Journal of Systematics JSE and Evolution doi: 10.1111/jse.12485 Research Article

Effective pollinia transfer by settling moths’ legs in an orchid Habenaria aitchisonii

Ying-Ze Xiong1, Li-Bing Jia1, Chang-Qiu Liu2, and Shuang-Quan Huang1*

1School of Life Sciences, China Central Normal University, Wuhan 430079, China 2Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, China *Author for correspondence. E-mail: [email protected]. Received 4 December 2018; Accepted 25 January 2019; Article first published online xx Month 2018

Abstract A great diversity of flower morphology in orchids has long been thought to be selected by diverse pollinators. Habenaria Willd. (Orchidaceae) species are generally characterized by long nectar spurs and pollinated by long-tongued insects (Lepidoptera), the mechanical fit between the spur and pollinator proboscis length being supposedly caused by “arms race” reciprocal selection. Here, we report that flowers of Habenaria aitchisonii Rchb. f. with nectar spurs (approximately 9 mm) were pollinated by three species of settling noctuid moths whose proboscises varied in length from 10 to 16 mm. When a settling moth crawled on the spikes and probed the flowers for nectar, pollinia were placed on the moths’ legs rather than on other body parts. Our 5-year survey of pollinia movement and 3-year supplemental pollination experiments indicated that fruit and seed production in this orchid were not often pollen-limited at flower level. In a natural population in Shangri-La, Southwest China, the proportions of pollinia removal and deposition on stigmas by moth legs were 93.8% and 83.5%, respectively. This finding of efficient pollen transfer by the pollinators’ legs in H. aitchisonii adds a new example of diverse pollinia placement on pollinators (here settling moths) in the Orchidaceae. Key words: leg placement, nectar spur, Orchidaceae, plant–pollinator interaction, pollen transfer, pollination mode, settling moth.

1 Introduction Habenaria is a species-rich genus of terrestrial orchids with more than 800 species, mainly distributed in tropical and Most flowering plants use animal vectors to deliver pollen for subtropical areas (Batista et al., 2006; Kurzweil, 2009), and sexual reproduction, developing diverse mechanisms to diversified in southern and central Africa and east Asia promote pollination. It has been shown that pollen is placed (Dressler, 1993; Batista et al., 2006, 2013). Flowers of on specific sites of the pollinator body that are likely to Habenaria species are characterized by their long spurs, contact conspecific stigmas, favoring pollination accuracy with nectar available at the tip. Previous investigations (Armbruster et al., 2009, 2014). For example, in a guild of 15 indicated that Habenaria species were predominantly polli- orchid species in South Africa that are pollinated by an oil- nated by lepidopterans (Singer & Cocucci, 1997; Singer, 2001; collecting bee Rediviva peringueyi Friese (Melittidae), pollinia Singer et al., 2007; Peter et al., 2009; Pedron et al., 2012; from different orchids are placed on different sites of the Ikeuchi et al., 2015; Xiong et al., 2015; Zhang & Gao, 2017; Tao bee’s body (Pauw, 2006). One of the most famous examples et al., 2018), which usually have long proboscises. The pollinia of pollinator-mediated selection on the evolution of floral in Habenaria species were observed to be placed on various traits is the extraordinary spur length of a Madagascan orchid sites on moths’ or butterflies’ bodies, such as on their eyes, Angraecum sesquipedale Thouars, first described by Darwin heads, or at the base of their proboscises, depending on the (1862), and its hawkmoth pollinator Xanthopan morganii match between the nectar spur and pollinator proboscis Rothschild & Jordan with a matching proboscis length length (Singer & Cocucci, 1997; Singer et al., 2007; Peter et al., (Wasserthal, 1997). Later studies on orchids including Disa 2009; Pedron et al., 2012; Xiong et al., 2015; Johnson et al., P.J. Bergius (Johnson & Steiner, 1997), Habenaria Willd. 2016; Zhang & Gao, 2017; Tao et al., 2018). In Habenaria (Johnson & Raguso, 2016) and Platanthera Rich. species limprichtii Schltr., pollinia were observed attached to (Nilsson, 1983, 1988; Schiestl & Schluter, 2009; Boberg et al., sphingids’ eyes but sometimes attaching to settling noctuid 2014), examining the mechanical fit between nectar spur moths’ legs (Tao et al., 2018). Although animal feet or legs length and pollinator mouthparts, indicated that a mismatch have been observed carrying and delivering pollinia (Frost, between pollinator proboscis and nectar spur length greatly 1965; Ollerton et al., 2003; Peter et al., 2009), few plants reduced pollinia removal and receipt, supporting the hypoth- whose pollinia are transferred only by the pollinators’ feet or esis of an effect of pollinator-mediated selection on floral legs are known (Eisikowitch, 1986; Liede & Whitehead, 1991; evolution. Johnson & Brown, 2004; Coombs et al., 2009).

