# Interspecific and Intersexual Differences in the Chemical Composition of Floral Scent inGlochidion Species (Phyllanthaceae) in South China

Authors: Daihong Huang; Fuchen Shi; Minwei Chai; Ruili Li; Houhun Li
Journal: Journal of Chemistry
 (2015)

---

## Abstract

Plants of theGlochidion (Phyllanthaceae) genus are pollinated exclusively by host-specificEpicephala(Gracillariidae) moths. Floral scent has been thought to play key role in the obligate pollination mutualism betweenGlochidion plants andEpicephala moths, but few studies have been reported about chemical variation in floral volatiles ofGlochidion species in China. Floral volatiles of male and female flowers of fiveGlochidion species in south China were collected by dynamic headspace absorption technique and then were chemically analyzed by using gas chromatography-mass spectrometry. A total of 69 compounds were identified from floral scents of five investigated species.Glochidion hirsutum andG.zeylanicumshowed no qualitative differences in floral scent, whereas there were clear variations of floral scent among other species (G. eriocarpum,G.daltonii, andG. sphaerogynum) and also they distinctly differed from these two species. Male flowers emitted significantly more scent than female flowers.Glochidion plants exhibited qualitative and quantitative differences in floral scent between two sexes of flowers. The findings suggest that the volatile variation of floral scent amongGlochidion species reflects adaptations to specific pollinators. Sexual dimorphism in floral scent has evolved to signal alternative rewards provided by each sex toEpicephala moths.


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## Body

## 1. Introduction

Mutualism is cooperative interactions in which each associated species benefits from its partner [1, 2]. Obligate pollination mutualism between plants and their seed-parasitic pollinators is known as one of the most specialized plant-insect interactions [3–6], where insects usually exclusively pollinate for the host flowers in which they lay eggs; in turn the hosts offer seeds to feed pollinator larvae [7, 8]. The classical examples of such nursery pollination systems are the fig-fig-wasp [4] and yucca-yucca-moth interactions [6]. Moreover, the obligate pollination mutualism betweenEpicephala moths (Gracillariidae) and tribe Phyllantheae plants (Phyllanthaceae) has been also documented, including the associations ofEpicephala moths withGlochidion,Phyllanthus,Breynia,Flueggea, andSauropus species [5, 9–11].Glochidion, the largest genus in the tribe Phyllantheae (Phyllanthaceae), contains around 318 species of monoecious shrubs/trees mainly distributed in tropical Asia, Pacific islands, Australia, and Madagascar [12], a few also in tropical America and Africa, and in China ranging from southwest China to Taiwan [13]. AllGlochidion plants rely on species-specific female moths of the genusEpicephala for obligate pollination [5, 14]. At night, a female moth actively collects pollen grains from the host male flower using unique proboscises equipped with numerous hairs and then deposits them on the female flower and subsequently lays an egg using the specialized acicular ovipositor. The hatched larva consumes only a subset of the maturing seeds within a single fruit to complete the development and drill out of the fruit. The rest of the seeds ensure that the hosts reproduce their offspring [5].The most intriguing characteristics amongGlochidion
-
Epicephala mutualism are extremely high taxonomic diversity and species specificity of both plants and moths [5, 7, 14]. Pollinators’ encounter with host plants is the key step in these specialized pollination interactions. Floral scents are complex blends of volatile organic compounds, which have been thought to be crucial chemical signals guiding obligate nocturnal moths to locate host plants inGlochidion
-
Epicephala associations. Interspecific variations of floral scents amongGlochidion plants are essential for maintaining species specificity ofGlochidion
-
Epicephala mutualism [15–17], andEpicephala moths are also capable of discriminating their hosts using floral odor [16, 18].So far, studies onGlochidion
-
Epicephala mutualism have focused on pollination biology and insect taxonomy in China [19, 20] and Japan [5, 9, 10]. However, little is reported about the chemical composition of floral scents fromGlochidion plants in China. Recently, two pollination relationships betweenGlochidion andEpicephala were found in south China: (1) oneEpicephala species exclusively pollinates oneGlochidion species (G. eriocarpum,G.daltonii, andG. sphaerogynum) (unpublished data); (2) oneEpicephala species jointly pollinates twoGlochidion species (G. hirsutum andG. zeylanicum) [12]. For maintaining extremely high species specificity ofGlochidion
-
Epicephala mutualism, floral scents may differ amongGlochidion species pollinated by differentEpicephala moths, whileGlochidion species pollinated by the sameEpicephala moth should produce similar floral signals. Therefore, it is speculated that variations in floral scent among differentGlochidion species should reflect differences inEpicephala moth species with which the plants are associated.The dioecious plants pollinated by animals often, but not always, transmit similar floral signals between the male and female flowers to attract the same pollinator to complete conspecific pollen transfer [21–23]. An implicit assumption in the fact is that pollinators use similar signals to seek the similar reward from flowers of both sexes (e.g., floral nectar) [21]. Conversely, if the plants offer different rewards to the specific pollinators on the male flowers (e.g., pollen) and female flowers (e.g., ovule), floral characters (e.g., shape, visual, and odor) may be selected to diverge between both sexes of the flowers to signal alternative rewards [23, 24]. Interestingly, inEpicephala-pollinated Phyllanthaceae plants, there was sexual dimorphism of floral scents between male and female flowers, which is in contrast to scent from non-Epicephala-pollinated Phyllanthaceae [18, 25]. FemaleEpicephala moths pollinate the host plants, involving distinctly different behaviors on male and female flowers: pollen collection from male flowers and then pollen deposition and oviposition on female flowers [5, 9–11]. Therefore, it was thought that sexual dimorphism in floral scent ofEpicephala-pollinated Phyllanthaceae plants has evolved to signal alternative floral rewards provided by each sex toEpicephala moths [18]. In our field observations onGlochidion
-
Epicephala mutualism,Epicephala moths also exhibited similar high specific pollination behaviors on male and female flowers as found in previous studies [5, 9–11].Based on the above discussion, we hypothesized that, in mutualism recently found in China betweenGlochidion andEpicephala, (1) variations in floral scent composition among fiveGlochidion species may reflect adaptations to species-specific pollinators with which the host plants are associated and (2)Glochidion plants should be selected to produce different fragrance blends between the male and female flowers to induceEpicephala moths to get the different rewards from flowers of each sex. Consequently, the floral volatiles of both male and female flowers in fiveGlochidion species were collected, respectively, by the headspace adsorption technique and chemically characterized using gas chromatography-mass spectrometry (GC-MS). Then, nonmetric multidimensional scaling (NMDS) was performed to investigate interspecific variations and intersexual differences in floral scent composition of fiveGlochidion species in south China.


## 2. Methods and Materials

### 2.1. Plants

Five studiedGlochidion species, includingG. eriocarpum,G.daltonii,G. sphaerogynum,G. hirsutum, andG. zeylanicum, generally inhabit forest margins of tropical and subtropical forests in south China [13]. Of these five species,G. hirsutum andG. zeylanicum grow together and occurred in sympatry at the study sites. In contrast, the other three species occurred in other different study sites and so their distributions did not overlap (Figure 1).Figure 1
Study sites of fiveGlochidion species from south China in this study.Glochidion species investigated in this study are monoecious trees with inconspicuous green female flowers and yellow male flowers which are aggregated on the axils of the leaves (Figure 2). Female flowers are sessile, more or less bud-like, and appear to have specialized morphology: the style fused into a column at the trip with a small opening where the stigma is concealed in the inner surface, which makes it difficult to touch for flower visitors other thanEpicephala moths. Male flowers are pedunculate with unfolded five or six sepals to expose the stamen.Glochidion flowers are small and are fragrant to the human nose only during the evening, and the floral scents ofGlochidion species may be one of the key signals attracting nocturnal obligate pollinatorEpicephala moths.Figure 2
Flowers of the fiveGlochidion species in this study. Female ofG.eriocarpum (a),G.daltonii (c),G.sphaerogynum (e),G. hirsutum (g), andG. zeylanicum (i). Male flowers ofG.eriocarpum (b),G.daltonii (d),G.sphaerogynum (f),G. hirsutum (h), andG. zeylanicum (j).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)The fiveGlochidion species are associated with fourEpicephala moth species in our study regions [20, 26]. Among these five species,G. hirsutum andG. zeylanicum share similar morphology except that the trichomes on the epidermis are produced in the female flowers ofG. zeylanicum rather than that ofG. hirsutum (Figures 2(g) and 2(i)) and pollinated by the sameEpicephala species,E. bipollenella. However, the other three species are, respectively, pollinated by other species-specificEpicephala moths.


