The floral scents of Nymphaea subg. Hydrocallis (Nymphaeaceae), the New World night-blooming water lilies, and their relation with putative pollinators Author Maia, Artur Campos Dália Departamento de Química Fundamental, Universidade Federal de Pernambuco, Recife 50740 - 560, Brazil Author Lima, Carla Teixeira de Author Navarro, Daniela Maria do Amaral Ferraz Author Chartier, Marion Author Giulietti, Ana Maria Author Machado, Isabel Cristina text Phytochemistry 2014 2014-07-31 103 67 75 http://dx.doi.org/10.1016/j.phytochem.2014.04.007 journal article 10.1016/j.phytochem.2014.04.007 1873-3700 10489776 2.1. Floral scent composition of Nymphaea subg. Hydrocallis The six species and two subspecies of Nymphaea subg. Hydrocallis investigated are all native to Brazil and three of them are known to be associated with pollinator cyclocephaline scarabs ( Fig. 1 ; Table 1 ). Fig. 1. Flowers of the investigated species of Nymphaea subg. Hydrocallis ( Nymphaeaceae ): (A) Nymphaea amazonum subsp. amazonum (staminate phase), (B) Nymphaea amazonum subsp. pedersenii (staminate phase), (C) Nymphaea gardneriana (staminate phase), (D) Nymphaea lasiophylla (pistillate phase), (E) Nymphaea lingulata (staminate phase), (F) Nymphaea rudgeana (staminate phase), (G) Nymphaea tenerinervia (staminate phase). To the human nose, flowers of the seven investigated taxa of Nymphaea subg. Hydrocallis were remarkably fragrant during the consecutive evenings of the pistillate ($) and staminate (#) phases of anthesis. While the flowers of N. rudgeana and N. gardneriana emitted a pungent, fermented fruity odor with solvent-like reminiscents, the scents of the remaining species all bore a strong, uncharacteristic solvent-like odor ( Table 1 ). The chemical analysis showed that the seven studied taxa emitted floral volatiles in different quantities and compositions. A total of 22 compounds were identified in the analyzed samples, ranging in molecular weight from 102 [methyl butanoate] ( 1 ) to 192 [benzyl 2-methylbutanoate] ( 2 ) ( Fig. 2 ; Table 2 ). The identified volatile compounds belong to three of the seven compound classes proposed by Knudsen et al. (2006) : aliphatics (9), C5-branched chain compounds (5) and aromatics (8). The number of compounds per species ranged from only two in N. lingulata , N. amazonum and N. tenerinervia , to 12 in N. lasiophylla ( Table 2 ). Dominant compounds reaching an overall relative percentage content of at least 10% in the analyzed scent samples were methyl hexanoate ( 3 ) in N. rudgeana (36.3–77.8% $; 81.0–97.7% #) and N. gardneriana (21.9% $); methyl 2-methylbutanoate ( 4 ) in N. lasiophylla (0.8–12.5% $; 0.4–18.0% #) and N. gardneriana (74.0% $); (methoxymethyl)benzene ( 5 ) in N. rudgeana (20.2–61.7% $; 1.2–15.7% #), N. lasiophylla (81.6–95.1% $; 75.1–97.2% #) and N. Table 1 The investigated species of Nymphaeae subg. Hydrocallis ( Nymphaeaceae ) with description of the floral scents and known biogeographical distribution and pollinator associations.