XXX 2018 | Volume 9999 | Issue 9999 | 1–8 © 2019 Institute of Botany, Chinese Academy of Sciences 2 Xiong et al.

Here we report a new case of pollinia transfer by pollinator EL200; Ocean Optics, Dunedin, FL, USA), with a fiberoptic legs in Habenaria aitchisonii Rchb. f. from the Hengduan reflection probe (QR400-7-SR; Ocean Optics) held at 45° to the Mountains, Southwest China. It is a terrestrial perennial sepals, petals, and lip surface (Wang et al., 2018) in the evening. orchid, having a relatively short nectar spur and greenish- yellow flowers with noticeable scent. To detect the potential 2.4 Floral scent pollinators, we measured floral traits including size, scent, and Floral scents were collected in the field station when smell spectral reflectance of flowers as well as nectar. In 2012 and was strong using dynamic head-space collection methods as 2013, we recorded the type of pollinators and their visitation described by Edens-Meier et al. (2014). An oven bag (Reynolds behavior to examine whether the pollinia are only transferred Oven Bag; Reynolds, Lake Forest, IL, USA) cut to a dimension by the pollinator legs. We measured the length of nectar spurs of 15 20 cm was used to cover each inflorescence bearing and pollinator proboscises to examine the mechanical fit fresh flowers and sealed at the bottom using a twist tie. An between flowers and pollinators. To investigate the pollen adsorbent tube was made from a glass pipette with 100 mg transfer efficiency of leg placement of pollinia in this species, porous polymer adsorbent (Porapak Q, 80–100 mesh; Sigma- we surveyed pollinia movement (percentage of flowers with Aldrich, Darmstadt, Germany) packed between plugs of glass pollinia removal and receipt per individual plant) and wool. One end of the adsorbent tube was attached to a reproductive success (fruit set per plant and seed set) over battery-operated vacuum pump (Libra-4; AP Buck, Orlando, 5 years in 2011–2013 and 2016–2017. To examine whether fruit FL, USA) with Teflon tubing (Norprene Chemical, Professional and seed production were limited by pollen availability, we Plastics, Fullerton, CA, USA). The other end of the tube was compared fruit and seed set between natural and supplemen- sealed into the top of the oven bag using a twist tie. Floral tal pollination treatments in 2011–2013. scent was collected for 2 h from 19:30 to 21:30 local time on 2016 at a flow rate of 150 mL air/min. Ambient air control was included to account for non-floral compounds. After sampling, 2 Material and Methods the compounds in the adsorbent tube were eluted into 2 mL Agilent autosampler vials (Agilent Technologies, Santa Clara, 2.1 Study species CA, USA) using 1500 mL gas chromatography–mass spectrom- Habenaria aitchisonii is a terrestrial orchid that usually grows etry (GC-MS) grade hexane. All collected sample vials were under forests and shrubs or in open meadows from 2200 to sent to Wuhan University Test Center (Wuhan, China) for GC- fl 3600 m a.s.l. in Southwest China. It has two eshy leaves lying MS analyses. We collected scent from two replicate fl – at on the ground, with a 15 30 cm long erect raceme bearing inflorescences and one ambient air control at the same time. fl multiple owers. These are greenish-yellow, with a strong and One microliter of each sample was injected into a GC system pleasant fragrance at night. The lateral sepals and lateral lip (Agilent GC6890; Agilent Technologies), with flame ionization fl lobes of H. aitchisonii are strongly re exed, allowing the viscidia detector as a detector. All analyses were done using splitless and stigma lobes to be touched easily during the pollination injections on a polar GC column (diameter, 0.25 mm; length, process (Fig. 1). Two separate viscidia lie below the anther sacs 30 m; film thickness, 0.25 mm [FFAP]; Agilent Technologies). containing the pollinia, which consist of many massulae. The Some samples were also analyzed on a non-polar column two lobes of the stigma are located on either side of the (diameter, 0.32 mm; length, 30 m; film thickness, 0.25 mm [DB- entrance to the spur, which contains visible nectar at the tip. It 5]). The carrier gas was nitrogen with a flow rate of 0.9 mL/ fl owers from early July to late August. The fruits mature in mid- min, a split ratio of 12, injection port temperature of 240 °C, October and each contains hundreds of dust-like seeds. and detector temperature of 250 °C. The oven program began Our investigation took place in a large population with with injection at 60 °C and a constant temperature for 5 min. hundreds of individuals in Shangri-La Alpine Botanical Garden Oven temperature increased by 10 °C per minute up to 250 °C, fi (SABG), a eld station of our university in northwestern where it was then held for 7 min. Compounds were identified 0 00 0 00 – Yunnan, China (27°54 5 N, 99°38 17 E, 3300 3350 m a.s.l.; see by comparing retention times with known standards bought Xiong et al., 2015). from Sigma-Aldrich (St. Louis, MO, USA).

