the Lepanthes clade, nothing is known males of the genus Forcipomyia (Diptera, Cera- about the pollination strategies of the mem- topogonidae). Flowers attract the insects with bers of the clade besides the pollination stud- the motile, pubescent, papillose surface of the ies of a few species of Lepanthes and Trichosal- lip blade, which secretes proteins and carbo- pinx, and this hampers the understanding of hydrates. The presence of females, the abso- evolutionary relationships within this group lute absence of males, and secretion of protein and the associated pollination shifts. Lepan- rewards indicate that Trichosalpinx imitates a thes flowers, which are mostly characterized model aimed at stimulating the protein collec- by a bilaminate lip with a central appendix, tion behavior of females for egg production exhibit a highly specialized pollination system through a complex deceptive system likely re- involving sexual deception. Flowers are specif- lated to kleptomyophyly (Bogarín et al., i2018). ically pollinated by male fungus gnats of the genus Bradysia (Diptera, Sciaridae) probably Some members of the clade exhibit similar attracted by a pheromone-mimicking strate- floral traits, suggesting they may share simi- gy (Blanco and Barboza 2005). On the other lar pollination syndromes. For example, pur- hand, the closely related Trichosalpinx s.s. (fur- ple flowers with motile lips attached to the ther referred to simply as Trichosalpinx), exhib- column foot by a thin ligament, as found in 390 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. Lepanthopsis flowers exhibit reduced petals, a glenion at the base of the lip, and a short col- umn with bilobed stigma, much more similar to what is found in many Pleurothallis R.Br. and Stelis Sw. species rather than any of its closest relatives. In Zootrophion, the flowers are fleshy, with the sepals fused at the base and apex and leaving only a window-like aperture at each side of the flower, features that are unique among its close relatives but found as well in a few unrelated genera, including Aci- anthera Scheidw., Phloeophila Hoehne & Schl- tr., Specklinia Lindl., and Stelis. In Trichosalpinx subgenus Tubella (further referred to simply as Tubella Archila), flowers are mostly white with an entire lip blade, which contrast with the purple pubescent lips of Trichosalpinx (Luer 1997, 2004, 2006). To make inferences about the pollination strategies of the remaining groups of the Lep- anthes clade and test the hypothesis of floral Fig. 2. Flower morphology of some representatives of Anath- allis, Lankesteriana, Trichosalpinx, and Tubella. A-C: Anathallis parallelism/divergence, we investigated the lewisiae. A. Flower; B. Papillose surface of the lip; C. Detec- anatomy, micromorphology, and possible re- tion of proteins with CBB in the epidermis of the lip; D-F: wards of flowers of some members of the clade Trichosalpinx reflexa. D. Flower; E. Papillose surface of the lip; F. Detection of proteins with CBB in the epidermis of the lip; by combining histochemistry, light microsco- G-I: Lankesteriana fractiflexa. G. Flower; H. Papillose surface py (LM), and scanning electron microscopy of the lip; I. Detection of proteins with CBB in the epidermis (SEM) techniques. In this study, we discuss of the lip; J-O: Tubella arbuscula. J. Flower; K. Papillose surface the anatomical features of the flowers among of the lip; L. Papillose apex of the lip with the median groove; M. Apex of the lip with positive detection of carbohydrates some of the members of the Lepanthes clade in (PAS); N. Papillose side of the lip with positive detection of order to assess 1) the characters shared among carbohydrates (PAS); O. Apex of the lip with negative detec- Anathallis, Lankesteriana, and Trichosalpinx that tion of proteins (CBB). (Photos: D. Bogarín) indicate adaptation to pollination by biting Trichosalpinx, are present in both Anathallis midges and thus parallelism, 2) pollination and Lankesteriana. These traits most likely syndromes in the Lepanthes clade that can be evolved to accommodate pollination by biting inferred from floral anatomy, and 3) micro- midges because these features are also pres- morphological characters that are useful in ent in Trichosalpinx and other angiosperms distinguishing the different groups within the pollinated by similar strategies such as Bulbo- Lepanthes clade. phyllum Thouars, Ceropegia L. spp. (Asclepia- daceae) and Theobroma cacao L. (Malvaceae) Material and methods (Bartareau 1994; O’Doherty and Zoll 2012; Ollerton et al. 2009; Bogarín et al. 2018). Study site and sample collection However, other genera of the Lepanthes clade Plant samples were collected in the wild and display floral traits that suggest pollination cultivated in the greenhouses of the Lankester through different syndromes. For example, Botanical