Bull. Natl. Mus. Nat. Sci., Ser. B, 44(1), pp. 41–51, February 22, 2018

Floral Scent Profiles and Visitors in Species of Series Sakawanum ()

Satoshi Kakishima and Yudai Okuyama*

Department of Botany, National Museum of Nature and Science, Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan *E-mail: [email protected]

(Received 18 August 2017; accepted 20 December 2017)

Abstract To understand the potential link between the variation in floral scents and pollinators in Asarum, a diverse genus of Japan, we conducted analyses of floral volatile compositions as well as field monitoring of flower visitors in species of the genus series Sakawanum. We detected a remarkably large number of floral volatile compounds, and found they are dominated by aliphatics and terpenoids but poor in benzenoids. However, despite a relatively intensive effort, we failed to identify specific species of flower visitors likely contributing well for the cross pollination of these . Contradicting to the genetic evidence that these species are generally outcrossing, the visi- tation frequency of the winged insects for their flowers was likely to be low and thus it remained enigmatic how they successfully cross-pollinate in the wild population.

Key words: Asarum, floral scent, Heterotropa, pollination, SPME.

The Japan archipelago harbors a rich endemic theless, only a few species are examined for the flora and has been designated as one of the biodi- plant-pollinator interactions in the genus (Suga- versity hotspots (Boufford et al., 2005; Mitter- wara, 1988; Mesler and Lu, 1993). The paucity meier et al., 2011). There are 1862 species and of the information on pollination system of Asa- 847 varieties of endemic land plants in Japan, rum in Japan is probably due to several reasons. accounting a quarter of the plant species native to First, the frequency of pollinator visitation is not Japan (Kato and Ebihara, 2011). Although these high, second, the floral organs are enclosed with endemic plants are scattered across 515 plant calyx tube so that the pollinator behaviors are genera and 161 families, with their close rela- invisible after they entered inside the flower, and tives are often found in the surrounding area of lastly, many of the species are now endangered Japan, some plant lineages are especially species and not abundant enough to conduct field obser- rich in Japan. vations. All of the documented pollination sys- Among these, the genus Asarum is a plant tem of the genus so far, including that of A. genus with the third highest number of the tamaense in Japan, involve mushroom-feeding endemic species in Japan, followed by Carex dipterans as the principal pollinator (Sugawara, (Cyperaceae) and Cirsium (Asteraceae) (Suga- 1988), thereby mushroom mimic has been sug- wara, 2006; Okuyama, 2010; Kato and Ebihara, gested for their pollination strategy (Sinn et al., 2011). The species richness of the genus is repre- 2015). However, we noticed that the floral scents sented by the marked diversity of floral forms of the species in Japan are markedly variable and including color, shape, and size, suggesting that most of them are far from the mushroom-like varieties of plant-pollinator interactions have note, which is expected for the mushroom-mim- shaped the diversification of the genus. Never- icking flowers. Nevertheless, very little is known 42 Satoshi Kakishima and Yudai Okuyama about the variation of the floral scent profile of column (30 m × 0.25 mm; film thickness, 250 the genus, with only those of 7 species in Japan µm; Restek, Bellefonte, PA, USA). Helium was and Taiwan are documented so far (Azuma et al., used as the carrier gas at a velocity of 48.1 cm s－1, 2010). and the injector temperature was 250°C. The Here, as the initial attempt to illustrate the injector was operated in the splitless mode for diversity of the pollination systems and associ- 1 min. Electron ionization mass spectra were ated floral scent profiles within the genus, we obtained at a source temperature of 200°C. The report the flower visitors and the floral scent pro- oven temperature was programmed to the follow- files of the species of the Asarum series Sakawa- ing sequence: 40°C for 5 min, followed by an num (hereafter Sakawanum species), a well char- increase of 5°C/min to 210°C and then 10°C/min acterized small subclade of the genus in Japan. to 280°C, at which the oven was held for 5 min. The rerative peak area in the total ion chromato- Materials and Methods gram (TIC) was used as a rough estimate of the relative content of each compound in the sam- Chemical analyses ples. We studied all the species and varieties of the For every volatile compound, retention indices genus Asarum series Sakawanum, i.e., Asarum were calculated with n-alkane (C6–C20) stan- costatum, A. sakawanum var. sakawanum, A. dards (Wako, Tokyo, Japan). Then identification sakawanum var. stellatum, and A. minamitania- was made by comparing the mass spectra with num, where 3, 2, 2, 3 individuals of each were those in the libraries (NIST14 and NIST14s, sampled for floral scent analyses, respectively. National Institute of Standards and Technology, For each plant individual, 1–2 flowers (0–5 days USA) at the cutoff of 90% similarity and the after opening) was collected and placed in a retention indices with those reported in the NIST 100 ml glass vial sealed with aluminum foil. Vol- Chemistry WebBook (Linstrom and Mallard, atile compounds were collected for 30 min using 2012). Where available, the mass spectra as well head space-solid phase microextraction (HS- as the retention times for the individual com- SPME) with fibers of 100 µm Divinylbenzene/ pounds were also compared with the authentic Carboxen/Polydimethylsiloxane (DVB/CAR/ standards. PDMS; Supelco, Bellfonte, PA, USA). To distin- guish volatile compounds of flowers from those Time-lapse photography of the ambient air, the volatiles from an empty To monitor flower visitors, time-lapse photog- vial was used as the control. To examine the vol- raphy using Optio WG-4 cameras (Ricoh, Tokyo, atile compounds emitted from the vegetative Japan) were conducted at a wild population of A. part, a of an individual of A. costatum costatum and A. minamitanianum. For A. costa- (TBG160624) was also cut from the and tum, a total of 12 individuals were monitored on was analyzed for the volatile using the same 20–21 April and 14–16 May, 2016 at Nahari site method as for the flower. All the plants used in in Kochi prefecture (N33° 25′ 26.9″, E134° 2′ this study are cultivated in Tsukuba Botanical 13.6″). For A. minamitanianum, 3 individuals Garden of the National Museum of Nature and were monitored on 24–25 March, 2014 at Hyuga Science, Japan. site in Miyazaki Prefecture (exact location of the The samples were subjected to gas chromatog- site is not shown for conservation reasons). The raphy/mass spectrometry (GC/MS) with the camera was set in front of a plant individual, equivalent settings to those reported previously keeping ＞5 cm distance from the plants. The (Okamoto et al., 2015). Specifically, we used time-intervals between the shots were set to GCMS-QP2010SE system (Shimadzu, Kyoto, 2 min, as this is almost the minimum time-inter- Japan) equipped with an Rtx-5SilMS capillary val for camera battery to be sustained overnight Floral Scent and Flower Visitor in Asarum spp. 43

