- Email: [email protected]
Floral scent chemistry of Nuphar japonica (Nymphaeaceae)
Biochemical Systematics and Ecology 48 (2013) 211–214
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Floral scent chemistry of Nuphar japonica (Nymphaeaceae) Hiroshi Azuma* Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
a r t i c l e i n f o Article history: Received 19 April 2012 Accepted 26 December 2012 Available online 30 January 2013 Keywords: 1,4-Dimethoxybenzene Lilac aldehydes Limonene Pentadecane Undecane Water-lily
1. Subject and source Floral scent is an important factor in many flowering plants for attracting pollinators and has undoubtedly played a key role in the evolution and diversification of angiosperms (Pellmyr and Thien, 1986). However, the chemical profiles for the floral scents of most wild plants remain unknown (Knudsen et al., 2006). The genus Nuphar Sm., Nymphaeaceae, is one such group. Nuphar (ca. 7–25 species) is a rhizomatous, perennial, aquatic plant distributed in the temperate region of the Northern Hemisphere and is a taxonomically confusing genus (Heslop-Harrison, 1955; Beal, 1956; Schneider and Williamson, 1993; Padgett, 1998; Padgett et al., 1999; Shiga and Kadono, 2004). In Japan, 4 species have been recognized (Ito and Kadono, 2006). Nuphar japonica, which is restricted to Japan and Korea, is the most widely distributed species throughout Japan. The flowers of N. japonica used for scent collection were obtained between 2004 and 2011 from individual plants growing in streams or ponds in Shiga and Gifu Prefectures, Japan, and the Botanical Garden of Kyoto University. 2. Previous work Although the floral scent chemistries of a few species, including ornamental cultivars in the related genera of Nymphaeaceae (i.e., Victoria Lindl. and Nymphaea L.), have been documented (Mookherjee et al., 1990; Kite et al., 1991; Ervik and Knudsen, 2003), such a study has not yet been conducted for Nuphar. 3. Present study Three flowers showing different floral stages were sampled from each locality. The floral stages included the following: (1) a flower in which almost all the stamens were tightly appressed to the gynoecium (stage 1, probably the first day of anthesis), * Tel./fax: þ81 (0)75 753 4125. E-mail address: [email protected]. 0305-1978/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.12.019
212
H. Azuma / Biochemical Systematics and Ecology 48 (2013) 211–214
(2) a flower in which about half of the stamens (outside) were bent backward (stage 2), and (3) a flower in which almost all the stamens were bent backward (stage 3). A picked flower with a peduncle (ca. 15–20 cm in length) was immediately put in a conical flask (100 mL) containing about 50 mL of water. Then, the flower was enclosed in a polyester bag (250 mm 250 mm). A glass cartridge (7 mm 50 mm) containing 35 mg of adsorbent (Tenax GR 60/80 mesh; GL Sciences, Tokyo, Japan) and connected to a flow tube was inserted into the bag. Air composed of volatiles emitted