### 2.2. Floral Scent Collections

To analyze floral scent profiles of both male and female flowers of fiveGlochidion species, odor collections were performed using the dynamic headspace adsorption technique [27]. The details of the study sites, dates, and sample sizes are given in Figure 1 and Table 1. Before scent collection, specimens of each studied species were gathered to save in our lab as a voucher. Female and male flowers of each species were removed from plants and put separately into the polyvinyl acetate bags for floral scent collection (Toppits; Melitta Scandinavia AB, Klippan, Sweden). Air was purified by active charcoal (Sigma-Aldrich, USA) and then introduced into the bag through Teflon tubes (VICI Jour Ltd., Sweden) with a mini air-pump. Air-containing floral volatiles were sucked through the small glass tube (outer diameter 6 mm, length 75 mm) filled with 100 and 50 mg of Tenax-TA (mesh 60/80; Sigma-Aldrich) to adsorb floral scent. Meanwhile, air samples were also collected from empty bags as controls to identify possible background contamination during volatile collections. The flow rate of air in the inlet and outlet of the sample bag was set to 400 mL min−1. Odor collection was done at ambient temperature (25–28°C) indoors and continued for 3 h during the night. After collection, the filters were sealed with Parafilm and then wrapped with silver paper and stored at −20°C until used for analysis.Table 1
Study sites, dates, and sample sizes of fiveGlochidion species headspace collections.
Species
Study sites
Dates
Samples
Number of flowers
Male
Female
Male
Female
G. eriocarpum
Pingxiang, Guangxi
Mar.-Apr. 2013
3
4
300–400
800–1000
G. daltonii
Sichuan, Panzhihua
Apr.-May 2013
7
6
200–500
500–1000
G. sphaerogynum
Wuzhishan, Hainan
Dec. 2013-Jan. 2014
4
4
100–200
300–350
G. hirsutum
Fujian, Xiamen
Sep.-Oct. 2013
3
3
75–150
125–300
G. zeylanicum
80–140
285–400Before analysis, the collected floral scents in Tenax-TA filters were eluted with 2 mL of hexane into brown glass vials, and then 10 μL of n-Eicosane (1 mg mL−1) was added as the internal standard to calculate release amount of individual scent compounds. The eluate containing the floral volatiles was carefully concentrated down to 100 μL under N2 flow.


### 2.3. Chemical Analysis of Floral Scents

To analyze chemical composition of floral scents, the headspace samples ofGlochidion species were subjected to GC-MS, using an HP 6890 gas chromatograph (Agilent Technologies Inc., USA), and coupled with an HP 5975 mass spectrometer (Agilent Technologies Inc.). The GC was equipped with HP-5MS capillary column (30 m × 0.25 mm × 250 μm film thickness; Agilent Technologies Inc.). Helium was used as the carrier gas at a flow rate of 1.0 mL min−1. For each sample, 2 μL was injected in a split mode (the split ratio was 1 : 1) for 1 min with an injector temperature of 250°C. After injection, the oven temperature was programmed to be kept at 40°C for 5 min, followed by an increase of 3°C min−1 to 150°C and 10°C min−1 to 200°C, where it was held for 5 min. Mass spectra were recorded at 70 eV in the electron impact ionization mode, and the ionic source temperature was 230°C.Compound identification was carried out using the NIST 08 mass spectral libraries and further confirmed by comparison of retention indices with those reported in the literature [28, 29], the NIST Chemistry WebBook (http://webbook.nist.gov/chemistry/) [30], and The Pherobase (http://www.pherobase.com/) [31]. Compounds in the samples presented at higher or similar percentages in controls were considered as contaminants and excluded from the analysis. For the quantities of compounds, the amount of each volatile compound was calculated as its peak area relative to the total peak area on gas chromatograms.


### 2.4. Statistical Analyses

Unpairedt
-test was used to determine if there were significant differences in the total amounts of floral scents emitted from single male and female flower of each species; one-way ANOVA was used to test the significant differences between relative content of four chemical classes compounds with each species; NMDS is a nonparametric dimension reduction technique that carries on without the assumption of linear response and escapes many of the distortions of eigenvector techniques [32]. In order to analyze the interspecific variation and sexual dimorphism of floral scent composition inGlochidion species, NMDS was first performed to visualize dissimilarities among the headspace samples of floral scents using Canoco 5.0 for Windows and then the Mann-Whitney U test was used to test for significant differences among them. All figures were plotted with Graphpad Prism 5.0.


## 2.1. Plants

Five studiedGlochidion species, includingG. eriocarpum,G.daltonii,G. sphaerogynum,G. hirsutum, andG. zeylanicum, generally inhabit forest margins of tropical and subtropical forests in south China [13]. Of these five species,G. hirsutum andG. zeylanicum grow together and occurred in sympatry at the study sites. In contrast, the other three species occurred in other different study sites and so their distributions did not overlap (Figure 1).Figure 1
Study sites of fiveGlochidion species from south China in this study.Glochidion species investigated in this study are monoecious trees with inconspicuous green female flowers and yellow male flowers which are aggregated on the axils of the leaves (Figure 2). Female flowers are sessile, more or less bud-like, and appear to have specialized morphology: the style fused into a column at the trip with a small opening where the stigma is concealed in the inner surface, which makes it difficult to touch for flower visitors other thanEpicephala moths. Male flowers are pedunculate with unfolded five or six sepals to expose the stamen.Glochidion flowers are small and are fragrant to the human nose only during the evening, and the floral scents ofGlochidion species may be one of the key signals attracting nocturnal obligate pollinatorEpicephala moths.Figure 2
Flowers of the fiveGlochidion species in this study. Female ofG.eriocarpum (a),G.daltonii (c),G.sphaerogynum (e),G. hirsutum (g), andG. zeylanicum (i). Male flowers ofG.eriocarpum (b),G.daltonii (d),G.sphaerogynum (f),G. hirsutum (h), andG. zeylanicum (j).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)The fiveGlochidion species are associated with fourEpicephala moth species in our study regions [20, 26]. Among these five species,G. hirsutum andG. zeylanicum share similar morphology except that the trichomes on the epidermis are produced in the female flowers ofG. zeylanicum rather than that ofG. hirsutum (Figures 2(g) and 2(i)) and pollinated by the sameEpicephala species,E. bipollenella. However, the other three species are, respectively, pollinated by other species-specificEpicephala moths.


## 2.2. Floral Scent Collections

To analyze floral scent profiles of both male and female flowers of fiveGlochidion species, odor collections were performed using the dynamic headspace adsorption technique [27]. The details of the study sites, dates, and sample sizes are given in Figure 1 and Table 1. Before scent collection, specimens of each studied species were gathered to save in our lab as a voucher. Female and male flowers of each species were removed from plants and put separately into the polyvinyl acetate bags for floral scent collection (Toppits; Melitta Scandinavia AB, Klippan, Sweden). Air was purified by active charcoal (Sigma-Aldrich, USA) and then introduced into the bag through Teflon tubes (VICI Jour Ltd., Sweden) with a mini air-pump. Air-containing floral volatiles were sucked through the small glass tube (outer diameter 6 mm, length 75 mm) filled with 100 and 50 mg of Tenax-TA (mesh 60/80; Sigma-Aldrich) to adsorb floral scent. Meanwhile, air samples were also collected from empty bags as controls to identify possible background contamination during volatile collections. The flow rate of air in the inlet and outlet of the sample bag was set to 400 mL min−1. Odor collection was done at ambient temperature (25–28°C) indoors and continued for 3 h during the night. After collection, the filters were sealed with Parafilm and then wrapped with silver paper and stored at −20°C until used for analysis.Table 1
Study sites, dates, and sample sizes of fiveGlochidion species headspace collections.
Species
Study sites
Dates
Samples
Number of flowers
Male
Female
Male
Female
G. eriocarpum
Pingxiang, Guangxi
Mar.-Apr. 2013
3
4
300–400
800–1000
G. daltonii
Sichuan, Panzhihua
Apr.-May 2013
7
6
200–500
500–1000
G. sphaerogynum
Wuzhishan, Hainan
Dec. 2013-Jan. 2014
4
4
100–200
300–350
G. hirsutum
Fujian, Xiamen
Sep.-Oct. 2013
3
3
75–150
125–300
G. zeylanicum
80–140
285–400Before analysis, the collected floral scents in Tenax-TA filters were eluted with 2 mL of hexane into brown glass vials, and then 10 μL of n-Eicosane (1 mg mL−1) was added as the internal standard to calculate release amount of individual scent compounds. The eluate containing the floral volatiles was carefully concentrated down to 100 μL under N2 flow.