Species	Description of floral scent	Biogeographical distribution *	Known association with pollinators †
Nymphaea amazonum pedersenii	Solvent-like	Central Brazil (Mato Grosso)	Cyclocephala epistomalis
Wiersema
Nymphaea amazonum amazonum	Solvent-like	Tropical South America and Caribbean Islands; Central	Cyclocephala verticalis
Mart. & Zucc.	America (up to Mexico)
Nymphaea gardneriana Planch.	Pungent, fermented, fruity,	South America (Venezuela, Brazil, Bolivia, Paraguay,	–
solvent-like	Argentina)
Nymphaea lasiophylla Mart. & Zucc.	Solvent-like	Coastal states of eastern Brazil	Cyclocephala nr. putrida
Nymphaea lingulata Wiersema	Solvent-like	Brazil (Maranhão, Goiás, Minas Gerais)	–
Nymphaea rudgeana G. Mey.	Pungent, fermented, fruity,	Eastern and northern South America; Central America;	Cyclocephala castanea ; C.
solvent-like	Caribbean islands	verticalis
Nymphaea tenerinervia Casp.	Solvent-like	Northern and northeastern Brazil	–

* Data from Wiersema (1987) and Amaral (2013) . † Data from Moore and Jameson (2013) . O Fig. 2. Structures of compounds 1–11 . Table 2 Chemical composition (amounts of each compound) of the floral scent of seven species of Nymphaea subg. Hydrocallis (Nymphaeaceae). Floral scent samples were obtained by dynamic headspace during the interval of highest perceivable odor emission in the course of the pistillate (day 1; $) and staminate (day 2; #) phases of anthesis.

Species list	RI	N. rudge	N. lasio	N. lingu	N. amaz	N. peder	N. gard	N. tener
$ ( n = 6)	# ( n = 2)	$ ( n = 2)	# ( n = 4)	$ ( n = 1)	# ( n = 2)	$ ( n = 4)	# ( n = 7)	$ ( n = 1)	# ( n = 1)	# ( n = 1)	# ( n = 1)
Total number of compounds	8	7	8	11	2	2	1	3	3	2	11	3
Total amount of scent per flower (µg h – 1)	1368–2123	973–1554	328–651	270–475	651	313–528	56–330	43–278	445	333	275	121
Aliphatics
Esters
Methyl butanoate	<800	–	–	–	–	–	–	–	–	–	–	0.53	–
Butyl acetate	815	–	–	–	–	–	–	–	–	0.04	–	–	0.05
Methyl hexanoate	928	36.28–77.78	81.03–97.69	0–0.05 a	0–0.42b	–	–	–	0–1.96b	–	–	21.94	–
Methyl pentanoate	824	–	–	–	–	–	–	–	–	–	–	0.02	–
( Z )-methyl hex-3-enoate	933	–	–	–	–	–	–	–	–	–	–	0.05	–
( E )-methyl hex-3-enoate	937	–	–	–	–	–	–	–	–	–	–	tr	–
( E )-methyl hex-2-enoate	966	0.03–0.47	0.10–0.20	–	–	–	–	–	–	–	–	0.03	–
Methyl heptanoate	1026	0.01–0.08	0.03–0.08	–	–	–	–	–	–	–	–	0.02	–
Methyl octanoate	1126	0.03–0.25	0.11–0.21	–	–	–	–	–	–	–	–	–	–
C5-branched chain compounds
Esters
Methyl 2-methylbutanoate	<800	–	–	0.83–12.54	0.36–17.96	–	–	–	–	–	–	74.02	–
Ethyl 2-methylbutanoate	849	–	–	–	0–0.21a	–	–	–	–	–	–	–	–
Methyl 2-hydroxy-2-methylbutanoate	849	–	–	–	0–0.04b	–	–	–	–	–	–	–	–
Methyl tiglate	865	–	–	–	–	–	–	–	–	–	–	0.28	–
Methyl 3-hydroxy-2-methylpropanoate	893	–	–	0–0.02 a	0–0.07b	–	–	–	–	–	–	–	–
Aromatics
Alcohols
Benzyl alcohol	1033	0–0.02 c	–	0.09–0.88	0–0.87b	0.22	0.09–0.25	–	–	–	–	–	–
2-(4-methoxyphenyl)ethanol	1244	0–0.16d	0.05–0.18	–	–	–	–	–	0–2.72b	–	–	–	–
Aldehyde
Benzaldehyde	959	–	–	–	0–0.10b	–	–	–	–	–	–	–	–
Esters
Methyl benzoate	1095	–	–	0–0.04 a	0–0.05b	–	–	–	–	–	–	–	–
Benzyl 2-methylbutanoate	1388	–	–	–	0–0.01 a	–	–	–	–	–	–	–	–
Ethers
Anisole	917	–	–	2.31–4.88	2.15–6.89	–	–	99.66–100.00	88.98–100.00	99.91	99.94	98.94