from the flower or an P empty bag (negative control) was suctioned with a mini-pump (MP– 30; Sibata Scientific Technology, Tokyo, Japan) at a flow rate of 100 ml/min for 2 h. The volatiles trapped on the adsorbent were eluted with 300 ml diethyl ether and 2 ml of this eluent was used for GC–MS analysis. The analysis was performed using a GC-17A gas chromatograph coupled with a GC–MS-QP5000 ver. 3 mass detector (Shimadzu, Kyoto, Japan). A DB-1 fused-silica capillary column (30 m; inner diameter, 0.25 mm; film thickness, 0.25 mm) was Table 1 Chemical profiles of the floral scents of Nuphar japonica. Flower stage
RIa
Number of samples
Stage 1
Stage 2
Stage 3
5
5
5
AV. ng/flower/hr (%) Hydrocarbons Nonane Decane Undecane Undecanal Dodecane Tridecane Tetradecane 1-Pentadeceneb Pentadecane Hydrocarbon (unsaturated) Hydrocarbon (unsaturated) 8-Heptadeceneb Hydrocarbon (unsaturated) Heptadecane Hydrocarbon (unsaturated) Nonadecane Heneicosane Subtotal
900 1000 1100 1184 1200 1300 1400 1478 1500 1592 1667 1677 1669 1700 1872 1900 2100
124.9 34.0 827.1 11.0 4.5 39.7 26.4 91.6 206.0 3.7 36.1 69.3 11.2 4.7 13.8 3.6 2.4 1510.0
(4.8) (1.3) (31.9) (0.4) (0.2) (1.5) (1.0) (3.5) (7.9) (0.1) (1.4) (2.7) (0.4) (0.2) (0.5) (0.1) (0.1) (58.2)
16.6 14.9 390.9 10.4 4.9 44.1 26.4 80.0 256.6 4.0 43.1 53.3 10.9 6.1 16.3 5.6 3.4 987.5
(0.8) (0.7) (17.7) (0.5) (0.2) (2.0) (1.2) (3.6) (11.6) (0.2) (2.0) (2.4) (0.5) (0.3) (0.7) (0.3) (0.2) (44.8)
0.2 1.1 171.0 11.7 3.0 16.0 20.3 24.7 142.2 3.6 18.9 22.9 4.8 4.2 7.1 2.7 2.2 456.7
(0.0) (0.1) (15.6) (1.1) (0.3) (1.5) (1.9) (2.3) (13.0) (0.3) (1.7) (2.1) (0.4) (0.4) (0.6) (0.2) (0.2) (41.8)
Terpenoids Monoterpene Limonene g-Terpinene Terpinolen Linalool Lilac aldehyde Lilac aldehyde Lilac aldehyde Lilac aldehyde Lilac alcohol Lilac alcohol Sesquiterpene Sesquiterpene b-Elemene Caryophyllene Humulene Subtotal
973 1017 1050 1076 1085 1111 1119 1120 1135 1176 1186 1303 1375 1382 1409 1442
4.3 440.0 4.1 24.9 2.8 42.9 32.5 33.0 33.8 9.7 4.7 6.7 4.6 76.3 232.9 7.3 960.6
(0.2) (17.0) (0.2) (1.0) (0.1) (1.7) (1.3) (1.3) (1.3) (0.4) (0.2) (0.3) (0.2) (2.9) (9.0) (0.3) (37.0)
5.5 459.4 3.2 22.8 3.9 44.4 32.7 33.9 32.2 6.9 6.3 7.2 5.8 97.6 276.6 9.6 1048.4
(0.3) (20.8) (0.1) (1.0) (0.2) (2.0) (1.5) (1.5) (1.5) (0.3) (0.3) (0.3) (0.3) (4.4) (12.5) (0.4) (47.5)
0.6 208.8 0.2 6.0 1.0 13.0 10.9 9.1 11.0 6.8 5.1 1.4 5.5 86.1 211.3 7.6 584.4
(0.1) (19.1) (0.0) (0.5) (0.1) (1.2) (1.0) (0.8) (1.0) (0.6) (0.5) (0.1) (0.5) (7.9) (19.3) (0.7) (53.4)
Benzenoids Benzaldehyde Benzyl alcohol 1,4-Dimethoxybenzene Subtotal
926 1002 1129
30.2 31.0 58.3 119.5
(1.2) (1.2) (2.3) (4.6)
54.6 28.4 66.5 149.5
(2.5) (1.3) (3.0) (6.8)
2.7 5.5 36.8 45.0
(0.2) (0.5) (3.4) (4.1)
Unknowns Unidentified Unidentified Unidentified Subtotal
811 995 1213
3.5 –c 1.3 4.8
(0.1) (0.0) (0.1) (0.2)
4.2 0.7 15.4 20.3
(0.2) (0.0) (0.7) (0.9)
4.7 –c 2.9 7.6
(0.4) (0.0) (0.3) (0.7)
Total a b c
Retention index relative to n-alkanes (DB-1 column). Tentatively identified. Not detected.