## 2.3. Chemical Analysis of Floral Scents

To analyze chemical composition of floral scents, the headspace samples ofGlochidion species were subjected to GC-MS, using an HP 6890 gas chromatograph (Agilent Technologies Inc., USA), and coupled with an HP 5975 mass spectrometer (Agilent Technologies Inc.). The GC was equipped with HP-5MS capillary column (30 m × 0.25 mm × 250 μm film thickness; Agilent Technologies Inc.). Helium was used as the carrier gas at a flow rate of 1.0 mL min−1. For each sample, 2 μL was injected in a split mode (the split ratio was 1 : 1) for 1 min with an injector temperature of 250°C. After injection, the oven temperature was programmed to be kept at 40°C for 5 min, followed by an increase of 3°C min−1 to 150°C and 10°C min−1 to 200°C, where it was held for 5 min. Mass spectra were recorded at 70 eV in the electron impact ionization mode, and the ionic source temperature was 230°C.Compound identification was carried out using the NIST 08 mass spectral libraries and further confirmed by comparison of retention indices with those reported in the literature [28, 29], the NIST Chemistry WebBook (http://webbook.nist.gov/chemistry/) [30], and The Pherobase (http://www.pherobase.com/) [31]. Compounds in the samples presented at higher or similar percentages in controls were considered as contaminants and excluded from the analysis. For the quantities of compounds, the amount of each volatile compound was calculated as its peak area relative to the total peak area on gas chromatograms.


## 2.4. Statistical Analyses

Unpairedt
-test was used to determine if there were significant differences in the total amounts of floral scents emitted from single male and female flower of each species; one-way ANOVA was used to test the significant differences between relative content of four chemical classes compounds with each species; NMDS is a nonparametric dimension reduction technique that carries on without the assumption of linear response and escapes many of the distortions of eigenvector techniques [32]. In order to analyze the interspecific variation and sexual dimorphism of floral scent composition inGlochidion species, NMDS was first performed to visualize dissimilarities among the headspace samples of floral scents using Canoco 5.0 for Windows and then the Mann-Whitney U test was used to test for significant differences among them. All figures were plotted with Graphpad Prism 5.0.