Garden (JBL) of the University of 391 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. Costa Rica (Cartago, Costa Rica) and the Hor- tus botanicus of Leiden University (Leiden, The Netherlands). We studied species of Ana- thallis, Lankesteriana, Lepanthes, Lepanthopsis, Trichosalpinx subgen. Trichosalpinx, Tubella, and Zootrophion. Due to unavailability of material, we did not include specimens of Draconanthes, Frondaria, Trichosalpinx subgenus Pseudolepan- thes Luer, and T. subgenus Xenia Luer. Vouch- ers of plant specimens were deposited at CR, JBL (spirit), L, and USJ. Digital imaging and microscopy Photographs were taken with a Nikon D7100 and AF-S VR Micro-Nikkor 105mm f/2.8G IF-ED lens and PB-6 bellows with a Nikon AF Nikkor 50mm f/1.8D lens and Broncolor® Siros 800 S flashes. Stacking was performed with Zeiss Stereo Discovery V20 and Axio- Cam MRc 5 Zeiss camera. Digital images of light microscopy were taken with a Zeiss® AXIO Imager.M2 motorized microscope with an AxioCam MRc 5 Zeiss camera. Final digital Fig. 3. SEM micrographs of the epidermis of the lip of: A. images and composite figures were processed Anathallis lewisiae; B. Lankesteriana barbulata; C. Trichosalpinx in Adobe Photoshop CS6®. chamaelepanthes; D. T. pergrata; E. T. reflexa; F. T. ringens; G. T. cf. patula; H. Tubella dura. (Photos: D. Bogarín) Microscopy fixation 70% ethanol for several days were also used for staining. Heavily pigmented tissue areas Samples were stored in FAA (ethanol 50%, of fresh flowers were cleared for 10-60 min acetic acid, and formalin at 18:1:1 v/v) or in 10% (v/v) commercial solution of sodium 70% ethanol for at least a week or more. For hypochlorite and rinsed in 30% ethanol for Epon and LR White embedding, dissected 1 hr before staining to avoid the interference fresh flowers were fixed for 3 hr in a modified of tissue coloration in staining results (Ruzin Karnovsky fixative (2.5% glutaraldehyde, 2% 1999). Neutral or acidic lipids, phospholipids, formaldehyde, pH 7.2) and rinsed three times and fatty acids were detected with a solution in 0.1 M sodium cacodylate buffer (pH 7.4) of Nile blue A 1% (NBA) (w/v, demi water) at prior to embedding. Staining was performed 37 C for 1 min and differentiated in 1% acetic for 2 hr in 2% osmium tetroxide and rinsed acid for 30 sec at 37 C and rinsed in demi wa- in 0.1 M sodium cacodylate buffer (pH 7.4). ter (Ruzin 1999). Sudan IV 0.5% (SIV) (w/v, 70% ethanol) and Sudan Black B (SBB) 0.07% Light microscopy (LM) and histochemistry (w/v, ethanol 70%) were used to detect lipids (fats, oils, and waxes) (Bronner 1975; Ruzin Entire fresh flowers and hand-cut sections 1999). Insoluble polysaccharides and starch of flowers were stained to detect lipids, poly- were detected with a periodic acid–Schiff saccharides, and proteins. Flowers fixed in reaction (PAS) by oxidizing the samples in aqueous solution of periodic acid (HIO4) 5% 392 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. Scanning electron microscopy (SEM) Fixed flowers were dehydrated for 20 min in a series of ethanol solutions (70%–96%– ≥99.9%) and twice in fresh ≥99.8% acetone. Critical-point-drying using ≥99.8% acetone and liquid CO2 as exchange fluids was per- formed in Automated Critical Point Dryer Leica EM CPD300 (Leica Microsystems, Wet- zlar, Germany). The drying protocol included a cooling step at 15 C, 50% stirrer speed with auto version, slow CO2 influx in the pressure chamber, with a delay of 120 sec after influx CO2 and before starting the exchange process, 18 exchange cycles (CO2: 99.8% acetone) at a speed of 5, a fast heating speed, and medi- um gas-out speed. Dried samples were mount- ed in stubs with adhesive carbon conductive tabs and sputter-coated with 20 nm of Pt/Pd in a Quorum Q150TS sputter-coater. Result- ing samples were observed with a JEOL JSM- 7600F field emission scanning electron micro- scope at an accelerating voltage of 10 kV. Fig. 4. SEM micrographs of the epicuticular secretions in the epidermis of the lip of: A. Anathallis funerea; B. Lankesteriana barbulata; C. Lepanthes chameleon; D. Trichosalpinx pergrata; E. Results T. reflexa; F. T. ringens; G. Tubella dura; H. Z. endresianum. (Pho- tos: D. Bogarín) Anathallis (m/v) for 10 min, rinsing 3 times in distilled water for 2 min and submerging for 15 min in As in Trichosalpinx and Lankesteriana, the lip Schiff’s reagent, and finally submerging in tap of the members of this genus is motile be- water at 50-60 C for 5 min (Ruzin 1999). Mu- cause it is hinged to the column foot by a cilage-secreting areas with acidic compounds, thin membranous ligament. The papillose pectic acids or hexuronic acids were detected lip shows slightly striate cuticles and various with a solution of ruthenium red 0.05% (RR) secretions such as lipids (SIV) and insoluble (w/v, tap water) for 15-20 min (Southworth polysaccharides (PAS) but most notably pro- 1973). Proteins were detected with Aniline teins (ABB, CBB) on the apex of the papillae Blue-Black (ABB) 1% in 7% acetic acid for 10 and occasionally prismatic crystals of calcium min at 50-60 C (Jensen, 1962; Fisher, 1968) oxalate, indicating a secretory function simi- and Coomassie Brilliant Blue R-250 (CBB) in lar to Trichosalpinx (Fig. 1A-C, 3A, 4A). Dif- a solution of 0.25% CBB, 50% ethanol, and ferences with Trichosalpinx and Lankesteriana 7% acetic acid for 3 min and rinsed in tap include the absence of elongated cells toward water (Jensen 1962; Fisher 1968). Areas of fra- the margins and the raised callus at the base grance emission were detected by submerging of the lip. In A. lewisiae (Ames) Solano & Soto the samples in a solution of neutral red 0.1% Arenas, the petals are also papillose, with stri- (NR) (w/v, tap water) for 15-20 min and differ- ate cuticles and secretions at the apices unlike entiated with tap water (Ruzin 1999). the petals of Trichosalpinx (Fig. 5E). Flowers of 393 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. some Anathallis species have purple colors, but 4D, E, F). Tests with (NR) were positive on other species have white or yellowish flowers. the apices of the papillae. In particular, the lip of T. ringens Luer has elongated hairs with Lankesteriana some striations in the cuticle unlike the short papillae of most Trichosalpinx species (Fig. 3F). Species belonging to the genus also show Crystals of calcium oxalate were exuded on similarities with the lip of Trichosalpinx and the apices of the papillae but were not present Anathallis species, including the papillose in all the specimens analyzed (Fig. 4E). The surface of the lip with striate cuticle and se- petals are oriented parallel to the column, cretory activity (Fig. 1G-I, 3B, 4B). We de- and they do not have papillose surfaces (Fig. tected lipids (SIV), insoluble polysaccharides 5E); no secretory activities were detected be- (PAS), and again proteins on top of the papil- cause histochemical tests yielded no positive lae (ABB, CBB; Fig. 1I). Also, the lip shows results. The column is arcuate with a rounded elongated cells with widened apices scattered foot where the lip is attached. At the base of towards the lip apex unlike the elongated the column foot, there are similar papillae to cells of Trichosalpinx that are restricted to the those observed on the sepals and lip. margins and not widened at the apex (Fig. 3B). The combination of brown-purple with Lepanthes white in the flowers of some Lankesteriana is similar to the flowers of Trichosalpinx (Fig. 1D, Flowers of Lepanthes are characterized in gen- G). The lip of Lankesteriana is grooved unlike eral by a bilobed lip with an appendix at the that of Trichosalpinx and Anathallis. Like Anath- base and an elongated column with apical an- allis, the apices of the petals are papillose and ther and stigma (Fig. 6A, B, F-H). Our stud- secretory with striate cuticles but sometimes ies indicate a generalized pattern in the epi- with elongated cells (Fig. 5A-B). Some Lank- dermal surface of the sepals and petals. The esteriana species have elongated, pubescent sepals have flattened, smooth cells and, in or papillose petals, ending in widened apices contrast, the petals and lip are always papillose such as L. fractiflexa (Ames & C.Schweinf.) (Fig. 5C, 6B, C). The sepals did not react to Karremans. Other species such as L. barbulata histochemical stains, but the petals reacted to (Lindl.) Karremans have Trichosalpinx-like pet- all the stains applied for carbohydrates (PAS, als without hairs but with irregular margins. RR), lipids (SBB, SIV), proteins (ABB, CBB), and scents (NR; Fig. 6D, E, I). The appendix Trichosalpinx subgen. Trichosalpinx of the lip is pubescent, hirsute or with a com- bination of elongated and flattened cells (Fig. In Trichosalpinx species [including the relatives 6F-H). Prismatic crystals and compounds on of T. berlineri (Luer) Luer], the base of the lip is the surface of the cells were observed (Fig. attached to the column foot by a membranous 4C). ligament. The lip blade is papillose with elon- gated cells appearing towards the margins. Lepanthopsis The papillae of the lip blade exhibit a notice- able striate cuticle, and toward the apices the This genus is easily recognized by the flattened cuticle is smooth and secretory. We detected flowers with reduced petals and the presence lipophilic compounds (SBB), polysaccharides of a glenion at the base of the papillose lip, (PAS), and proteins (CBB and ABB) within a feature not found in any of the sister gen- and on the apex of the papillae, indicating era (Fig. 7). In L. floripecten (Rchb.f.) Ames, a secretory function (Fig. 1D-F, 3C, E, F, G, the glenion is made up of an aggregation of 394 