2594.8
(100)
2205.7
(100)
1093.7
(100)
H. Azuma / Biochemical Systematics and Ecology 48 (2013) 211–214
213
used. The injection temperature was held at 40 C for the first 15 min, programmed to increase by 5 C/min to 250 C, and held at 250 C for 5 min. Helium was used as the carrier gas. After each initial analysis, a 1-ml aliquot of nonyl acetate solution (0.5 mg/ml dichloromethane) was added to the same sample as an internal standard and then immediately reanalyzed under the same conditions to calculate the rough amounts of the volatiles detected based on their peak areas (Azuma and Toyota, 2012). The volatile compounds were identified by comparing their GC retention times and MS spectra with those of the authentic compounds, or tentatively identified by MS spectra in the NIST 02 mass spectral library and retention indexes reported elsewhere (Adams, 2009; NIST Chemistry WebBook, http://webbook.nist.gov/chemistry/). The chemical profile of the floral scents is presented in Table 1. In total, 39 compounds were detected. The major scent compounds included undecane, limonene, caryophyllene, pentadecane, and nonane, as well as minor amounts of oxygenated monoterpenes, e.g. lilac aldehydes, and benzenoids, e.g. 1,4-dimethoxybenzene. There was no substantial difference among the chemical profiles of the different flower stages. However, undecane, the main volatile detected at stage 1, drastically decreased with the age of the flower. 4. Chemotaxonomic significance Most of the scent volatiles detected in the floral headspace of N. japonica are commonly found in many flowering plants (Knudsen et al., 2006). For example, limonene (the second most abundant compound at stage 1) and caryophyllene (the third most abundant compound at stage 1) are known to be the most commonly occurring scent volatiles in the class of monoterpenes and sesquiterpenes, respectively (Knudsen et al., 2006). Several n-alkanes, e.g. undecane (the most abundant compound at stage 1) and pentadecane (the fourth most abundant compound at stage 1) also commonly occur in floral scents; however, they are usually detected as minor or trace elements. Cases in which n-alkanes are dominant scent volatiles are rarely observed. For example, flowers of Magnolia acuminata (L.) L. and Magnolia denudata Desr., deciduous tree species, dominantly emitted pentadecane, which was not detected (or was a minor element) in other Magnolia species that were examined (Azuma et al., 1997). Interestingly, several cultivars of Nymphaea sp. emitted pentadecane and heptadecadiene dominantly with small amounts of benzenoid compounds (Mookherjee et al., 1990). In Nymphaea lotus, the main scent volatiles were 2,5-dimethoxytoluene and methyl 2-methoxy-3-methylbutanoate with small amounts of several n-alkanes (Ervik and Knudsen, 2003). Likewise, floral scent of Victoria amazonica cruziana (Nymphaeaceae) was mainly composed of methyl 2-methylbutanoate and methyl tiglate (methyl 2-methyl-2-butenoate) with trace amounts of n-alkanes (Kite et al., 1991). Although additional data of other species are required, the exclusive emission of common volatile compounds (i.e., n-alkanes, limonene, and caryophyllene) may give some insights into understanding the ancestral features of