Conifers are distributed worldwide from the tropics to the Arctic regions. Many species are economically important because they provide essential oils (EOs) and pine extracts, which are used in cosmetics as fragrances, in aromatherapy, or as food and beverage ingredients. In addition, the pharmaceutical industry uses EOs, resin, and the bark of certain conifer species. Juniper oil has a herbal monograph in the European Union, products containing pine tar have been used to treat different skin conditions (e.g., eczema, psoriasis, and skin inflammation), pine bark extracts have been investigated in a variety of chronic diseases, and the effects of pine resin have shown promise in the treatment of malignant ulcerating wounds (Barnes and Greive, 2017; Robertson et al., 2020; Batista et al., 2022).

EOs are mainly plant metabolites of terpenoids, i.e., mixtures of volatile organic compounds (VOCs). The building block of terpenoids is the isoprene unit, which comprises an isopentenyl skeleton. Monoterpenes, the most important essential oil components, consist of two isoprene units, while sesquiterpenes comprise three isoprene units. EOs and VOCs are important candidates for drug development. For example, monoterpenes are subjected to structural modifications in medicinal chemistry, a promising way to synthesize novel biologically active substances. According to Zielinska et al., borneol-based polymers have recently been investigated to improve cell adhesion, borneol isomers improve surface bacterial resistance properties, and α-pinene derivatives have been synthesized as potential antitumor agents, among others (Zielinska-Blajet and Feder-Kubis, 2020). Nootkatone, which is a sesquiterpene and has been used as a safe and effective active substance in a commercial insect repellent, was first isolated from Chamaecyparis nootkatensis (D.Don) Spach (Dietrich et al., 2006). Other economically important sesquiterpenes (e.g., β-farnesene, santalol, β-caryophyllene) are also available on the market. They are used in the food and fragrance industries due to their pleasant flavor and smell (Jiang and Wang, 2023). Sundar and Parikh, authors of a recent publication, have summarized the available knowledge on encapsulation techniques of essential oils. This process contributes to increasing the shelf life and the bioavailability of essential oils. Therefore, it is a promising technique for the development of EO-containing drugs (Sundar and Parikh, 2023). VOCs can also serve as initial molecules for the synthesis of other substances in the chemical industry. Oxygenated terpenoids may play an important role in the synthesis of biofuels (e.g., 1,8-cineole as the potential bio-jet fuel precursor) (Xie et al., 2020). Current applications of non-wood pine products are discussed in a study by Neis et al.; for example, sustainable thermoplastic elastomers are made from terpene-derived monomers (Neis et al., 2019). The wood of Cupressaceae is durable; therefore, it is a suitable source of timber and construction materials, particularly species from the Calocedrus, Chamaecyparis, Cupressus, and Thuja genera. Additionally, within the Cupressaceae family, Chamaecyparis, Juniperus, Platycladus and Thuja species are valued as ornamental trees. Cryptomeria japonica is the major plantation tree in the Far East. Furthermore, conifers are also used in horticulture, contributing significantly to their economic importance as cultivated species (Farjon, 2018; Mao et al., 2019).

The aim of this work was to investigate the monoterpene and sesquiterpene profiles of conifer species and to explore possible chemotaxonomic relationships. According to current gymnosperm phylogeny (Yang et al., 2022), extant gymnosperms are classified into three Linnaean classes, Cycadopsida, Ginkgoopsida, and Pinopsida - the latter including three subclasses, Gnetidae, Pinidae, and the Cupressidae. Conventionally, conifers are the last two subclasses. The classification of Gymnosperm (including subclasses and families) is presented in Table 1 . Our current study focuses on two families of conifers, namely Pinaceae and Cupressaceae. The classification of these two families (i.e., a list of corresponding genera) and their habitats are presented in the Supplementary Material in Supplementary Table S1 (WFO, 2024). Pinaceae is the most prominent gymnosperm family, with 11 genera and 272 species. It is widely distributed and restricted to the Northern Hemisphere (except for P. merkusii). The family is traditionally subdivided into two groups: the pinoid subfamily (Pinoideae) includes Cathaya, Larix, Picea, Pinus, and Pseudotsuga, whereas the abietoid subfamily (Abietoideae) includes Abies, Cedrus, Keteleeria, Nothotsuga, Pseudolarix, and Tsuga (Ran et al., 2018). Cupressaceae is also rich, comprising 31 genera and 169 species. It is distributed on all continents (except for Antarctica) from the sea level to the timberline. Gadek et al. identified seven subfamilies of the family: Cunninghamioideae, Taiwanioideae, Athrotaxidoideae, Sequoioideae, Taxodioideae, Callitroideae, and Cupressoideae. This classification was supported also by genome-based phylogeny studies (Gadek et al., 2000; Yang et al., 2012; Liu et al., 2022; Yang et al., 2022). Several samples investigated in the present study originate from species of the Cupressoideae subfamily, the largest subfamily within the Cupressaceae family, encompassing approximately 100 species distributed among 13 genera.

