Variations in Essential Oil Composition and Chemotype Patterns of Wild Thyme ( Thymus ) Species in the Natural Habitats of Hungary

: A comprehensive study was conducted on the diversity and characteristics of five Thymus species native to Hungary, concerning frequency of occurrence, habitat preferences, essential oil content of the dried flowering shoots, and chemotype patterns determined by GC/MS. Our main aims were to provide an overview of the essential oil diversity of thyme resources and select the best genotypes with potential for cultivation and utilization. Based on the results obtained in 74 populations of 63 localities belonging to 15 regions of the Transdanubian and Northern Hungarian Mountains, considerable essential oil diversity was found. Thymus pannonicus (TPA), of generalist character, was proven to be the most frequent species (38 populations), while T. serpyllum (TSE) occurred only in two habitats. High average amounts of essential oil (EO) were shown for T. pannonicus (0.46 mL/100 g DW), T. pulegioides (TPU: 0.47 mL/100 g DW), and T. serpyllum (0.59 mL/100 g DW), while low EO accumulating ability was detected in T. glabrescens (TGL: 0.26 mL/100 g DW) and in T. praecox (TPR: 0.10 mL/100 g DW). In general, the thymol chemotype was the most frequent (34 populations), found together with the related molecules (p-cymene: 26; γ -terpinene: 15), while numerous other monoterpenes (M: geraniol: 12, linalool: 7) or sesquiterpenes (S: germacrene D: 25, β -caryophyllene: 21) were dominant, as well as combined (MS) chemotypes, which were also described in the Eos of Thymus species in Hungary. Our findings confirmed that T. pannonicus shows potential for cultivation with homogenous drug quality, adequate amounts of essential oil, and stability in EO composition. Data from original habitats also supports its high tolerance and adaptability to diverse environmental conditions, which is advantageous when facing climate change and extremities.


Introduction
The genus Thymus (Lamiaceae, Nepetoideae) involves diverse essential oil-bearing species with high intraspecific variability [1].Besides garden thyme (Thymus vulgaris L.), further wild thyme species attract regional or broader interest, where flowering shoots are collected and used for various therapeutic purposes or applied as ornamental plants in gardens [2].Essential oils and extracts of wild thyme species (T.sepyllum L. s. l.) have a long history and relevance in therapy.Active substances of expectorant and spasmolytic preparations have been used in European traditional medicine for centuries [3].Recent findings have verified that essential oil and polyphenol-rich extracts of Thymus species collected in natural habitats show significant antioxidant potential as well as antibacterial and antifungal activity against various foodborne and human pathogens, respectively.Moreover, the cytotoxic activity of T. serpyllum extract and the antitumor potential of the essential oil have also been proven [4][5][6].Thus, wild Thymus species provide high-quality raw materials that can be applied in various formulations in self-medication as well as in the pharmaceutical, food, cosmetic, and chemical industries [3,6].
Dried flowering shoots of wild thyme (Serpylli herba) are listed in the Pharmacopoea Europaea, with a specification for the essential oil (EO) level (min.3 mL/kg DW) [7].In addition, bitter compounds, tannins, flavonoids, and phenolic acids have also been determined in the dried aerial parts of wild thyme [3,8].Essential oil (Serpylli aetheroleum) is produced by distillation of collected flowering shoots of various native species and occurs in different parts of Europe, with two gene centers found in Turkey and in the Iberian Peninsula [9].Monographs [3] refer to the collected plant material as Thymus serpyllum s. l. (sensu lato: in a broader sense), so the main terpenoid compound of the EO can also be different (p-cymene, carvacrol, thymol, linalool, or borneol) in the dried aerial parts of wild thyme plants collected.However, it is to be considered that the application of the drug in phytotherapy is attributed to the phenolic monoterpene molecules thymol and/or carvacrol [6].
According to the current taxonomic concept of Jalas (1972) [10], Thymus species are considered collective taxa, 'species aggregata', involving 2-3 microspecies each.Wild thyme taxa with Eurasian distribution, belonging to the Section Serpyllum of 120 chamaephyte species, were divided into 6 subsections and possess the highest chromosomal variability within the genus [9].The Hungarian Thymus species can be classified into the following four subsections:
Previous studies have reported a significant level of essential oil polymorphism concerning our five indigenous Thymus species from various habitats in Western Europe, the Nordic and Baltic countries, the Iberian and Balkan Peninsulas, Turkey, Italy, Iran, Slovakia, and Bulgaria, as well as from Ukraine.However, only sporadic data were available about the essential oil compositions of the drug collected from Hungarian wild thyme populations [11][12][13][14][15][16][17][18][19][20][21][22][23].
Based on the relevant literature data, considerable essential oil polymorphism could be expected for Hungary, owing to the quite variable habitat conditions, diverse plant communities, and the existence of five collective species and a few hybrids.
The main aim of our studies was to provide a general overview concerning the occurrence, environmental preference, essential oil properties, and chemotype patterns of the populations of five Thymus spp.occurring among diverse habitat conditions in the Hungarian Mountain Range.In addition, a general classification of EO chemotypes, their distribution, and frequency in Hungary are also discussed, along with outlines of future applications and cultivation prospects.Our further purpose was to point out the most valuable resources available in the natural habitats and select the high-yielding genotypes with excellent drug quality.With our findings, our aim was to support specific data on proper wild crafting practices, breeding, growing, and applications, as well as grounding the basis of the gene reservation of native Thymus taxa found in various habitats in Hungary.