## 3. Results

### 3.1. Chemical Composition of Floral Volatiles

The chemical compositions of floral volatiles of both male and female flowers of five investigatedGlochidion plants are summarized in Table 2. The compounds were categorized into four chemical classes by biosynthetic pathways [33, 34]: aliphatics, monoterpenes, sesquiterpenes, and aromatics.Table 2
Chemical composition of floral scents of both male flowers and female flowers of fiveGlochidion species.
G. eriocarpum
G. daltonii
G. sphaerogynum
G. hirsutum
G. zeylanicum
Compounds
RIa
Maleflowers
Femaleflowers
Maleflowers
Femaleflowers
Maleflowers
Femaleflowers
Maleflowers
Femaleflowers
Maleflowers
Femaleflowers
(N
b = 3)
(N = 4)
(N = 7)
(N = 6)
(N = 4)
(N = 4)
(N = 3)
(N = 3)
(N = 3)
(N = 3)
Aliphatics
1
(E)-2-Hexenal
854
0.04 ± 0.01
0.39 ± 0.18
0.04 ± 0.02
0.25 ± 0.24
1.23 ± 0.21
0.46 ± 0.17
—
—
—
—
2
(Z)-3-Hexen-1-ol
857
0.12 ± 0.13
5.77 ± 3.43
0.03 ± 0.04
0.19 ± 0.15
0.19 ± 0.16
0.58 ± 0.33
0.60 ± 0.42
0.38 ± 0.18
0.52 ± 0.22
0.25 ± 0.10
3
(E)-2-Hexen-1-ol
862
0.05 ± 0.06
11.55 ± 3.49
—
—
7.76 ± 5.59
—
—
—
—
—
4
1-Hexanol
867
—
—
—
0.19 ± 0.11
2.28 ± 1.07
0.64 ± 0.52
4.06 ± 2.15
0.23 ± 0.12
1.51 ± 0.72
0.12 ± 0.04
5
Butyl butyrate
994
—
—
—
—
—
0.19 ± 0.11
—
—
—
—
6
(Z)-3-Hexenyl acetate
1007
0.23 ± 0.18
—
0.12 ± 0.08
1.30 ± 0.62
0.16 ± 0.05
0.60 ± 0.36
—
—
—
—
7
Hexyl butyrate
1011
0.02 ± 0.02
—
0.14 ± 0.18
0.49 ± 0.33
0.13 ± 0.05
0.07 ± 0.02
—
—
—
—
8
(Z)-2-Hexenyl acetate
1012
0.03 ± 0.05
—
0.39 ± 0.19
1.09 ± 0.84
0.52 ± 0.10
0.08 ± 0.03
—
—
—
—
9
(Z)-6-Nonenal
1099
—
—
0.10 ± 0.09
—
—
—
—
—
—
—
10
(Z)-3-Nonen-1-ol
1158
—
—
—
0.18 ± 0.08
1.32 ± 0.46
0.12 ± 0.09
1.07 ± 0.07
—
1.86 ± 0.41
—
11
(E)-2-Nonenal
1167
0.05 ± 0.03
—
0.22 ± 0.19
0.98 ± 0.65
—
0.22 ± 0.06
36.60 ± 3.04
—
23.50 ± 0.99
—
12
(E)-2-Nonen-1-ol
1170
0.05 ± 0.04
—
0.16 ± 0.15
0.70 ± 0.85
1.27 ± 0.42
0.17 ± 0.07
14.14 ± 2.04
—
11.17 ± 3.83
—
13
(Z)-6-Nonen-1-ol
1171
0.03 ± 0.01
—
0.13 ± 0.06
—
—
—
—
—
—
—
14
(Z)-3-Hexenyl butyrate
1185
0.02 ± 0.01
—
0.04 ± 0.04
0.92 ± 0.56
—
1.11 ± 0.40
—
—
—
—
15
Hexyl butyrate
1192
0.01 ± 0.01
—
0.08 ± 0.06
7.43 ± 6.55
—
—
—
—
—
—
16
(E)-2-Hexenyl butyrate
1195
0.02 ± 0.01
—
0.25 ± 0.25
0.71 ± 0.22
1.35 ± 0.45
0.52 ± 0.23
—
—
—
—
17
(Z)-3-Hexenyl isovalerate
1238
—
—
—
1.00 ± 1.27
—
0.04 ± 0.03
—
—
—
—
18
(Z)-3-Hexenyl valerate
1239
—
—
—
0.41 ± 0.32
—
0.08 ± 0.03
—
—
—
—
19
Tetradecane
1400
—
3.61 ± 3.27
—
—
—
—
—
—
—
—
20
Selinane
1464
—
4.42 ± 3.46
—
—
—
—
—
—
—
—
21
Pentadecane
1500
—
6.92 ± 5.81
—
—
—
—
—
—
—
—
Monoterpenes
22
α-Pinene
939
—
0.70 ± 0.47
—
—
—
—
—
—
—
—
23
2-Pentylfuran
992
—
—
0.04 ± 0.07
0.50 ± 0.27
—
—
—
—
—
—
24
(Z)-β-Ocimene
1038
0.06 ± 0.03
0.88 ± 0.09
—
0.08 ± 0.05
0.03 ± 0.01
0.07 ± 0.10
—
0.77 ± 0.20
—
0.75 ± 0.17
25
(E)-β-Ocimene
1048
6.58 ± 3.67
42.6 ± 23.82
0.50 ± 0.47
3.47 ± 1.64
0.2 ± 0.03
1.49 ± 1.61
2.14 ± 1.53
59.4 ± 2.91
1.60 ± 0.97
62.74 ± 2.50
26
(Z)-Linalool oxide(furanoid)
1076
0.14 ± 0.15
—
0.11 ± 0.08
0.13 ± 0.05
0.67 ± 0.20
0.19 ± 0.17
—
—
—
—
27
(E)-Linalool oxide(furanoid)
1088
0.35 ± 0.12
2.93 ± 1.14
0.95 ± 0.82
0.73 ± 0.30
2.86 ± 0.99
0.59 ± 0.45
—
—
—
1.23 ± 0.72
28
Linalool
1098
4.86 ± 5.05
4.61 ± 2.36
1.02 ± 1.09
44.85 ± 9.15
59.76 ± 11.44
23.61 ± 9.68
—
12.43 ± 0.69
—
12.26 ± 2.23
29
Hotrienol
1109
—
—
0.13 ± 0.12
3.07 ± 1.19
—
—
—
—
—
—
30
(E,E)-2,6-Dimethyl-1,3,5,7-octatetraene
1130
—
—
—
—
—
—
—
0.46 ± 0.08
—
0.40 ± 0.04
31
(E,Z)-2,6-Nonadienal
1155
0.14 ± 0.07
—
0.14 ± 0.07
0.37 ± 0.18
1.64 ± 0.57
0.14 ± 0.04
19.19 ± 2.86
—
17.46 ± 1.21
—
32
2,5-Dimethyl-1,6-heptadien
1161
—
—
—
—
2.6 ± 1.46
—
—
—
—
—
33
α-Terpineol
1186
0.01 ± 0.01
—
—
0.10 ± 0.08
—
—
—
0.65 ± 0.21
—
0.63 ± 0.19
Sesquiterpenes
34
α-Copaene
1376
—
—
—
—
—
—
—
0.62 ± 0.19
—
0.68 ± 0.32
35
β-Elemene
1391
28.22 ± 3.26
—
27.13 ± 5.62
7.54 ± 4.77
—
22.64 ± 1.89
—
1.36 ± 1.11
1.95 ± 1.90
—
36
(Z)-Jasmone
1393
—
—
—
0.16 ± 0.06
5.63 ± 0.98
3.26 ± 2.61
—
—
—
—
37
β-Caryophyllene
1418
1.32 ± 0.31
—
0.44 ± 0.12
3.28 ± 1.41
1.4 ± 0.44
3.26 ± 2.61
1.13 ± 0.61
7.344 ± 2.37
3.31 ± 2.16
6.90 ± 2.34
38
γ-Elemene
1430
—
—
—
—
0.04 ± 0.03
1.21 ± 0.47
—
—
—
—
39
β-Copaene
1433
—
—
—
—
—
—
—
0.43 ± 0.31
0.87 ± 0.93
0.30 ± 0.14
40
(E)-α-Bergamotene
1439
—
—
0.04 ± 0.02
—
—
—
—
—
—
—
41
α-Caryophyllene
1451
0.12 ± 0.08
—
0.06 ± 0.06
1.10 ± 0.35
0.13 ± 0.11
0.18 ± 0.08
—
3.53 ± 0.98
1.45 ± 0.97
1.71 ± 0.64
42
β-Humulene
1454
—
—
—
—
—
0.33 ± 0.12
—
—
—
—
43
Z,Z,Z-1,5,9,9-Tetramethyl-1,4,7-cycloundecatriene
1456
—
—
—
—
0.37 ± 0.37
0.21 ± 0.20
—
—
—
—
44
α-Patchoulene
1457
0.49 ± 0.26
—
1.42 ± 0.62
—
—
2.93 ± 0.83
—
—
—
—
45
(E)-β-Farnesene
1458
—
—
0.08 ± 0.09
—
—
—
—
—
—
—
46
4,5-Di-epi-aristolochene
1467
0.14 ± 0.03
—
0.06 ± 0.06
—
—
—
—
—
—
—
47
γ-Selinene
1470
8.97 ± 1.64
—
4.78 ± 0.72
2.13 ± 0.85
—
5.64 ± 1.21
—
—
—
—
48
Germacrene D
1481
0.08 ± 0.03
—
—
—
—
—
—
—
—
—
49
β-Selinene
1485
14.54 ± 4.13
—
7.04 ± 1.00
4.37 ± 1.71
—
12.03 ± 2.85
—
—
—
—
50
δ-Selinene
1487
0.20 ± 0.12
—
0.18 ± 0.10
—
—
0.38 ± 0.22
—
—
—
—
51
α-Muurolene
1493
—
—
—
4.59 ± 2.85
—
—
—
—
—
—
52
α-Selinene
1498
16.09 ± 3.37
—
7.70 ± 1.31
4.84 ± 2.18
—
11.87 ± 2.98
—
—
—
—
53
Eremophilene
1502
—
—
—
1.24 ± 0.72
—
—
—
—
—
—
54
(E,E)-α-Farnesene
1506
1.47 ± 1.15
5.1 ± 5.96
40.30 ± 9.92
1.66 ± 1.03
0.78 ± 1.34
0.14 ± 0.04
—
—
—
—
55
α-Panasinsene
1518
0.75 ± 0.15
—
0.31 ± 0.10
0.43 ± 0.73
—
0.38 ± 0.10
—
1.64 ± 2.05
—
2.50 ± 1.31
56
Longiborneol
1592
14.16 ± 2.09
—
3.99 ± 1.92
—
—
6.98 ± 2.63
—
—
—
—
Aromatics
57
Benzaldehyde
962
0.07 ± 0.04
0.51 ± 0.33
0.03 ± 0.02
—
—
—
—
—
—
—
58
Benzeneacetaldehyde
1043
—
—
—
—
—
—
0.58 ± 0.29
—
1.08 ± 0.56
—
59
Methyl benzoate
1090
—
—
—
—
—
—
—
0.42 ± 0.07
—
0.61 ± 0.29
60
Benzoyl cyanide
1094
—
—
—
0.14 ± 0.15
—
—
—
—
—
—
61
Phenylethyl alcohol
1116
0.10 ± 0.07
—
0.06 ± 0.08
—
—
—
10.04 ± 4.05
1.10 ± 0.26
20.76 ± 3.56
0.09 ± 0.05
62
Phenylacetonitrile
1143
0.17 ± 0.18
—
0.23 ± 0.33
—
—
—
1.92 ± 0.72
—
3.28 ± 0.54
—
63
Methyl salicylate
1190
0.04 ± 0.02
—
—
—
—
0.72 ± 0.16
1.02 ± 0.15
0.92 ± 0.35
3.34 ± 2.82
0.42 ± 0.04
64
Benzothiazole
1227
—
1.48 ± 0.72
—
—
—
—
—
—
—
—
65
2-Phenylethyl acetate
1256
—
—
0.07 ± 0.10
—
—
—
—
—
—
—
66
Indole
1287
—
—
—
—
—
—
1.54 ± 1.75
3.51 ± 0.7
1.38 ± 0.17
2.96 ± 1.73
67
β-Phenyethyl butyrate
1447
—
—
0.06 ± 0.06
—
—
—
—
—
—
—
68
Butylated hydroxytoluene
1512
—
4.61 ± 4.84
—
—
—
—
—
—
—
—
69
Mellein
1546
—
—
—
—
4.73 ± 2.03
0.63 ± 0.17
—
—
—
—
Unknown
0.17 ± 0.04
3.93 ± 3.51
0.64 ± 0.60
0.25 ± 0.17
2.2 ± 1.13
0.36 ± 0.08
6.08 ± 0.81
4.68 ± 1.47
5.12 ± 0.44
5.22 ± 1.8
aRetention index.
bThe number of headspace samples.
“—” presents that the compound was not detected.
The boldface compounds present the main compounds of each species (relative amounts >10%).A total of 69 volatile compounds were detected and identified from the five species, comprising 35 terpenoids (12 monoterpenes and 23 sesquiterpenes), 21 aliphatics, and 13 aromatics. Dominant compound classes were monoterpenes and sesquiterpenes. Of the 69 detected compounds, 46 were found in at least two species, while only 10 were commonly shared by all five species. Notably, two compounds, (Z)-3-hexen-1-ol (compound 2) and (E)-
β
-ocimene (compound 25), occurred frequently in both male and female flowers of all species; and 23 (G. zeylanicum) to 48 (G.daltonii) compounds were found in each species. There was a high degree of overlap in floral compounds produced betweenG. hirsutum andG. zeylanicum, and only one compound, (E)-linalool oxide (furanoid) (compound 27), was found only inG. zeylanicum; thus the two species were thought to have similar composition pattern in floral volatiles. IfG. hirsutum andG. zeylanicum were considered as one species, 5-6 compounds were unique to a single species. For instance, butyl butyrate (compound 5), (Z,Z,Z)-1,5,9,9-tetramethyl-1,4,7-cycloundecatriene (compound 47), γ
-elemene (compound 38), β
-humulene (compound 42), and mellein (compound 69) were species-specific compounds forG.sphaerogynum, while (E,E)-2,6-dimethyl-1,3,5,7-octatetraene (compound 30), α,β
-copaene (compounds 39, 34), benzeneacetaldehyde (compound 58), and methyl benzoate (compound 59) were only produced in bothG. hirsutum andG. zeylanicum and not in any other species. Most of the compounds were detected in relatively small amounts (between trace amounts and <10%) in flowers of each sex; the floral scent of each species studied was dominated by only 1–4 compounds in relatively high amounts as follows. In male flowers,G. eriocarpum was dominated by β-elemene (compound 35, 28.22%), α-selinene (compound 52, 16.09%), β-selinene (compound 49, 14.54%), and longiborneol (compound 56, 14.16%);G. daltonii was dominated by (E,E)-α-farnesene (compound 54, 40.3%) and β-elemene (compound 35, 27.13%);G. sphaerogynum was dominated by linalool (compound 28, 59.75%);G. hirsutum andG. zeylanicum were dominated by (E)-2-nonenal (compound 11, 30.6 and 23.5%, resp.), (E,Z)-2,6-nonadienal (compound 31, 19.19 and 17.46%, resp.), and (E)-2-nonen-1-ol (compound 12, 14.14 and 11.17%, resp.). In female flowers,G. eriocarpum was dominated by (E)-β-ocimene (compound 25, 42.6%) and (E)-2-hexen-1-ol (compound 3, 11.55%);G. daltonii was dominated by linalool (compound 28, 44.85%);G. sphaerogynum was dominated by linalool (compound 28, 23.61%), β-elemene (compound 35, 22.64%), α-selinene (compound 52, 12.03%), and β-selinene (compound 49, 11.87%);G. hirsutum andG. zeylanicum were dominated by (E)-β-ocimene (compound 25, 59.4 and 62.74%, resp.) and linalool (compound 28, 12.43 and 12.26%, resp.).The relative contents of four chemical classes of compounds from male and female flowers among the respective five species are shown in Figure3. For male flowers, the floral scents emitted by the five species could be divided into four groups:G.eriocarpum (86.65%) andG.daltonii (94.76%) dominated by sesquiterpenoids;G.sphaerogynum (67.87%) dominated by monoterpenoids; aliphatics with monoterpenoids inG. hirsutum (56.46 and 23.33%, resp.); aliphatics with aromatics inG. zeylanicum (38.56 and 29.85%, resp.). Likewise, the floral scents produced by female flowers of the five species were also divided into four groups:G.eriocarpum (51.73 and 32.68%, resp.) dominated by monoterpenoids with aromatics;G.sphaerogynum (67.95%) dominated by sesquiterpenoids;G. hirsutum (73.71%) andG. zeylanicum (78.02%) dominated by monoterpenoids. The findings indicated degrees of difference in chemical composition of floral scents among the five species.Figure 3
Relative amounts (%) of four chemical classes volatile compound of both male and female flowers from fiveGlochidion species. (a)G.eriocarpum; (b)G.daltonii; (c)G.sphaerogynum; (d)G. hirsutum; (e)G. zeylanicum (one-way ANOVA: the different lowercase letters on the bars indicate significant difference at P
=
0.05 level).
(a)
(b)
(c)
(d)
(e)In addition, there were clear differences in scent composition pattern of four different chemical classes between the male and female flowers of the five species. For example, inG.daltonii, the major components of male flowers were sesquiterpenoids, whereas those of the female flowers were monoterpenoids.