floral scent chemistry in Nymphaeaceae. That is, because Nuphar is the basal lineage and sister to a clade containing all the rest genera (Barclaya, Euryale, Nymphaea, Victoria) of Nymphaeaceae (Les et al., 1991; Borsch et al., 2011), and if the other species of Nuphar also show almost the same scent characteristics, the results in this study may suggest the floral scent characteristics in the ancestor of Nymphaeaceae. In addition, emission of n-alkanes as major scent volatiles has rarely known. Although they were recorded as minor or trace elements in many flowering plants (Knudsen et al., 2006), exclusive emission of these volatiles has only known in some Magnolia species (Azuma et al., 1997) and Nymphaea cultivars (Mookherjee et al., 1990). It is interesting to know whether emission of n-alkanes show any interaction with insects and, if so, which kinds of insect involved. It is known that various kinds of insects, i.e., flies, bees, and beetles, were recorded as flower-visitors in other species of Nuphar (Schneider and Moore, 1977; Ervik et al., 1995; Lippok and Renner, 1997; Lippok et al., 2000), however, pollinators of N. japonica are not known. But, overall floral structure together with sepal movement and protogyny indicates that Nuphar species seem to be adapted for beetle-pollination (Schneider and Moore, 1977). I am now interested in revealing which volatile compound is associated with beetles and/or flies and bees in Nuphar. Acknowledgments I thank Dr. Hiroshi Takahashi for giving access to the plant materials used in this study. This study was supported by Grantin-Aid for Scientific Research (KAKENHI 16770064 and 23570116) from JSPS (Japan Society for the Promotion of Science). References Adams, R.P., 2009. Identification of Essential Oil Components by Gas Chromatography, Mass Spectrometry, fourth ed. Azuma, H., Toyota, M., 2012. Biochem. Syst. Ecol. 41, 55. Azuma, H., Toyota, M., Asakawa, Y., Yamaoka, R., García-Franco, J.G., Dieringer, G., Thien, L.B., Kawano, S., 1997. Pl. Species Biol. 12, 69. Beal, E.O., 1956. J. Elisha Mitchell Sci. Soc. 72, 317. Borsch, T., Löhne, C., Mbaye, M.S., Wiersema, J., 2011. Telopea 13, 193. Ervik, F., Knudsen, J.T., 2003. Biol. J. Linn. Soc. 80, 539. Ervik, F., Renner, S.S., Johanson, K.A., 1995. Flora 190, 109. Heslop-Harrison, Y., 1955. J. Ecol. 43, 342. Ito, M., Kadono, Y., 2006. Flora of Japan vol. IIa, 356. Kite, G., Reynolds, T., Prance, G.T., 1991. Biochem. Syst. Ecol. 19, 535. Knudsen, J.T., Eriksson, R., Gershenzon, J., Ståhl, B., 2006. Bot. Rev. 72, 1.
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Les, D.H., Garvin, D.K., Wimpee, C.F., 1991. PNAS 88, 10119. Lippok, B., Gardine, A.A., Williamson, P.S., Renner, S.S., 2000. Am. J. Bot. 87, 898. Lippok, B., Renner, S.S., 1997. Pl. Syst. Evol. 207, 273. Mookherjee, B.D., Trenkle, R.W., Wilson, R.A., 1990. Pure Appl. Chem. 62, 1357. Padgett, D.J., 1998. Can. J. Bot. 76, 1755. Padgett, D.J., Les, D.H., Crow, G.E., 1999. Am. J. Bot. 86, 1316. Pellmyr, O., Thien, L.B., 1986. Taxon 35, 76. Schneider, E.L., Moore, L.A., 1977. Brittonia 29, 88. Schneider, E.L., Williamson, P.S., 1993. The Families and Genera of Vascular Plants II, p. 486. Shiga, T., Kadono, Y., 2004. Acta Phytotax. Geobot. 55, 107.