(＞20 h). Any animal individual touching the Flower visitors in the wild populations of A. upper surface of the calyx was count as a single costatum and A. minamitanianum visit, and the animal on the same flower taken in Using time-lapse photography, we monitored the subsequent shot was not count. flower visitors of A. costatum and A. minamitani- anum for 19–44 h (586–1338 photographs) per plant individual (Table 2). As the result, we Results detected 8–54 flower visits per plant individual Floral volatile compounds in Sakawanum species for A. costatum while detected only 5–8 flower We detected 35–73 volatile compounds in the visits per plant individual for A. minamitania- individual headspace samples. Overall, 133 floral num. Most (＞88%) of the flower visitors were volatile compounds were found from three Asa- flightless or ground dwelling animals (Table 3). rum species and one variety in the series Judging from the contiguous shots of the photo- Sakawanum, of which 12 were aliphatics, 11 graphs, some of the pictured animals clearly were C5-branched chain compounds including entered inside the calyx tube. This suggests their hemiterpenes, 30 were monoterpenes, 71 were potential as pollinators, although it was unclear sesquiterpenes, two were diterpenes, and one was whether the pollen attached on their body (Fig. the other class of compound, while the remaining 1). six were unidentified compounds (Table 1). Among these, methyl angelate, α-pinene, cam- Discussion phene, β-pinene, limonene, trans- and cis-β- ocimene, β-caryophyllene, trans-α-bergamotene, We detected 156 floral volatile compounds, and β-selinene were common among all samples. which is a remarkably large number for only We did not detect any benzenoids from the sam- three closely-related plant species, i.e., A. costa- ples, although they constitute a major class of tum, A. minamitanianum, and A. sakawanum. volatile compounds in most of the insect-polli- This was somewhat unexpected because the vol- nated flowers. Although the volatile composition atile compounds usually detected in a set of represented by the TIC peak area ratios was vari- closely related species are usually far less. For able among the plant individuals, there was a example, Okamoto et al. (2015) found only 27 clear tendency among the species. The TIC peak volatile compounds from the headspace of 13 area of A. costatum is dominated by the aliphatic species of the genus Mitella (Saxifragaceae), methyl angelate (37–87% of the total), while that although the results are not directly comparable of A. minamitanianum is dominated by sesquiter- as absorbents used for volatile collection are dif- penes such as humulene (16–18%) and ferent [SPME with fibers of DVB/CAR/PDMS in γ-curcumene (7–18%). The TIC peak area of A. the present study vs. Tenax-TA in Okamoto et al. sakawanum is dominated by both methyl angel- (2015)]. ate (6–22%) and sesquiterpenes such as In the present study, we could not discriminate β-caryophyllene (6–26%), somewhat an interme- the floral volatiles from volatiles of the damaged diate pattern between the former two species. tissue such as the cut . However, the The headspace volatile compositions of a young contents of volatile compounds detected in the leaf of A. costatum were dominated by sesquiter- present study are generally congruent with the penes, and the major compounds were trans-α- previous study using seven Asarum species (A. bergamotene (28%), unidentified compound 5 yaeyamense, A. lutchuense, A. hypogynum, A. (13%), trans-β-farnesene (9%), sesquicineole fudsinoi, A. dissitum, A. senkaku-insulare, and A. (6%), sesquiterpene 5 (5%), and methyl angelate tokarense) in that C5-branched chain ester and/or (5%). sesquiterpenes were the dominant compounds (Azuma et al., 2010). In Azuma et al. (2010), the 44 Satoshi Kakishima and Yudai Okuyama — — — — — — — — — — — — — — — — — — — — — — — 4.62 2.09 0.36 0.60 2.88 0.31 6.51 