Principal Component and Hierarchical Cluster Analysis of Major Compound Variation in Essential Oil among Some Red Oregano Genotypes in Albania

Abstract

Red oregano (Origanum vulgare L. subsp. vulgare) is native to the mountainous slopes of Albania, thriving at altitudes ranging from 400 to 1300 m above sea level. The aerial components of oregano have been found to address a spectrum of health concerns. However, this subspecies presents intriguing characteristics that require comprehensive exploration and analysis. This study extensively analyzes the ex −situ collection of various genotype populations of red oregano in Albania. Essential oils were extracted by employing the hydrodistillation method. At the same time, their chemical analyses were carried out using gas chromatography coupled with a flame ionization detector (GC−FID) and gas chromatography coupled with a mass−spectrometer detector (GS−MS). We employed two statistical techniques, namely hierarchical cluster analysis (HCA) and principal component analysis (PCA), which allowed for a comprehensive examination of the relationships within the data set and more profound insights into the compositional patterns and interrelationships within the essential oils to be gained. The results revealed significant qualitative distinctions at the intraspecific level, particularly for sesquiterpenes, of populations originating from seven diverse geographic locations. The study’s findings enhance our understanding of the chemical composition of Albania’s red oregano and its chemical variation among different populations, which will potentially contribute to identifying the most suitable clones for breeding programs within red oregano populations.

1. Introduction

Red oregano (Origanum vulgare L. subsp. vulgare), belonging to the Lamiaceae family, is locally referred to as Çaj Mali in the North of Albania, where it flourishes in its natural habitat on mountain slopes. This subspecies, meticulously identified in 1980 [1], is particularly abundant in Albania’s central and northern areas. It thrives from 400 to 1300 m above sea level, establishing itself in diverse habitats such as cliffs, rocks, ravines, and alpine meadows.

According to the Albanian Flora taxonomy, two subspecies of Origanum vulgare are recognized in Albania: Origanum vulgare L. subsp. hirtum, known as white oregano, and Origanum vulgare L. subsp. vulgare, known as red oregano. White oregano is rich in essential oil, about 5%, with carvacrol as the main compound, followed by its precursors p−cymene and γ−terpinene. It is primarily used as a spice, and its essential oils have stimulant, carminative, antispasmodic, and anticancer properties. They are also used for the treatment of gastrointestinal disorders. Red oregano contains less essential oil, up to 2%, and is characterized by Germacrene D as the main compound, followed by α−Cadinol, Elemol, and Bornyl acetate. In Albania, red oregano is commonly used to prepare tea. Both subspecies contain considerable amounts of non−volatile phenolic compounds such as flavonoids and phenolic acids [2,3,4,5,6,7].

The tea derived from this plant is crucial in Albanian folk medicine practices in the Alps. Rooted in the cultural heritage of Albania, this fragrant herb boasts a rich history with applications in both culinary and medicinal domains, celebrated for its capacity to enhance the flavors of diverse dishes and be crafted into a fragrant tea [8,9]. The aerial parts of oregano serve a multifaceted role in addressing various health issues. These encompass respiratory disorders, such as alleviating symptoms associated with the common cold, including fever, flu, and bronchitis during episodes of colds and coughs, acting as a natural antibiotic. Additionally, it has been proven efficacious in addressing stomachaches, arthritis, pain relief, digestive disturbances, and urinary problems. Importantly, it functions as a diuretic and antiurolithic agent in urinary health [8,10,11]. However, beyond its prevalent applications, this subspecies exhibits intriguing qualities that necessitate a more thorough investigation. Regarding the yield of essential oil and the quantity and quality of sesquiterpenes, research on red oregano has previously been reported from various countries, such as Moldova [12,13], Albania [12], Romania [14,15], Bulgaria [16], Poland [17,18], Turkey [19], Italy [20] Lithuania [7], Montenegro, etc. [21].