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Floral scent chemistry of Nuphar japonica (Nymphaeaceae) Hiroshi Azuma* Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
a r t i c l e i n f o Article history: Received 19 April 2012 Accepted 26 December 2012 Available online 30 January 2013 Keywords: 1,4-Dimethoxybenzene Lilac aldehydes Limonene Pentadecane Undecane Water-lily
1. Subject and source Floral scent is an important factor in many flowering plants for attracting pollinators and has undoubtedly played a key role in the evolution and diversification of angiosperms (Pellmyr and Thien, 1986). However, the chemical profiles for the floral scents of most wild plants remain unknown (Knudsen et al., 2006). The genus Nuphar Sm., Nymphaeaceae, is one such group. Nuphar (ca. 7–25 species) is a rhizomatous, perennial, aquatic plant distributed in the temperate region of the Northern Hemisphere and is a taxonomically confusing genus (Heslop-Harrison, 1955; Beal, 1956; Schneider and Williamson, 1993; Padgett, 1998; Padgett et al., 1999; Shiga and Kadono, 2004). In Japan, 4 species have been recognized (Ito and Kadono, 2006). Nuphar japonica, which is restricted to Japan and Korea, is the most widely distributed species throughout Japan. The flowers of N. japonica used for scent collection were obtained between 2004 and 2011 from individual plants growing in streams or ponds in Shiga and Gifu Prefectures, Japan, and the Botanical Garden of Kyoto University. 2. Previous work Although the floral scent chemistries of a few species, including ornamental cultivars in the related genera of Nymphaeaceae (i.e., Victoria Lindl. and Nymphaea L.), have been documented (Mookherjee et al., 1990; Kite et al., 1991; Ervik and Knudsen, 2003), such a study has not yet been conducted for Nuphar. 3. Present study Three flowers showing different floral stages were sampled from each locality. The floral stages included the following: (1) a flower in which almost all the stamens were tightly appressed to the gynoecium (stage 1, probably the first day of anthesis), * Tel./fax: þ81 (0)75 753 4125. E-mail address: [email protected]. 0305-1978/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.12.019
212
H. Azuma / Biochemical Systematics and Ecology 48 (2013) 211–214
(2) a flower in which about half of the stamens (outside) were bent backward (stage 2), and (3) a flower in which almost all the stamens were bent backward (stage 3). A picked flower with a peduncle (ca. 15–20 cm in length) was immediately put in a conical flask (100 mL) containing about 50 mL of water. Then, the flower was enclosed in a polyester bag (250 mm 250 mm). A glass cartridge (7 mm 50 mm) containing 35 mg of adsorbent (Tenax GR 60/80 mesh; GL Sciences, Tokyo, Japan) and connected to a flow tube was inserted into the bag. Air composed of volatiles emitted from the flower or an P empty bag (negative control) was suctioned