### 3.2. Interspecific Variation of Floral Scents

The NMDS analysis of floral scent profiles of both male and female flowers of the fiveGlochidion species is shown in Figure 4. For male flowers, the floral samples were divided into four groups:G.daltonii;G.eriocarpum;G.sphaerogynum; andG. hirsutum andG. zeylanicum. The floral samples ofG. hirsutum andG. zeylanicum greatly overlapped (χ
2
=
0.048, P
=
0
.
827), whereas those of the other three species were evidently distinguished from each other, as well as being obviously separated fromG. hirsutum andG. zeylanicum (χ
2
=
17.50, P
=
0.0
01). A similar separation pattern was also observed in the floral samples of female flowers (G. hirsutum andG. zeylanicum: χ
2
=
1.19, P
=
0
.
275;G.daltonii,G.eriocarpum,G.sphaerogynum andG. hirsutum, orG. zeylanicum: χ
2
=
12.752, P
=
0
.
005). Altogether, the results suggested thatG. hirsutum and G. zeylanicum had no difference in floral scent composition, whereas there were clear variations in floral scent among the other three species and it greatly differed from these two species.Figure 4
Score plots from nonmetric multidimensional scaling (NMDS) of floral scents in male and female flowers of fiveGlochidion species.


### 3.3. Emission of Floral Scents

Total emissions of floral scents emitted by male and female flowers of the fiveGlochidion species are shown in Figure 5. In all investigated species, a male flower produced on average significantly greater amounts of scents than a female flower (G.daltonii: t
=
9.18, P
<
0.001;G.eriocarpum: t
=
11.61, P
<
0.001;G.sphaerogynum: t
=
3.63, P
<
0.05;G. hirsutum: t
=
7.88, P
<
0.001;G. zeylanicum: t
=
12.35, P
<
0.001). In addition, regardless of male or female flower,G. hirsutum andG. zeylanicum produced similar amounts of floral odor. Altogether, the results showed evident quantitative differences in floral scents between male and female flowers of theGlochidion species.Figure 5
Total emission (Mean ± SD) of scent compounds from male and female flowers of fiveGlochidion species (unpaired t-test: “∗” and “∗
∗
∗” on the bars indicate significant difference at P
=
0.05 and 0.001 level).


### 3.4. Sexual Dimorphism of Floral Scents

The NMDS analysis of floral scents in male and female flowers of eachGlochidion species is shown in Figure 6. In each investigatedGlochidion species, the headspace samples of male flowers were clearly separated from those of female flowers (G.daltonii: χ
2
=
6, P
=
0.034;G.eriocarpum: χ
2
=
9, P
=
0.03;G.sphaerogynum: χ
2
=
5.33, P
=
0.0
21;G. hirsutum andG. zeylanicum: χ
2
=
3.857, P
=
0.0
5). The results indicated a qualitative difference between floral scents produced by male and femaleGlochidion flowers, which is so-called sexual dimorphism of floral scents.Figure 6
Score plots from NMDS of floral scents in male and female flowers of fiveGlochidion species. The headspace samples with each species are represented by different numbers sharp symbols as shown. The headspace samples within each species are boxed and coloured white for male flowers but grey for female flowers.