Most conifers are evergreen, although exceptions exist within certain genera, such as Larix and Pseudolarix, which are deciduous. The leaves of the Pinaceae are needle-like, whereas scale leaves are predominantly found in the Cupressaceae family. The morphological properties of the bark are characteristic of the species. Seeds are enclosed within cones, originating from the female cone, while the male cone produces pollens (Debreczy et al., 2011). Throughout various parts of conifer trees, oleoresin is present within resin ducts, resin vesicles, resin blisters, resin glands, and resin cells. Oleoresin is produced due to physical damage to the plant. Its primary purpose is chemical and physical defense against herbivores and to prevent the damaged part of the plant. Recent investigations have highlighted the involvement of terpenes in chemo-ecological interactions, as they influence insect behavior and serve as signal molecules for bark beetles. Additionally, alterations in the terpene profile contribute to enhanced resistance against microbial pathogens and insect-induced damage (Celedon and Bohlmann, 2019; Whitehill et al., 2019; Ninkuu et al., 2021).

In conifers, typical monoterpenes are α-pinene, β-pinene, camphene, 3-carene, myrcene, α-terpineol, bornyl acetate, and typical sesquiterpenes are β-caryophyllene and germacrene D (Bhardwaj et al., 2020). However, the similarities and differences in the essential oil composition of conifers have not been exhaustively investigated by chemometric methods. In our work, essential oil samples of 30 species of the Pinaceae and Cupressaceae families were analyzed chemically and chemometrically. Mapping the chemotaxonomic relationships of conifers can contribute to a better understanding of taxonomic relationships and provide valuable data for industrial exploitation of VOCs. In addition, this study may support other research on the terpene synthase system of conifers. A better understanding of the conifer terpene synthase system may contribute to the identification of new pathways for microbial production of terpenes and the design of key enzymes involved in the biosynthesis process (Zhang et al., 2017; Zhang and Hong, 2020).

Terpenes represent a major group of plant metabolites in conifers. Monoterpenes and sesquiterpenes are essential oil components, whereas terpenoids with higher molecular weight, such as diterpenes, are usually not volatile. The volatile profiles of species are mainly determined by the terpene synthase (TPS) family (Alicandri et al., 2020); however, the actual amounts of volatiles in the plant part depend on different factors. These factors include developmental factors, stress, and other stimuli (temperature, biotic stress, pollution, season, etc.) (Kopaczyk et al., 2020).

We acknowledge that there are certain constraints on the results and conclusions because the sample tissues were collected from two artificially managed arboreta in the same region (Hungary). Whereas such a sampling scheme is not suitable for evaluating overall geographic variability in the volatile oil composition of any plant, it ensures that the trees were grown under very similar environmental conditions, thereby guaranteeing that our observations are less dependent on the environment. In this way, differences among species are more likely attributable to their own metabolism.