0.18 2.57 0.94 0.53 2.26 0.27 TBG160626 stellatum — — — — — — — — — — — — — — — — — — — — — — — — — 4.66 6.78 1.74 4.35 0.37 7.02 3.63 0.46 4.51 1.44 var. A. sakawanum var. 23.46 TBG160620 — — — — — — — — — — — — — — — — — — — — — — — 0.98 0.39 0.98 2.68 0.07 6.75 0.36 0.11 5.91 0.42 9.23 0.08 10.57 TBG160619 sakawanum — — — — — — — — — — — — — — — — — — 0.18 0.11 0.45 3.46 0.12 0.07 0.09 3.49 1.79 0.64 3.80 0.17 0.09 0.47 0.07 0.12 var. A. sakawanum var. 16.50 15.80 TBG146306 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.67 0.22 1.40 1.11 1.08 0.41 0.35 TBG152619 Asarum species of the series Sakawanum — — — — — — — — — — — — — — — — — — — — — — — — — — 0.15 0.09 1.67 0.51 0.33 0.13 0.31 0.22 3.85 0.44 TBG152618 A. minamitanianum — — — — — — — — — — — — — — — — — — — — — — — — — 0.90 0.31 1.96 1.14 0.99 0.54 0.39 2.31 0.19 0.14 0.77 TBG152617 — — — — — — — — — — — — — — — — 0.04 0.12 0.32 0.07 0.06 0.38 0.07 0.09 0.03 1.13 0.73 1.14 0.81 2.75 0.03 0.19 0.04 0.78 0.12 87.35 Acos1701 — — — — — — — — — — — — — — — — 0.12 0.72 5.06 0.55 0.11 0.20 0.07 7.16 3.42 6.17 7.70 1.01 2.70 0.08 9.77 1.15 0.07 4.16 0.15 37.73 A. costatum TBG160624 — — — — — — — — — — — — — — — 0.26 1.35 0.15 0.05 0.28 0.10 1.49 0.80 1.37 1.52 3.04 4.46 0.06 0.03 2.86 0.15 0.06 4.53 0.07 0.43 71.31 TBG160623 737 918 765 822 863 969 924 930 946 970 974 608 597 600 601 620 630 665 676 755 796 903 997 608 653 671 728 733 591 RI 1005 1014 1022 1027 1029 1036 1003 * A B A A A A A B A A B A A A A A A B A B B A B A B B C B B B Identification Table Table 1. Relative amount (%) of volatile compounds detected from the flower Compound C5-branched chain compounds 1 compounds chain C5-branched 3-Carene 4-Carene p-Cymene Limonene Eucalyptol trans- β -Ocimene Tricyclene Methyl 2-methylbutyrate Methyl angelate Methyl tiglate 3-Methyl-2(5H)-furanone α -Thujene α -Pinene Camphene Sabinene β -Pinene α -Phellandrene 2-Methylfuran 2-Butanone 1,2-Epoxybutane 2-Butanol 2-Methyl-1-propanol Aliphatics 1 Aliphatics 2 Methyl isobutyrate Aliphatics 3 Aliphatics 4 Aliphatics 5 3-Octanol 2-Methyl-3-buten-2-ol 2-Methyl-butanal sec-Butyl formate 2-Methyl-1-butanol 2-Methyl-2-butenal 2,3-Epoxybutane Monoterpenes C5-branched chain compounds Aliphatics Floral Scent and Flower Visitor in Asarum spp. 45 — — — — — — — — — — — — — — — — — — — — — — — 0.95 0.66 0.46 1.74 0.38 3.10 2.52 0.53 0.51 0.45 0.55 0.57 1.97 1.57 0.24 24.53 TBG160626 stellatum — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.57 9.64 0.71 1.62 1.69 0.44 2.71 1.75 1.40 1.31 1.56 var. A. sakawanum var. TBG160620 — — — — — — — — — — — 0.71 0.07 0.18 0.21 0.57 0.31 2.30 0.44 0.05 0.22 0.14 0.27 0.23 0.57 2.10 0.05 0.14 0.88 0.44 0.45 3.52 0.08 0.11 0.11 2.36 0.23 0.27 26.95 TBG160619 sakawanum — — — — — — — — — — — — — — — — — — — 0.35 0.11 6.32 0.46 0.26 1.01 0.62 0.20 0.26 0.05 0.45 0.16 0.86 0.89 0.07 2.05 0.09 0.13 1.53 0.10 var. A. sakawanum var. TBG146306 — — — — — — — — — — — — — — — — — — — — — — — — — — 0.75 2.73 0.73 0.58 4.67 6.83 8.86 0.96 0.58 0.50 0.96 2.80 0.41 TBG152619 — — — — — — — — — — — — — — — — 0.26 0.47 6.11 1.23 0.77 3.26 8.64 0.11 0.04 0.19 0.31 0.21 0.06 0.06 0.52 0.11 0.07 0.57 1.22 3.08 0.39 0.57 0.38 TBG152618 A. minamitanianum — — — — — — — — — — — — — — — — — — — — — 0.28 1.02 2.05 0.28 0.47 6.43 9.13 0.81 0.36 0.31 0.17 0.32 0.47 1.53 3.37 3.60 0.13 2.43 TBG152617 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.07 0.58 0.86 0.44 0.43 0.10 0.11 Acos1701 Table Table 1. (Continued) — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.22 0.85 0.09 2.69 0.78 0.68 0.06 0.17 1.73 0.08 0.38 A. costatum TBG160624 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.10 1.02 0.04 1.32 0.83 0.07 0.49 0.17 TBG160623 RI 1128 1128 1139 1143 1149 1169 1176 1186 1193 1405 1408 1412 1414 1418 1422 1425 1428 1431 1046 1056 1083 1098 1330 1228 1282 1345 1352 1334 1345 1352 1367 1374 1381 1382 1386 1388 1392 1401 1403 * B B B A C B A A B A