Several studies have indicated that plants with diminished essential oil levels often show elevated levels of sesquiterpenes [7,21,22,23,24,25]. Its restricted volatile compound profile, marked by a significant prevalence of sesquiterpenes, distinguishes it from other subspecies. These sesquiterpenes, functioning as essential oils, contribute to the distinctive aroma of the plant and may possess therapeutic properties, prompting inquiries into its potential benefits.

Furthermore, this subspecies’ natural habitat in Albania, marked by diverse altitudes, introduces a captivating ecological dimension. Investigating this impact on the chemical composition of oregano in distinct regions can offer valuable insights into the plant’s adaptation and variability [26]. The chemical components of seven red oregano accessions and their constituents were shown [26]. However, this study does not thoroughly evaluate the chemical relationships between different genotypes.

In an era when traditional remedies and herbal medicine garner renewed interest, we hypothesize that red oregano, valued for its cultural, medicinal, and ecological attributes, holds bioactive richness. Its multifunctional role in health and sesquiterpene−rich profile suggest therapeutic potential. Investigating the efficacy of oregano in these applications can serve as a link between traditional knowledge and contemporary medical science. The rich cultural heritage and chemical uniqueness of oregano make it a captivating subject of study, promising insights into its culinary, medicinal, and ecological significance. Given the species’ endangerment, urgent germplasm research is expected to guide conservation efforts and sustainable management. This study aimed to investigate the essential oil composition, utilizing similarity analysis, principal component analysis (PCA), and hierarchical cluster analysis (HCA), of red oregano sourced from seven diverse sites in Albania.

2. Materials and Methods

Seven accessions of red oregano (Origanum vulgare subsp. vulgare) were sourced from the Albanian GenBank, originating from seven distinct geographic locations across Albania following the FAO regulation rules [27] (see Figure 1).

2.1. Experimental Design and Plant Characterization

Fifteen individuals from seven distinct accessions of red oregano, collected from various natural sites, were evaluated for agronomic and chemical traits in an experimental site. This site is located at the Agricultural University of Tirana, Municipality of Tirana, Albania (41°39′445″ N, 19°72′92″ E, at an altitude of 38 m). Data from 15 individuals per accession were analyzed. From 15 to 30 June of summer 2017, we harvested the aerial parts from each plant from all accessions during the blooming time. Then, we dried them in a well−ventilated, shaded area under controlled conditions within a consistent environmental milieu.

2.2. Harvesting and Sample Preparation

From 15 to 30 June of summer 2017, we harvested the aerial parts from each plant from all accessions during the blooming time and then dried them in a well−ventilated, shaded area under controlled conditions. The temperature was approximately 25 °C, and the relative humidity was around 40%. The Herbarium specimen was deposited with voucher numbers in the Genetics and Plant Breeding Working Group of the Agricultural University of Tirana.

2.3. Essential Oil Extraction

Hydrodistillation involving the use of a Clevenger apparatus was used to extract the essential oil. Ten grams of dried oregano plants, including flowers, stems, and leaves, were finely cut and transferred into a 1 −L flask containing 0.5 L of distilled water. Distillation lasted for three hours at three milliliters per minute. The oil yield was calculated as a percentage of volume by weight (% v/w) relative to the dry weight of the plant material. The essential oils were extracted from three replications from 15 individuals per accession. The replicates were performed independently to reduce the impact of any potential variability in the plant material and extraction process. The essential oils were stored in a light−protected environment at −18 °C in a deep freezer for subsequent analysis.

2.4. Gas Chromatography Analysis (GC/FID)

The gas chromatography system (Agilent 7890A, Agilent Technologies, Palo Alto, CA, USA) with a flame ionization detector (GC−FID) was used to analyze the essential oils quantitatively. Separation was performed in an HP−5MS column (Agilent Technologies, Palo Alto, CA, USA) (30 m × 0.25 mm with a 0.25 μm film thickness). Helium gas was utilized as the carrier medium, initially flowing at 0.6 mL/min and then maintained at a constant pressure of 50.0 psi. The front inlet was kept at 250 °C with a split ratio of 50:1. The GC oven temperature was ramped from 60 °C to 260 °C at a rate of 5 °C/min, and the FID operated at 250 °C with an airflow of 350 mL/min and a hydrogen flow of 35 mL/min. The injection volume was maintained at 1.0 μL. The essential oil’s percentage composition was calculated using the normalization method based on GC peak areas without applying correction factors.