Materials and Methods
Study areas: Samples were collected from 74 populations of 5 native Thymus species (Figure 1) found on different substrata of 63 natural habitats belonging to 15 regions of Hungary, as summarized in Table 1 and in Figure 2, respectively.Data obtained by species during our studies conducted in the past twenty years (2000-2020) have partly been published [19][20][21]; however, numerous new findings are also included in this paper to achieve a more complete overview.Fifteen new localities (4_14; 6_20; 8A_23; 8A_28; 8B_34; 8B_35; 9_36; 9_38; 9_39; 14_52; 14_53; 15B_59; 15B_60; 15C_61; 15C_63) with seventeen thyme populations (6_20: a and b) were explored and examined in the last period (unpublished data).A wide range of Thymus habitats with diverse parent rocks, soil types, and climatic conditions were designated as model areas based on floristic and coenological data recorded by botanists about the occurrence of different thyme populations.Field trips were timed to the flowering period of the species when comprehensive surveys were performed: the exact identification of species and microtaxa, sampling of flowering shoots and soils, habitat description, and preparation of herbarium specimens (Table 1).
Plant material: Identification of species was based on the relevant literature, reference book [24], and field data (morphological and coenological traits), as well as on accurate reviews of herbarium specimens.In general, approximately 25-30 g of fresh flowering shoots in Thymus populations with 3 to 6 replicates were collected per site, and essential oil data from 2 subsequent years were also evaluated in order to describe the true chemotypes of the localities.Plant samples were dried naturally under indoor conditions and shaded, where room temperature ranged between 15 and 25 • C, directly after collection into marked paper sacks, for approx.14 days.
When evaluating the EO quality obtained from wild thyme species in our studies, the broader interpretations of Thymus serpyllum (s.l.) and Serpylli herba (s.l.) were considered, involving all the 5 taxa belonging to the Sect.Serpyllum in the genus Thymus.Voucher specimens were deposited in the Herbarium of the Department of Medicinal and Aromatic Plants, Buda Campus, Hungarian University of Agriculture and Life Sciences, Budapest.
Isolation of essential oil: Dried plant material was hydro-distilled using a Clevengertype apparatus for 2 h, according to the pharmacopoeial standard, in 3-6 repetitions per population.The essential oil (EO) samples were stored in sealed vials under refrigeration prior to analysis.The EO content of each sample was expressed in ml/100 g on a dried weight basis (DW) and compared with the relevant standard for Serpylli herba of Ph.Eur.[7], which specified the minimum level of EO as 3 mL/kg.
Gas chromatography: A new analytical method was developed for identifying the exact percentage composition of the essential oil samples.A GC-FID analysis was carried out using an Agilent Technologies 6890N GC System (Santa Clara, California, USA) instrument equipped with an HP-5 (5% phenyl methyl siloxane) capillary column (30 m × d = 350 µm, film thickness: 0.25 µm), programmed as follows: initial temperature of 50 • C (10 min), from 50 to 150 • C at a rate of 4 • C min −1 ; from 150 to 220 • C at a rate of 12 • C min −1 and 220 • C (10 min).Carrier gas: helium (constant flow rate of 0.5 mL min −1 ), injector and detector temperatures: 250 • C, split ratio: 22.6:1.Injected quantity: 0.2 µL.The percentage composition of the essential oil was computed from the GC peak areas.
Gas chromatography-mass spectrometry (GC-MS) analyses were carried out using an Agilent Technologies 6890N GC System instrument equipped with an HP-5 (5% phenyl methyl siloxane) capillary column (30 m × d = 350 µm, film thickness: 0.25 µm) and connected to an Agilent Technologies MS 5975 inert mass selective detector.The temperature program was the following: initial temperature of 60 • C, then by a rate of 3 • C/min up to 240 • C; the final temperature was maintained for 5 min.The carrier gas was helium (1 mL min −1 ); injector and detector temperatures were 250 • C. Split ratio: 30:1.Injected quantity: 0.2 µL (1%, solvent: n-hexane).Ionization energy was 70 eV.The MS (mass spectra) were recorded in full scan mode, which revealed the total ion current (TIC) chromatograms (mass range m/z 50-550 uma).The compounds were identified by linear retention indices, which were calculated using the generalized equation of Van Den Dool and Kratz [25], literature data, and by matching their recorded mass spectra with those in mass spectral library references (NIST MS Search 2.0 library, Wiley 275, John Wiley & Sons, Inc., Hoboken, NJ, USA), as well as with a home-made database [26].The calculations of the retention indices were made on a homologous series of alkanes.Relative percentages (%) of compounds were presented in tables and figures in their order of elution in the column.
Chemotype determination: In general, chemotypes were characterized on the basis of the main essential oil compounds with a relative percentage of over 10%; however, in exceptional cases, further significant compounds of slightly lower GC% (over 8%) were also indicated.In addition, chemotypes were grouped by the chemical structure of the main compounds into monoterpene (M), sesquiterpene (S), or mixed (MS) classes.
Statistical analysis: Data originating from 3 to 6 repetitions of collected samples by populations were involved in statistical analyses.A one-way ANOVA was applied to show significant differences among Thymus species concerning essential oil-producing ability as well as to evaluate the effect of habitat conditions provided by regions included in the study.The IBM SPSS Statistics 29.0 software package was used for univariate analysis, where homogeneous groups were separated using the Tukey HSD or Duncan post hoc test, and the mean difference was significant at the p < 0.05 level.As a multivariate method, a hierarchical cluster analysis was performed on the basis of the percentage composition of major compounds in individual volatile samples (with compounds over 8%).A dendrogram was obtained using complete linkage with Euclidean distances using the TIBCO Statistica™ 14.0.0 (TIBCO Software Inc., Palo Alto, CA, USA) software package.