We reviewed the literature to compare our GC/MS results with the previously reported analyses. The major VOCs identified by our study in the abietoid group were α-pinene, β-pinene, β-caryophyllene, β-phellandrene and bornyl acetate, which is consistent with the literature. The major components found in our samples of Abies concolor (needles) were α-pinene, camphene, β-pinene, and bornyl acetate. In contrast to the analysis by Swor et al., the amount of δ-3-carene (0-1%) and limonene (0-2%) were negligible in our samples, but the other VOCs were found in similar amounts (Swor et al., 2022). In this analysis by Swor et al., the authors also provided the main chemical compounds of A. grandis, such as α-pinene, camphene, β-pinene, β-phellandrene, and bornyl acetate. They concluded that the investigated compositions were qualitatively similar to other Abies essential oils examined in the literature. The main components from our A. grandis samples were qualitatively similar. Sarria et al. identified an α/β pinene synthase in A. grandis, which produces about 40% α-pinene and about 60% of β-pinene (Sarria et al., 2014). This can also be a reasonable cause of the larger amount of β-pinene (31%) compared to α-pinene (10%) in our samples. According to Na-Hyun Lee at al., the major compounds of Abies firma were bornyl acetate, δ-3-carene, α-pinene, β-caryophyllene, and camphene (Lee et al., 2015). According to Satou at al. the main constituents of A. firma were α-pinene and δ-3-carene (Satou et al., 2011). The main VOCs of our Abies firma samples were consistent with the reported analyses, however a major difference was our samples containing only a low amount of δ-3-carene (0.7%). Based on the reported analysis of Lee and Hong, the main components in A. holophylla essential oil were bicyclo[2.2.1]heptan-2-ol (28%), δ-3-carene, α-pinene, camphene, limonene, β-myrcene (Lee and Hong, 2009). Bornyl acetate (bicyclo[2,2,1]-heptan-2-ol-1,7,7-trimethyl-acetate) was not detected by Lee and Hong, however our samples contained a considerable amount of this VOC (13%). Based on the above detailed chemical profile data of previous studies we can conclude that our results of Abies species align with those found in the literature. According to Boudarene et al., the main VOCs of Cedrus atlantica oils were α-pinene and β-caryophyllene. Our results are in line with these data, however we detected β-myrcene in considerable amount (18%). In contrast to other analyses reported in the literature, himachalenes were negligible in our samples (Boudarene et al., 2004; Ainane et al., 2019; Ailli et al., 2023). Bornyl acetate and α-pinene were reported as the most abundant VOCs of Tsuga heterophylla and T. canadensis species in the study of Lagalante (Lagalante and Montgomery, 2003). Correspondingly, our Tsuga samples contained a high percentage of α-pinene and bornyl acetate, however, the ratio of β-phellandrene was also high (17%) in our T. heterophylla samples. A study by Ankney et al. reported that one of the major components in T. heterophylla essential oil is β-phellandrene (6.6–19.3%) (Ankney et al., 2024). In summary, our findings are consistent with the literature.

The major VOCs in the pinoid group identified by us were α-pinene, β-pinene, bornyl acetate, β-phellandrene, β-caryophyllene and β-myrcene, which is consistent with the literature. Nikolic et al. reported that the main compounds of Picea omorika were bornyl acetate (29.2%), camphene (18.7%), and a-pinene (12.9%) (Nikolic et al., 2009). In our samples, the main components were the same, and their quantity closely matches the results of Nikolic et al. Furthermore, our results are consistent with the findings of Hall et al., as the main components of P. sitchensis were α-pinene, β-myrcene, limonene, β-phellandrene (Hall et al., 2011). However, in our samples, the amount of α-farnesene was high (19%). Farnesenes are reported to influence the behavior of insects, for example, Picea sitchensis, when attacked by pine weevils, releases α- and β-farnesenes (Kännaste et al., 2008). The main components of our P. nigra samples were α-pinene, germacrene D, and β-caryophyllene, which is in accordance with the results of Sarac et al. (2013); Koutsaviti et al. (2015), and Mitic et al. (2018), however Mitic et al. identified a relatively high percentage of β-pinene (10%) when compared to that of our samples (5%). Koutsaviti reported that their analyses were in line with the previous studies on the chemical composition of P. nigra needle oils. The most abundant VOCs of our P. cembra needle samples were α-pinene, β-pinene, and β-phellandrene, which is in line with the findings of Dormont et al. (1998). The major components of our samples in P. heldreichii were limonene, α-pinene, germacrene D, and β-caryophyllene. These data align with the results of Mitic et al. (2019) and Basholli-Salihu et al. (2017). Pinus aristata proved to contain the highest amount of δ-3-carene among our samples (42%), which is in good agreement with the findings described by Faiola et al. (2015). Despite the high variability, their analysis also suggests that the same seven compounds can be found in the largest amount in Pinus aristata, Abies grandis, and Pseudotsuga menziesii. The major components, as stated in the report, were: α-pinene, limonene, δ-3-carene, β-pinene, β-myrcene, camphene, and β-phellandrene. Our results do not confirm this statement, as P. aristata also contained terpinolene (10%) while P. menziesii and A. grandis contained bornyl acetate (28% and 16%, respectively). Major VOCs in our P. peuce samples were α-pinene, germacrene D, β-phellandrene and β-pinene and their quantities were similar as described in the literature (Nikolic et al., 2008; Basholli-Salihu et al., 2017; Mitic et al., 2018). However, δ-3-carene and β-caryophyllene were also considerable in our samples (15% and 8%, respectively). Corresponding to our results P. strobus contains high percentage of α-pinene, β-pinene, δ-3-carene, and β-caryophyllene. According to Koutsaviti et al. a considerable amount of β-pinene, α-pinene, and β-caryophyllene were identified in P. strobus. They concluded that quantitative differences were observed between their analysis and other analyses in the literature; however, qualitative similarities were observed (Krauze-Baranowska et al., 2002; Koutsaviti et al., 2015). The primary VOCs of our P. menziesii samples were bornyl acetate, camphene, and β-pinene. Based on the findings of Swor et al. (2023), the chemical profile of P. menziesii var. glauca is characterized by high levels of bornyl acetate and camphene, which is consistent with our results.

