Pollination and Essential Oil Production of Lavandula angustifolia Mill. (Lamiaceae)

Abstract

Lavandula angustifolia Mill., lavender, is an aromatic plant in the Lamiaceae family. Lavender, which is native to the Mediterranean region but cultivated throughout the world, is an important economic plant. Several studies have investigated two aspects of this aromatic plant: (1) which pollinators, particularly bees, pollinate lavender, and (2) the composition of lavender essential oil. However, little research has been conducted to investigate how pollination affects either the yield or phytochemistry of lavender. The current study, which was conducted in North America, investigates which bee species visit lavender and how pollination affects plant chemistry, specifically the essential oil produced by lavender. Over the course of the 5-week observational period, a total of 12 species (across 10 genera) of bees were identified visiting lavender. Compared to previous studies on cultivated lavender at the same site (Mt. Nebo Botanical Farm, Mona, UT), four bee species not previously observed on lavender were identified. These included Hoplitis producta, Nomada sp., Osmia trevoris, and Megachile snowi. Pollinated lavender, compared to lavender excluded from pollinators, produced more essential oil (yield (w/w) = 1.49% vs. 1.07%), lower relative amounts of linalool (35.4% vs. 39.9%), and higher relative amounts of linalyl acetate (21.3% vs. 16.8%). The findings of this study demonstrate the ecological interactions between pollinators and lavender, and how those interactions impact phytochemistry.

1. Introduction

Lavandula angustifolia Mill, lavender, is an aromatic plant in the Lamiaceae (mint) family [1]. While native to the Mediterranean region, lavender is cultivated throughout the world and is an important economic plant used for cosmetics, flavors, fragrances, and medicines [2,3,4,5].

Previous studies, conducted both in lavender’s native growing region and in North America, have shown that the primary pollinators of lavender are bee species [6,7,8,9]. Of the long list of bee species observed visiting lavender, Apis mellifera and Bombus spp. have been demonstrated to be the most prominent and proficient pollinators [6,7,8,9,10,11,12]. The pollination of lavender is an important process in seed production. While lavender has been shown to be capable of self-pollination, both seed production and seed viability are increased through pollinator events [8]. It is important to understand which insects are pollinating lavender and which of those pollinators are the most prevalent and efficient.

Much research has been conducted on the essential oil production and profiling of lavender. Several studies have demonstrated that different plant structures of lavender produce different phytochemicals [13,14,15,16]. The volatile compounds found in the corolla are associated with attracting pollinators and those found in the calyx with protection from herbivory [14]. However, little research has been conducted on how pollinators (or the lack of pollinators) impact the yield and phytochemical profile of lavender essential oil.

The current study investigates (1) which bee species are visiting North American cultivated lavender and (2) how pollination impacts the essential oil yield and phytochemical profile of lavender essential oil. The findings of this study demonstrate the ecological interactions between pollinators and lavender and are of interest to the fields of both environmental conservation and agronomy.

2. Materials and Methods

The study took place at the Mt. Nebo Botanical Farm in Mona, UT, USA (39.868498, −111.840540; 1500 m elevation). A single rectangular plot was used (Figure 1), which contained approximately 480 Lavandula angustifolia (lavender) plants (16 rows × 30 plants per row). Lavender plants in this section of the farm were five years old at the time of the study, containing cover crops (Trifolium sp.) between the rows of lavender, and were irrigated by a drip system. A representative voucher sample of lavender is held in the Young Living Aromatic Herbarium (YLAH): Lavandula angustifolia Mill., Wilson 2024-01 (YLAH).

Lavender plants were studied in four different groups (A-C, C₀1–5). Groups A and B each consisted of 60 lavender plants, group C consisted of 20 lavender plants, and group C₀1–5 consisted of 5 individual lavender plants. At the culmination of the study, groups A-C were each distilled into individual essential oil samples (n = 3), while group C₀1–5 was distilled into five individual samples (n = 5) (

Table 1. Lavandula angustifolia (lavender) study group details.