1.
The occurrence of native Thymus populations was various, depending on their ecological tolerance, habitat preferences, and social behavior types.Among 74 populations of 5 species surveyed in the Hungarian Mountain Range, T. pannonicus (TPA) was found with the highest frequency (38 populations, 51.35%), followed by T. glabrescens (TGL) (17 populations, 22.98%) (Tables 1 and 2).Both species were explored at new sites in the last few years, which also verifies their broad ecological tolerance and generalist character (Tables 1 and 2).
Populations of T. serpyllum (TSE), however, were found only in two habitats in our studies, as natural competitor species in plant communities developed on acidic (Somogy Hills) or calciferous sandy soils (Bakony Hills) (Tables 1 and 2).6.
Where the habitat conditions were favorable, the coexistence of 1-2 thyme species was also observed in the combination of TPA/TGL/TPR (dry conditions, calciferous rocks, and grassland communities) or of TPA/TPU (humid conditions and meadows).
In our studies, the most important plant communities involving wild thymes were grasslands on sand, loess, silicate stones, dolomite, and limestones (Table 1).

Essential Oil Production of Thymus Species in Different Habitats
In the case of T. pannonicus, a high overall mean essential oil content was detected (0.46 mL/100g DW), and the values ranged between the minimum of 0.01 mL/100g (4_14: Szőc, Bakony Hills) and the maximum of 1.90 mL/100 g (8A_27a: Nagykovácsi, Nagy-Szénás, Buda Hills) (Figure 3, Table 3).The latter value represented the highest drug quality with respect to TPA as well as all the Thymus samples examined in this experiment.Considering regional mean EO values of TPA samples, almost all the study areas can be recommended for collection, with the exception of the populations surveyed in the Gerecse, Zemplén, and Aggtelek Hills, which showed lower EO values than expected by Ph.Eur.(<0.3 mL/100 g DW) (Figures 3 and 4; Table 3).The average essential oil content (0.26 mL/100 g) of T. glabrescens samples surveyed was lower than the minimum value of the pharmacopoeial standard; however, in some cases (Bakony, Gerecse, and Buda Hills), the collected drug may fulfil the requirements.The highest essential oil content (1.71 mL/100 g) of T. glabrescens was measured in the sample originating from Csesznek, Castle Hill (4_13, Bakony Hills), while the poorest quality (0.01 mL/100 g) was obtained among the extreme habitat conditions of Stone Hill, Szentbékkálla (3_8B: Balaton Uplands) (Figure 3, Table 3).
Concerning the essential oil content of T. praecox, our results always indicated substandard drug quality, where the values ranged between 0.06 mL/100 g (4_15a: Várpalota, Bakony Hills) and 0.18 mL/100 g (8A_31: Diósd, Buda Hills), with an average of 0.1 mL/100 g.None of these populations are suggested for collecting flowering shoots of TPR to obtain dried drugs (Figure 3, Table 3).

The Role of the Genetic Factor in the Essential Oil Accumulating Ability
In our studies, the effect of the genotype was significant for the essential oil content detected in populations belonging to different wild thyme species, according to the ANOVA (p < 0.001).This phenomenon seemed to be highly evident when 1-3 species occur among equal circumstances in the same habitat, but rather different EO levels can be detected in the drug (e.g., see Table 3: 8A_24a: TPA:1.10 mL/100 g; 8A_24b: TPR: 0.01 mL/100 g).In our experiment, natural populations belonging to TSE, TPA, and TPU species provided adequate amounts of essential oil.TSE: 0.59 ± 0.17 mL/100g TPA: 0.50 ± 0.39 mL/100 g, TPU: 0.47 ± 0.32 mL/100 g, while TGL (0.26 ± 0.28 mL/100 g) and TPR (0.11 ± 0.03 mL/100 g) accumulated rather low levels (Figures 3 and 4).T. praecox showed the poorest drug quality (group 'a'); the difference was statistically proven with respect to T. pannonicus and T. serpyllum, both of which belonged to group 'b'.T. glabrescens and T. pulegioides represented a transitional group ('ab') that is quite variable in essential oil levels (Figure 4).
When comparing the performance of the 5 species studied in essential oil accumulating, we can conclude that most of the samples ( 35) can be categorized into the range of 0.1-0.5 mL/100 g or into the group of 0.5-1.0mL/100 g (18) (Figure 5).Only a few populations showed excellent levels of essential oils (1.0-1.5 mL/100 g: 5; 1.5 < mL/100 g: 3), while the number of cases with very low EO quantities was higher (13).As far as the species is concerned, TPA, TPU, and TSE possessed appropriate essential oil contents with higher frequency, while TGL was very diverse, and TPR can be completely excluded from collection based on low accumulation levels (Figure 5).