(methoxymethyl)benzene	988	20.20–61.66	1.16–15.69	81.57–95.12	75.10–97.24	99.78	99.75–99.91	–	0–9.06b	0.01	0.02	3.10	1.00
1.4-dimethoxybenzene	1164	1.23–3.65	0.61–2.85	0–0.01 a

Fig. 3. Odor profiles of ten species of Nymphaea and two species of Victoria ( Nymphaeaceae ). Stacked bars represent the scent composition for each individual sample based on the relative amounts of the main chemical subclasses (top) and individual VOCs (bottom). ($) Sample collected during the pistillate phase of anthesis; (#) sample collected during the staminate phase of anthesis. ‘‘Other’’: chemical subclasses or individual VOCs that do not reach more than 3% relative abundance in any of the analyzed samples. Fig. 4. The ‘‘scent space’’ representation of ten species of Nymphaea and two species of Victoria ( Nymphaeaceae ) visualized in two dimensions by non-metric multidimensional scaling (NMDS) based on Bray-Curtis similarities of the floral scent composition: (A) VOCs accounting for À 1% of the overall floral scent composition of any of the species pooled into chemical subclasses (11 chemical subclasses; S = 0.06); (B) individual VOCs accounting for À 1% of the overall floral scent composition of any of the species (28 VOCs; stress values: S = 0.08). Characteristic VOCs or chemical subclasses in the area plots are highlighted by small red dots. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) lingulata (99.8% $; 99.8–99.9% #); and anisole ( 6 ) in N. tenerinervia (98.9% $), N. amazonum subsp. amazonum (99.7–100.0% $; 89.0– 100.0% #) and N. amazonum subsp. pedersenii (99.9% $; 99.9% #) ( Figs. 2 and 3 ; Table 2 ). No marked differences in floral scent composition between pistillate and staminate phases of anthesis could be evidenced, with the sole exception of the analyzed samples of N. rudgeana ($, n = 4; #, n = 2), in which the emission of (methoxymethyl)benzene ( 5 ) was dramatically reduced during the staminate phase ( Fig. 3 ; Table 2 ). The rates of floral scent emission differed between species (PERMANOVA: df = 6, F = 46.709, R 2 = 0.9, P = 0.031) and phases of anthesis (PERMANOVA: df = 1, F = 5.043, R 2 = 0.016, P = 0.031). The interaction between these two factors had no significant effect (PERMANOVA: df = 4, F = 1.477, R 2 = 0.019, P = 0.262), meaning that variations of scent between the pistillate and the staminate phases showed the same pattern for each species: floral scent discharge was higher at the pistillate phase (865 ± 194 µg h – 1 , n = 16) than at the staminate phase (392 ± 97 µg h – 1 , n = 16). Among the different species from subg. Hydrocallis , only N. amazonum subsp. amazonum (144 ± 29 µg h – 1 , n = 11), N. lasiophylla (436 ± 54 µg h – 1 , n = 6), and N. rudgeana (1654 ± 138 µg h – 1 , n = 8) exhibited significantly different rates of floral scent emission. N. lingulata (497 ± 99 µg h – 1 , n = 3) and N. amazonum subsp. pedersenii (389 ± 56 µg h – 1 , n = 2) did not show any significant difference in floral scent emission rates when compared to any of the other sampled species, and only a single sample of N. tenerinervia (121 µg h – 1 ) and N. gardneriana (275 µg h – 1 ) each were analyzed.