B C B C A C B B B A C B B B B B B Identification Compound 4-Acetyl-1-methylcyclohexene α -Gurjunene Sesquiterpene 6 cis- α -Bergamotene α -Cedrene β -Caryophyllene Sesquiterpene 7 β -Caryophyllene isomer Sesquiterpene 8 trans- α -Bergamotene cis- β -Ocimene γ -Terpinene or Isoterpinolene Terpinolene Linalool trans-Alloocimene Elemene isomer Alloocimene isomer Camphor Ectocarpene endo-Borneol Menthol Monoterpene 1 Monoterpene 2 Thymol methyl ether Bornyl acetate Monoterpene 3 Monoterpene 4 Elemene isomer α -Cubebene Sesquiterpene 1 Sesquiterpene 2 α -Copaene Sesquiterpene 3 β -Bourbonene 7-epi-Sesquithujene β -Elemene Sesquiterpene 4 Sesquiterpene 5 Italicene Sesquiterpenes 46 Satoshi Kakishima and Yudai Okuyama — — — — — — — — — — — — — — — — — — — — — — — 0.65 1.22 0.49 0.74 3.03 0.49 0.18 7.10 6.08 0.19 2.81 0.70 8.80 0.25 0.42 1.48 0.28 TBG160626 stellatum — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 1.22 0.81 1.75 3.90 5.09 1.11 2.67 1.03 0.59 var. A. sakawanum var. TBG160620 — — — — — — — — — — — — — — — — — — — — 0.33 1.27 0.05 0.19 0.36 0.16 1.82 1.04 0.09 0.12 1.33 3.32 0.22 1.44 4.32 0.13 0.90 0.14 0.15 0.05 TBG160619 sakawanum — — — — — — — — — — — — — — 0.56 0.14 0.10 0.36 0.09 0.28 0.20 0.44 0.87 0.35 0.07 0.46 2.49 3.09 0.15 1.01 0.61 1.53 0.15 0.35 0.08 0.42 0.66 0.33 0.14 0.09 var. A. sakawanum var. TBG146306 — — — — — — — — — — — — — — — — — — — — — — — — — 1.47 1.88 0.24 0.28 1.03 1.16 8.38 0.73 0.39 0.67 3.45 0.76 16.16 16.68 10.10 TBG152619 — — — — — — — — — — — — — 1.61 1.00 0.06 0.21 0.38 0.08 0.56 0.95 0.33 0.20 2.25 0.22 0.10 1.16 7.72 0.35 1.90 1.32 5.68 0.52 0.35 0.81 1.74 0.97 0.03 18.29 14.61 TBG152618 A. minamitanianum — — — — — — — — — — — — — — — — — — — — — 0.62 0.89 0.09 0.76 0.34 0.14 0.65 0.43 0.51 6.90 0.39 0.41 4.82 0.09 0.40 2.96 0.33 18.21 18.26 TBG152617 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.07 0.15 0.41 0.05 0.08 0.04 0.14 Acos1701 Table Table 1. (Continued) — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.25 0.44 1.35 0.07 0.18 0.17 0.13 0.69 A. costatum TBG160624 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.15 0.22 0.38 0.07 0.10 0.07 0.20 0.16 0.05 0.06 0.11 TBG160623 RI 1511 1522 1525 1530 1535 1539 1539 1436 1439 1443 1445 1447 1447 1449 1452 1454 1459 1459 1466 1466 1467 1473 1476 1479 1482 1487 1489 1490 1493 1496 1499 1503 1506 1508 1512 1516 1542 1556 1581 1582 * B B B B B B C C B B B B B B B B B B B B B B B B B C B Identification Compound α -Curcumene or Germacrene D α -Muurolene or -Amorphene 7-epi-trans-Sesquisabinene hydrate β -Sesquiphellandrene Sesquiterpene 20 Cadina-1,4-diene α -Cadinene Sesquiterpene 21 α -Bisabolene Aromandendrene Sesquisabinene isomer Cadina-3,5-diene Sesquiterpene 9 Seychellene Sesquiterpene 10 Sesquiterpene 11 trans- β -Farnesene Humulene cis-Muurola-4(15),5-diene 9-epi-Caryophyllene Sesquiterpene 12 Sesquiterpene 13 Sesquiterpene 14 γ -Muurolene γ -Curcumene Sesquiterpene 16 β -Selinene Viridiflorene γ -Amorphene Sesquiterpene 17 Sesquiterpene 18 α -Farnesene Sesquiterpene 19 β -Curcumene γ -Cadinene Sesquicineole δ -Cadinene Germacrene B Sesquiterpene 22 Sesquiterpene 23 Floral Scent and Flower Visitor in Asarum spp. 47 — — — — — — — — — — — — — — — — — 0.22 TBG160626 stellatum — — — — — — — — — — — — — — — — — — var. A. sakawanum var. TBG160620 — — — — — — — — — — — — — — — — — 0.04 TBG160619 sakawanum — — — — — — — — — 0.38 0.14 0.55 0.51 0.11 0.07 0.18 0.39 var. A. sakawanum var. 19.28 TBG146306 — — — — — — — — — — — — — — — — — — TBG152619 — — — — — — — — — — — — — — — 0.04 0.04 0.18 TBG152618 A. minamitanianum — — — — — — — — — — — — — — — — — — TBG152617 — — — — — — — — — — — — — — — — 0.08 0.16 Acos1701 Table Table 1. (Continued) — — — — — — — — — — — — — — 0.18 0.11 0.12 0.48 A. costatum TBG160624 — — — — — — — — — — — — — — — — — — TBG160623 965 RI 1152 2005 1588 1594 1594 1605 1644 1652 1667 1684 1721 2013 1395 1583 1658 1673 1882 * B B C C

