Consistent Pollination Services to Cypripedium Macranthos Var. Rebunense (Orchidaceae) by Bombus Pseudobaicalensis

Received: 28 September 2018 Revised: 30 November 2018 Accepted: 10 December 2018 DOI: 10.1111/1442-1984.12232

NOTES AND COMMENTS

Consistent pollination services to Cypripedium macranthos var. rebunense (Orchidaceae) by Bombus pseudobaicalensis

Naoto Sugiura

Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto Abstract University, Kumamoto, Japan Bombus pseudobaicalensis queens are known pollinators of the threatened orchid Correspondence Cypripedium macranthos var. rebunense. However, whether other bumblebee spe- Naoto Sugiura, Faculty of Science, Kumamoto cies also pollinate this orchid has not yet been ascertained. A 10-year observation University, Kumamoto 860-8555, Japan. Email: [email protected] provided convincing evidence that B. pseudobaicalensis is the only consistent pollinator. B. beaticola moshkarareppus queens were newly found to rarely carry pollen smears of the orchid, whereas B. hypocrita sapporoensis and B. diversus queens were practically mere flower visitors. Why these three species do not equal B. pseudobaicalensis in pollination effectiveness remains unclear but differences in body size and individual abundance between B. pseudobaicalensis and the other species are considered as possible primary causes. Given the almost complete dependence of C. macranthos var. rebunense on B. pseudobaicalensis for pollination and its potential risk, conservation managers should take care not to reduce the bumblebee colonies.

KEYWORDS body size, bumblebee, conservation, individual abundance, orchid

1 | INTRODUCTION through an opening on its dorsal surface, an insect of the appropriate body size is forced to rub its thoracic dorsum The Orchidaceae is one of the largest families of angiosperms, against the stigma on its way to either of two apertures at the comprising approximately 27,000 species (Dressler, 1981; base of the labellum and then against the anther just before Zotz, 2013). However, many orchid species are now threatened escaping through the aperture (Nilsson, 1979; Sugiura & with extinction in the wild (e.g., for the present status of Japa- Yumiyama, 2016). As with many other rewardless orchids, nese taxa, see Ministry of the Environment Government of identifying the pollinators of Cypripedium spp. often Japan 2018). For such endangered species, a significant decline demands long hours in the field. For example, Li, Pember- in their pollinators may have a great impact on population per- ton, Zheng, and Luo (2012) required 25-hr and 30-hr obser- sistence, because Orchidaceae includes many pollinator- vations to find a single pollinator of C. micranthum Franch. oriented species, whose flowers are pollinated primarily by and two pollinators of C. sichuanense Perner, respectively. insects such as bees and moths (Dressler, 1981); therefore, an In the present study, I investigated the pollinator assem- extensive knowledge of insect pollinators is sometimes benefi- blage of pale-cream-flowered Cypripedium macranthos cial to in-situ conservation of endangered orchids (Pierson, Sw. var. rebunense (Kudô) Miyabe & Kudô, an endangered Tepedino, Sipes, & Kuta, 2001; Roberts, 2003). taxon (EN) on the Japanese Red List of vascular plants The genus Cypripedium is a group of lady’s slipper (Ministry of the Environment Government of Japan 2018), orchids containing approximately 50 species (Cribb, 1997). on Rebun Island, Hokkaido, Japan. Although pre-nesting The labellum is deeply pouched and functions as a queens of Bombus pseudobaicalensis are already reported to pollinator-trapping device; after entering the labellum pollinate this taxon (Sugiura, Fujie, Inoue, & Kitamura,

2001), whether other bumblebee species also engage in pol- queen was rarely observed, because pre-nesting queens wan- lination of this orchid has not yet been ascertained. Our pre- der from place to place to seek a suitable nesting place vious studies (Sugiura et al., 2001; Sugiura & Yumiyama, (Heinrich, 2004; Wilson, 1971). (b) In the protected areas, I 2016) revealed that three bumblebee species (B. beaticola inspected several hundreds of C. macranthos var. macranthos moshkarareppus, B. diversus, and B. hypocrita sapporoen- flowers one by one, although the success rate was rather low. sis) had the ability to remove pollen smears; that is, when When finding a bumblebee, I recorded its flower-visiting artificially introduced into the labellum, the queen could behavior, which consists of four components (approaching, escape through the exit aperture, with a pollen smear on its alighting, entering and exiting [Nilsson, 1979]). thoracic dorsum. Nevertheless, no queens with pollen smears I measured the following body parts of individual queens were found in orchid habitats during a 2-year study period of B. pseudobaicalensis (n = 4), B. beaticola moshkararep- (Sugiura et al., 2001). To better understand the pollinator pus (n = 3), B. diversus (n = 3) and B. hypocrita sappor- community of this orchid and its temporal variability, I con- oensis (n = 6), all of which were road kills in 2009–2013, tinued to research for 10 years, and the results obtained are with a digital caliper: (a) head width; (b) thoracic width; reported here. Given the rarity of C. macranthos var. rebu- (c) thoracic height; (d) abdominal width; and (e) unextended nense, the decadal data on pollinator assemblage will be body length. The dataset was analyzed by a principal com- helpful for the conservation management of this orchid. ponent analysis (PCA) to investigate the extent of differences in body size among the taxa.