The Role of the Environmental Factors on the Essential Oil Accumulating Ability
Environmental conditions highly affected the essential oil levels measured, as shown by the average values originating from different regions involved (Figure 6).The statistical analysis verified that the differences among the regions were significant (p = 0.008).In general, Somogy Hills (TPA, TSE), Buda Hills (TPA), Börzsöny Hills (TPU), Gödöllő Hills (TPA), and Cserehát Hills (TPA) have proven to be the most valuable areas (EO > 0.50 mL/100 g DW) to collect and produce wild thyme drugs.If considering the EO requirement of (>0.3 mL/100 g DW) of the Ph.Eur.standard, seven regions (Mecsek, Velence, Vértes, Gerecse, Medves, Bükk, and Aggtelek Hills) are to be discarded according to the substandard (<0.30mL/100 g DW) mean values (Figure 6).However, the data obtained from smaller locations and populations belonging to certain thyme species may represent proper drug quality.
Very low essential oil levels have been detected in thyme populations situated on exposed rock surfaces (e.g., gray limestone in Mecsek Hills; granite rocks in Velence Hills; Pannonian sandstone rocks at Szentbékkálla or andesite cliffs at Dömös, etc.), covered sometimes only by thin bare soil layers, which are considered an extreme condition.In these places, secondary metabolite production can be highly restricted.

Essential Oil Chemotypes in Native Thymus Populations 3.3.1. Thymus pannonicus
A high level of essential oil diversity in T. pannonicus was proven on the basis of samples collected from wild populations.According to the chief compounds of their essential oils, the Hungarian thyme populations investigated could be classified into 18 well-defined chemotypes (Figure 7, Table 3).Chemotypes were basically monoterpenedominated (28 populations, 73.70%), while the proportions of sesquiterpene types (15.80%) and combined ones (10.53%) were rather low.
The chemotype patterns detected in TPA essential oil samples with the respective data are summarized as follows (new data are of bold type): Monoterpene chemotype (M): 1.
According to the chief compounds, their essential oil samples could be classified into well-defined monoterpene, sesquiterpene, and mixed chemotypes, which are closely related to the different habitats surveyed.Apart from those known from the previous literature (thymol, linalool/geraniol/linalyl acetate, and geraniol/linalyl acetate), the following five new chemotypes are described below (Figure 10): Monoterpene chemotype (M) of phenolic character: 1.

Frequency of Chief Volatiles Detected in Thymus Chemotypes
Concerning the therapeutic aspects of the drugs collected, not only should the essential oil contents be considered, but also the appropriate proportion of effective compounds detected by GC/MS as relative percentages.In the case of the Thymus species, utilization of the drugs and industrial raw materials is generally based on monoterpene phenol compounds (thymol, carvacrol) and derivatives (thymol methyl ether, carvacrol methyl ether), as well as biosynthetic intermediates (p-cymene, γ-terpinene) that are detectable in typical thyme essential oils, where high thymol percentages are expected.
In the EO samples of native Thymus populations studied, altogether 30 terpene molecules were identified as the main compounds of essential oil constituting different chemotypes, listed in Table 4 in the order of elution (1-30) during GC analysis.Supplementary data concerning retention time (RT) and linear retention indices (LRI) are also included.Half of these terpenoid molecules (15) were found to belong to the monoterpene group (M), where a wider variability of oxygenated monoterpenes (MO: 12), including thymol, linalool, or geraniol, were detected more than in the non-oxygenated monoterpene class (MH: 3).On the contrary, most of the chemotype-determining sesquiterpenes (S) can be grouped with the non-oxygenated sesquiterpenes (SH: 11), while fewer oxygenated (SO: 4) ones were detected in wild thyme oil.The former included the very abundant germacrene D found in 25 EOs and β-caryophyllene, which was present in 21 EO samples (Table 4).In our studies, thymol was the most frequent chemotype-determining monoterpene due to the wide distribution and occurrence of T. pannonicus populations in Hungary, where this compound dominated the essential oils.Concerning all of the EO samples, thymol (34) and the relative compounds, p-cymene (29) and γ-terpinene (15), could be detected with the highest frequency.Geraniol was also important among oxygenated monoterpenes, represented by three species (TPA, TPU, and TGL).β-caryophyllene was the only terpenoid compound that was detected as a chemotype-determining molecule in all of the five species involved in our studies (Table 4).
The widest spectrum of terpenoids, as possible molecules of the essential oil chemotypes, was found in T. pannonicus (19 compounds), followed by T. pulegioides (14 compounds), while the lowest variability of chemotypes was shown in T. praecox (5 compounds) (Table 4).
If considering the total number of chemotypes detected in all of the samples collected in the populations of Thymus species, the number of cases (28) at monoterpene-dominated chemotypes of T. pannonicus was proven to be outstanding (Figure 10).We can also add that this high value is principally due to the highly abundant thymol chemotype at TPA.The occurrence of monoterpene chemotypes was much lower in other species (TPU: 4; TGL: 4), while completely absent in T. praecox.The dominance of sesquiterpene (S) or combined (MS) chemotypes was characteristic in the samples of T. praecox and T. glabrescens (Figure 12).

