bs_bs_banner Plant Species Biology (2014) 29, 294–299 doi: 10.1111/1442-1984.12016 NOTES AND COMMENTS Pollination biology of the endangered orchid Cypripedium japonicum in a fragmented forest of Japan KENJI SUETSUGU* and SHIGEKI FUKUSHIMA† *Graduate School of Human and Environmental Studies, Kyoto University, Kyoto, and †Chiba Prefectural Agriculture and Forestry Research Center, Sanbu, Japan Abstract Pollination biology studies of the endangered orchid Cypripedium japonicum were conducted in its natural habitat using pollinator observation and hand-pollination experiments. The observed fruit set was as follows: artificial outcross-pollinated, 100%; artificial self-pollinated, 100%; pollinator-excluded, 0%; and emasculated flowers, 0%. These results show that this species, although self-compatible, is neither autogamous nor agamospermous. The fruit set for open-pollinated flowers was 14.9%, which sug- gests that the study population was subject to pollinator limitation. The nectarless flowers of C. japonicum were exclusively visited and pollinated by the queens of two bumblebee species (Bombus ardens and B. diversus diversus). It is probable that the nectarless flowers of C. japonicum attract pollinators through a generalized food decep- tive system. Keywords: Bombus, Cypripedium, deceptive pollination, pollination biology. Received 26 October 2012; revision received 25 February 2013; accepted 1 March 2013 Introduction (Li et al. 2008). As the pollinator passes through the basal orifice, it is forced to pass the stigma and under the The Orchidaceae is one of the most species-rich plant anthers where it picks up some of the pollinium (Li et al. families, and their floral diversity and pollination biology 2008). If the pollinator has already picked up pollen from have long intrigued evolutionary biologists (Cozzolino a previous visit, pollination can also occur under passing & Widmer 2005). Of the estimated 18 500 Orchidaceae the stigma. Despite this fixed pollination route, there is a species, around one-third are thought to employ decep- wide diversity of pollinators and pollination mechanisms tive pollination systems (Jersáková et al. 2006; Renner in this subfamily (Stoutamire 1967; Sugiura et al. 2001, 2006). While deceptive flowers offer no rewards such as 2002; Bänziger et al. 2005; Li et al. 2008; Sun et al. 2009; nectar and pollen, they can nonetheless attract pollinators Ren et al. 2011). The genus Cypripedium is a group of by the use of visual and/or olfactory deception (Jersáková lady’s slippers comprising approximately 45 species et al. 2006; Renner 2006). found throughout the northern temperate zones that are The lady’s slippers subfamily (Cypripedioideae) of the generally pollinated by wild bee species (Stoutamire north temperate and certain tropical zones is regarded as 1967; Sugiura et al. 2001; Sugiura et al. 2002; Bänziger a model lineage for food-deceptive orchids. This subfam- et al. 2005; Li et al. 2008; Sun et al. 2009; but also see Ren ily, comprising five genera, contains a total of 158 species et al. 2011). (Pridgeon et al. 2001). Their flowers are designed as one- Most wild orchid species in Japan have recently expe- way traps; the pollinator passes through a front entrance rienced a drop in their population, primarily as a result into the labellum and then proceeds to exit through one of habitat destruction and overharvesting (Tsuji & Kato of the pair of basal orifices that are formed by the stami- 2010). An understanding of the reproductive biology of node blocking the opening at the bottom of the labellum these species could help us determine whether a failure in Correspondence: Kenji Suetsugu the recruitment cycle is limiting the success of their repro- Email: [email protected] duction, leading to their constrained population growth © 2013 The Society for the Study of Species Biology CYPRIPEDIUM JAPONICUM POLLINATION BIOLOGY 295 (Gale 2007). This information, when integrated with data Materials and methods regarding other factors of their life history, could provide Study species and study sites an estimate of the persistence of a population in a given location (Gale 2007). It has been emphasized by Roberts Cypripedium japonicum is a terrestrial orchid found in (2003) that this approach is necessary when planning China, Japan, and Korea (Kitamura et al. 1986). This intervening measures to conserve endangered orchid species has long, creeping rhizomes with rather stout species, given their taxonomic diversity and the intri- roots forming at the nodes, and is characterized by its cacies and inefficiencies of their pollination systems 10–15 cm long, 12–20 cm wide, nearly opposite, flabellate (Tremblay et al. 2005). The current study focused on the suborbicular leaves (Kitamura et al. 1986; Sun et al. 2009). pollination biology of Cypripedium japonicum, a species Its large, solitary yellow-green flower opens in late April threatened both by overharvesting, and by habitat distur- and remains in flower through early May. Individual bance and fragmentation. In Japan, C. japonicum is catego- flowers remain open for 2–3 weeks, even