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. salpinx, and Lankesteriana but with a median groove of flattened cells and without hairs along the margins (Fig. 2K-M). Some areas of the lip at the sides and toward the apex con- tain insoluble polysaccharides (PAS), but the blade does not react for proteins (ABB, CBB) and lipids (SIV; Fig. M-O). Some species such as T. arbuscula (Lindl.) Luer are fragrant. The petals are flattened, not pubescent and with- out papillae. The sepals are elongated and also entire. Some species classified have smooth papillose surfaces on the lip, such as T. cha- maelepanthes (Rchb.f.) Luer and T. pergrata (Ames) Luer (Fig. 3C, D). Also, the apex of the petals of T. pergrata are papillose in contrast of those of T. arbuscula and T. dura (Lindl.) Luer (Fig. 5E). Fig. 5. SEM micrographs of the epidermis of the petals of: A. Anathallis lewisiae; B. Lankesteriana fractiflexa; C. Lepanthes Zootrophion chameleon; D. Lepanthopsis prolifera; E. T. reflexa; F. T. pergrata. (Photos: D. Bogarín) papillose and secretory cells just in front of Flowers of this genus are unique within the the reduced column. Flowers of some species group. The basally and apically fused sepals resemble those of Platystele Schltr. (Fig. 7B-C, form a closed flower with only two open sides, 8A-D). In L. astrophora, the glenion is also pa- resembling windows. The color of the flower pillose but sunken (Fig. 8B). The papillose lip varies from white and yellow to pinkish and reacts for proteins on top of the papillae over purple, sometimes with spotted sepals and the lip (ABB, CBB) but most notably in the petals (Fig. 9A). The inner surface of the syn- glenion (Fig. 7D). SEM images show several sepal is made up of papillose or rugose surfac- compounds in the surface of the glenion that es that react to carbohydrates (PAS) and lipids also react positively for lipids (SIV) and insol- (SB). In Z. vulturiceps (Luer) Luer, a species uble polysaccharides (PAS; Fig. 7 D, E). Petals with white flowers, we did not detect proteins and sepals have flattened cells and do not re- (CBB) on the rugose surface (Fig. 9B-F). How- act for proteins and carbohydrates but proba- ever, the papillose area of the synsepal of the bly contain epicuticular waxes (SIV). Howev- purple-spotted Zootrophion sp. shows a positive er, other species such as L. prolifera Garay have reaction for proteins (CBB). The dorsal sepal epidermal cells with characteristic projections is smooth and does not react with the stains. (Fig. 5D). We observed prismatic crystals on The lip is reduced and motile and is attached the surface of the cells but mostly concentrat- to the column foot by a membranose tissue, ed on the glenion (Fig. 7G). securing mobility. The surface is papillose and rugose with various evident secretions on the Tubella epidermal cells (Fig. 4H, 9G-H). The column is footed and elongated as in Anathallis, Tricho- Flowers of this group are generally white-green- salpinx, and Lankesteriana. The petals are par- ish to yellowish (Fig. 1, 2J). The lip is papillose allel to the column and enclose the column with striate cuticles as in Anathallis, Tricho- and lip. 395 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. of the lip and petals, whereas the whitish or Discussion translucent areas have flattened and smooth epidermal cells. Floral micromorphology and histochemistry The papillose epidermal areas of flowers The epidermal secretory papillae of the lip of might increase the area of emission of scents Anathallis, Lankesteriana, and Trichosalpinx is or “emission layer” (Vogel 1990). In Lepanthes, also present in other myophilous species of the active parts of the flower in terms of com- Bulbophyllum and fly-pollinated Pleurothallid- pound synthesis are the papillose epidermis inae (De Pádua Teixeira at al. 2004; Nunes at of petals and scattered colleters of sepals. The al. 2014; Nunes et al. 2015; Pridgeon and Stern role of this tissue in the production of phero- 1985). Secretion of proteins on the lip occurs mone-like odors that attract male fungus gnats in species of Bulbophyllum sect. Racemosae as pollinators has to be tested experimentally, Benth. & Hook.f., possibly as floral rewards but our histochemical evidence indicates that for female flies (Davies and Stpiczyńska 2014). the papillose petals are involved in scent syn- In addition, flowers of B. wendlandianum con- thesis. In addition, this papillose epidermis is tained protein secretions in the epithelium mostly not striate and does not concentrate (Kowalkowska et al. 2014), as observed previ- the secretions on the apex of the papillae as ously in Trichosalpinx and the species of Ana- observed in Anathallis, Lankesteriana, and thallis and Lankesteriana studied here. Positive Trichosalpinx, indicating that Lepanthes flow- reaction with NR and lipophilic compounds ers do