Classification of Thymus Chemotypes by Major Terpene Compounds
A hierarchical cluster analysis was performed on the basis of major essential oil compounds in samples belonging to seventy-three populations, resulting in nine clusters, which were primarily separated into two main groups.The first cluster included chemotypes with thymol (T) chief compounds, mainly including T. pannonicus; however, T. glabrescens and T. pulegioides were also represented, irrespectively of the place of origin (Figure 13).Chemotypes, where the role of sesquiterpene compounds was significant, were classified into the second-biggest cluster of the dendrogram.Non-phenolic monoterpenes and mixed chemotypes represented smaller subclasses.Germacrene D (GD) occurred in the essential oils grouped into a well-separated cluster and comprised samples belonging mainly to T. praecox and T. glabrescens.Further smaller groups could also be distinguished according to the main terpene compounds, as follows: third: β-caryophyllene (CAR); fourth: p-Cymene (CY) and thymol (T); fifth: geraniol (G); sixth: linalool (L); seventh: β-Cubebene (CU); and eighth: carvacrol ©.T. serpyllum, with its special essential oil composition (geranylisobutyrate: GEB), could be found in an extreme position in cluster 9 (Figure 13).Based on the classification of chemotypes shown by the dendrogram, our results on the essential oil chemotypes of Thymus species native to Hungary were confirmed by a cluster analysis.