Lavender Group	Study Group Details	# of Plants	# of EO Samples
A	large group, open to pollination	60	1
B	large group, closed to pollination	60	1
C	small group, open to pollination	20	1
C01–5	individual plants, open to pollination	5	5

). To reduce the possibility of bias, plants were assigned into groups using random number generation. Group A, C, and C₀1–5 were left open to pollination, while group B was covered by a netted cage to exclude pollinator visits.

For group B, pollinator exclusion enclosures were constructed from wood frames using 5 cm × 5 cm pine material (dimensions h/d/w: 76 cm × 89 cm × 167 cm). The wood-framed cages were covered with fine mesh fabric (openings 3 mm × 3 mm), which allowed for sun, wind, and rain to penetrate and reach the plants, but excluded pollinators. Eye screws were put on each 5 cm × 5 cm post at the bottom of the frame to insert stakes to secure the cages to the ground around the plants (Figure 2). Each pollinator exclusion enclosure covered three adjacent lavender plants.

Weekly observations (18 June 2024 through 23 July 2024) of selected plants were conducted to ensure the pollinator exclusion enclosures were intact, and to catalog and identify which pollinators were visiting the lavender plants (groups A, C, C₀1–5). Bee specimens were identified by experts at Utah State University (USU) and at the USDA-ARS Pollinating Insect Research Unit. Bee specimens are housed at Utah State University. Additionally, the inter-row area of cover crops was mowed each week by hand by researchers.

Once plants reached floral maturity (>50% of corolla emerged), plants were harvested, separated according to group (A-C, C₀1–5), bagged, and frozen until distillation. At the time of harvest, only 50% of each plant was cut for distillation purposes (Figure 3, left) and, in order to reduce the potential for bias, either the east or west half of each plant was harvested using random number generation. Given that lavender plants are long-living perennials, and that stem-leaf materials grow during the early growing season, only flowering tops were harvested (Figure 3, right). The flowering tops were selected since their aromatic profiles are most influenced by pre- and post-pollination factors [14].

Laboratory-scale distillation was conducted as follows: 3 L of water was added to the bottom of a distillation chamber (Albrigi Luigi S.R.L., Italy), the plant material was accurately weighed and added to the distillation chamber, distillation was conducted for 2 h by direct steam, and essential oil was separated by a cooled condenser and Florentine flask. Essential oil samples were filtered and stored in a sealed amber glass bottle in a cool, dark location until analysis.

Essential oils were analyzed in triplicate, and volatile compounds identified by GC/MS using an Agilent 7890B GC/5977B MSD and J&W DB-5, 0.25 mm × 60 m, 0.25 μm film thickness, fused silica capillary column. Operating conditions: 0.1 μL of sample (neat essential oil, 0.1% soln. for C7-C30 alkanes in hexane), 150:1 split ratio, initial oven temperature of 40 °C with an initial hold time of 5 min, oven ramp rate of 4.5 °C per minute to 310 °C with a hold time of 5 min. The electron ionization energy was 70 eV, the scan range was 35–650 amu, the scan rate was 2.4 scans per second, the source temperature was 230 °C, and the quadrupole temperature was 150 °C. Volatile compounds were identified using the Adams volatile oil library [17] using the Chemstation library search in conjunction with retention indices (KIs). When identifications could not be made with the Adams library, the NIST Mass Spectral Library (version 2.3) was used and the KIs calculated using C7–C30 alkane standards. Volatile compounds were quantified and reported as a relative area percentage by GC/FID using an Agilent 7890B and J&W DB-5, 0.25 mm × 60 m, 0.25 μm film thickness, fused silica capillary column. Operating conditions: 0.1 μL of sample (50% soln. for essential oils in ethanol, 10% soln. for reference compounds in ethanol, 0.1% soln. for C7–C30 alkanes in hexane), 25:1 split injection, initial oven temperature at 40 °C with an initial hold time of 2 min, oven ramp rate of 3.0 °C per minute to 250 °C with a hold time of 3 min. Compounds were identified using retention indices coupled with the retention time data of reference compounds.