not produce collectable rewards. This also indicate scent synthesis in the papillose is consistent with the hypothesis of sexual epidermis. Vogel (1990) documented fra- deception and behavior of pollinators in the grance emission in the epithelium of the distal flowers that do not search for rewards. On the lobed ends of the corolla of Ceropegia, which other hand, papillose or verrucose areas of the is mostly pollinated by biting midges. These synsepal of Zootrophion are secretory, and the structures are purple, papillose or hairy, like epicuticular compounds on the cells of the the lip of some species of Anathallis, Lankesteri- epidermis of the lip observed with SEM may ana, and Trichosalpinx. The striate cuticle of the indicate that Zootrophion flowers offer rewards. papillae is another feature shared by species of these genera and also with some myophilous The parallel position of the petals with respect Bulbophyllum species (Davies and Stpiczy´ska to the column and the lack of rewards in the 2014; Kowalkowska et al. 2014; Nunes et al. species with smooth epidermis indicate that 2014, 2015; Stpiczyńska et al. 2015). the function of the petals is to keep the in- sects directed toward the base of the lip, pre- Striate cuticles have been associated with light venting them from exiting at the sides. This is diffraction, producing more intense "struc- probably true for Trichosalpinx and some Lank- tural colors" acting as a visual effect on pol- esteriana and Zootrophion. However, in Anath- linators (Antoniou Kourounioti et al. 2012). allis and other species of Lankesteriana, some Nunes et al. (2015) postulated that the striate areas of the petals are papillose and secretory, cuticular patterns in Bulbophyllum sect. Na- probably acting as visual/olfactory attractants pellii Rchb.f. are related to these visual cues. for pollinators as in some Bulbophyllum species Our findings on Anathallis and Lankesteriana (Pridgeon and Stern 1983; Vogel 1990; Kowal- support previous observations in Trichosalpinx kowska et al. 2014; Nunes et al. 2014). and Bulbophyllum in which the striate cuticles are present only in the purple-colored areas The detection of proteins and carbohydrates 396 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. tened cells (sometimes sunken) of secretory activity. The anatomy of the glenion varies across these genera, and more ultrastructur- al and histochemical comparative studies are needed to characterize the micromorphology and its role in pollination (Bogarín et al., un- publ.). Crystals occur in the sepals, petals, and lip of many Pleurothallidinae (pers. obs.). The func- tion of these non-protoplasmic inclusions is not entirely clear, and little is known about their role (if any) in pollination. Chase and Peacor (1987) proposed that the refractile properties of crystals in Stelis might mim- ic nectar droplets (or pseudonectar), which act as visual attractants that lure pollinators. Nunes et al. (2015) attributed to them a pos- sible function as a visual signal, enhancing the reflection of light emitted in conjunction with the vacuoles containing pigments. Other Fig. 6. Flower anatomy and histochemistry of: A-H: Lepanthes studies suggest that they may be involved in horichii. A. Flower; B. SEM of the lip and petals; C. Papillose regulation of high levels of calcium ions and surface of petals; D. LM of cells of the epidermis of the petal stained with SBB showing lipid droplets (black); E. LM of the calcium oxalate that eventually precipitate in epidermal cells the petal stained with PAS (carbohydrates); F. epicuticular crystals (Franceschi and Horner SEM micrographs of the hairy appendix of the lip; G. Detail 1980). of the appendix; H. Detail of the elongated cells (hairs) of the appendix; I. Histochemistry of Lepanthes bradei showing the positive reaction with stains, from left to right: flower with Pollination syndromes in the Lepanthes clade natural pigmentation, CBB, ABB, NR (Photos: D. Bogarín and M.M. Chabert) The recent discovery of the pollination of on the apex of the papillae of the lip and Trichosalpinx by biting midges allows us to particularly in the glenion of Lepanthopsis make inferences about the pollination systems floripecten indicates that pollinators are guid- of other members of the Lepanthes clade (Bog- ed toward this point. The glenion has been arín et al. in press). Trichosalpinx, Lankesteriana, defined as a circular structure or callus at the and the T. berlineri group have a close affinity base of the labellum, placed right in front of according to the latest phylogenetic analyses the reduced column; it occurs in several unre- of the Pleurothallidinae and unpublished data lated genera of Pleurothallidinae, namely Bra- (Karremans 2016; Karremans 2014; Pérez-Es- chionidium Lindl., Lepanthopsis, Platystele, Pleu- cobar et al. 