2.2 Spur and nectar measurement 2.5 Pollinator observations In 2012, we labeled 41 plants and selected one flower on each Pollinator visits to flowers were observed in the field from July raceme at random in SABG. We measured the length of spur to August in 2012 and 2013. In the daytime, pollinators were (the length of the floral spur from its entrance under the observed for more than 20 h from 10:00 to 17:00 on sunny days column to its tip) and nectar (the nectar length inside the spur (see Fang & Huang, 2012, 2013). Night-time observations took from the upper surface of the nectar to the tip of the spur) place from 18:30 to 22:00; one observer focused on a plot with digital calipers to an accuracy of 0.01 mm. After that, the containing approximately 10 flowering individuals. After dusk spur tip was cut off with microscissors and nectar volume was we used a small flashlight covered with thick red plastic film to estimated using a 20-mL micropipette. Nectar concentration assist night-time observation (see Xiong et al., 2015). was measured with a hand-held sucrose refractometer Nocturnal pollinators were observed for at least 10 h per (Bellingham and Stanley, 0–45%; Xylem, Rye Brook, NY, USA). person on four or five nights each year. To examine the mechanical fit between nectar spurs and 2.3 Spectral reflectance of flowers pollinator proboscides, we caught 5–7 individuals per species We measured the spectral reflectance of sepals, petals, and lips of visitor to H. aitchisonii using insect nets. The caught moths of H. aitchisonii.Fiveflowers from different individuals were were anesthetized by chilling for approximately 30 s in a examined at 300–700 nm range using a spectrometer (JAZ- 20 °C refrigerated cabinet, and then the proboscides were

