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Research Article
Can floral volatile organic compounds contribute to the taxonomy of the Rhamnus sect. Alaternus?
expand article infoLeonardo Llorens‡, Pere Ferriol‡, Joan Tomàs‡, María Trinidad García‡, Lorenzo Gil‡
‡ University of the Balearic Islands (UIB), Palma de Mallorca, Spain

Corresponding author: Leonardo Llorens ( lleonard.llorens@uib.es )

Academic editor: Lorenzo Peruzzi
© 2023 Leonardo Llorens, Pere Ferriol, Joan Tomàs, María Trinidad García, Lorenzo Gil.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Llorens L, Ferriol P, Tomàs J, García MT, Gil L (2023) Can floral volatile organic compounds contribute to the taxonomy of the Rhamnus sect. Alaternus? Italian Botanist 16: 149-164. https://doi.org/10.3897/italianbotanist.16.116255
Open Access

Abstract

The chemistry of plants represents a taxonomic and phylogenetic value. Researchers have recently used volatile organic compounds (VOCs) for taxonomic studies. The present work analysed and determined, for the first time, the usefulness of floral volatile compounds in the taxonomy of two species of Rhamnus sect. Alaternus, as well as the hybrid between these taxa. The two species show significant quantitative and qualitative differences. The terpenes linalool, ocimene, caryophyllene, and green volatiles are exclusive to Rh. alaternus, while farnesene (terpene), methyl salicylate and methyl benzoate (benzoids) are obtained from Rh. ludovici-salvatoris. Both volatilomes were attractive to Hymenoptera pollinators; therefore, these pollinators could serve as the main hybridisation vector. In addition, Rh. alaternus shows greater chemical and genetic heterogeneity than Rh. ludovici-salvatoris. Hybrids between the two species, Rh. × bermejoi, are closer in chemical similarity to Rh. alaternus than Rh. ludovici-salvatoris.

Keywords

Balearic Islands, hybrids, Rhamnus, taxonomy, VOCs

Introduction

Plant chemistry has repeatedly been used in systematics (Hegnauer 1986a; Tundis et al. 2014 It has long been known that chemical traits and characteristics, notably the secondary metabolites (such as alkaloids, terpenoids, and flavonoids) contained in vegetative parts of the plants, help to establish relationships between plant taxa (Cronquist 1977; Singh R 2016). Moreover, they have wide applications in plant systematics (see Seigler 1981; Roma-Marzio et al. 2017); however, their use presents some limitations (Hegnauer 1986b; Hadacek 2002). It is also known that the variability of volatile secondary metabolites in the vegetative parts of plants can be high (Ahmad et al. 2014). For this reason, several authors consider the construction of phylogenies on this basis too labile (Whitehead and Peakall 2009). The chemistry of floral biogenic volatile organic compounds (BVOCs) has been well studied as it relates to communication (with pollinators) and defence (mainly herbivore-related) (Bouwmeester et al. 2019). The analysis of BVOCs also enables the evaluation of their important role in the adaptation, environmental adequacy, and medicinal interest of plants (Goswami et al. 2016).Their stability and repeatability can also allow accurate identification of natural or cultivated closely-related species and varieties (Nogueira et al. 2001; Kong et al. 2012; Yang et al. 2014; Carta et al. 2015; Jaeger et al. 2016; Peruzzi et al. 2019). Recently, BVOCs have also been studied from the standpoint of taxonomy (e.g., Levin et al. 2003; Raguso et al. 2006; Meekijjaroenroj et al. 2007; Feulner et al. 2011; Tóth et al. 2016; Stocki et al. 2020), showing their utility as discriminating characteristics. BVOCs have also been used to recognise hybrids, in which new compounds, absent in the parents, can appear (Orians 2000; Cheng et al. 2011), together with others that are not taxonomically relevant (Georgescu et al. 2015). Additionally, some VOC-related phylogenetic patterns have been recognised in some taxa, such as Ophrys (Gögler et al. 2009).