Discussion
While the variability of T. vulgaris (garden thyme) has been extensively studied [28], less is known about the Central European wild thyme taxa.As proven in T. vulgaris, the chemotype patterns were a result of an adaptation process to diverse and well-defined environmental conditions [29].According to Thompson (2002), essential oil polymorphism was considered a marker of adaptation to a particular environment.Long-term adaptation processes led to a genetically controlled essential oil biosynthesis with different monoterpenes (thymol, carvacrol, geraniol, terpineol, linalool, or thujanol) at the end of the six branches of the biosynthetic chain (regulated by the epistatic series of five loci with the following sequence: G > A > U > L > C > T) [30].Nevertheless, no theory for the evolution of numerous further known chemotypes of taxa belonging to the sect.Serpyllum has been verified yet, despite the numerous data reported on the essential oil diversity.
Based on the data presented in the literature available, a considerable level of infraspecific and interspecific diversity could be predicted for the five collective species native to Hungary as well.According to our hypothesis, the drug quality of collected wild thyme samples is likely to be variable owing to the diverse habitat conditions and plant communities distributed in our country.As the occurrence of T. serpyllum is rather scarce, the raw material of Serpylli herba is to be supplied by other, widely distributed, productive taxa with high essential levels and adequate EO compositions.Consequently, the main objectives of our experiments were to explore the native Thymus populations in diverse regions of Hun-gary, record taxonomical, ecological, and coenological observations, and evaluate the drug quality with respect to the essential oil content and composition.We aimed to determine the factors influencing the occurrence of different taxa and the essential oil properties as well.Valuable areas and taxa were then selected for collection, and outstanding, productive chemotypes were subjected to gene reservation and introduction into cultivation.
The identification of the species exclusively using morphological traits was a rather difficult task in natural populations.Ontogeny (flowering period), habitat features, and the type of plant community provided additional information to recognize the existing taxa.Moreover, we observed further morphological variability in dry grassland associations where populations of the same species (e.g., T. glabrescens, T. pannonicus) may appear with hairy and naked shoots or both.Among the humid conditions of mountain meadows (Mátra Hills, Zemplén Hills, etc.), the occurrence of different odors and color varieties was detected within the same population of T. pulegioides or T. pannonicus.
Concerning the frequency of occurrence of the indigenous populations of the Thymus species, we could conclude that T. pannonicus (Hungarian thyme) was found to have the highest frequency (38 sites), followed by T. glabresbcens (common thyme: 17 sites), T. pulegioides (mountain thyme: 9 sites), and T. praecox (creeping thyme: 8 sites), while T. serpyllum (wild thyme: 2 sites) was the least abundant among the model areas.The highest frequency of occurrence of TPA, TGL, and TPU in quite distinct habitats is likely to be related to the generalist character of the species, showing high adaptability to different circumstances.
Our results on the chemotype pattern of five Thymus species are partly in accordance with previous findings originating from diverse localities in the surrounding countries.In the case of T. pannonicus, previously available data indicate the existence of thymol, carvacrol, thymol/carvacrol, geraniol, and carveole chemovarieties from Ukraine [31], while thymol and α-terpinyl-acetate chemotypes were reported from Bosnia [11].Further αterpynyl-acetate, thymol, thymol/p-cymene and p-cymene/thymol, linalool, geraniol, and γ-muurolene/β-caryophyllene chemovarieties were noted in Slovakia [13,32].A chemotype containing a high level of geranyl acetate in the essential oil was also described in a German population [33].Moreover, Serbian authors have found a population in Vojvodina Province (Pannonian lowlands) with