Received: 19 September 2018 Revised and accepted: 11 December 2018 DOI: 10.1111/1442-1984.12231 ORIGINAL ARTICLE Specialized pollination by honeybees in Cymbidium dayanum, a fall–winter flowering orchid Yoshiyuki Matsuda | Naoto Sugiura Department of Biological Sciences, Graduate School of Science and Technology, Abstract Kumamoto University, Kumamoto, Japan Floral biology and pollination mechanisms of Cymbidium dayanum, an endangered Correspondence epiphytic orchid, were investigated in south Kyushu, Japan. The flowering period Naoto Sugiura, Faculty of Science, Kumamoto spanned approximately 4 months from fall to winter, with the inflorescences often University, Kumamoto 860-8555, Japan. Email: [email protected] blooming asynchronously on each plant and individual flowers commonly lasting for a month. The nectarless flowers are self-compatible but cannot autonomously self-pollinate; the orchid needs to attract pollinators by deceit for capsule produc- tion. The field observations showed that the flowers were pollinated exclusively by the Japanese honeybee Apis cerana japonica, despite its long flowering period. The worker bees pollinated flowers and/or received the pollinarium on the thorax, while escaping from the labellum chamber. The natural fruit-set ratios at the popu- lation level, an estimate of pollination success, varied interannually but were gener- ally low, as the result of infrequent flower visits by honeybee workers owing to the cold winter climate and the lack of a nectar reward. Because most flowers that were artificially pollinated in the winter successfully developed into capsules, the cold- ness was not considered a direct cause of the low fruit-set ratios. Our results explic- itly indicate that the bee pollination niche could be available to temperate plant species even during mid-winter. We inferred advantages for the winter flowering of C. dayanum. KEYWORDS Apis, cymbidium, deceptive pollination, flowering season, winter climate 1 | INTRODUCTION pollination, generalized food deception, and Batesian food-source mimicry. To date, autonomous self- Cymbidium Sw. (Orchidaceae: Epidendroideae) includes pollination has been reported in C. macrorhizon Lindl, approximately 52 species naturally distributed in Asia and C. nipponicum (Franch. et Sav.) Rolfe [as C. aberrans Oceania (Du Puy & Cribb, 2007; Pridgeon, Cribb, Chase, & (Finet) Schltr.], and C. nagifolium Masam. (as C. lancifo- Rasmussen, 2009). This taxon includes epiphytic, lithophy- lium Hook.), all of which lack a rostellum that functions tic and terrestrial species, which thrive in a variety of habi- as the physical barrier between the anther and stigma tats, such as open savannas, tropical rain forests and (Suetsugu, 2015). Reward-based pollination takes place in high-elevation grasslands (Pridgeon et al., 2009). Most C. mandidum Lindl., in which flowers are pollinated by species are autotrophic and rarely mycoheterotrophic the stingless bee Trigona kockingsi, which collects viscid (Motomura, Selosse, Martos, Kagawa, & Yukawa, 2010; substances on the labellar surface (Macpherson & Rupp, Suetsugu, 2015). 1935, cited in Du Puy & Cribb, 2007). The substance is In Cymbidium orchids, there are four known modes of probably used as a nest-building material. A similar mode pollination: autonomous self-pollination, reward-based is also thought to occur for C. lowianum (Rchb.f.) Rchb., Plant Species Biol. 2019;34:19–26. wileyonlinelibrary.com/journal/psbi © 2019 The Society for the Study of Species Biology 19 20 MATSUDA AND SUGIURA in which the labellar surface has protein-containing papil- 2 | MATERIALS AND METHODS lae that may function as food hairs (Davies, Stpic- zynska, & Turner, 2006). Generalized food deception has 2.1 | Study sites been found for some species, including C. lancifolium Because Japanese C. dayanum is now considered nearly (Cheng, Liu, He, Wei, & Luo, 2007), C. goeringii (Rchb. extinct in the wild, mainly as a result of overexploitation f.) Rchb.f. (Tsuji & Kato, 2010; Yu, Luo, & Dong, 2008) (Inoue, 2015), we did not find a natural population appropri- and C. kanran Makino (Sugawara & Tsutsui, 1998; ate for study. Thus, field observations were conducted at Tsuji & Kato, 2010). The nectarless flowers attract polli- two seminatural habitats (sites N and K) located near rem- nating bees by visual and olfactory stimuli. Finally, nant broad-leaved evergreen forests (past habitats) in south Batesian food-source mimicry occurs in C. insigne subsp. Kyushu, Japan. At both sites, orchid seedlings of various seidenfadenii P.J. Cribb & Du Puy. The plant relies exclu- sizes were present, suggesting that the series of events neces- sively on the bumblebee Bombus eximius, which also pol- sary for sexual reproduction, including