Rhamnus is a wide-ranging genus of the Temperate and (sub)Tropical regions of the Northern Hemisphere that comprises more than 100 species (Hauenschild et al. 2016). In the Mediterranean region it is represented by 37 species, where Rh. cathartica L. and Rh. alaternus L. are the most widespread (Henning and Raab-Straube 2016). Molecular studies performed on Rhamnus, excluding Oreoherzogia W.Vent (Hauenschild et al. 2016), recognise the existence of at least two sections: sect. Rhamnus and sect. Alaternus (Mill.) DC. The latter includes different species of Mediterranean and Macaronesian distribution (such as Rh. alaternus L. and related taxa, Rh. lojaconoi Raimondo, R. ludovici-salvatoris Chodat for Mediterranean, as well Rh. glandulosa Ait. and Rh. integrifolia DC. for Macaronesia. Generally, hybrids are uncommon or rare; however, Rh. cathartica (Gil-ad and Reznicek 1997; Kurylo et al. 2007) and, occasionally, Rh. alaternus (Llorens 1979; Fraga and Bermejo 2008) have shown a notable capacity to hybridise.

Rhamnus alaternus is an evergreen tree native to the Mediterranean region and is often cultivated as an ornamental garden shrub in Mediterranean-climate regions. For this purpose, this tree was introduced to many areas of the Australasian-Pacific region, where it became an invasive tree along coastlines (Muyt 2001; GISD 2018). This wide distribution and the different types of plant communities in which it thrives agree with its remarkable morphological variability (López-González 2006; Rivas-Martínez and Pizarro 2011, 2013). On the other hand, although Rh. alaternus is a defined species, its differentiation from other species of the Rh. sect. Alaternus, especially the hybrids, is problematic (Ferriol et al. 2009). This differentiation occurs with Rh. ludovici-salvatoris (Fig. 1), from which it differs basically in its larger size and the arrangement of the leaf teeth (approximately perpendicular to the margin in Rh. ludovici-salvatoris and obliquely oriented upwards in Rh. alaternus). This difficulty has taxonomic and nomenclatural consequences, especially in plants that develop in unfavorable conditions. This difficulty occurs with the definition and identification of some specimens, such as Rh. jacobi-salvadorii O.Bolòs & Vigo or Rh. alaternus var. ferruginea Pourr. ex Texidor, making it difficult to establish a more precise distribution range. This is the case, for example, in the confusing or erroneous indications of Rh. ludovici-salvatoris in the eastern Iberian Peninsula or the actual distribution of the hybrid of this species with Rh. alaternus (i.e., Rh. × bermejoi Fraga & Rosselló).

Figure 1. 

Inflorescences, flowers and major pollinator (Apis mellifera) of Rhamnus A–C Rh. alaternus D, E Rh. ludovici-salvatoris F Rh. × bermejoi G leaves of the three species.

The Balearic Islands are the largest archipelago of continental islands in Spain. These islands display a complex palaeogeography, although they have been isolated relatively recently. The eastern islands (Gymnesian) and western islands (Pithyusian) have been separate groups from the early Pliocene. Nevertheless, the Gymnesian Islands (Majorca and Minorca) have only been completely isolated from each other since the Würm glaciation, approx. 15,000 ya (Cardona and Contandriopoulos 1979). In these islands, only two species from Rh. sect. Alaternus grow: the Mediterranean Rh. alaternus and the Gymnesian endemic Rh. ludovici-salvatoris. However, both species are related, and some hybrid plants have been identified in one locality of Minorca (Tirant) (Llorens 1979; Fraga and Rosselló 2008). Despite this, phenological and ecological barriers establish good isolation of the populations (Ferriol et al. 2009).

The chemical profile of the floral scent of the taxa of Rhamnus sect. Alaternus present in Majorca, Minorca, Ibiza, and eastern Iberian Peninsula was analysed in order to address the following three goals: 1) to recognise the volatile compounds of the two species, 2) to establish analogies and chemical differences between the hybrid plants and the parental species, and 3) to determine their value in taxonomy and their usefulness as discriminating characteristics in these species and their hybrids, as well as to establish their possible application to the taxonomy of other Rhamnus species.