lemon-scented essential oil containing geranial and neral, and α-pinene and germacrene-D were also detected as leading monoterpenes, where the role of the latter sesquiterpene in a chemotype pattern was emphasized for the first time [5,34].Recently, a germacrene D/β-caryophyllene chemovariety was found in the Eastern Rodope Mountains, Bulgaria [22].Numerous new data were found in our studies as well, where wider spectra of chief essential oil compounds have already been presented in Hungary rather than the above-mentioned sources [19,21,35].In addition, several new chemotype patterns were detected in our studies from new locations with combinations of thymol, geraniol, geranyl acetate, p-cymene, and carvacrol as major monoterpenes, while sesquiterpenes were represented by germacrene D, β-caryophyllene, farnesol, caryophyllene oxide, β-farnesene, and δ-cadinene.A special chemotype with a combination of mono-and sesquiterpenes was recorded in Jósvafő, Aggtelek Hills, including geraniol, geranyl acetate, and β-bisabolene as chief compounds.The wide variety in the EO composition reflects the diverse ecological conditions that exist in the mountainous areas of Hungary.Recent findings have confirmed that T. pannonicus has a perspective in cultivation, as promising experimental data were reported on homogenous drug quality, adequate amounts of essential oil, and stability in EO composition [36].Our data from original habitats also supports the fact that Hungarian thyme has a high tolerance for diverse environmental conditions, which can be an advantageous trait when facing climatic changes and raising incidences of extremities.
A high diversity of major terpene compounds was also detected in the essential oils of T. glabrescens populations distributed widely among various habitat conditions [35].Most of the essential oil samples contained sesquiterpenes (germacrene D, β-caryophyllene, τ-cadinol or β-cadinene germacrene D, β-caryophyllene, caryophyllene oxide, etc.), while the occurrence of monoterpenes (thymol, geraniol) was rare, such as major compounds.Our results contradict the known data in the literature, as a geraniol/neryl acetate chemotype was recorded in Romania [37], while the major essential oil compounds of Serbian and Bulgarian T. glabrescens populations were found to be thymol and γ-terpinene [22,38], respectively.
The essential oil diversity of T. pulegioides has already been thoroughly examined as an element of the habitats of alpine or boreal regions [36,39].The European populations of the species are predominantly described as having phenolic (thymol/carvacrol) chemotypes.On the contrary, Bulgarian authors have found a new chemotype with α-terpinyl acetate in the Vlahina Mts., while another paper recently reported a wild mountain thyme population with α-terpineol, geraniol, and carvacrol as major EO compounds from the Carpathians/Ukraine [22,23].Moreover, we have also pointed out the role of sesquiterpenes in the formation of these chemotypes.In the case of lemon-scented varieties, not only did geraniol, geranial (citral B), and neral (citral A) appear in the volatile oils, but also linalool and an ester derivative, linalyl acetate, were detected.Our data were supplemented with the literature data on mountain thyme chemotypes.
The chemotype patterns revealed that the examined Thymus serpyllum populations were unique because the essential oil of the Bakony (Fenyőfő) was dominated by geraniol and geranyl isobutyrate, while the others in the Somogy Hills (Nagybajom) contained τ-cadinol, caryophyllene oxide, and β-cubebene, respectively.In previous studies, however, thymol and related compounds were found to be major essential oil compounds that met the requirements of the respective standards [16,18].Recently, new chemotypes with linalool as well as α-terpineol and geraniol were also found in native populations of the Carpathians in Ukraine [23].
Further research is needed to evaluate the role of different genetic and/or environmental factors in determining chemotype patterns and their distribution in different areas.Correlations between relative percentages of several essential oil compounds and edafic conditions have already been found, but there are still open questions in this field.