pollination, were linates the nectar-producing flowers of Rhododendron occurring successfully. One site was in the grounds of the ciliicalyx subsp. lyi (H. Lév.) R.C. Fang Noborio Elementary School (site N), Minami-Osumi Town, (as Rhododendron lyi Leveille) (Kjellsson, Rasmussen, & Kagoshima Prefecture, where six adult C. dayanum grew on Dupuy, 1985). Because both plants have a similar floral six trees (but one was seriously damaged by a typhoon in appearance, the bumblebees occasionally mistakenly visit late September 2016). Approximately 20 km east-northeast of site N, the second site was in the grounds of Kishira Ele- the nectarless flowers of the orchid. In addition to the mentary School (site K), Kimotsuki Town, Kagoshima Pre- above-mentioned modes, insect pheromone mimicry may fecture, where three adult plants grew on three trees. At both be used by C. floribundum Lindl. (as C. pumilum Rolfe) sites, the trees were well isolated from each other, resulting under cultivated conditions, which has flowers that are in the orchids usually receiving sunlight daily for moderately exclusively pollinated by the Japanese honeybee Apis cer- long hours. No nectariferous flowers that resembled C. daya- ana japonica (Apoidea: Apiformes: Apidae) (Sasaki, num flowers were found at the study sites or nearby broad- Ono, & Fukuda, 1992; Sasaki, Ono, & Yoshida, 1991). leaved evergreen forests. The nectarless flowers emit a scent that includes two man- dibular gland components of the honeybee, 2.2 | Flowering habits and functional morphology of 3-hydroxyoctanoic acid and 10-hydroxy-(E)-2-decenoic flowers acid, to deceive individual bees or bee swarms into engag- At site N, the number of opening flowers on inflorescences ing in pollination activity (Sugahara, Izutsu, Nishimura, & (n = 126 inflorescences in the 2016–2017 flowering season; Sakamoto, 2013). n = 155 inflorescences in the 2017–2018 season) was C. dayanum Rchb.f. is distributed in Asia, from the recorded intermittently to clarify the flowering phenology. Himalayas of northern India to Japan and south through We also carefully studied functional features of flowers and Indo-China and the Philippines to Indonesia, and prefers inflorescences, which may be important for pollinator attrac- cooler, higher elevations of about 1,000–1,500 m in its tion and manipulation, with natural plants at site N and four southern distribution area (Du Puy & Cribb, 2007). How- cultivated plants on the campus of Kumamoto University, ever, despite its wide distribution, to our knowledge, no Kumamoto City, central Kyushu. To examine the lifespan of information on the pollination strategy is available. individual flowers, some floral buds of the cultivated plants Recently, we investigated the floral and pollination biol- were individually marked, and the opening and wilting dates ogy of this epiphytic cymbidium in south Kyushu, Japan, for each were recorded during the two flowering seasons where it flowers between October and December (n = 24 flowers of 10 inflorescences in the 2016–2017 sea- (Yukawa, 2015). The main aims of our study were: (a) to son; n = 69 flowers of 12 inflorescences in the 2017–2018 investigate the floral features of C. dayanum from a season). functional perspective, an aspect rarely examined thus To examine whether C. dayanum is self-compatible and far, (b) to identify the pollinator and characterize its whether it is pollinator dependent, 33 intact flowers were intrafloral behavior, and (c) to examine the natural fruit- selected randomly on October 21, 2016 at site K. Each set ratio, which is an indicator of pollination success. On flower was subjected to one of the following treatments: the basis of the results, we discuss the pollination strat- (a) self-pollination with one pollinarium from the same egy of Japanese C. dayanum, including possible reasons flower; (b) cross-pollination with one pollinarium from a why this orchid continues to flower in winter when flower of a different plant; or (c) no pollination. The perianth almost all bee species have already ceased their extra- segments of all the treated flowers were then trimmed off to nest activities (Ikudome, 1992; Y. Matsuda, personal prevent natural pollination (Li, Zheng, Dafni, & Luo, 2010; observation, 2016–2018). Sugiura, 2013; Sugiura, 2014). On January 18, 2017, the MATSUDA AND SUGIURA 21 flowers were checked to determine whether their ovaries had asynchronously. Within an inflorescence, about 12 flowers developed into swollen green capsules. For each treatment, (11.4 ± 2.2, n = 126 in the 2016–2017 season; 12.1 ± 3.3, we calculated the fruit-set ratio (i.e., the ratio of swollen cap- n = 155 in the 2017–2018 season) opened sequentially, but sules to treated flowers). Furthermore, 16 flowers were self- all of