2.5. Gas Chromatography−–Mass Spectrometry Analysis (GC/MS)

GC/MS analysis involved the employment of an Agilent instrument 7890A GC system in conjunction with an Agilent i5975C MSD. The GC system operated in identical conditions as programmed for GC/FID analysis. The MS ion source temperature was set to 230 °C, the MS quadrupole temperature was set to 150 °C, and the transfer line temperature was set to 280 °C. The scan rate covered a mass range from 50 to 550 Da.

2.6. Identification of Compounds

The essential oil components were identified by comparing their Kovats retention indexes to those found in the literature [28]. We used linear interpolation of n−alkane (C9–C32) retention time in a homologous series under the same operating conditions to calculate the Kovats index. Additionally, mass spectra for each component were compared to those stored in the NIST 08. L and WILEY MS 9th databases.

2.7. Principal Component and Cluster Analysis

Principal component analysis (PCA) was used to simplify and summarize important information from the data, performing a linear transformation of original variables into orthogonal principal components, with the first capturing the maximum variance [29,30]. This process aids in reducing dimensionality and discerning underlying patterns. Advanced PCA techniques, such as 3D PCA and multiblock PCA, were also considered for enhanced data exploration [31]. This facilitated the subsequent use of hierarchical cluster analysis (HCA) to group samples based on similarities determined through various distance metrics such as Euclidean, Manhattan, and Pearson. Other authors have detailed the HCA’s algorithm and methodological considerations [32]. Considering the influence of the chosen distance metric and the presence of outliers, HCA was applied strictly for exploratory purposes [33].

2.8. Statistical Analyses

The data obtained from the GC−FID were subjected to multivariate analysis. Open−source statistical software, R (Rstudio version 2022.07.2 Build 576), was used to identify patterns and relationships within the data presented in Table S1, incorporating 15 individuals per accession. The aim was to determine whether the identified essential oil constituents can reflect the chemical relationships between different accessions.

Bivariate analysis was conducted using statistical software R to calculate Pearson and Spearman correlation coefficients to identify relationships between different factors. The data were compared with R statistical software, Rstudio version 2022.07.2 Build 576, using cluster and labDSV to perform principal component analysis (PCA) and hierarchical cluster analysis (HCA).

3. Results and Discussion

3.1. Biodiversity, Microclimates, Ecological Importance, and Conservation Implications

Figure 1 shows the geographic distribution of oregano populations from the center to the country’s northeastern region, along with the corresponding latitude and longitude.

The mean annual temperature at the experimental site at the Agricultural University of Tirana, Albania, was 23.3 °C, and the annual precipitation was 1345 mm during the experimental year.

Table S1 reports the essential oil yield of 15 individuals from seven different red oregano accessions collected from various Albanian locations. The essential oil content ranged from 0.05% to 1.3%, with the highest yield recorded for accession 5, followed by accession 1 (0.5%) and accession 6 (0.5%). The lowest yield belonged to accession 4 (0.05%).

The main compounds consisting of the essential oil of red oregano in the analyzed samples were: Germacrene D (28.34%), E−Caryophyllene (15.7%), and several other compounds, including α−Cadinol, Carvacrol, Thymol, Elemol, Bornyl acetate, Z−β−OcimeneZ−b−Ocimene, γ−Terpineneg−Terpinene, γ−Muuroleneg−Muurolene, E, E−a−Farnesene, γ−Cadinened−Cadinene, Caryophyllene oxide, α−Muurolola−Muurolol, and Sabinene [26].