2017; Rykaczewski et al. 2017; Boga- rothallis R.Br.. Stelis Sw., and Teagueia (Luer) rín et al., unpubl.; Fig. 1). Species of Lankesteri- Luer (Pridgeon et al. 2005; Fig. 8). The func- ana and the T. berlineri group have a mobile, tion of the glenion in the pollination of spe- pubescent lip that is almost indistinguishable cies of these genera is discussed in further de- from that of Trichosalpinx and some Bulbophyl- tail by Karremans and Díaz elsewhere in these lum (Bartareau 1994; Luer 2006). Although no Proceedings. Initial evidence indicates that this data on pollination of Lankesteriana and T. ber- structure is an aggregation of papillose or flat- lineri group are available, our findings indicate 397 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. pollination by biting midges. The papillose epidermis with a striate cuticle and secretion of proteins are consistent with the anatomical features found previously in the Trichosalpinx species pollinated by females of Forcipomyia that search for proteins (Bogarín et al., 2018). In addition, this hypothesis is strengthened by floral traits present in other angiosperm groups pollinated by biting midges such some Aristolochia L. and Pararistolochia Hutch. & Dalziel in Aristolochiaceae, Caralluma R.Br., Ceropegia L. in Apocynaceae, and Abroma Jacq., Herrania Goudot, and Theobroma L. in Malvaceae (Davies and Stpiczyńska 2014; Kow- alkowska et al. 2014; Nunes et al. 2014, 2015; Stpiczyńska et al. 2015; Vogel 1990). In Or- Fig. 7. Flower anatomy and histochemistry of: A-I: Lepan- chidaceae, the flowers of the distantly related thopsis floripecten. A. Flowers; B. SEM of the petals, lip, and column; C. Papillose surface of the glenion; D. LM of cells Australian Bulbophyllum macphersonii Rupp., a of the glenion stained with SIV showing lipids surrounding species pollinated by biting midges, are simi- the cell walls of the papillae; E. LM of cells of the glenion lar to those of Trichosalpinx, Lankesteriana, and stained with PAS detecting carbohydrates; F. LM of cells of the glenion stained with CBB detecting proteins; G. Detail of some species of Bulbophyllum section Hybochi- the papillae of the glenion with some epicuticular secretions; lus Schltr., B. sect. Oxysepalum Schltr., and B. H. Detail of the smooth surface of the petals; I. LM of the sect. Polyblepharon Schltr. (Bartareau 1994). papillose lip stained with CBB detecting proteins (Photos: D. Bogarín and M. M. Chabert) Common features among these species are again the pubescent, purple, mobile lip with striate papillae and secretion of proteins in the two basal auricles and the purple sepals and epidermis of the lip represent another case of petals. Trichosalpinx and B. macphersonii are ex- evolutionary parallelism to attract the same amples of evolutionary convergence toward a type of pollinator guild as Lankesteriana and common mechanism of pollination, and this some Bulbophyllum and Trichosalpinx species. is likely occurring in Lankesteriana and the spe- cies of the T. berlineri group as well. Floral morphology of Trichosalpinx pergrata (Ames) Luer, T. ringens Luer, and T. sanctuarii Similarity in floral traits are also present Mel.Fernández & Bogarín is different from in some Anathallis species, such as A. lew- the species of Trichosalpinx s.s. For example, isiae (Fig. 24A), A. microgemma (Schltr. ex the absence of a trembling lip with a flexible Hoehne) Pridgeon & M.W. Chase, and A. membrane and the Acianthera-like flowers of nanifolia (Foldats) Luer as noted by Luer T. ringens and T. sanctuarii indicate that these (1997). Anathallis appears to be related to species may be pollinated by different pollina- species of Tubella in the phylogenetic anal- tor groups. The red flowers of T. pergrata with ysis and are not embedded within the Tricho- papillose apices of petals and two nectary-like salpinx clade in the strict sense (Fig. 1). How- structures in the column also indicate an- ever, some species have purple flowers and a other, yet unknown, pollination mechanism mobile lip (though not pubescent) hinged by a (Luer 1997; Fernández and Bogarín 2011). membrane at the bottom of the column foot. Pollinators of Anathallis are not yet known, The species of Tubella are not strictly embed- but we hypothesize that some species showing ded within the Trichosalpinx clade (Pridgeon 398 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. with white flowers and a brownish lip. As already noted by Luer (1997), white flowers with caudate petals are present in species of Specklinia subgen. Hymenodanthae, such as S. calyptrostele (Schltr.) Pridgeon & M.W.Chase, which resemble flowers of Tubella. Karremans et al. (2016) recorded pollination of S. calyp- strotele by a Ceratopogonidae species, possibly Atrichopogon. Therefore, the pollinators of Tu- bella may be biting midges as well, but