2 | MATERIALS AND METHODS 3 | RESULTS In Cypripedium spp., the viscid pollen is pulled out in a mass when touched (Cribb, 1997), and this enables us to During the flowering season of C. macranthos var. rebu- examine whether bumblebees visited the flowers: cypripe- nense, by far the most abundant bumblebees were dium pollen smears on the body surface of bumblebees B. hypocrita sapporoensis (n = 1,247 observations), followed could be easily identified because of their characteristic by B. pseudobaicalensis (n =571), B. diversus (n = 265) appearance (viscid or paste-like mass, fig. 1F of Sugiura and B. beaticola moshkarareppus (n = 200) in the protected et al., 2001). On Rebun Island, another variety of C. areas and in neighboring areas. The preponderance of macranthos, purplish-pink-flowered var. macranthos, also B. hypocrita sapporoensis and/or B. pseudobaicalensis was occurs and is pollinated by B. pseudobaicalensis queens also found in most of the study years (Table 1). (Sugiura & Takahashi, 2015), so not all queens with pollen As already mentioned, in 1,602 observations I could smears of Cypripedium can be necessarily regarded as determine whether or not the individual bore pollen smears flower visitors of var. rebunense. However, because var. on its thoracic dorsum. In 18% (73 observations) of 406 obser- macranthos flowers were very rare (8–48 flowers per year in vations, B. pseudobaicalensis was found to bear pollen two protected areas for var. rebunense [Sugiura & Takaha- smears. In addition to this species, B. beaticola moshkararep- shi, 2015]), compared to several thousand var rebunense pus and B. hypocrita sapporoensis also carried pollen smears, flowers (Kosaka, Kawahara, & Takahashi, 2014; N. Sugiura, the former in approximately 9% (11 observations) of personal observation, 2004–2013), and their annual fruit-set 121 observations and the latter only once (0.1% of 903 obser- ratios were also generally low (Sugiura & Takahashi, 2015), vations), with the difference being significant between almost all pollen-smear-laden queens were considered to be B. beaticola moshkarareppus and B. pseudobaicalensis (chi- flower visitors of var. rebunense. squared test, P < 0.05). In B. diversus (172 observations), no In each flowering season of C. macranthos var. rebu- pollen-smear-laden queens were found. Eventually, I nense (usually late May to mid-June) from 2004 to 2013, observed bumblebees with pollen smears 85 times, with field studies (10–27 days per year, mean 16.0 days, n = 10) B. pseudobaicalensis queens comprising about 86% of them. were conducted in the northern portion of Rebun Island, Figure 1 shows numbers of observations of pollen-smear- where the two protected areas were. I adopted two different laden queens in each year from 2004 to 2013. The result ways to find pollen-smear-bearing bumblebees. (a) At vari- shows that only B. pseudobaicalensis was consistently ous places within and around the protected areas, I tried to engaged in pollination of C. macranthos var. rebunense. ascertain whether bumblebee queens were laden with pollen In the protected areas, I found flower-visiting bumble- smears when finding the bees on flowers other than bees only 34 times in 10 years. The most frequent species C. macranthos var. macranthos or on the ground. Ultimately, were B. pseudobaicalensis (n = 17 observations, 22 flowers), bumblebees were found 2,283 times during the study period, followed by B. hypocrita sapporoensis (n = 11 observa- and in approximately 70% of cases (n = 1,602), I could deter- tions, 18 flowers), B. diversus (n = 4 observations, four mine whether or not the individual bore pollen smears on its flowers), B. beaticola moshkarareppus (n = one observa- thoracic dorsum. Although I was not able to discriminate tion, one flower), and either B. hypocrita sapporoensis or among individual bumblebees, it seemed likely that the same B. beaticola moshkarareppus (n = one observation, one 40 SUGIURA

TABLE 1 Numbers of observations of bumblebees in each year from 2004 to 2013

Year Bombus hypocrita sapporoensis B. pseudobaicalensis B. diversus B. beaticola moshkarareppus 2004 86 52 8 17 2005 54 38 4 5 2006 140 43 22 33 2007 434 201 118 75 2008 13 13 12 8 2009 31 56 19 18 2010 186 28 24 6 2011 35 86 15 5 2012 203 37 18 24 2013 65 17 25 9 Total 1,247 571 265 200