Materials and methods

Plant material

The two species under study and their hybrids were collected from the Balearic Islands and the eastern Iberian Peninsula (Fig. 2, Suppl. material 1). Samples of natural hybrid plants reported as Rh. × bermejoi (Llorens 1979; Fraga and Rosselló 2008) were collected in the locality of Tirant (N of Minorca), where they coexist with the parental species. Hybrid plants obtained by manual artificial hybridisation, cultivated and vegetatively propagated were also sampled. A total of 35 plants (14 female and 21 male) were screened for floral VOCs. The wild plants were collected from 13 sites, and the cultivated hybrids were collected from two sites.

Figure 2. 

Location of the Rhamnus populations included in the study. (See Suppl. material 1 for population abbreviations). Rh. alaternus open squares; Rh. ludovici-salvatoris filled triangles; Hybrids filled circle.

Floral scent trapping

The volatiles emitted by flowers were obtained by the headspace solid phase extraction (HS-SPME) sampling technique, adapted from Friberg et al. (2013) and Tomas et al. (2022). Volatile compounds were extracted in a manual SPME holder together with 10 mL vials and PDMS-DVD fibres (Supelco Inc., Bellefonte, USA). First, ten flowers of each plant were placed in a vial. Next, these vials were placed at 25 °C for 20 min; then, the SPME fibre was exposed in the upper space (headspace) of the sealed vial for 30 min at 25 °C to adsorb the analytes. Three collections and their respective controls (empty vials) were prepared simultaneously. In addition, to distinguish between BVOCs from flowers and those from flower pedicels, volatiles emitted by ten pedicels in both parental species were analysed.

Evaluation of scent composition

Analyses were conducted no longer than 24 hours after collection. HS-SPME-GC-MS analyses were performed on an Agilent 6980 GC - MDS 5975 inert XL (Agilent Technologies, USA), using a Supelcowax 10 gas capillary column (60 m × 0.25 mm × 0.25 mm; Supelco, Bellefonte, Pennsylvania, USA). The PDMS-DVB fibre of the SPME was aged for 3 min in the inlet of the GC. Helium was used as a carrier gas at a flow of 1.3 mL min-1. The splitless sample injection mode was used. The injector temperature was 220 °C. The initial oven temperature was programmed as follows: 45 °C for 2 min, increasing to 250 °C at a rate of 5 °C min-1, which was continued for 5 min. These settings were sufficient for the quantitative desorption of all analytes studied. Mass spectra (MS) were obtained in electron ionisation (EI) mode at 70 eV, while the ionisation source was 180 °C. The MS scan range was set between 45–300 amu. The chromatograms and spectra of the samples were processed using the GC-EM software Turbomass version 5.1 (Perkin-Elmer, Inc.).

The main isolated volatile compounds were annotated by comparing the mass spectra with mass spectral libraries (Wiley 7th edition and NIST08) and by comparing the calculated retention indices with those provided by NIST08, Adams (2007) and El-Sayed (2012). The annotation of the main compounds was verified using an in-house-developed mass spectra/RI library. The main compounds were verified by comparison with synthetic standards. All compounds that were not identified immediately as floral/plant volatiles were verified in the literature to determine if they were accepted as floral BVOCs or at least as potential volatiles.

Statistical analysis of data

Differences in BVOCs composition were analysed using the statistical software R version 4.1.2 (R Project for Statistical Computing). Since the total amount of volatiles released strongly fluctuated between individuals, we used the relative amounts taken as the peak area of each compound in relation to the total peak area. Those compounds considered as likely artefacts were excluded from the analysis. The volatiles from three plants were analysed for each population, averaged, and then treated as a single data set.