J. Syst. Evol. 9999 (9999): 1–8, 2018 www.jse.ac.cn Pollinia transfer by moths’ legs 3

Fig. 1. Flowers and visitors of Habenaria aitchisonii. A, Greenish-yellow flowers on a raceme; the green nectar spur is marked by an arrow. B, A moth Autographa gamma (Noctuidae) probing for nectar on an inflorescence. C, A moth Heliophobus sp. (Noctuidae) with pollinia attached to its legs (arrows). D, A moth Thysanoplusia intermixta (Noctuidae) sucking nectar on the inflorescence, with several pollinia attached to each of its legs (arrows). carefully unrolled, pinned and measured using digital calipers deposited (ms) on two stigma lobes on each of five flowers to an accuracy of 0.01 mm, without killing the moth. from 25 individuals. The number of massulae per pollinium (m) was determined from 30 pollinia from different flowers, 2.6 Pollinia removal and receipt following the method described by Johnson & Brown (2004). To estimate pollination success, we examined pollinia removal The pollen transfer efficiency (PTE) equals the number of and receipt in the late flowering stages of H. aitchisonii in 2011– massulae per stigma divided by the total number of massulae 2013, 2016, and 2017 (Xiong et al., 2015). We recorded pollinia per flower (PTE ¼ ms/(m pr), Johnson et al., 2005). receipt by checking for massulae on stigmas using a 20 hand magnifier. The percentage of pollinia removal and receipt was 2.7 Pollination experiments calculated as the number of flowers with pollinia removed and To test whether fruit and seed production were limited by received divided by the number of flowers examined. In 2012 pollen availability in H. aitchisonii, we undertook supplemental to 2013, we counted pollinia removed (pr) and massulae pollination treatment over 3 years from 2011 to 2013. Twenty www.jse.ac.cn J. Syst. Evol. 9999 (9999): 1–8, 2018 4 Xiong et al.

flowers on different individuals were hand-pollinated with pollinia collected from other individuals at least 10 m away (outcross pollination). One month later, we calculated fruit set as the number of expanded fruits divided by the number of flowers hand-pollinated. Fifteen fruits resulting from supple- mentary treatment and another 15 fruits from open-pollinated flowers (control) were harvested when the fruits were nearly mature. We counted developed embryos in mature fruits and calculated seed set per fruit (Xiong et al., 2015). Fruit set and seed set of open-pollinated plants were estimated for 5 years from 2011 to 2013 and 2016 to 2017.

2.8 Data analysis To examine the mechanical match between flowers and pollinators, the length of pollinator proboscises and nectar spurs of flowers were compared using a generalized linear model (GLM) with normal distribution and identity-link function. To investigate pollinia transfer efficiency in H. aitchisonii, we used Pearson’s correlation analysis to test whether pollinia receipt correlates with pollinia removal. To Fig. 2. Spectral reflectance curves of flowers (sepal, petal, examine whether fruit and seed production are limited by and labellum) of Habenaria aitchisonii. pollen availability, fruit and seed set in 2011 to 2013 were compared between natural and supplemental pollination treatments using a GLM with binary distribution and logistic- three species of settling noctuid moths: Autographa gamma link function. Because of numerical problems, fruit set L., Thysanoplusia intermixta Warren, and Heliophobus sp. comparison in 2012 was carried out using one-way anova Boisduval, with proboscis lengths of 15.08 0.18, 16.03 0.13, instead of GLM analysis. All analyses were undertaken in IBM and 10.78 0.28 mm, respectively. On the three moth species, spss 20.0 and data are presented as mean standard error. pollinia were observed only adhering to their legs (Fig. 1). In all cases, the noctuid moth approached and slowly landed on the raceme and then inserted its proboscis into the nectar spur of a flower (Fig. 1). Noctuid moths usually spent a long time 3 Results searching the entrance of the spur and crawling on the 3.1 Floral morphology and nectar measurement inflorescence, taking approximately 2–3 min per inflores- Each plant produced a single raceme with 11–36 flowers cence. Several flowers on one raceme could be visited and (11.76 1.12 flowers/plant, n ¼ 25). The spur length ranged single flowers could be probed more than once and then the from 7.11 to 12.11 mm (8.94 0.12 mm) with an average of noctuid moth might move to nearby inflorescences to 2.97 0.13 mm length of nectar at the tip. The mean nectar continue foraging for nectar. volume was 2.43 0.15 mL and the mean sugar concentration While probing for nectar on a raceme, the settling noctuid was 27.11 0.60%. The nectar spur of Habenaria aitchisonii was moths needed to stand stably on the raceme and were shorter than the proboscises of all three species of noctuid observed placing their legs on the stigma lobes, viscidia, moths (Wald’s x2 ¼ 699.233, d.f. ¼ 3, P < 0.001), indicating a dorsal sepals, or lateral lip lobes. The leg standing on the mechanical mismatch between the length of nectar spur and viscidia or stigma lobes might have pollinia attached to it, or the pollinator proboscis. might deposit already-attached pollinia onto the stigma (Fig. 1). During a sequence of probes on the raceme, 3–6 3.2 Spectral reflectance of flowers pollinia were generally attached to each leg of the noctuid Spectral reflectance of H. aitchisonii revealed that the sepals, moth (Fig. 1), whose foraging behavior seemed hardly petals, and lip appeared similar in color to pollinators (Fig. 2). disturbed. The flower had an obvious spectral reflectance starting from 400 to 700 nm, with a peak at approximately 550 nm, 3.5 Pollinia removal and receipt corresponding with the green appearance to human eyes. We observed frequent pollinia movement in 2011–2013 and 2016–2017 (Fig. 3). Our 5-year survey showed that 93.8 1.2% 3.3 Floral scent of flowers had at least one pollinium removed, and 83.5 2.1% We identified 29 compounds from two headspace samples. of stigmas had massulae deposited on them (Fig. 3). The One aromatic benzenoid, o-xylene, and one aliphatic hydro- percentage of pollinia receipt was not significantly correlated carbon, 2, 6, 10, 14-tetramethylheptadecane, were found in with that of pollinia removal (Pearson’s correlation, 2011: both samples comprising a relatively high percentage of the r ¼ 0.271, P ¼ 0.147; 2012: r ¼ 0.396, P ¼ 0.050; 2013: r ¼ 0.276, total emission (Table 1). P ¼ 0.181; 2016: r ¼ 0.283, P ¼ 0.170; 2017: calculation was unnecessary because the percentage of pollinia removal 3.4 Pollinator behavior constantly equaled 100%). One pollinium contained on average All floral visits occurred in the dark after sunset between 20:30 97.5 4.7 massulae. Pollen transfer efficiency was 4.4% in and 22:30 local time. A total of 110 visits were recorded by 2012, 19.1% in 2013, 8.3% in 2016, and 6.5% in 2017.