Identification Compound 2-Methoxy-3-methyl-pyrazine Manool oxide Sesquiterpene 24 Sesquiterpene 25 Sesquiterpene 26 Sesquiterpene 27 Sesquiterpene 28 Sesquiterpene 29 Sesquiterpene 30 α -Bisabolol Sesquiterpene 31 Verticilla-4(20),7,11-triene unidentified 1 unidentified 2 unidentified 3 unidentified 4 unidentified 5 unidentified 6 Identification are based on A: mass spectrum and retention index of authentic compound, B: similarity of mass spectrum to those in the libraries and previously reported RI index in the NIST Diterpenes Other compounds Unidentified compounds * For C, identification are based solely on mass spectrum to those in the libraries and thus it is regarded as temporary identification. WebBook. Chemistry 48 Satoshi Kakishima and Yudai Okuyama

Table 2. The detailed information of time-lapse photography in the present study Plant Number of Photographing Number of Plant species Time of day Note Individual flowers date photographs A. costatum 1 2 April 20–21, 2016 12:19–12:55 741 Rainy for the last 3 h 2 2 April 20–21, 2016 12:13–13:27 758 Rainy for the last 3 h 3 1 April 20–21, 2016 11:59–12:25 734 Rainy for the last 2 h 4 2 April 20–21, 2016 11:55–12:01 724 Rainy for the last 2 h 5 1 April 20–21, 2016 12:25–13:27 752 Rainy for the last 3 h 6 1 May 14–15, 2016 15:29–13:27 660 May 15–16, 2016 13:37–11:27 656 Rainy for the last 8 h 7 1 May 14–15, 2016 14:59–14:21 702 May 15–16, 2016 14:32–11:40 635 Rainy for the last 8 h 8 2 May 14–15, 2016 14:36–13:40 693 May 15–16, 2016 14:03–11:31 645 Rainy for the last 8 h 9 1 May 14–15, 2016 15:13–14:39 704 May 15–16, 2016 14:44–11:36 627 Rainy for the last 8 h 10 1 May 14–15, 2016 14:22–13:04 682 May 15–16, 2016 13:12–11:18 664 Rainy for the last 8 h 11 1 May 14–15, 2016 14:18–13:16 690 May 15–16, 2016 13:23–11:19 659 Rainy for the last 8 h 12 2 May 14–15, 2016 14:52–12:58 664 May 15–16, 2016 14:38–9:40 572 Rainy for the last 8 h A. minamitanianum 1 2 March 24–25, 2014 14:25–13:21 689 2 4 March 24–25, 2014 14:32–10:02 586 3 1 March 24–25, 2014