Figure 2 shows a two-dimensional PCA ordination of the size data for five body parts of the four bumblebee species. The first principal component accounted for all measured characters with positive coefficients and the axis represented 94.7% of the total variance. Although no distinct differences in body size between B. pseudobaicalensis and B. beaticola moshkarareppus were apparent, the two species had smaller bodies than either B. hypocrita sapporoensis or B. diversus. In addition to the above-mentioned three bumblebee species, a queen of the yellow jacket Dolichovespula media media bore a pollen smear on its thoracic dorsum. However, FIGURE 1 Numbers of observations of pollen-smear-bearing bumblebees the queen was considered to be an incidental flower visitor, in each year from 2004 to 2013. Bp, Bombus pseudobaicalensis; Bbm, B. beaticola moshkarareppus; Bhs, B. hypocrita sapporoensis because this was the only record of flower visitation by the yellow jacket obtained during the 10-year study period. flower) (Table 2). Of the four species, only B. pseudobaicalensis was observed to carry pollen smears of C. macranthos 4 | DISCUSSION var. rebunense on its thoracic dorsum: on five occasions, queens already bore pollen smears when I found them, and The decadal bumblebee survey reported here provided con- queens were observed six times to remove pollen smears vincing evidence that B. pseudobaicalensis queens are the from flowers. In the other three species, queens were occa- primary pollinator of C. macranthos var. rebunense; they sionally observed to alight on the labellum but none entered it (Table 2).

TABLE 2 Behavior components of four bumblebee species at Cypripedium macranthos var. rebunense and their frequencies

No. No. No. No. approaching alighting entering exiting Bumblebee species (%) (%) (%) (%) Bombus 18 (100) 15 (83.3) 10 (55.6) 6 (33.3) pseudobaicalensis B. hypocrita 16 (100) 7 (43.8) 0 0 sapporoensis B. diversus 4 (100) 2 (50.0) 0 0 B. beaticola 1 (100) 0 0 0 moshkarareppus B. hypocrita 1 (100) 1 (100) 0 0 sapporoensis or B. beaticola FIGURE 2 Two-dimensional principal component analysis ordination of moshkarareppus size data of five body parts of Bombus spp. Bd, Bombus diversus; Bp, a Of 34 bumblebee observations on 46 flowers for 10 years, only complete Bombus pseudobaicalensis; Bbm, B. beaticola moshkarareppus; Bhs, B. behavioral records (30 observations, 40 flowers) were used. hypocrita sapporoensis SUGIURA 41 were the most abundant and the only consistent pollen car- its potential risk, conservation managers need to know about riers during the study period. According to Sakagami and the nutritional, provisional and nesting requirements of Katayama (1977), B. pseudobaicalensis is an open land B. pseudobaicalensis to make sexual reproduction and con- dweller and nests on the ground of grassy areas. Such habitat tinued existence of this rare orchid possible (Pierson et al., preference may be viewed as an appropriate trait for pollen 2001). For example, herbs with different flowering times vectors of the orchid, because C. macranthos var. rebunense must coexist within the orchid reserves and in neighboring is a coastal grassland species (Kosaka et al., 2014; Sugiura areas, because the foraging activity of B. pseudobaicalensis et al., 2001). My long-term survey also clarified that queens lasts usually from May to October on Rebun Island (Sugiura of B. hypocrita sapporoensis and B. diversus are practically N., unpubl. data). B. pseudobaicalensis nests on the ground mere flower visitors, whereas B. beaticola moshkarareppus surface in grassy areas (Sakagami & Katayama, 1977), so queens rarely carry pollen smears. Why the three bumblebee grasses, although they usually offer no floral rewards to the species do not equal B. pseudobaicalensis in pollination bees, must also be present. Ultimately, these considerations effectiveness, although they have the ability to remove pol- highlight the need for community-based management of “ len smears (Sugiura et al., 2001; Sugiura & Yumiyama, C. macranthos var. rebunense. Applying such extended ” 2016), remains unclear. However, two possible primary care to the orchid would also help to preserve many other causes are considered. (a) In Cypripedium spp., morphomet- community members, including other endangered plants ric fit between labella and pollinating insects has been docu- (Roberts, 2003; Tepedino, Sipes, Barnes, & Hicker- mented to be necessary for pollination (e.g., Bänziger, son, 1997). Sun, & Luo, 2005; Edens-Meier, Arduser, Westhus, & Bern- hardt, 2011; Li et al., 2012; Nilsson, 1979). Because ACKNOWLEDGMENTS B. hypocrita sapporoensis and B. diversus are distinctly larger than B. pseudobaicalensis and B. beaticola moshkar- I am grateful to K. Furuya, K. Kozawa, Y. Miura and areppus, both species seldom entered the labellum even S. Sasaki for their kind help during my stays on Rebun though they alighted on flowers of the orchid. This interpre- Island, and M. Goubara for field assistance. S. Miyamoto, tation is also congruent with my own experience; I often had A. Shioguchi and H. Takahashi generously informed me of difficulty in inducing each queen of B. hypocrita sapporoen- their observations on bumblebee visits to C. macranthos var. sis and B. diversus, unlike those of the other two species, to rebunense. This study was supported by grants from the Japan Society for the Promotion of Science (JSPS voluntarily enter the labellum, when artificially introducing KAKENHI grant no. 16310157) and the Ministry of the them into the labellum (Sugiura et al., 2001; Sugiura & Environment, Government of Japan (Global Environment Yumiyama, 2016). (b) Bombus beaticola moshkarareppus Research Fund). was consistently much less abundant than B. pseudobaicalensis within and around the protected areas, thereby remaining a minor pollen carrier. 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