The percent yield was calculated as the ratio of the mass of the processed plant material immediately before distillation to the mass of the essential oil produced, multiplied by 100.

3. Results

3.1. Visitation by Bees

Over the course of the 5-week observational period, a total of 12 species of bees were identified visiting Lavandula angustifolia (lavender). These 12 species of bees were distributed among 10 genera. The observed species were: Agapostemon angelicus, Anthophora urbana, Apis mellifera, Bombus huntii, Bombus griseocollis, Halictus rubicundus, Halictus tripartitus, Hoplitis producta, Melissodes communis, Nomada sp., Megachile snowi, and Osmia trevoris.

3.2. Lavender Distillation

Lavender plant material was distilled in part to determine essential oil yield. The distillation details are presented in

Table 2. Lavandula angustifolia (lavender) distillation details, including plant material mass (g), yield (g), and calculated yield % (w/w).

Lavender Group	Plant Material (g)	Yield (g)	Yield (%)
A	2077.37	30.85	1.49
B	3981.72	42.60	1.07
C	1907.80	25.68	1.35
C01	47.19	0.34	0.72
C02	53.24	0.66	1.24
C03	59.69	0.57	0.95
C04	51.47	0.34	0.66
C05	130.15	1.28	0.98

. Lavender samples C₀1–C₀5 were each distilled from individual plants to demonstrate potential natural variation in both essential oil yield and profile. Lavender samples A and C were produced from the distillation of lavender plants open to pollination, and their yields are similar (1.49% and 1.35%, respectively). Lavender sample B was produced from the distillation of lavender plants closed off from pollination and had a lower yield than samples A and C (yield = 1.07%).

3.3. Lavender Essential Oil Profile

Lavender essential oil samples were analyzed by GC/MS to identify the compounds present and by GC/FID for the quantification of said compounds. A total of 46 compounds were detected and identified in the lavender samples (n = 8) and these profiles are presented in

Table 3. Lavandula angustifolia (lavender) essential oil profiles. Reported values represent averages from each sample analyzed in triplicate, which was carried out to ensure the repeatability of the values (standard deviation ≤ 0.1 for all compounds). Values less than 0.1% are denoted as trace (tr) and those not detected as nd. KI is the Kovat’s Index value and was previously calculated by Robert Adams using a linear calculation on a DB-5 column [17]. Relative area percentage was determined by GC/FID.