Data were transformed using the decostand function in the vegan library (Oksanen et al. 2019). To assess similarities between individual samples, a permutational multivariate analysis of variance (PERMANOVA) was performed with the adonis2 function on the vegan library using the Bray-Curtis dissimilarity indices (Oksanen et al. 2019). Finally, as a post hoc test to detect differences in volatile composition between each of the species, a pairwise contrast for multilevel comparison (pairwise.adonis function, Martínez-Arbizu 2020) was used.

Significant p-values in a PERMANOVA indicate a significant difference between groups in the centroid or spread of objects in a multivariate space, so non-metric multidimensional scaling (NMDS, metaMDS function with Bray-Curtis distance, performing a Wisconsin double standardisation and also an sqrt transformation) was used. (Clarke et al. 2014).

To test whether there is a separation between groups in relation to the sex of the flowers, an analysis of similarities (anosim function, vegan package) was performed. To identify the percentage contributions of each volatile compound to the average dissimilarity amongst the three Rhamnus species, the similarity percentage analysis (SIMPER, the simper function in vegan) was used (Clarke 1993).

Results

Floral scent chemistry

No relevant traces of BVOCs were detected in floral pedicel samples. In the flowers, the entire GC–MS dataset allowed the detection of 41 compounds, of which 22 had greater presence and abundance (with percentages higher than 0.1%; Suppl. material 2). Chromatographic profiles of R. alaternus showed high heterogeneity. Thus, some plants exhibited the presence of certain components in high proportions (up to 89.9%), e.g., ionones (dihydro-β-ionone and α-ionone), 3-hexen-1-ol, acetate (Z), benzaldehyde 4-dimethoxy, caryophyllene, and linalools (cis-linaloxide, (E)-linalool oxide (furanoid), linalool, hotrienol). However, some of these components were absent in other plants. By contrast, despite including a larger number of samples, R. ludovici-salvatoris showed a remarkable uniformity. Thus, the major compounds, such as methyl salicylate, methyl benzoate, or α-farnesene, were found in 85–100% of the samples. Similarly, the largest number of minority compounds, such as ethyl benzoate, 1-butanol, and 3-methyl-benzoate, were present in more than 75% of the samples.

Floral scent species relations

The chromatographic profiles of Rh. alaternus and Rh. ludovici-salvatoris showed significant qualitative and quantitative differences between the species (Suppl. material 2 and Fig. 3). Thus, the following components were exclusive to Rh. alaternus: acetic acid hexyl ester, certain terpenoids and ocimene including cis- and trans-β-ocimene (6.73%); linalools including linalool, cis-linaloxide, trans-linalool oxide furanoid, linalyl formate, and hotrienol (9.10%); caryophyllenes including β- and α-caryophyllene (15.71%), and ionones including α-ionone and dihydro-β-ionone (48.28%). On the other hand, in Rh. ludovici-salvatoris, unique compounds were found, namely benzoic acid derivatives (methyl salicylate, methyl benzoate, 1-butanol, 3-methyl benzoate) and farnesenes (farnesene and α-farnesene). Among the quantitatively differential products, 3-hexen-1-ol acetate (Z) emerged because in Rh. alaternus, it was present in high amounts (25.92 ± 7.55%), while in Rh. ludovici-salvatoris it was only found in low quantities (0.65 ± 0.37%) and in less than half of the samples.

Figure 3. 

VOCs similarity histogram of Rhamnus alaternus (violet), Rh. × bermejoi (blue) and Rh. ludovici-salvatoris (green). Colour intensity is related to the level of presence of a component in each sample.

The results of the SIMPER analysis (Table 1 and Fig. 4) clearly highlight the differences in the aroma profile of the two species. They show that the two species have a distinct floral aroma profile and that four compounds (methyl salicylate, 3-hexen-1-ol acetate, ionone complex, and b-caryophyllene) account for 75% of these differences. On the other hand, no differences were found between the BVOC profiles in relation to sex (ANOSIM statistic R = -0.03533, p = 0.8222). According to the chemical composition of the hybrid samples and their uniformity, it is noteworthy that they exhibited a great affinity with Rh. alaternus (Table 1, Figs 3, 4, Suppl. material 2).