## 3.1. Chemical Composition of Floral Volatiles

The chemical compositions of floral volatiles of both male and female flowers of five investigatedGlochidion plants are summarized in Table 2. The compounds were categorized into four chemical classes by biosynthetic pathways [33, 34]: aliphatics, monoterpenes, sesquiterpenes, and aromatics.Table 2
Chemical composition of floral scents of both male flowers and female flowers of fiveGlochidion species.
G. eriocarpum
G. daltonii
G. sphaerogynum
G. hirsutum
G. zeylanicum
Compounds
RIa
Maleflowers
Femaleflowers
Maleflowers
Femaleflowers
Maleflowers
Femaleflowers
Maleflowers
Femaleflowers
Maleflowers
Femaleflowers
(N
b = 3)
(N = 4)
(N = 7)
(N = 6)
(N = 4)
(N = 4)
(N = 3)
(N = 3)
(N = 3)
(N = 3)
Aliphatics
1
(E)-2-Hexenal
854
0.04 ± 0.01
0.39 ± 0.18
0.04 ± 0.02
0.25 ± 0.24
1.23 ± 0.21
0.46 ± 0.17
—
—
—
—
2
(Z)-3-Hexen-1-ol
857
0.12 ± 0.13
5.77 ± 3.43
0.03 ± 0.04
0.19 ± 0.15
0.19 ± 0.16
0.58 ± 0.33
0.60 ± 0.42
0.38 ± 0.18
0.52 ± 0.22
0.25 ± 0.10
3
(E)-2-Hexen-1-ol
862
0.05 ± 0.06
11.55 ± 3.49
—
—
7.76 ± 5.59
—
—
—
—
—
4
1-Hexanol
867
—
—
—
0.19 ± 0.11
2.28 ± 1.07
0.64 ± 0.52
4.06 ± 2.15
0.23 ± 0.12
1.51 ± 0.72
0.12 ± 0.04
5
Butyl butyrate
994
—
—
—
—
—
0.19 ± 0.11
—
—
—
—
6
(Z)-3-Hexenyl acetate
1007
0.23 ± 0.18
—
0.12 ± 0.08
1.30 ± 0.62
0.16 ± 0.05
0.60 ± 0.36
—
—
—
—
7
Hexyl butyrate
1011
0.02 ± 0.02
—
0.14 ± 0.18
0.49 ± 0.33
0.13 ± 0.05
0.07 ± 0.02
—
—
—
—
8
(Z)-2-Hexenyl acetate
1012
0.03 ± 0.05
—
0.39 ± 0.19
1.09 ± 0.84
0.52 ± 0.10
0.08 ± 0.03
—
—
—
—
9
(Z)-6-Nonenal
1099
—
—
0.10 ± 0.09
—
—
—
—
—
—
—
10
(Z)-3-Nonen-1-ol
1158
—
—
—
0.18 ± 0.08
1.32 ± 0.46
0.12 ± 0.09
1.07 ± 0.07
—
1.86 ± 0.41
—
11
(E)-2-Nonenal
1167
0.05 ± 0.03
—
0.22 ± 0.19
0.98 ± 0.65
—
0.22 ± 0.06
36.60 ± 3.04
—
23.50 ± 0.99
—
12
(E)-2-Nonen-1-ol
1170
0.05 ± 0.04
—
0.16 ± 0.15
0.70 ± 0.85
1.27 ± 0.42
0.17 ± 0.07
14.14 ± 2.04
—
11.17 ± 3.83
—
13
(Z)-6-Nonen-1-ol
1171
0.03 ± 0.01
—
0.13 ± 0.06
—
—
—
—
—
—
—
14
(Z)-3-Hexenyl butyrate
1185
0.02 ± 0.01
—
0.04 ± 0.04
0.92 ± 0.56
—
1.11 ± 0.40
—
—
—
—
15
Hexyl butyrate
1192
0.01 ± 0.01
—
0.08 ± 0.06
7.43 ± 6.55
—
—
—
—
—
—
16
(E)-2-Hexenyl butyrate
1195
0.02 ± 0.01
—
0.25 ± 0.25
0.71 ± 0.22
1.35 ± 0.45
0.52 ± 0.23
—
—
—
—
17
(Z)-3-Hexenyl isovalerate
1238
—
—
—
1.00 ± 1.27
—
0.04 ± 0.03
—
—
—
—
18
(Z)-3-Hexenyl valerate
1239
—
—
—
0.41 ± 0.32
—
0.08 ± 0.03
—
—
—
—
19
Tetradecane
1400
—
3.61 ± 3.27
—
—
—
—
—
—
—
—
20
Selinane
1464
—
4.42 ± 3.46
—
—
—
—
—
—
—
—
21
Pentadecane
1500
—
6.92 ± 5.81
—
—
—
—
—
—
—
—
Monoterpenes
22
α-Pinene
939
—
0.70 ± 0.47
—
—
—
—
—
—
—
—
23
2-Pentylfuran
992
—
—
0.04 ± 0.07
0.50 ± 0.27
—
—
—
—
—
—
24
(Z)-β-Ocimene
1038
0.06 ± 0.03
0.88 ± 0.09
—
0.08 ± 0.05
0.03 ± 0.01
0.07 ± 0.10
—
0.77 ± 0.20
—
0.75 ± 0.17
25
(E)-β-Ocimene
1048
6.58 ± 3.67
42.6 ± 23.82
0.50 ± 0.47
3.47 ± 1.64
0.2 ± 0.03
1.49 ± 1.61
2.14 ± 1.53
59.4 ± 2.91
1.60 ± 0.97
62.74 ± 2.50
26
(Z)-Linalool oxide(furanoid)
1076
0.14 ± 0.15
—
0.11 ± 0.08
0.13 ± 0.05
0.67 ± 0.20
0.19 ± 0.17
—
—
—
—
27
(E)-Linalool oxide(furanoid)
1088
0.35 ± 0.12
2.93 ± 1.14
0.95 ± 0.82
0.73 ± 0.30
2.86 ± 0.99
0.59 ± 0.45
—
—
—
1.23 ± 0.72
28
Linalool
1098
4.86 ± 5.05
4.61 ± 2.36
1.02 ± 1.09
44.85 ± 9.15
59.76 ± 11.44
23.61 ± 9.68
—
12.43 ± 0.69
—
12.26 ± 2.23
29
Hotrienol
1109
—
—
0.13 ± 0.12
3.07 ± 1.19
—
—
—
—
—
—
30
(E,E)-2,6-Dimethyl-1,3,5,7-octatetraene
1130
—
—
—
—
—
—
—
0.46 ± 0.08
—
0.40 ± 0.04
31
(E,Z)-2,6-Nonadienal
1155
0.14 ± 0.07
—
0.14 ± 0.07
0.37 ± 0.18
1.64 ± 0.57
0.14 ± 0.04
19.19 ± 2.86
—
17.46 ± 1.21
—
32
2,5-Dimethyl-1,6-heptadien
1161
—
—
—
—
2.6 ± 1.46
—
—
—
—
—
33
α-Terpineol
1186
0.01 ± 0.01
—
—
0.10 ± 0.08
—
—
—
0.65 ± 0.21
—
0.63 ± 0.19
Sesquiterpenes
34
α-Copaene
1376
—
—
—
—
—
—
—
0.62 ± 0.19
—
0.68 ± 0.32
35
β-Elemene
1391
28.22 ± 3.26
—
27.13 ± 5.62
7.54 ± 4.77
—
22.64 ± 1.89
—
1.36 ± 1.11
1.95 ± 1.90
—
36
(Z)-Jasmone
1393
—
—
—
0.16 ± 0.06
5.63 ± 0.98
3.26 ± 2.61
—
—
—
—
37
β-Caryophyllene
1418
1.32 ± 0.31
—
0.44 ± 0.12
3.28 ± 1.41
1.4 ± 0.44
3.26 ± 2.61
1.13 ± 0.61
7.344 ± 2.37
3.31 ± 2.16
6.90 ± 2.34
38
γ-Elemene
1430
—
—
—
—
0.04 ± 0.03
1.21 ± 0.47
—
—
—
—
39
β-Copaene
1433
—
—
—
—
—
—
—
0.43 ± 0.31
0.87 ± 0.93
0.30 ± 0.14
40
(E)-α-Bergamotene
1439
—
—
0.04 ± 0.02
—
—
—
—
—
—
—
41
α-Caryophyllene
1451
0.12 ± 0.08
—
0.06 ± 0.06
1.10 ± 0.35
0.13 ± 0.11
0.18 ± 0.08
—
3.53 ± 0.98
1.45 ± 0.97
1.71 ± 0.64
42
β-Humulene
1454
—
—
—
—
—
0.33 ± 0.12
—
—
—
—
43
Z,Z,Z-1,5,9,9-Tetramethyl-1,4,7-cycloundecatriene
1456
—
—
—
—
0.37 ± 0.37
0.21 ± 0.20
—
—
—
—
44
α-Patchoulene
1457
0.49 ± 0.26
—
1.42 ± 0.62
—
—
2.93 ± 0.83
—
—
—
—
45
(E)-β-Farnesene
1458
—
—
0.08 ± 0.09
—
—
—
—
—
—
—
46
4,5-Di-epi-aristolochene
1467
0.14 ± 0.03
—
0.06 ± 0.06
—
—
—
—
—
—
—
47
γ-Selinene
1470
8.97 ± 1.64
—
4.78 ± 0.72
2.13 ± 0.85
—
5.64 ± 1.21
—
—
—
—
48
Germacrene D
1481
0.08 ± 0.03
—
—
—
—
—
—
—
—
—
49
β-Selinene
1485
14.54 ± 4.13
—
7.04 ± 1.00
4.37 ± 1.71
—
12.03 ± 2.85
—
—
—
—
50
δ-Selinene
1487
0.20 ± 0.12
—
0.18 ± 0.10
—
—
0.38 ± 0.22
—
—
—
—
51
α-Muurolene
1493
—
—
—
4.59 ± 2.85
—
—
—
—
—
—
52
α-Selinene
1498
16.09 ± 3.37
—
7.70 ± 1.31
4.84 ± 2.18
—
11.87 ± 2.98
—
—
—
—
53
Eremophilene
1502
—
—
—
1.24 ± 0.72
—
—
—
—
—
—
54
(E,E)-α-Farnesene
1506
1.47 ± 1.15
5.1 ± 5.96
40.30 ± 9.92
1.66 ± 1.03
0.78 ± 1.34
0.14 ± 0.04
—
—
—
—
55
α-Panasinsene
1518
0.75 ± 0.15
—
0.31 ± 0.10
0.43 ± 0.73
—
0.38 ± 0.10
—
1.64 ± 2.05
—
2.50 ± 1.31
56
Longiborneol
1592
14.16 ± 2.09
—
3.99 ± 1.92
—
—
6.98 ± 2.63
—
—
—
—
Aromatics
57
Benzaldehyde
962
0.07 ± 0.04
0.51 ± 0.33
0.03 ± 0.02
—
—
—
—
—
—
—
58
Benzeneacetaldehyde
1043
—
—
—
—
—
—
0.58 ± 0.29
—
1.08 ± 0.56
—
59
Methyl benzoate
1090
—
—
—
—
—
—
—
0.42 ± 0.07
—
0.61 ± 0.29
60
Benzoyl cyanide
1094
—
—
—
0.14 ± 0.15
—
—
—
—
—
—
61
Phenylethyl alcohol
1116
0.10 ± 0.07
—
0.06 ± 0.08
—
—
—
10.04 ± 4.05
1.10 ± 0.26
20.76 ± 3.56
0.09 ± 0.05
62
Phenylacetonitrile
1143
0.17 ± 0.18
—
0.23 ± 0.33
—
—
—
1.92 ± 0.72
—
3.28 ± 0.54
—
63
Methyl salicylate
1190
0.04 ± 0.02
—
—
—
—
0.72 ± 0.16
1.02 ± 0.15
0.92 ± 0.35
3.34 ± 2.82
0.42 ± 0.04
64
Benzothiazole
1227
—
1.48 ± 0.72
—
—
—
—
—
—
—
—
65
2-Phenylethyl acetate
1256
—
—
0.07 ± 0.10
—
—
—
—
—
—
—
66
Indole
1287
—
—
—
—
—
—
1.54 ± 1.75
3.51 ± 0.7
1.38 ± 0.17
2.96 ± 1.73
67
β-Phenyethyl butyrate
1447
—
—
0.06 ± 0.06
—
—
—
—
—
—
—
68
Butylated hydroxytoluene
1512
—
4.61 ± 4.84
—
—
—
—
—
—
—
—
69
Mellein
1546
—
—
—
—
4.73 ± 2.03
0.63 ± 0.17
—
—
—
—
Unknown
0.17 ± 0.04
3.93 ± 3.51
0.64 ± 0.60
0.25 ± 0.17
2.2 ± 1.13
0.36 ± 0.08
6.08 ± 0.81
4.68 ± 1.47
5.12 ± 0.44
5.22 ± 1.8
aRetention index.
bThe number of headspace samples.
“—” presents that the compound was not detected.
The boldface compounds present the main compounds of each species (relative amounts >10%).A total of 69 volatile compounds were detected and identified from the five species, comprising 35 terpenoids (12 monoterpenes and 23 sesquiterpenes), 21 aliphatics, and 13 aromatics. Dominant compound classes were monoterpenes and sesquiterpenes. Of the 69 detected compounds, 46 were found in at least two species, while only 10 were commonly shared by all five species. Notably, two compounds, (Z)-3-hexen-1-ol (compound 2) and (E)-
β
-ocimene (compound 25), occurred frequently in both male and female flowers of all species; and 23 (G. zeylanicum) to 48 (G.daltonii) compounds were found in each species. There was a high degree of overlap in floral compounds produced betweenG. hirsutum andG. zeylanicum, and only one compound, (E)-linalool oxide (furanoid) (compound 27), was found only inG. zeylanicum; thus the two species were thought to have similar composition pattern in floral volatiles. IfG. hirsutum andG. zeylanicum were considered as one species, 5-6 compounds were unique to a single species. For instance, butyl butyrate (compound 5), (Z,Z,Z)-1,5,9,9-tetramethyl-1,4,7-cycloundecatriene (compound 47), γ
-elemene (compound 38), β
-humulene (compound 42), and mellein (compound 69) were species-specific compounds forG.sphaerogynum, while (E,E)-2,6-dimethyl-1,3,5,7-octatetraene (compound 30), α,β
-copaene (compounds 39, 34), benzeneacetaldehyde (compound 58), and methyl benzoate (compound 59) were only produced in bothG. hirsutum andG. zeylanicum and not in any other species. Most of the compounds were detected in relatively small amounts (between trace amounts and <10%) in flowers of each sex; the floral scent of each species studied was dominated by only 1–4 compounds in relatively high amounts as follows. In male flowers,G. eriocarpum was dominated by β-elemene (compound 35, 28.22%), α-selinene (compound 52, 16.09%), β-selinene (compound 49, 14.54%), and longiborneol (compound 56, 14.16%);G. daltonii was dominated by (E,E)-α-farnesene (compound 54, 40.3%) and β-elemene (compound 35, 27.13%);G. sphaerogynum was dominated by linalool (compound 28, 59.75%);G. hirsutum andG. zeylanicum were dominated by (E)-2-nonenal (compound 11, 30.6 and 23.5%, resp.), (E,Z)-2,6-nonadienal (compound 31, 19.19 and 17.46%, resp.), and (E)-2-nonen-1-ol (compound 12, 14.14 and 11.17%, resp.). In female flowers,G. eriocarpum was dominated by (E)-β-ocimene (compound 25, 42.6%) and (E)-2-hexen-1-ol (compound 3, 11.55%);G. daltonii was dominated by linalool (compound 28, 44.85%);G. sphaerogynum was dominated by linalool (compound 28, 23.61%), β-elemene (compound 35, 22.64%), α-selinene (compound 52, 12.03%), and β-selinene (compound 49, 11.87%);G. hirsutum andG. zeylanicum were dominated by (E)-β-ocimene (compound 25, 59.4 and 62.74%, resp.) and linalool (compound 28, 12.43 and 12.26%, resp.).The relative contents of four chemical classes of compounds from male and female flowers among the respective five species are shown in Figure3. For male flowers, the floral scents emitted by the five species could be divided into four groups:G.eriocarpum (86.65%) andG.daltonii (94.76%) dominated by sesquiterpenoids;G.sphaerogynum (67.87%) dominated by monoterpenoids; aliphatics with monoterpenoids inG. hirsutum (56.46 and 23.33%, resp.); aliphatics with aromatics inG. zeylanicum (38.56 and 29.85%, resp.). Likewise, the floral scents produced by female flowers of the five species were also divided into four groups:G.eriocarpum (51.73 and 32.68%, resp.) dominated by monoterpenoids with aromatics;G.sphaerogynum (67.95%) dominated by sesquiterpenoids;G. hirsutum (73.71%) andG. zeylanicum (78.02%) dominated by monoterpenoids. The findings indicated degrees of difference in chemical composition of floral scents among the five species.Figure 3
Relative amounts (%) of four chemical classes volatile compound of both male and female flowers from fiveGlochidion species. (a)G.eriocarpum; (b)G.daltonii; (c)G.sphaerogynum; (d)G. hirsutum; (e)G. zeylanicum (one-way ANOVA: the different lowercase letters on the bars indicate significant difference at P
=
0.05 level).
(a)
(b)
(c)
(d)
(e)In addition, there were clear differences in scent composition pattern of four different chemical classes between the male and female flowers of the five species. For example, inG.daltonii, the major components of male flowers were sesquiterpenoids, whereas those of the female flowers were monoterpenoids.