with a mini-pump (MP– 30; Sibata Scientific Technology, Tokyo, Japan) at a flow rate of 100 ml/min for 2 h. The volatiles trapped on the adsorbent were eluted with 300 ml diethyl ether and 2 ml of this eluent was used for GC–MS analysis. The analysis was performed using a GC-17A gas chromatograph coupled with a GC–MS-QP5000 ver. 3 mass detector (Shimadzu, Kyoto, Japan). A DB-1 fused-silica capillary column (30 m; inner diameter, 0.25 mm; film thickness, 0.25 mm) was Table 1 Chemical profiles of the floral scents of Nuphar japonica. Flower stage
RIa
Number of samples
Stage 1
Stage 2
Stage 3
5
5
5
AV. ng/flower/hr (%) Hydrocarbons Nonane Decane Undecane Undecanal Dodecane Tridecane Tetradecane 1-Pentadeceneb Pentadecane Hydrocarbon (unsaturated) Hydrocarbon (unsaturated) 8-Heptadeceneb Hydrocarbon (unsaturated) Heptadecane Hydrocarbon (unsaturated) Nonadecane Heneicosane Subtotal
900 1000 1100 1184 1200 1300 1400 1478 1500 1592 1667 1677 1669 1700 1872 1900 2100
124.9 34.0 827.1 11.0 4.5 39.7 26.4 91.6 206.0 3.7 36.1 69.3 11.2 4.7 13.8 3.6 2.4 1510.0
(4.8) (1.3) (31.9) (0.4) (0.2) (1.5) (1.0) (3.5) (7.9) (0.1) (1.4) (2.7) (0.4) (0.2) (0.5) (0.1) (0.1) (58.2)
16.6 14.9 390.9 10.4 4.9 44.1 26.4 80.0 256.6 4.0 43.1 53.3 10.9 6.1 16.3 5.6 3.4 987.5
(0.8) (0.7) (17.7) (0.5) (0.2) (2.0) (1.2) (3.6) (11.6) (0.2) (2.0) (2.4) (0.5) (0.3) (0.7) (0.3) (0.2) (44.8)
0.2 1.1 171.0 11.7 3.0 16.0 20.3 24.7 142.2 3.6 18.9 22.9 4.8 4.2 7.1 2.7 2.2 456.7
(0.0) (0.1) (15.6) (1.1) (0.3) (1.5) (1.9) (2.3) (13.0) (0.3) (1.7) (2.1) (0.4) (0.4) (0.6) (0.2) (0.2) (41.8)
Terpenoids Monoterpene Limonene g-Terpinene Terpinolen Linalool Lilac aldehyde Lilac aldehyde Lilac aldehyde Lilac aldehyde Lilac alcohol Lilac alcohol Sesquiterpene Sesquiterpene b-Elemene Caryophyllene Humulene Subtotal
973 1017 1050 1076 1085 1111 1119 1120 1135 1176 1186 1303 1375 1382 1409 1442
4.3 440.0 4.1 24.9 2.8 42.9 32.5 33.0 33.8 9.7 4.7 6.7 4.6 76.3 232.9 7.3 960.6
(0.2) (17.0) (0.2) (1.0) (0.1) (1.7) (1.3) (1.3) (1.3) (0.4) (0.2) (0.3) (0.2) (2.9) (9.0) (0.3) (37.0)
5.5 459.4 3.2 22.8 3.9 44.4 32.7 33.9 32.2 6.9 6.3 7.2 5.8 97.6 276.6 9.6 1048.4
(0.3) (20.8) (0.1) (1.0) (0.2) (2.0) (1.5) (1.5) (1.5) (0.3) (0.3) (0.3) (0.3) (4.4) (12.5) (0.4) (47.5)
0.6 208.8 0.2 6.0 1.0 13.0 10.9 9.1 11.0 6.8 5.1 1.4 5.5 86.1 211.3 7.6 584.4
(0.1) (19.1) (0.0) (0.5) (0.1) (1.2) (1.0) (0.8) (1.0) (0.6) (0.5) (0.1) (0.5) (7.9) (19.3) (0.7) (53.4)
Benzenoids Benzaldehyde Benzyl alcohol 1,4-Dimethoxybenzene Subtotal
926 1002 1129
30.2 31.0 58.3 119.5
(1.2) (1.2) (2.3) (4.6)
54.6 28.4 66.5 149.5
(2.5) (1.3) (3.0) (6.8)
2.7 5.5 36.8 45.0
(0.2) (0.5) (3.4) (4.1)
Unknowns Unidentified Unidentified Unidentified Subtotal
811 995 1213
3.5 –c 1.3 4.8
(0.1) (0.0) (0.1) (0.2)
4.2 0.7 15.4 20.3
(0.2) (0.0) (0.7) (0.9)
4.7 –c 2.9 7.6
(0.4) (0.0) (0.3) (0.7)
Total a b c
Retention index relative to n-alkanes (DB-1 column). Tentatively identified. Not detected.