Compound Name	KI	A	B	C	C01	C02	C03	C04	C05
		Area %
α-thujene	924	0.2	0.2	0.2	nd	tr	tr	nd	0.1
α-pinene	932	0.3	0.3	0.3	tr	0.1	tr	tr	0.3
camphene	946	0.1	0.1	0.1	tr	0.1	tr	tr	tr
sabinene	969	0.3	0.4	0.2	0.1	0.2	0.1	0.9	0.4
1-octen-3-ol	974	nd	nd	nd	nd	nd	nd	0.2	nd
3-octanone	979	1.1	1.0	1.5	0.5	0.6	0.7	nd	0.7
myrcene	988	1.1	1.0	1.0	0.7	0.5	0.6	0.6	0.8
butyl butanoate	993	nd	nd	nd	0.3	nd	nd	nd	nd
hexyl acetate	1007	nd	nd	nd	0.6	nd	nd	0.6	nd
δ-3-carene	1008	0.3	0.2	0.2	0.1	0.2	0.2	nd	0.1
p-cymene	1020	0.3	0.3	0.3	0.1	0.2	0.1	nd	0.4
limonene	1024	1.0	0.7	0.9	0.3	0.7	0.6	0.3	0.3
1,8-cineole	1026	1.0	0.8	0.8	1.5	1.0	0.9	0.7	1.4
(Z)-β-ocimene	1032	2.8	3.5	3.9	0.6	0.8	0.7	1.3	3.5
(E)-β-ocimene	1044	3.3	2.8	2.7	0.8	0.4	0.4	0.5	0.6
γ-Terpinene	1054	0.2	0.2	0.2	nd	tr	nd	nd	tr
(Z)-sabinene hydrate	1065	nd	nd	nd	nd	0.4	0.1	nd	0.6
(Z)-linalool oxide	1067	0.2	0.1	0.1	nd	nd	nd	nd	nd
(E)-linalool oxide	1084	nd	tr	nd	nd	nd	nd	nd	nd
terpinolene	1086	0.1	0.2	0.2	nd	nd	nd	nd	nd
linalool	1095	35.4	39.9	35.7	32.4	29.2	39.4	29.2	18.4
1-octen-3-yl-acetate	1110	0.9	1.2	1.3	0.2	0.3	nd	1.7	nd
3-octanol acetate	1120	tr	tr	tr	nd	nd	nd	nd	0.1
camphor	1141	0.2	0.2	0.2	0.4	0.2	0.1	0.3	0.2
lavandulol	1165	1.3	1.9	1.5	0.2	6.7	2.7	1.2	nd
borneol	1165	0.9	0.9	0.9	1.5	nd	nd	1.2	1.2
terpinen-4-ol	1174	8.2	7.7	7.8	0.8	8.1	2.2	1.0	15.3
cryptone	1183	0.4	0.3	0.4	0.3	tr	0.4	0.2	nd
α-terpineol	1186	0.4	0.3	0.3	1.3	0.4	0.2	0.7	0.6
hexyl butanoate	1191	1.2	1.3	1.2	0.4	0.5	0.2	0.3	nd
nerol	1227	tr	tr	tr	0.1	nd	nd	nd	nd
isobornyl formate	1235	0.2	0.3	0.2	0.1	nd	nd	tr	nd
geraniol	1249	0.1	0.1	0.1	0.2	nd	nd	nd	tr
linalyl acetate	1254	21.3	16.8	19.5	28.8	19.6	29.2	28.1	32.4
lavandulyl acetate	1288	4.9	5.7	5.9	7.3	11.9	8.9	8.7	6.6
neryl acetate	1359	0.5	0.5	0.4	0.7	0.2	0.3	0.4	0.5
geranyl acetate	1379	0.8	0.8	0.8	1.3	0.4	0.7	0.6	1.5
(E)-caryophyllene	1417	2.8	2.8	2.8	1.9	3.3	1.4	3.0	1.7
α-(E)-bergamotene	1432	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
(Z)-β-farnesene	1440	1.7	1.6	2.1	nd	2.4	1.4	2.2	1.0
α-humulene	1452	0.1	0.1	0.1	0.1	0.2	0.1	0.2	0.1
germacrene D	1480	0.2	0.2	0.2	0.1	0.1	tr	0.1	0.1
γ-cadinene	1513	0.6	0.4	0.6	0.4	0.5	0.4	0.8	0.1
caryophyllene oxide	1582	0.3	0.3	0.3	3.5	1.9	0.8	2.5	1.4
1-epi-cubenol	1627	0.1	0.1	0.1	0.1	nd	nd	0.1	nd
epi-α-cadinol	1638	0.8	0.6	0.9	1.8	0.9	0.7	1.6	0.2
		95.8	96.1	95.8	89.8	92.1	93.7	89.1	90.6

. The complete dataset (repeat injections, average values, etc.) can be found in the supplemental data table (Table S1: GC data).