Table 3. A list of the flower visitors photographed on each Asarum plant individual Plant individual Total Category of visitor A. costatum A. minamitanianum count 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 Diptera 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 2 Ants 1 0 1 3 5 8 3 38 3 0 0 4 0 0 0 66 Curculionidae 2 0 0 0 4 0 0 0 0 0 1 0 1 0 3 11 Other Coleoptera 1 0 0 2 0 0 4 1 5 0 1 0 0 0 0 14 Other Hexapoda 1 7 4 1 0 1 4 7 2 9 22 9 6 6 1 81 Isopoda 0 0 0 1 0 0 2 1 4 1 1 2 0 0 0 12 Myriapoda 0 1 0 2 0 1 0 1 2 0 0 3 0 0 0 10 (millipedes and centipedes) Araneae and other arachnids 0 1 0 0 0 0 3 1 2 1 2 4 1 0 1 16 Land snail 2 0 0 0 1 3 0 2 0 0 1 1 0 0 0 10 Land planarians 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 2 Unknown 2 1 3 1 3 0 2 3 3 2 3 4 0 0 0 27 Total 9 11 8 10 13 15 18 54 21 13 31 28 8 6 5 250

flowers were not removed from the plant body cific volatile compounds. and therefore the volatiles from the damaged tis- To understand the potential association sue were unlikely to be included. We also found between the variation of floral scents and the pol- that the leaf of A. costatum emitted sesquiter- lination systems in Asarum species, we also con- pene-dominated volatiles, the compositions of ducted monitoring of flower visitors in two spe- which are mostly shared with the flower of the cies of Asarum series Sakawanum. Especially in same plant individual (Fig. 2). Therefore, mono- A. costatum, a relatively intensive effort was terpenes and C5-branched chain compound, made for the monitoring, with a total of 12 plant methyl angelate could be the major flower-spe- individuals observed for ＞40 h each. Neverthe- Floral Scent and Flower Visitor in Asarum spp. 49