the op- erating mechanism is probably different and similar to the anthophilous nectar-seeking flies pollinating the white flowers of Hevea or M. indica. Although more pollination obser- vations and anatomical and histological stud- ies are needed in this genus, the presence of carbohydrates found in the papillae of the lip instead of proteins supports these hypotheses. Of the other genera belonging to the Lepan- Fig. 8. SEM micrographs of the column and glenion at the base of the lip of: A. Lepanthopsis astrophora; B. Detail of the thes clade, there is no information available glenion of Lepanthopsis astrophora; C. Platystele sp.; D. Detail on pollinators yet. The floral morphology of of the glenion of Platystele sp.; E. Stelis sp.; F. Detail of the these groups is unlike those already studied, glenion of Stelis sp. (Photos: D. Bogarín) indicating that at least two additional mecha- et al. 2001; Karremans 2016; Rykaczewski et nisms may be in place. Lepanthopsis is unique al. 2017). Species of Tubella have white or yel- among its close relatives in having a papillose lowish flowers, and the lip is not pubescent. secretory glenion of the lip. The sepals and pet- Besides the preference of biting midges for als are generally flat, and the sepals are caudate. flowers with purple and hirsute structures, The column is short, broad, and footless with they have also been documented to visit plants an apical anther and bilobed stigma (except for such as Hevea brasiliensis (Willd. ex A.Juss.) a few species). This type of column indicates Müll.Arg. (Euphorbiaceae) and Mangifera in- that the pollinarium is positioned either on the dica L. (Anacardiaceae) with white flowers head, antenna or legs but most likely not on (Borkent and Spinelli 2007). Males and fe- the dorsal part of the thorax or abdomen of males seek nectar in these small white flowers the pollinator. Undoubtedly, this represents an- to meet their energy needs. Art Borkent (pers. other pollination mechanism yet unknown but comm.) observed biting midges of the genera different from the currently documented cases Atrichopogon Kieffer and Dasyhelea Kieffer, a in Lepanthes and Trichosalpinx. Unrelated gen- group with reduced mouthparts and without era such as Brachionidium, Platystele, Pleurothallis, blood sucking behavior, visiting flowers of Stelis, and Teagueia exhibit similar floral traits, Epidendrum piliferum Rchb.f. in Monteverde, specifically flat flowers with a short column Costa Rica, an orchid with white flowers and with a bilobed stigma and glenion at the base purple nectar guides on the blade of the lip. of the lip (Luer 1990). Some of these groups Pedersen (1995) recorded biting midges of are pollinated by Mycetophilidae and Sciari- the genus Forcipomyia as a visitor to Dendroch- dae (Duque-Buitrago et al. 2014), and these ilum longibracteatum Pfitzer, an orchid species families may be involved in the pollination 399 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. of Lepanthopsis as well, as further discussed by Karremans and Díaz elsewhere in these Pro- ceedings. Some species of the aberrant group T. subgenus Xenia such as Trichosalpinx ballatrix Luer & Esco- bar, T. escobarii Luer, and T. tenuiflora (Schltr.) Luer are florally similar to some Teagueia (such as T. bar- beliana L.Jost & A.Shepard and T. puroana L.Jost & A.Shepard). Unfortunately, we do not have any anatomical, phylogenetic or pollination data avail- able for these groups yet (Luer 1997). Because of the unique morphology of the flowers of Zootrophion, there is no doubt that a different pollinating mechanism operates in this genus. Flowers probably attract pollinators that enter through one of the so-called lateral “windows” in the synsepal. In other Pleurothallidinae such as Dracula, Masdevallia, and Specklinia, the papil- lose, warty sepals attract pollinators, which initial- Fig. 9. Flower anatomy and histochemistry of Zootrophion. ly land on these surfaces and spend most of the A. Flower of Zootrophion vulturiceps (upper), Zootrophion sp. time collecting floral rewards (Endara et al. 2010; (lower); B. Inner view of the flower of Zootrophion sp. showing the papillose surface of sepals and the shorter column, lip, Karremans et al. 2015). Later they are guided to and petals; C. Inner view of the flower of Z. vulturiceps show- the entrance of the tiny lip, initiating pollination. ing the rugose surface of sepals and the shorter column, lip, The combination of a footed column and motile and petals; D-F: Histochemistry of cells of the epidermis of the synsepal of Z. vulturiceps. D. Staining with PAS (carbohy- lip that acts as a hinge in Anathallis, Lankesteriana, drates); E. Staining with SBB showing lipids (black); F. Stain- Tubella, and Zootrophion is similar to some Bulbo- ing with CBB showing proteins (blue). G-I: SEM micrographs phyllum