4. Discussion

4.1. Visitation by Bees

In 2022, a study was conducted at the same site (Mt. Nebo Botanical Farm) where bees were collected and identified throughout the farm [9]. Compared to the said study, four additional bee species (Hoplitis producta, Nomada sp., Osmia trevoris, and Megachile snowi) were collected and identified in the current study, expanding the list of bees known to visit lavender at this site (Figure 4). All four of these bee species (Hoplitis producta, Nomada sp., Osmia trevoris, and Megachile snowi) are native to North America [18,19,20]. Nomada sp. was only identified to the genus level as no scientific revisions of this group in western North America have been made of late [21]. While relatively little is known about the pollination of lavender in regions outside the Mediterranean, such as North America, these four bee species are likely foraging on a plant outside its native region. Many bees are thought to use lavender as a nectar source and not necessarily for pollen, making it an important resource for other generalist and specialist bees [9]. It is likely that the list of bees known to visit lavender will continually grow as similar studies are conducted in the future.

The current study focused on the general diversity of bee species, and overall abundance was not a focus. In the previous study conducted at this site [9], the top five most abundant bee species identified were Apis mellifera (45%), Halictus ligatus (11%), Lasioglossum incompletum (7%), Melissodes communis (5%), and Anthopera urbana (3%). While similar trends were observed (Apis mellifera being the predominant species collected), neither Halictus ligatus nor Lasioglossum incompletum were observed at all in the current study. Within 1 km of the study site, 20 beehives (Apis mellifera) are maintained. Healthy beehives typically contain between 35,000 and 50,000 bees [22,23], suggesting that the current site has approximately 840,000 supplemented bees (avg. 42,500 × 20 = 840,000). Given the diversity of wild bees observed in the current study, the diversity and abundance of wild and honey bees previously observed at the same site [9], and the potential number of supplemented A. mellifera, pollinators were not limited at the study site. While it can be argued that flowering lavender was highly active with foraging bees throughout the current study, the actual abundance was not investigated and should be the focus of future studies.

4.2. Lavender Distillation

Given that the Mt. Nebo Botanical Farm cultivates population lavender (non-clonal lavender grown from seed), plant-to-plant variation in phytochemistry is to be expected. This is observed here, with distillation yields (w/w) varying in individual plant samples (C₀1–C₀5), from as low as 0.66% (C₀4) to as high as 1.24% (C₀2). The purpose of including sample C, which was produced from distilling plant material from 20 lavender plants, was to demonstrate that a larger sample set is likely representative of the overall population of non-clonal lavender, with yields averaging out. When looking at essential oil yield, this is demonstrated here with sample C (yield = 1.35%) and suggests that sample A (yield = 1.49%), which was produced from distilling plant material from 60 lavender plants, is an even better representation of the overall population of lavender. Lavender sample B, which was also produced by distilling plant material from 60 lavender plants, had a lower yield (yield = 1.07%) than either samples A or C. The low yield of sample B could potentially be explained by the fact that these plants were excluded from pollination.

4.3. Lavender Essential Oil Profile

For the GC/FID analysis, samples were analyzed in triplicate to ensure reproducibility. The small standard deviation for each compound on repeat injections (≤0.1) does just this and allows for a proper discussion of the difference in values between the samples.

As mentioned previously, samples C₀1–C₀5 were produced by distilling plant material from individual plants. This was carried out to investigate the potential variability in plant chemistry from plant to plant. Several of the prominent compounds (defined as ≥2.0% in at least one sample) from these samples demonstrate high standard deviations between samples. For instance, linalool values range from 18.4% to 39.4% (σ = 6.8), lavandulol from not detected (nd) to 6.7% (σ = 2.5), terpinen-4-ol from 0.8% to 15.3% (σ = 5.6), and linalyl acetate from 19.6% to 32.4% (σ = 4.3). Distilling multiple lavender plants together averages the essential oil profile for the population.