if the flowers rized as an endangered species (Environment Agency of are pollinated (Sun et al. 2009; Suetsugu pers. obs.). The Japan 2000) which means that the risk of its extinction is flower has a large, pendent, sac-like labellum (ca 5.0– increasing. The extinction probability for this species in 6.5 cm long and ca 2·5–3.5 cm wide) with a crimson Japan over the next 100 years is estimated to be almost mouth on its upper surface. Despite the large size of its 100% (Environment Agency of Japan 2000). flower and its apparent nectar guides, the plant produces The workers of two bumblebee species, Bombus no nectar (Sun et al. 2009). The flowers have two anthers, remotus and B. Imitator, have recently been shown to be each situated above an exit from the labellum. Each anther the effective primary pollinators of C. japonicum in China bears a pollinium, a mass of sticky pollen that is usually (Sun et al. 2009). However, it has previously been shown removed as a unit when a pollinator squeezes through the that other members of the genus Cypripedium can use exit (Sun et al. 2009). different pollinators depending on their location. For The pollination experiments and pollinator observa- example, in Vermont, USA the main pollinator of C. re- tions were conducted in late April to early May 2012 at a ginae is the syrphid fly, Syrphus torvus Osten et Sacken, fragmented coniferous forest dominated by C. japonica with occasional pollination by beetles (Vogt 1990), while that are surrounded by agricultural landscapes and urban in Canada leaf-cutter bees, Megachile lanophaea Smith and zones in Sanbu City, Chiba Prefecture. The entire study M. centuncularis L., are the main pollinators (Guignard site contained approximately 600 flowering individuals. 1886). In addition, Edens-Meier et al. (2011) revealed six As far as we know, there are no C. japonicum flowering medium-sized bees (Anthophora, Apis and Megachile spp.) populations within a 1 km radius. The investigated popu- carried segments of massulate pollinia in Missouri, USA. lation is situated within the landed estate of Chiba Pre- Information concerning the breeding systems of Japa- fectural Agriculture and Forestry Research Center. The nese C. japonicum and its interactions with pollinators population has been preserved by prohibiting collection is therefore essential for both conservation of Japanese of any plants except for research. habitats and to enhance our understanding of the evo- lution of the diverse deceptive systems employed by the genus Cypripedium. However, the life history in its Pollination experiments and pollinator observations natural habitats has been poorly understood in Japan. Tanaka (1997, 2009) noted that C. japonicum was visited Hand-pollination experiments were performed to deter- by bumblebees. This observation suggested that the mine whether fruit set was limited by the receipt of Japanese population of C. japonicum was also pollinated pollen. Each flower in the experiment was assigned to by some bumblebees. However, it remains unknown (i) one of four treatments. (i) Bagged treatment: flowers which bumblebee species visits, (ii) whether they actu- were bagged with a fine meshed net before anthesis to ally work as the pollinator, and (iii) if so, whether they exclude pollinators (n = 5). This treatment was used to are the exclusive pollinator. test whether fruit set could occur by autonomous self- The reproductive biology of C. japonicum was investi- pollination. (ii) Agamospermy treatment: the pollinaria gated in the present study to clarify its deception mecha- were removed before anthesis using forceps, and the nism and plan comprehensive conservation strategies. flowers were then bagged (n = 5). This treatment was used Pollination experiments were conducted to characte- to test for agamospermy. (iii) Artificial self-pollinated rize the breeding system, such as the capacity for self- treatment: the pollinaria were removed and used to hand- fertilization. The plants were also observed under field pollinate the same flower before being bagged (n = 5). (iv) conditions and the flower visitors recorded and iden- Artificial cross-pollinated treatment: same as treatment tified to determine which insects might be candidate (iii) but using the pollinia from a different plant (n = 5). To pollinators. avoid sampling within a clonal plant, all the plants were Plant Species Biology 29, 294–299 © 2013 The Society for the Study of Species Biology 296 K. SUETSUGU AND S. FUKUSHIMA kept at least 10 m from their nearest neighbour during without embryo. The effect of pollination treatment on the the cross-pollination experiments. In addition to the four seed mass and proportion of seeds with embryo were treatment regimens, flowering individuals were ran- tested by student t-test. domly tagged and allowed to develop fruit by natural The observations of flower visitors were made for pollination (n = 215). Only a small number of plants were a total of approximately 10 h during periods of high used in this study to minimize the impact on these endan- diurnal pollinator activity (09.00–18.00 h).