species (Bartareau 1994; Borba and Semir of the rugose surface of the lip of Z. endresianum. G. Epicutic- ular compounds on the epidermis; H. View of the papillose 1998; Humeau et al. 2011). The mobility of the mid-part of the lip; I. Rugose surface of the lip. lip is crucial in the pollination mechanism, in which the insect normally walks toward the base the evolution toward a pseudocopulation sys- of the lip, where its weight activates a lever move- tem involving a diverse group of Diptera under- ment. Consequently, the lip pushes the body of pins the astonishing diversification of Lepanthes the insect onto the column, thereby sticking the (Valente et al. 2012; Bogarín et al. 2016). pollinarium to the scutellum. This has been ob- served for Trichosalpinx and likely also occurs in Conclusions Anathallis, Lankesteriana, Tubella, and Zootrophion (Bogarín et al. 2018) In addition to macromorphological similari- ties of the flowers of Lankesteriana and Tricho- In Lepanthes the combination of apical anthers salpinx and some Anathallis, the species of and sticky viscidium are morphological traits these genera share micromorphological and linked to pollination by pseudocopulation in histological characters that support a hy- which the insect visits the flowers to mate with pothesis of pollination by biting midges and them but not to collect compounds. It is still thus parallelism. One of the most important unclear whether the pollination shift per se or shared characters is the secretion of proteins 400 CONFERENCE PAPERS / SYSTEMATICS / BOGARÍN ET AL. in the papillae of the lip and the striate cu- Acknowledgments ticle of their epidermis. Species of Trichosal- pinx employ this strategy to attract females of We acknowledge the Ministerio de Ambi- Forcipomyia for pollination, and this might ente y Energía (MINAE) and Sistema Nacio- occur in Lankesteriana and some Anathallis as nal de Áreas de Conservación (SINAC) of well. Costa Rica for issuing the scientific permits: 026-2011-SINAC, 073-2012-SINAC, R-SINAC- Two different families of Diptera -- Sciaridae DE-077, SINAC-SE-GASP-PI-R-019-2015 un- and Ceratopogonidae -- carry out the polli- der which plant and insect material was col- nation of Lepanthes and Trichosalpinx, respec- lected. Specimens were exchanged by CITES tively. It is likely that other members of the Institution Numbers CR001 (JBL) and group are pollinated by Diptera. In Tubella, NL008 (L). The Comisión Institucional de Lepanthopsis, Zootrophion, and possibly other Biodiversidad of UCR granted the permit genera, the pollination systems are probably for the access to the genetic and biochemi- different from those already known. Apart cal resources under resolution No. 56. Spe- from the pollination system, in Anathallis, cial thanks are extended to Marie Madeleine Frondaria, Lankesteriana, Tubella, Trichosal- Chabert for helping with documentation pinx s.l., and Zootrophion, the pollinarium is and DNA barcoding and microscopy. Ber- deposited on the thorax of the pollinator tie-Joan van Heuven, Rob Langelaan, Marcel because the columns are long and arcuate Eurlings (Naturalis Biodiversity Center) and with incumbent anthers and pollinaria with Jaime Aguilar, Mario Blanco, Miguel Bena- sticky caudicles. In contrast, in Lepanthopsis vides, Maricruz Bonilla, Marco Cedeño, Isler the pollinarium is likely not deposited on the Chinchilla, Melissa Díaz; and Jorge Warner thorax of the pollinator because the column (Lankester Botanical Garden) for helping is short and bilobed, and the flower there- with documentation, cultivation, and mate- fore does not allow for an entrance and exit rial shipments. We thank Gerson Villalobos as described for the genera mentioned above. for providing information and material. Jaco Therefore, Lepanthopsis might employ a sim- Kruizinga and Rogier van Vugt of Hortus ilar pollination strategy as Platystele, Stelis or botanicus Leiden, Jorge Brenes, and Giovan- Pleurothallis. ni Meza of JBL helped with the cultivation of plants. This research is part of the Ph.D Among the most important micromorpho- project of the first author enabled by Leiden logical characters to characterize the groups University and NBC, The Netherlands, the in the Lepanthes clade are the location of pa- Office of International Affairs and External pillose tissues, the striations of the cuticle of Cooperation, UCR and the research proj- the lip, and the secretion of proteins or car- ect: 814-B6-140 supported by Vicerrectoría bohydrates at the apex of the papillae. The de Investigación, UCR. The Alberta Men- presence of a papillose, secretory glenion is nega Stichting financed the presentation of unique in Lepanthopsis; this feature does not this research by the first author during IOCC occur in other members of the clade. 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