Pollination Biology of the Endangered Orchid Cypripedium Japonicum in a Fragmented Forest of Japan

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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 deceptive 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

(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 evolution 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). Pollinators gered populations. The experimental plants were moni- were defined as species that were observed not only to tored intermittently over the subsequent 6 weeks, and visit the flower, but also to carry away pollinia. Some of scored for fruit set once the capsules had formed. To these insect visitors were captured after and identified, determine pollen limitation, we compared the fruit set referring to Washitani et al. (1997) under natural conditions with artificial self- and cross- pollinated treatments by Fisher’s exact test. In addition, in late September, all the mature but inde- Results hiscent fruits were picked up and silica dried from both Bombus pollination and pollen limitation artificial self- and cross-pollinated individuals. Following this, the mass of dry seeds freed from the capsule was The nectarless flowers of C. japonicum were visited and obtained to the nearest 0.0001 g. After this, all of the seeds pollinated exclusively by the queens of the bumblebees from each capsule were mixed and 500 seeds were exam- Bombus ardens (seven visits) and B. diversus diversus (three ined per capsule under the dissecting microscope to visits) (Fig. 1b–f). Since the study was conducted during assess the ratio between seeds with embryo and seeds early spring, the worker bees of these species had still not

(a) (b)

(f) (g) (h)

Fig. 1 Flowers and pollinators of Cypripe- dium japonicum. (a) Flowering habitat, (b–e) flower visits of Bombus ardens, (f) flower visits of Bombus diversus diversus, (g) B. diversus diversus visiting a co-occurring Disporum sessile flower, and (h) B. ardens visiting a co-occurring D. sessile flower. White arrows indicate pollinia attachment.

© 2013 The Society for the Study of Species Biology Plant Species Biology 29, 294–299 CYPRIPEDIUM JAPONICUM POLLINATION BIOLOGY 297 emerged. The queens of both species were also observed 1996; Sugiura & Yamaguchi 1997; Suetsugu & Tanaka visiting the white bell-shaped flower of the co-occurring 2013). There was no difference in the ratio of fruit set species Disporum sessile (Fig. 1g,h). Pollen removal between artificial self-pollination and cross-pollination of C. japonicum occurred when a bumblebee squeezed treatments, indicating that C. japonicum is a highly self- past one of the lateral anthers, excepting only one visit of compatible species. In addition, although Tremblay et al. Bombus ardens. No pollinium removal in a Bombus ardens (2005) found that self-pollination tends to result in signifi- visitation occurred because no pollinium was left in the cantly lower levels of embryo formation as compared passed anther. In addition, we confirmed pollinium depo- with that following cross-pollination, such effects were sition actually occurred by inspecting stigma after two not detected in C. japonicum (student t-test, P = 0.49). This species of bumblebees, which had carried a pollinium, information also confirmed full self-compatibility in this exited. Deceived bumblebees were found to leave nonre- species. This trend is consistent with a previous study warding clusters immediately after escaping from the (Edens-Meier et al. 2010) that showed self–compatibility in labellum. Cypripedium appears more common than in other decep- Only 32 of the 215 natural flowering individuals bore tive orchids. Although C. japonicum showed very low fruit fruits, giving a pollination frequency of 14.9%. The artifi- set in natural conditions, a significant increase in fruit set cial pollination experiment revealed that the fruit set for was achieved through hand-pollination (Fisher’s exact the four treatments: outcross-pollinated, self-pollinated, test, P < 0.01), suggesting that pollinator limitation exists pollinator-excluded and emasculated flowers, were 100%, under natural conditions. However, it should be noted 100%, 0% and 0%, respectively. Therefore, although that the study site was in a fragmented forest dominated C. japonicum showed very low fruit set in natural condi- by C. japonica, which could have exacerbated the pollina- tions, a significant increase in fruit set can be achieved tor limitation, since forest fragmentation is known to through hand-pollination (Fisher’s exact test, P < 0.01). result in declining bee populations. It is therefore possible The seed mass sampled from artificial self-pollinated that the low rate of successful pollination for C. japonicum individuals was 165.7 Ϯ 22.1 mg (average Ϯ SD) and was could be a consequence of both its nectarless flowers and not significantly different from those taken from arti- forest fragmentation. ficial cross-pollinated individuals (171.3 Ϯ 22.3 mg; The genus Cypripedium does not produce food rewards, average Ϯ SD; P = 0.70). Additionally, the number of either nectar or edible pollen, which infers that a decep- viable seeds sampled from artificial self-pollinated tive pollination system is being employed when insect- individuals (497.0 Ϯ 2.0; average Ϯ SD) was also not mediated pollination does occur. Indeed it has been significantly different from those taken from artificial reported previously that most Cypripedium species attract cross-pollinated individuals (497.8 Ϯ 1.5; average Ϯ SD; pollinators through deception, although not by mimick- P = 0.49). ing a specific model plant (Stoutamire 1967; Nilsson 1979; Bänziger et al. 2005; Li et al. 2008; Sun et al. 2009). For example, Nilsson (1979) suggested that the flowers of Discussion C. calceolus attract pollinators through a generalized food The fruit set of C. japonicum in natural conditions (14.9%) deception (color, odor, and false nectar guides), as well was very low, which was in agreement with the general as nest site resemblance (odor and cavity), while the trend for nectarless orchids, excepting autogamous flowers of C. guttatum evidently exploit the innate forag- species (Suetsugu 2013), which have an average fruit set of ing preferences of halictid bees through a generalized ca 20% (Tremblay et al. 2005). It should be noted that the food deception (Bänziger et al. 2005). To date, C. macran- small sample size may not be enough to precisely deter- thos var. rebunense is the only example of a Cypripedium mine the fruit set ratio on each treatment. However, the species that exploits a specific model plant. In this case, its results obtained showed clear tendency that there are no flower color is in a similar range of the bumblebee’s visual fruits developing under both pollinator exclusion and the spectrum as that of the co-occurring species P. schistostegia emasculated treatment, whereas there is a high propor- (Sugiura et al. 2001, 2002). The two plants also have a tion of fruit developed by both self- and cross-pollination. similar flowering season and spatial distribution (Sugiura Bumblebee pollinators appeared to be essential for fruit et al. 2001, 2002). set in natural conditions since neither agamospermy Although the queens of B. ardens and B. diversus diver- nor spontaneous autogamy was detected. Bombus is able sus were the pollinators of C. japonicum in the population to regulate its body temperature, and pollinate flowers investigated (Fig. 1b–f), it is unlikely that sexual deception located in shaded understories of forests. Because is being employed since these bee species are phyloge- C. japonicum typically inhabits understories of dense netically distinct subgenera that utilize different sexual forests, the endothermic character of Bombus may be pheromones (Kubo & Ono 2010). Further evidence sup- favored as the pollinator in such shaded habitats (Sugiura porting this view is the observation that the workers of

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