## 3.2. Interspecific Variation of Floral Scents

The NMDS analysis of floral scent profiles of both male and female flowers of the fiveGlochidion species is shown in Figure 4. For male flowers, the floral samples were divided into four groups:G.daltonii;G.eriocarpum;G.sphaerogynum; andG. hirsutum andG. zeylanicum. The floral samples ofG. hirsutum andG. zeylanicum greatly overlapped (χ
2
=
0.048, P
=
0
.
827), whereas those of the other three species were evidently distinguished from each other, as well as being obviously separated fromG. hirsutum andG. zeylanicum (χ
2
=
17.50, P
=
0.0
01). A similar separation pattern was also observed in the floral samples of female flowers (G. hirsutum andG. zeylanicum: χ
2
=
1.19, P
=
0
.
275;G.daltonii,G.eriocarpum,G.sphaerogynum andG. hirsutum, orG. zeylanicum: χ
2
=
12.752, P
=
0
.
005). Altogether, the results suggested thatG. hirsutum and G. zeylanicum had no difference in floral scent composition, whereas there were clear variations in floral scent among the other three species and it greatly differed from these two species.Figure 4
Score plots from nonmetric multidimensional scaling (NMDS) of floral scents in male and female flowers of fiveGlochidion species.


## 3.3. Emission of Floral Scents

Total emissions of floral scents emitted by male and female flowers of the fiveGlochidion species are shown in Figure 5. In all investigated species, a male flower produced on average significantly greater amounts of scents than a female flower (G.daltonii: t
=
9.18, P
<
0.001;G.eriocarpum: t
=
11.61, P
<
0.001;G.sphaerogynum: t
=
3.63, P
<
0.05;G. hirsutum: t
=
7.88, P
<
0.001;G. zeylanicum: t
=
12.35, P
<
0.001). In addition, regardless of male or female flower,G. hirsutum andG. zeylanicum produced similar amounts of floral odor. Altogether, the results showed evident quantitative differences in floral scents between male and female flowers of theGlochidion species.Figure 5
Total emission (Mean ± SD) of scent compounds from male and female flowers of fiveGlochidion species (unpaired t-test: “∗” and “∗
∗
∗” on the bars indicate significant difference at P
=
0.05 and 0.001 level).


## 3.4. Sexual Dimorphism of Floral Scents

The NMDS analysis of floral scents in male and female flowers of eachGlochidion species is shown in Figure 6. In each investigatedGlochidion species, the headspace samples of male flowers were clearly separated from those of female flowers (G.daltonii: χ
2
=
6, P
=
0.034;G.eriocarpum: χ
2
=
9, P
=
0.03;G.sphaerogynum: χ
2
=
5.33, P
=
0.0
21;G. hirsutum andG. zeylanicum: χ
2
=
3.857, P
=
0.0
5). The results indicated a qualitative difference between floral scents produced by male and femaleGlochidion flowers, which is so-called sexual dimorphism of floral scents.Figure 6
Score plots from NMDS of floral scents in male and female flowers of fiveGlochidion species. The headspace samples with each species are represented by different numbers sharp symbols as shown. The headspace samples within each species are boxed and coloured white for male flowers but grey for female flowers.