Table 1.
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Results of SIMPER analysis indicate the cumulative contributions of most influential compounds. AvA: Average in Rhamnus alaternus; AvB: Average in Rhamnus ludovici-salvatoris; Cumsum: cumulative contribution (in%).

AvA AvB Cumsum
Methyl salicylate 0.00 82.32 44.4
3-Hexen-1-ol, acetate (Z)- 24.88 0.64 57.6
Ionone complex 16.09 0.00 66.4
b-Caryophyllene 15.18 0.00 74.6
Methyl benzoate 0.00 9.25 79.6
Linalool and related 9.11 0.00 84.4
Benzaldehyde 4-dimethoxy 5.23 0.00 87.1
(Z)-b-Ocimene 4.69 0.00 89.7
1-Butanol, 3 methyl, acetate 4.26 0.54 92.0
Figure 4. 

Non-metric multidimensional scaling ordination (NMDS) of floral scent profiles for Rhamnus alaternus (violet and empty circles), Rhamnus × bermejoi (blue and half black circles) and Rhamnus ludovici-salvatoris (green and black points).

Discussion

Volatile compounds of the green parts of the plants have been used repeatedly as taxonomic characteristics (see Hegnauer 1986a, Nwafor et al. 2018). By contrast, the analysis of volatile biogenic compounds of flower aromas (floral BVOCs) has been scarcely studied. Despite this, most recently, the qualitative and quantitative variations of these compounds have shown their value as discriminating characteristics in related species or in ecotypes, such as in the genus Clusia (Nogueira et al. 2001), Nicotiana (Raguso et al. 2006), Lilium (Kong et al. 2012) or Artemisia (Jaeger et al. 2016). The results obtained in Rh. alaternus and Rh. ludovici-salvatoris and their hybrids were consistent with these observations.

The main differences between Rh. alaternus and Rh. ludovici-salvatoris were found in the types of terpenoids and benzoids emitted, as well as in their emission rates (Suppl. material 2). Thus, among the terpenoids, R. alaternus exhibited the emission of monoterpenes ((Z)- and (E)-β-ocimene and linalool and related compounds) and ionones; all these products are absent in Rh. ludovici-salvatoris. There were also different types of sesquiterpenes, farnesenes being characteristic of Rh. ludovici-salvatoris and caryophyllenes being characteristic of Rh. alaternus. On the other hand, the uniformity and richness of benzoids in Rh. ludovici-salvatoris (specifically, methyl salicylate and methyl benzoate) were very prevalent. This finding contrasts with its absence in Rh. alaternus, because in this species, only the rare presence of benzaldehyde 4-dimethoxy has been recognised. Methyl salicylate (83.3%) and methyl benzoate (9.2%) constitude the primary components of the floral aroma in Rh. ludovici-salvatoris. These compounds are also present in hybrids but notably absent in Rh. alaternus. Both esters are biosynthesised by the action of carboxyl methyltransferases and have been associated with pollinator attraction (Negre et al. 2003), but they also play an important role as aerial signals involved in inter- and intra-plant communication to activate disease (Shulaev et al. 1997) and, as also seems plausible in this case, signalling an abiotic stress situation for this species (Munné-Bosch and Peñuelas 2003; Karl et al. 2008; Liu et al. 2018; Gondor et al. 2022).

The high intra-population heterogeneity of chemical compounds of Rh. alaternus was evidenced by the fact that not all differential compounds were found in all the plants; however, some of them were always present. This observation stands in contrast with Rh. ludovici-salvatoris, which has a high chemical similarity with all plants, as evidenced by the presence of its main differential compounds, methyl salicylate and methyl benzoate. These chemical diversity profiles followed a pattern similar to that of genetic diversity (Ferriol et al. 2009; Hauenschild et al. 2016), which recognizes much higher levels of genetic variation in the populations of Rh. alaternus than in those of endemic Rh. ludovici-salvatoris.