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Table 1 Floral scent analyses using gas chromatography–mass spectrometry from two headspace samples of Habenaria aitchisonii Sample Compound Retention time (min) 1 2 Ethyl benzene 4.38 3.49 1.33 o-Xylene 4.51 22.32 5.76 a,b-Dimethylbenzeneethanol 4.81 13.20 a-Pinene 5.35 4.46 1-Chlorooctane 5.35 9.18 2,5-Dimethylhexane 5.47 0.28 7-Hydroxynorlumiflavin 5.47 0.59 Sabinene 5.89 2.74 b-Myrcene 5.95 4.29 (3,3-Dimethylbutyl)oxirane 5.98 4.07 3-Propyltoluene 6.22 9.85 2-Ethylhexanol 6.37 6.95 1.35 l-Limonene 6.43 11.53 1,8-Cineole 6.49 24.47 (2-Phenyl-1,3-dioxolan-4-yl)methyl octadecanoate 6.84 0.99 0.44 1,2,3,4-Tetramethylbenzene 6.92 0.98 0.84 7-Methoxymethyl-2,7-dimethylcyclohepta-1,3,5-triene 7.54 4.50 6.62 (2-Decyldodecyl)benzene 7.62 1.69 Benzyl oleate 7.64 0.64 Benzyl acetate 7.66 3.16 3-(1-Cyclopentenyl)furan 7.91 5.71 a-Terpineol 7.99 5.24 2,6-Dimethylheptadecane 8.17 0.99 Carvacrol 8.34 10.45 Mesitylacetic acid 8.88 2.47 rel-9-Octadecenoic acid 9.08 0.57 2,6,10,14-Tetramethylheptadecane 9.78 15.96 9.08 Behenyl behenate 10.30 2.17 2,6-Di-tert-butyl-4-ethylphenol 11.71 1.46 Number of compounds 16 20 Compounds are listed in order of increasing retention time. Sample values represent the percentage of total emission for each compound.