Conclusions
Based on the diverse ecological conditions existing in different localities of Hungary, considerable essential oil polymorphism was found, possibly due to the outstanding adaptability of T. pannonicus, T. glabrescens, and T. pulegioides, respectively.Numerous chemotypes have been detected producing major volatiles of monoterpene or sesquiterpene structures.The role of sesquiterpene compounds was also proven to be important in determining the chemical character of the volatile oils, either representing independent sesquiterpenic chemovarieties or accompanying other major monoterpenes.
As the occurrence of T. serpyllum is rather scarce in Hungary, the raw material of Serpylli herba Ph.Eur. is important to be supplied by other, widely distributed, productive taxa with high essential levels and adequate EO compositions.It was established that T. pannonicus could be suggested for this purpose, as its essential oil content is generally high in natural habitats and, in most cases, rich in either thymol and/or its precursors (p-cymene/γ-terpinene), like those of Thymus vulgaris.The other species are either less abundant in the country (TSE), linked to special ecological conditions (TPU, TPR), or provide substandard drug quality (TGL, TPR) with respect to the essential oil content and composition.
Further efforts suggested introducing the most valuable taxa into the horticultural systems where homogeneous drug quality with high amounts of thymol containing essential oil can be ensured.Chemotypes with special essential oil patterns are proposed to be subjected to gene reservation.
The summarized data on the occurrence, essential oil properties, and chemotype patterns of Hungarian Thymus species may serve as a supplement to the knowledge base about the chemical diversity of Thymus essential oils.

Figure 1 .
Figure 1.Thymus species found in natural plant communities in Hungary (Photos by Zs.Pluhár).

Figure 7 .
Figure 7.The chemotype pattern of Thymus pannonicus essential oils originating from native populations exists in different habitats in Hungary.(Legends: see site codes in Table1).

Figure 8 .
Figure 8. Chemotype pattern of Thymus glabrescens essential oils originating from native populations exist in different habitats of Hungary.(Legends: see site codes in Table1).

Figure 9 .
Figure 9. Chemotype pattern of Thymus praecox essential oils originating from native populations in different habitats of Hungary.(Legends: see site codes in Table1).

Figure 10 .
Figure 10.Chemotype pattern of Thymus pulegioides essential oils originating from native populations exist in different habitats of Hungary.(Legends: see site codes in Table1).

Figure 11 .
Figure 11.Chemotype pattern of Thymus serpyllum essential oils originating from native populations exist in different habitats of Hungary.(Legends: see site codes in Table1).

Table 1 .
Summary of the geographical locations where populations of Thymus species were studied in Hungary with base rocks and soil types (2000-2020).

Table 2 .
[27]uency of occurrence (number, %) of Thymus populations surveyed in Hungary and classification of Thymus species according to the social behavior types, according to Borhidi, 1995[27].

Table 4 .
Frequency of occurrence, analytical data and classification of chief essential oil compounds of chemotypes found in native Thymus populations in Hungary.