Fig. 1. A part of the flower visitors photographed in the present study. A: a ground-dwelling weevil visiting on A. costatum, B: a sciarid fly visiting on A. costatum, C: two weevils on A. minamitanianum, D: a collembolan visiting on A. minamitanianum. less, we failed to observe repeated visits by to address how the plant-pollinator interactions winged insects that are presumably effective as are associated with the floral scent profiles of the pollinator. Thus we could not identify a specific, Sakawanum species. Mushroom-mimic or other potential pollinator species in this study. The vis- type of brood-site mimicry is considered as the itation frequency of the flowers of the two Asa- pollination system of the Asarum species in gen- rum species were low, and most of the flower eral (Vogel, 1973; Sinn et al., 2015), and a few visitors were flightless animals (Table 3), which documented examples of the pollinators are are unlikely to contribute to the long-distance indeed mushroom-feeding flies (Sugawara, 1988; transportation of the pollen. Therefore, we failed Mesler and Lu, 1993), yet we could not find any 50 Satoshi Kakishima and Yudai Okuyama

Fig. 2. Chromatograms of the volatile samples collected from a flower and a leaf of A. costatum (TBG160624). The scale for signal intensity of the two chromatograms are adjusted. The peaks marked with numbers are the major volatile compounds. 1: 2-butanone. 2: 2-butanol. 3: 2-methyl-3-buten-2-ol. 4: 2-methyl-butanal. 5: 2-methyl-1-butanol. 6: methyl angelate. 7: α-pinene. 8: camphene. 9: β-pinene. 10: α-phellandrene. 11: p-cymene. 12: limonene. 13: eucalyptol. 14: cis-β-ocimene. 15: terpinolene or isoterpinolene. 16: bornyl ace- tate. 17: β-caryophyllene. 18: trans-α-bergamotene. 19: trans-β-farnesene. 20: unidentified compound 4. evidence that Sakawanum species are mimicking the floral scents were typically characterized by mushrooms [i.e., no mushroom-feeding insects the C8 alcohols and ketones, which are common were observed visiting on the flowers (Fig.1, in true mushroom fruiting bodies (Johnson and Table 3)]. Floral scent profiles of these species Schiestl, 2016) and we did not find any in the further suggest that they are not mushroom-mim- present study. icking flowers. In mushroom mimicking flowers, Recently, Takahashi et al. (2017) developed 17 Floral Scent and Flower Visitor in Asarum spp. 51