Sabinene is a typical component of the Cupressaceae family (Foster et al., 2013), and our results support this. In addition, α-thujone and β-thujone were detected in the Cupressaceae family but were absent in the samples of the Pinaceae family. Thujone is formed from sabinene, and sabinol is an intermediate product in thujone biosynthesis (Nemeth and Nguyen, 2020). In most cases where thujone was detected in our samples, sabinol was also present; see Table 5 for all samples containing thujone.

Our research indicates differences in the terpene profiles of the plant organs of species from Pinaceae family (see Figure 9 ), which is in line with the literature (Dormont et al., 1998; Chizzola and Müllner, 2021; Butnaru et al., 2022). According to previous studies, the proportional amounts of several terpenes show strong positive correlations among different plant parts in conifers, or the same main VOCs could be identified in them (Dormont et al., 1998; Manninen et al., 2002). In addition, terpenoid production might be controlled not only by genetics, but also by environmental factors or plant organ development (de Simon et al., 2021; Bai, 2022). It is important to mention that although some research has observed significant differences in diterpenes (Chizzola and Müllner, 2021; de Simon et al., 2021; Nantongo et al., 2022), we did not investigate them in our study. The limitation of our study is that the collection of samples was not completely consistent (i.e., we did not always collect and analyze the same plant parts and did not consider the developmental stages of the plant organ). For future trials, we recommend using standardized and more comprehensive sample collections to improve the reliability of the data and reflect a more accurate composition of VOCs.

Terpenoid metabolites are synthesized in plants by specific terpene synthase enzymes. This is an enormous and intensively researched area (Karunanithi and Zerbe, 2019; Alicandri et al., 2020), and it is particularly important to investigate the relationships between the phylogeny and the terpene profiles of the species. This knowledge is necessary to refine metabolic engineering and synthetic biology tools to optimize the efficient production of economically valuable terpenoid compounds in the future. Our data on conifer VOCs can provide valuable scientific information for further research in this area.

In this comprehensive study, monoterpenes, sesquiterpenes, and their derivatives were analyzed by SPME-GC/MS from samples of 30 conifer species collected from arboreta in Hungary. A total of 151 samples (needles, cones, barks, and resin samples) were measured. Our gas chromatographic analyses in the Pinaceae family (103 samples) reveal that the most abundant components are α-pinene, β-pinene, bornyl acetate, limonene, and β-caryophyllene. In the Cupressaceae family (50 samples), the most important components are α-pinene, limonene, sabinene, β-myrcene, and δ-3-carene. In addition, further multivariate analyses (PCA and CVA) were performed on the analytical results to explore the correlation between chemical composition and species or plant organs. The detected VOCs showed similarities and differences within species and also among organs or higher taxonomic order levels such as genera. Characteristic chemical components were identified, such as sabinene for the cupressoid group, camphene and bornyl acetate for the abietoid group, and longifolene and β-pinene for the pinoid group.

Our present research performed on several conifer species provides comprehensive data on VOCs in the Pinaceae and Cupressaceae families. Furthermore, these data can complement our understanding of the biodiversity of conifer species and support other research on the production of terpenes by synthetic biology.