¼ ¼ ’ x2 ¼ ¼ 3.6 Pollination experiment F1, 53 5.758, P 0.020; 2013: Wald s 0.324, d.f. 1, Fruit set per plant did not differ significantly between open- P ¼ 0.569). Similarly, seed set per fruit did not differ pollinated flowers (2011–2013, mean standard error: significantly between open-pollinated flowers (74.2 2.5%) 86.5 1.5%) and those in the supplemental pollination and supplemental hand-pollinated flowers (86.5 1.8%) over treatment (2011–2013: 95.5 3.3%) over 3 years except in the 3 years except in 2013 (Fig. 4) (2011: Wald’s x2 ¼ 3.491, 2012 (Fig. 4) (2011: Wald’s x2 ¼ 0.597, d.f. ¼ 1, P ¼ 0.440; 2012: d.f. ¼ 1, P ¼ 0.062; 2012: Wald’s x2 ¼ 2.803, d.f. ¼ 1, P ¼ 0.094; 2013: Wald’s x2 ¼ 4.198, d.f. ¼ 1, P ¼ 0.040). These results indicated that fruit and seed production were not pollen- limited in this orchid in most years.

4 Discussion Our study provided a case of pollinia transfer by settling noctuid moths’ legs in an orchid with nectar spur, unlike other Habenaria species in which pollinia were usually attached to the head, eyes, or proboscis of pollinators. We observed that all removed pollinia were only attached to legs of settling noctuid moths (Fig. 1), which might be attributed to the combination of special flower structure and the mechanical Fig. 3. Percentage of pollinia removal and receipt in Habenaria mismatch: the proboscises of noctuid moths are longer than aitchisonii in 2011–2013 and 2016–2017. the nectar spurs. High percentages of pollinia movement www.jse.ac.cn J. Syst. Evol. 9999 (9999): 1–8, 2018 6 Xiong et al.