Sample C was produced by distilling plant material from 20 lavender plants and was included to determine what sample size (number of lavender plants) is representative of the cultivated population. Sample A, which is the actual study group, was produced by distilling plant material from 60 lavender plants. When comparing compound values between sample A and C, all standard deviations for compounds are ≤0.3, with the exception of (Z)-β-ocimene (σ = 0.5), linalyl acetate (σ = 0.9), and lavandulyl acetate (σ = 0.5). Comparing the variability observed between samples produced from individual lavender plants (samples C₀1–C₀5) and those produced from distilling either 20 plants (sample C) or 60 plants (sample A), we can conclude that at least 20 plants are needed to provide a sample set representative of the population of lavender.

Up to this point, it has been determined that (1) individual lavender plants (C₀1–C₀5) display high variability in essential oil profiles, and (2) that at least 20 lavender plants need to be distilled together to produce an essential oil with a yield and profile representative of the overall population of lavender. Samples A and B, which are the focal point of this study, were both distilled from groups of 60 lavender plants. The difference between these samples, as previously mentioned, is that group A lavender was left open to pollination while group B lavender was covered with enclosures that excluded pollinators from accessing plants. When comparing compound values between these samples, all standard deviations for compounds are ≤0.3, with the exception of two volatile compounds that displayed high variability between these samples, linalool (σ = 2.2) and linalyl acetate (σ = 2.3) (Figure 5). Given the sample size (the number of lavender plants) in group A and group B, it can be confidently concluded that lavender pollination results in an overall decreased production of linalool and an increased production of linalyl acetate.

4.4. Differences in Essential Oil Yield and Profile

Researchers have established that once plants are pollinated, plant resources are reallocated for seed production [24,25]. This allocation of a plant’s resources suggests that essential oil production (yield and profile), which are produced based on the needs of the plant, will also change during a plant’s maturation and associated needs.

Radev [26] found, similar to the current study, that pollinated plants in the Lamiaceae family produced a significantly higher essential oil yield than unpollinated plants. Additionally, Nazem and associates [27] found that the profile of mint essential oil, once a plant was pollinated, changed, which is also supported by the current study. Given that flowers contain the reproductive organs of a plant and that each yearly growth cycle of lavender culminates in seed production through flower pollination, it is likely that essential oil production mirrors the needs of the lavender plant. It is herein hypothesized that the yield is higher in pollinated lavender in order to safeguard the seeds that are produced. Additionally, the profile of pollinated lavender (a lower abundance of linalool and a higher abundance of linalyl acetate) best serves for the protection of seeds from herbivory. Interestingly, the single study previously conducted on this same topic found similar trends with linalyl acetate content, but different trends with linalool content (a higher linalool content in pollinated lavender) [28]. The topic reviews conducted by several researchers concluded that linalool is a seemingly ubiquitous compound that displays a diversity of biological functions, ranging from attracting pollinators to deterring herbivory [29,30]. Conflicting trends in linalool content may be tied to the specific variety of lavender used in each study and both abiotic and biotic factors inherent to the growing conditions of lavender. Interestingly, little research has apparently been conducted on the ecological interplay of linalyl acetate. Additional research is needed to investigate how shifts in essential oil production benefit the lavender plant.

5. Conclusions

Lavender is often recommended as one of the main plants that should be included in gardens to support bees and other pollinators. Growing evidence suggests that lavender can indeed support diverse bee communities. The current study expands the growing list of bees observed visiting lavender (Hoplitis producta, Nomada sp., Osmia trevoris, and Megachile snowi).

Furthermore, several studies have shown that pollinators, particularly bees, are important for lavender seed production. The current study investigates how pollination affects the quality and quantity of essential oil produced in these plants. These findings show that pollinated plants had higher essential oil yields and different chemical profiles than non-pollinated plants. This highlights the importance of pollinators, particularly bees, in lavender cultivation.

The focal samples of this study, samples A and B, were each distilled from lavender plant material collected from 60 plants. While the large number of plants included to produce each sample ensured that samples were representative of the population, the overall number of samples (n = 2) was low and eliminates the possibility of determining the statistical significance of the findings. Future studies should include more samples, thus allowing for concrete conclusions.