EST-SSR markers for the Asarum series Sakawa- and da Fonseca, G. A. B. (eds.), Hotspots Revisited: Earth’s Biologically Richest and Most Endangered num in Japan. Using these markers, they calcu- Ecoregions. University of Chicago Press, Chicago, IL, lated a basic genetic diversity metrics for all of USA. the Sakawanum species and found that they sel- Johnson, S. D. and Schiestl, F. P. 2016. Floral Mimicry. dom deviated from Hardy-Weinberg equilibrium. Oxford University Press, Oxford, UK. This indicates that they are largely outcrossing, Kato M. and Ebihara A. (eds.) 2011. Endemic Plants of or at least, only the produced by outcross- Japan. A Book Series from the National Museum of Nature and Science No. 11. Tokai University Press, ing could give rise to the flowering individuals. Hadano (in Japanese). Therefore, it remained enigmatic how these Linstrom, P. J. and Mallard, W. G. 2012. NIST Chemistry plants sustain their reproduction with the very- WebBook, NIST Standard Reference Database Number low pollinator visitation frequency. 69. Gaithersburg, MD: National Institute of Standards It might be still early to conclude that their and Technology, 20899. http://webbook.nist.gov. Mesler, M. R. and Lu, K. L. 1993. Pollination biology of pollinator visitation frequency is extremely low, (Aristolochiaceae): An evaluation of because the efficient observation of flower visi- Vogel’s mushroom-fly hypothesis. Madroño 40: 117– tors could be hindered by the floral characteristic 125. of Asarum. Asarum flowers often bloom at the Mittermeier, R. A., Turner, W. R., Larsen, F. W., Brooks, ground and the floral organs are hidden inside the T. M. and Gascon, C. 2011. Global biodiversity conser- vation: the critical role of hotspots. In: Zachos, F. E. calyx tube, which could discount the possibility and Habel, J. C. (eds.), Biodiversity Hotspots, pp. of the time-lapse photography to detect the 3–22. Springer, Heidelberg. flower visitors. Therefore, further research effort Okamoto, T., Okuyama, Y., Goto, R., Tokoro, M. and might be necessary to elucidate their reproduc- Kato, M. 2015. Parallel chemical switches underlying tive system. Such information will also help pollinator isolation in Asian Mitella. Journal of Evolu- designing the strategy for conservation of the all tionary Biology 28: 590–600. Okuyama, Y. 2010. Which genus to study? In search of four taxa of Asarum series Sakawanum, given plant genera underrepresented or overrepresented in the that they are listed in the Red List of Japan (A. research from the flora of Japan. Bulletin of the costatum: NT, A. minamitanianum: CR, A. National Museum of Nature and Science Series B 36: sakawanum var. sakawanum: VU, A. sakawanum 81–89. var. stellatum: EN). Sinn, B. T., Kelly, L. M. and Freudenstein, J. V. 2015. Putative floral brood-site mimicry, loss of autonomous selfing, and reduced vegetative growth are significantly Acknowledgment correlated with increased diversification in Asarum (Aristolochiaceae). Molecular Phylogenetics and Evo- We thank Masami Saito for guiding YO to the lution 89: 194–204. population of A. minamitanianum, and Tomoko Sugawara, T. 1988. Floral biology of Heterotropa tamaensis (Aristolochiaceae) in Japan. Plant Species Okamoto for critical comments on the prelimi- Biology 3: 7–12. nary version of the manuscript. Sugawara, T. 2006. Asarum. In Iwatsuki, K., Boufford, D. E. and Ohba, H. (eds.), Flora of Japan IIa. Kodansha, Tokyo. References Takahashi, D., Sakaguchi, S. and Setoguchi, H. 2017. Azuma, H., Nagasawa, J. I. and Setoguchi, H. 2010. Flo- Development and characterization of EST-SSR markers ral scent emissions from Asarum yaeyamense and in Asarum sakawanum var. stellatum and cross-amplifi- related species. Biochemical Systematics and Ecology cation in related species. Plant Species Biology 32: 38: 548–553. 256–260. Boufford, D. E., Hibi, Y. and Tada, H. 2005. Japan In: Vogel, S. 1973. Fungus gnat flowers and fungus mimesis. Mittermeier, R. A., Robles Gil, P., Hoffmann, M., Pil- In Brantjes, N. B. M. and Linskens, H. F. (eds.), Polli- grim, J., Brooks, T., Mittermeier, C. G., Lamoreux, J. nation and Dispersal. Nijimegan, Netherland.