Habenaria species (Zhang & Gao, 2017; Tao et al., 2018). An analysis of floral scent from 15 moth-pollinated species showed that the typical floral scent usually contained terpenoids or their derivatives (Knudsen & Tollsten, 1993). Both benzenoids and terpenoids were observed in H. aitchisonii, suggesting that these odor components are attractive to moth pollinators. In contrast to the placement of pollinia on the head, eyes, and proboscis in previously studied Habenaria species, we observed pollinia placement on the legs of settling moths in H. aitchisonii.Whichflower features might facilitate pollinia transfer by legs? First, the strongly reflexed lateral sepals and lateral lip lobes expose the viscidia and stigma lobes to the pollinators, providing a structure on which a moth could place its legs and then undergo pollinia attachment when standing on the raceme (Fig. 1). Second, H. aitchisonii has a dense inflorescence tightly packed with dozens of flowers, allowing settling moths to move between flowers easily without flight. This situation is similar to the sunbird- pollinated orchid, Disa chrysostachya Sw., with a dense inflorescence and jutting-out viscidia and stigmas, so that pollinia would be deposited on the feet of a small bird Fig. 4. Fruit set per inflorescence (%) (A) and seed set per fruit standing on the inflorescence (Johnson & Brown, 2004). (%) (B)inHabenaria aitchisonii under natural pollination (open Actually, pollinia placement on animal feet or legs has been circle) in 2011–2013 and 2016–2017 and supplementary out- observed in several species, exemplified by some hymenop- crossing pollination treatment (closed circle) in 2011–2013. teran-pollinated species, such as Diascia longicornis Druce Stars indicate significant differences in fruit set or seed set (Vogel & Michener, 1985), Calotropis procera W.T. Aiton between treatments (P < 0.05). (Eisikowitch, 1986), Sarcostemma viminale (L.) R. Br (Liede & Whitehead, 1991), Disa bivalvata T. Durand & Schinz, Disa atricapilla Bolus (Steiner et al., 1994), Disa sankeyi Rolfe (removal and receipt) and generally no pollen limitation in (Johnson, 2005), Pterygodium catholicum Sw., P. alatum Sw., fruit and seed production at flower level were observed in this P. caffrum Sw., P. inversum Sw. (Pauw, 2006), and orchid, H. aitchisonii, indicating efficient pollinia transfer on Gomphocarpus physocarpus E. Mey. (Coombs et al., 2009). settling noctuid moths’ legs. To date, pollinia placement on legs has been observed only No diurnal insect was observed visiting flowers of H. in two moth-pollinated orchid species, Habenaria epipactidea aitchisonii over 40 h of daytime observations, perhaps Rchb.f.(Peteretal.,2009)andH. limprichtii (Tao et al., because of the inconspicuous flower color. It is believed 2018). The former orchid was pollinated by sphingids that flower color serves as a visual signal to attract diurnal (Sphingidae) when hovering in front of flowers to probe pollinators in various plants (Waser & Price, 1981; Delph & for nectar, trying to grapple the flower with the forelegs, to Lively, 1989; Newman et al., 2010; Sletvold et al., 2016). For which the pollinia became attached. In the latter species, a example, in Habenaria radiata Thunb. Spreng, the showy white number of pollinia were deposited on the thorax and legs of color of the petals and lips attracted diurnal butterflies as settling noctuid moths; however, these pollinia showed little pollinators (Ikeuchi et al., 2015). In our studied orchid, sign of erosion, implying that these pollinia placed on legs however, sepals, petals, and lip of flowers, with a relatively might contribute little to plant reproductive success. high reflectance in visible light (400–700 nm) and a peak Outside the Orchidaceae, pollinia have also been observed reflectance at 550 nm (Fig. 2), present a green color similar to on the legs of noctuid moth pollinators when these crawled the background of the habitat. on the inflorescence of Hoya carnosa (L. f.) R. Br. The three species of settling noctuid moths were observed (Apocynaceae) (Mochizuki et al., 2017). visiting flowers of H. aitchisonii with pollinia attached on their The nectar spurs of H. aitchisonii are much shorter than their legs (Fig. 1). This orchid, like other moth-pollinated species, pollinators’ proboscises, in contrast to previous studies on has inconspicuous flowers without showy colors (Fig. 2), but Habenaria, which indicated that only pollinators with probos- emits noticeable scent (Table 1) and provides nectar of cises roughly equal to or slightly shorter than the nectar spurs medium concentration (Peter et al., 2009; Tao et al., 2018). No could ensure efficient pollinia transfer and reproductive single compounds were predominant in the floral scent of H. success (Singer & Cocucci, 1997; Singer et al., 2007; Peter aitchisonii, but two aromatic benzenoids (ethyl benzene and et al., 2009; Pedron et al., 2012; Xiong et al., 2015; Johnson o-xylene) and four terpenoids (sabinene, b-myrcene, l- et al., 2016; Zhang & Gao, 2017; Tao et al., 2018). In other limonene, and 1,8-cineole) together accounted for approxi- words, the mechanical mismatch could result in an opposite mately 38% of the total floral scent emission, in accordance situation in which visitors probe flowers without touching the with floral scent examined in other moth-pollinated species. column and removing pollinia. For example, in Habenaria For example, benzenoids, such as benzyl acetate, benzalde- pleiophylla Hoehne & Schltr., ineffective sphingid visitors with hyde, and linalool, were dominant in some moth-pollinated much longer mouthparts could not remove pollinia from the

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flowers and consequently made no contribution to pollination Delph LF, Lively CM. 1989. The evolution of floral color change: (Singer et al., 2007). Because of the mismatch between the Pollinator attraction versus physiological constraints in Fuchsia length of the nectar spurs and the pollinator proboscis in H. excorticata. Evolution 43: 1252–1262. aitchisonii, pollinia placement on the legs of pollinators rather Dressler RL. 1993. Phylogeny and classification of the orchid family. than on their mouthparts or eyes is favored, given that the six Portland: Dioscorides. legs could all deliver pollinia effectively. Edens-Meier R, Raguso RA, Westhus E, Bernhardt P. 2014. Floral Pollen transfer efficiency in H. aitchisonii was relatively high, fraudulence: Do blue Thelymitra species (Orchidaceae) mimic with 9.6 2.8% over 4 years. This figure is similar to most orchids Orthrosanthus laxus (Iridaceae)? 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