## 4. Discussion

In total, 69 volatiles are detected from the floral scents of the fiveGlochidion species. Most of these reported volatiles are quite common in floral odors of many flowering plants [33, 34]. In particular, 26 compounds also occur in floral fragrances of other Phyllantheae species, close relatives of the investigatedGlochidion species, which are also exclusively pollinated byEpicephala moths [16, 18, 35]. For instance, (E)-
β
-ocimene (compound 25) and linalool (compound 28) occurred together in the five studiedGlochidion species and are also produced by the flowers of four other reportedGlochidion species [16, 18]. In addition, these two compounds can attract a wide range of pollinators of flowering plants, such as moths [35, 36], butterflies [37], and bees [38]. Therefore, our results suggest that these interactions betweenGlochidion species andEpicephala moths may be mediated by conventional floral compounds. This is similar to findings in most obligate insect-plant mutualism, where identified floral volatiles are also regarded as general floral volatiles [16, 25, 39]. However, Chen et al. [40] showed that an unusual compound, 4-methylanisole, mediated the species specificity inFicus semicordata andCeratosolen gravelyi wasp mutualism through a “private channel.”In highly specific plant-insect interactions, the floral scent profile of host plants is sometimes dominated by one or few compounds [16, 39, 41, 42], and these dominant compounds may account for variation in floral scents among species [16]. Similarly, our GC-MS showed the floral odor of each of the five species was also dominated by one compound at least, such as, for male flowers, β
-elemene (compound 35, 28.22%) inG. eriocarpum, (E,E)-
α
-farnesene (compound 54, 40.3%) inG.daltonii, linalool (compound 28, 59.75%) inG.sphaerogynum, and (E)-2-nonenal inG. hirsutum andG. zeylanicum (compound 11, 36.6 and 23.5%, resp.). The PCA results revealed that both male and female flowers of the five species could be divided into four distinct groups:G. zeylanicum andG. hirsutum clustered together and the other species were spaced with each other. Thus, floral odors of investigatedGlochidion species could be clearly discriminated mainly due to the dominant compounds. This is in contrast to findings for the odor profile of five otherGlochidion species that were distinguished by minor compounds [16]. These results suggest that the presence of dominant compounds in floral scents ofGlochidion species may be crucial for attracting specificEpicephala pollinators. However, electroantennographic detection analysis and behavioral tests with authentic chemicals are needed for further screening of specific compounds responsible for this host specificity.Highly interspecific variation in floral scent has been observed in many plant species [16, 39, 43–50]. It is likely that variation in floral odor composition between species is due to adaptation to host-specific pollinators [16, 50]. The species specificity in theGlochidion
-
Epicephala mutualism is extremely high, where each host plant is usually exclusively pollinated by specific pollinator species. Thus, to facilitate host specificity in these interactions, the floral scent should be selected to be strongly divergent among species. Interspecific variation in floral scent profile ofGlochidion species observed in this study could contribute to the maintenance of the species specificity of the mutualism and may reflect adaptation to specificEpicephala moths. Pollinators can also find and locate their hosts by discriminating the difference in floral odor among species [16, 40]. Our NMDS analysis showed a similar floral scent profile betweenG. hirsutum andG. zeylanicum. InGlochidion-Epicephala mutualism,E. bipollenella moths jointly pollinatedG. hirsutum andG. zeylanicum [20]. Similarly,G.obovatum andG.rubrum, two parapatric species pollinated jointly by the sameEpicephala species, produced similar floral scents, more similar than those emitted by other cooccurringGlochidion species pollinated by otherEpicephala species [16]. In addition,Ficus natalensis andF.burkei jointly sharing the same pollinator wasp species emitted similar floral scent profiles, which are different from those produced by other AfricanFicus species [51]. When different plant species are pollinated by the same group of pollinators and especially by closely related pollinator taxa, they may be expected to have floral volatiles of similar chemical composition [43]. This implies that the similarity in odor of differentGlochidion species in this study may suggest evolutionary convergence to attract the sameEpicephala pollinators [51].Generally, but not always, the animal-pollinated dioecious plants produce similar floral signals between male and female flowers. This is because attraction of the same pollinator to flowers of both sexes is essential for successful conspecific pollen transfer; thus the hosts should be selected to transmit such similar floral scents in the flowers of both sexes [21–23]. Underlying the fact above is that pollinators use similar cues to find the same floral rewards (e.g., floral nectar) from male and female flowers [21, 24]. Selection for similarity in signals from male and female flowers is thought to be particularly strong when one sex (often the female) does not produce any reward and effectively mimics flowers of the other sex, so deceptive pollination occurs in the female [52]. For instance, chemical mimicry in floral scent occurs in fig-fig-wasp highly species-specific pollination systems. Although fig wasps are deceived into pollinating female figs but do not obtain any reward from female figs, the pollinated female figs can produce seeds to ensure the successful reproduction of fig species. Therefore, for fig-fig-wasp attraction, selection for chemical mimicry between sexes is suggested to be particularly critical to maintain the stability of obligate pollination mutualism between fig and fig wasp [40, 52, 53]. In addition, chemical mimicry between sexes of the flowers has also been documented in cycad species [50, 54–57]. Hemborg and Bond [24] proposed that different rewards offered by male and female flowers of some plant species to pollinators could promote the evolution of sexually dimorphic floral signals of flowers. Svensson et al. [25] and Okamoto et al. [18] found thatEpicephala
-pollinated Phyllanthaceae plants consistently showed clear differences in scent between male and female flowers. Similarly, our NMDS results also revealed inGlochidion-Epicephala a remarkable sexual dimorphism in floral scent between sexes of the five investigatedGlochidion species. InEpicephala
-pollinated Phyllanthaceae, whenEpicephala moths pollinate for the host, male flowers can offer pollen to pollinators as food, whereas female flowers can provide the ovule forEpicephala adults as egg-laying sites and seeds forEpicephala larvae as foods [5, 9–12], and it was thought thatEpicephala moths could get access the different and essential rewards from the flowers of both sexes of hosts; thus sexual dimorphism in floral scent of two sexes inEpicephala
-pollinated Phyllanthaceae is selected to signal an alternative reward provided by each sex toEpicephala moths.Epicephala can also distinguish male and female flowers by odor and is crucial for successful reproduction of the moths and plants [18]. Our results also provide strong support that different rewards in male and female flowers result in the sexual dimorphism in floral scent of flowers ofGlochidion species in this study [24].Our results showed quantitative differences in floral scent emission between the sexes in each studiedGlochidion species. In addition, there was a similar finding forB. vitis-idaea, a close relative ofGlochidion, which is also obligately pollinated by species-specificEpicephala moths [25]. Okamoto et al. [18] showed thatEpicephala moths preferred the scent of male flowers over that of female flowers inG. zeylanicum. According to sexual selection theory, the males are limited in their reproductive success by access to mates, while females are mostly limited by available resources [58]. In animal-pollinated plants, attraction of pollinators and successful pollination are crucial for reproductive success; thus males in monoecious plants are expected to be under selection to increase their attractiveness to pollinators by investing more than females in floral traits that enhance pollinator visitation. Generally, male plants should produce more floral scent than the females to increase pollinator attraction. Waelti et al. [59] suggested that, in theSilene latifolia
-
Hadena bicruris nursery pollination system, male flowers emitted significantly more fragrance, especially of the pollinator-attracting compounds, than female flowers, and pollinator-moths preferred male over female flowers in behavioral tests, implying that evolution of the divergence in the floral signal ofS. latifolia is shaped by sexual selection. Ashman et al. [60] also showed that the smaller hermaphroditic flowers in the gynodioecious strawberryFragaria virginiana produced larger amounts of foliage volatiles, which resulted in attraction of more pollinators compared to female flowers. Therefore, more probably, the observed quantitative difference in floral scent of flowers between the sexes in the five species ofGlochidion may be an outcome of sexual selection. It is possible that the stronger odor emission of male flowers inGlochidion species resulted in increasing attractiveness forEpicephala moths compared with female flowers.


## 5. Conclusions

Our findings suggest that the volatile variation of floral scent amongGlochidion species reflects adaptations to specific pollinators. Sexual dimorphism in floral scent has evolved to signal alternative rewards provided by each sex toEpicephala moths. It is concluded that floral scent has been thought to play key role in the obligate pollination mutualism betweenGlochidion plants andEpicephala moths.



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*Source: 865694-2015-11-08.xml*