The qualitative and quantitative patterns of VOC expression in hybrids are heterogeneous (López-Caamal 2014). In some species, hybridization could lead to the production of new secondary metabolites that are not present in parental species, due to the obstruction of biosynthetic pathways (Vereecken et al. 2010; Kong et al. 2012). In other hybrid species, highly variable proportions of several compounds are present. In some, the emission rates of some of these may be interpreted as transgressive with the parents; however, they are not always consistently intermediate. In others, only the emission of specific compounds was intermediate (Bischohff et al. 2014). In general, whenever hybridization occurs, a large qualitative variation in secondary metabolites is expected. In the studied hybrids, compounds shared with parental strains were produced at different levels, but no new products were recognized (Suppl. material 2). In the amounts of shared compounds, there was a clear tendency to be more like Rh. alaternus (Fig. 4).

Both personal observations and those of Canale et al. (2016) showed that the main pollinators of the two Rhamnus species were Hymenoptera (mainly Apis mellifera Linnaseus, 1758). This finding was consistent with the fact that the two groups of BVOCs that are dominant in the flowers of the two species of Rhamnus attract honey bees: terpenoids for Rh. alaternus (Dobson 2006; Twidle et al. 2015) and benzoids for Rh. ludovici-salvatoris (Negre et al. 2003; Mallinger et al. 2011; Dötterl et al. 2014). Thus, this could be the pollination vector that mediates the hybridization process.

When the diversity of BVOCs is greater in hybrids than in the parentals, their heterogeneity and progression are encouraged, since this could help in attracting more pollinator species (Ayasse et al. 2011), breaking the reproductive isolation of hybrids. However, this increase does not occur in the studied hybrids; therefore, their VOC content, as in their parent R. ludovici-salvatoris, was not a factor that favours pollination and reproduction.

Under these conditions, compared to Rh. alaternus, lower photosynthetic efficiency of Rh. ludovici-salvatoris and its hybrids in the current climatic Mediterranean circumstances (Yll et al. 1997; Gulias et al. 2002) would be, together with the inability of regrowth, the main cause that would explain both the progressive decrease in the populations of this species as well as the rarity of hybrids and the relictual character of the current population of Rh. ludovici-salvatoris and its hybrids in Minorca (Fraga and Bermejo 2008).

Conclusions

The analysis of the floral volatile compounds in the studied taxa reveals both qualitative and quantitative differences in the chemical profile of R. alaternus and Rh. ludovici-salvatoris. Among them, the discriminatory nature of various compounds stands out, such as methyl salicylate and methyl benzoate (present in Rhamnus ludovici-salvatoris and absent in Rh. alaternus), linalool and related compounds, ionones, β-caryophyllene, 4-dimethoxy benzaldehyde, and (Z)-β-ocimene (present in Rh. alaternus and absent in Rh. ludovici-salvatoris). In contrast, the chemical profile does not allow differentiation between Rh. alaternus and the hybrid Rh. × bermejoi. According to these results, it is suggested that floral volatile compounds could be an effective discriminatory trait among species in Rh. sect. Alaternus. This finding should be confirmed through the study of plants from other populations of Rh. alaternus, as well as samples from Rh. lojaconoi and the Macaronesian endemic species Rh. glandulosa and Rh. integrifolia.

Acknowledgements

The authors are especially grateful to L. Cortés and F. Roma-Marzio for their valuable comments on the manuscript, to S. Biondi for the English revision of the text, and to C. Cardona for providing plant material.

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Supplementary materials

Supplementary material 1 

Geographical location and sexuality of Rhamnus sampled plants

Leonardo Llorens, Pere Ferriol, Joan Tomàs, María Trinidad García, Lorenzo Gil

Data type: xlsx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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Supplementary material 2 

Floral scent chemistry

Leonardo Llorens, Pere Ferriol, Joan Tomàs, María Trinidad García, Lorenzo Gil

Data type: docx

Explanation note: Floral scent chemistry of Rhamnus alaternus (blue), R. ludovici-salvatoris (yellow) and hybrids (green). For each species, compounds with presence in >10% of samples.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (21.17 kb)
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