2594.8
(100)
2205.7
(100)
1093.7
(100)
H. Azuma / Biochemical Systematics and Ecology 48 (2013) 211–214
213
used. The injection temperature was held at 40 C for the first 15 min, programmed to increase by 5 C/min to 250 C, and held at 250 C for 5 min. Helium was used as the carrier gas. After each initial analysis, a 1-ml aliquot of nonyl acetate solution (0.5 mg/ml dichloromethane) was added to the same sample as an internal standard and then immediately reanalyzed under the same conditions to calculate the rough amounts of the volatiles detected based on their peak areas (Azuma and Toyota, 2012). The volatile compounds were identified by comparing their GC retention times and MS spectra with those of the authentic compounds, or tentatively identified by MS spectra in the NIST 02 mass spectral library and retention indexes reported elsewhere (Adams, 2009; NIST Chemistry WebBook, http://webbook.nist.gov/chemistry/). The chemical profile of the floral scents is presented in Table 1. In total, 39 compounds were detected. The major scent compounds included undecane, limonene, caryophyllene, pentadecane, and nonane, as well as minor amounts of oxygenated monoterpenes, e.g. lilac aldehydes, and benzenoids, e.g. 1,4-dimethoxybenzene. There was no substantial difference among the chemical profiles of the different flower stages. However, undecane, the main volatile detected at stage 1, drastically decreased with the age of the flower. 4. Chemotaxonomic significance Most of the scent volatiles detected in the floral headspace of N. japonica are commonly found in many flowering plants (Knudsen et al., 2006). For example, limonene (the second most abundant compound at stage 1) and caryophyllene (the third most abundant compound at stage 1) are known to be the most commonly occurring scent volatiles in the class of monoterpenes and sesquiterpenes, respectively (Knudsen et al., 2006). Several n-alkanes, e.g. undecane (the most abundant compound at stage 1) and pentadecane (the fourth most abundant compound at stage 1) also commonly occur in floral scents; however, they are usually detected as minor or trace elements. Cases in which n-alkanes are dominant scent volatiles are rarely observed. For example, flowers of Magnolia acuminata (L.) L. and Magnolia denudata Desr., deciduous tree species, dominantly emitted pentadecane, which was not detected (or was a minor element) in other Magnolia species that were examined (Azuma et al., 1997). Interestingly, several cultivars of Nymphaea sp. emitted pentadecane and heptadecadiene dominantly with small amounts of benzenoid compounds (Mookherjee et al., 1990). In Nymphaea lotus, the main scent volatiles were 2,5-dimethoxytoluene and methyl 2-methoxy-3-methylbutanoate with small amounts of several n-alkanes (Ervik and Knudsen, 2003). Likewise, floral scent of Victoria amazonica cruziana (Nymphaeaceae) was mainly composed of methyl 2-methylbutanoate and methyl tiglate (methyl 2-methyl-2-butenoate) with trace amounts of n-alkanes (Kite et al., 1991). Although additional data of other species are required, the exclusive emission of common volatile compounds (i.e., n-alkanes, limonene, and caryophyllene) may give some insights into understanding the ancestral features of floral scent chemistry in Nymphaeaceae. That is, because Nuphar is the basal lineage and sister to a clade containing all the rest genera (Barclaya, Euryale, Nymphaea, Victoria) of Nymphaeaceae (Les et al., 1991; Borsch et al., 2011), and if the other species of Nuphar also show almost the same scent characteristics, the results in this study may suggest the floral scent characteristics in the ancestor of Nymphaeaceae. In addition, emission of n-alkanes as major scent volatiles has rarely known. Although they were recorded as minor or trace elements in many flowering plants (Knudsen et al., 2006), exclusive emission of these volatiles has only known in some Magnolia species (Azuma et al., 1997) and Nymphaea cultivars (Mookherjee et al., 1990). It is interesting to know whether emission of n-alkanes show any interaction with insects and, if so, which kinds of insect involved. It is known that various kinds of insects, i.e., flies, bees, and beetles, were recorded as flower-visitors in other species of Nuphar (Schneider and Moore, 1977; Ervik et al., 1995; Lippok and Renner, 1997; Lippok et al., 2000), however, pollinators of N. japonica are not known. But, overall floral structure together with sepal movement and protogyny indicates that Nuphar species seem to be adapted for beetle-pollination (Schneider and Moore, 1977). I am now interested in revealing which volatile compound is associated with beetles and/or flies and bees in Nuphar. 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