The values of these main components for the 15 individuals evaluated within each of the seven accessions in our study exhibited significant diversity. In

Table 1. Content in [%] of 15 major compounds chosen for scrutiny within the extracted essential oil leaves of red oregano collected in Bicaj, Kukes, Albania (Accessions 1), cultivated in the experimental Agricultural University of Tirana field.

Major Compounds	KIcal	KIlit	Individuals/Plants
			P1	P2	P3	P4	P5	P6	P7	P8	P9	P10	P11	P12	P13	P14	P15	Means
Sabinene	977	975	2.48	0.01	0.82	1.49	1.40	1.79	2.15	0.98	3.94	0.01	0.01	10.96	2.40	3.33	0.01	2.12
Z−β−Ocimene	1041	1037	1.71	0.55	1.42	0.84	0.01	1.37	1.33	0.45	2.78	0.01	0.01	5.09	1.15	1.44	0.01	1.21
γ−Terpinen	1063	1059	1.28	1.22	1.67	0.54	2.15	0.01	0.83	0.82	2.41	0.01	12.69	1.77	2.27	0.56	0.01	1.88
Bornyl acetate	1286	1285	1.30	0.01	2.82	1.16	3.80	0.84	2.15	2.29	5.30	0.01	0.01	2.98	0.47	2.09	0.01	1.68
Thymol	1293	1290	0.26	0.01	0.34	0.22	0.86	0.81	0.56	0.71	0.15	0.01	0.01	0.01	1.47	0.72	0.01	0.41
Carvacrol	1303	1299	22.27	0.01	17.38	4.93	2.51	2.90	2.91	1.99	2.86	0.01	0.01	0.01	1.19	0.72	0.01	3.98
E−Caryophyllene	1423	1419	15.89	27.68	5.87	23.61	17.60	21.57	13.41	19.12	14.55	16.95	21.17	19.89	23.62	23.89	57.70	21.50
γ−Muurolene	1482	1479	0.25	0.01	0.30	0.36	0.35	0.35	0.39	0.01	0.01	0.01	0.01	0.22	0.32	0.28	0.01	0.19
Germacrene D	1484	1481	25.14	27.07	35.95	39.51	36.84	31.21	45.21	37.78	41.7	38.95	27.04	25.71	39.30	34.99	42.30	35.25
E,E−α−Farnesene	1509	1505	2.85	0.01	2.87	2.33	3.22	4.66	2.99	3.91	1.13	0.01	0.01	1.95	2.19	2.87	0.01	2.07
γ−Cadinene	1518	1513	3.79	0.01	2.53	4.02	1.08	0.74	2.72	6.48	3.98	8.35	0.01	1.92	2.23	7.86	0.01	3.05
Elemol	1550	1548	2.99	0.01	2.67	2.37	3.43	4.32	3.46	3.48	2.53	13.68	4.67	1.75	1.74	2.21	0.01	3.29
Caryophyllene oxide	1588	1583	2.92	0.01	2.36	1.91	4.63	5.38	3.62	3.12	2.04	0.01	0.01	1.95	2.27	2.68	0.01	2.19
α−Muurolol	1648	1646	1.72	0.01	1.47	1.45	2.00	3.27	1.74	2.14	1.39	6.79	0.01	0.99	1.22	1.40	0.01	1.71
α−Cadinol	1661	1655	3.61	0.01	4.24	2.88	3.77	5.43	3.41	4.16	2.23	0.01	0.01	1.95	2.06	2.13	0.01	2.39

KI_cal = Kovats index, calculated; KI_lit = Kovats index from the literature, according to Adams [28].

, we present the data on major compounds in red oregano for 15 individuals of seven accession collected in Bicaj, Kukes, Albania, cultivated in the experimental field of the Agricultural University of Tirana. It was observed that the values of Germacrene D, the most abundant component, vary among individuals in the population from 25.14% to 45.21%. The values of E−Caryophyllene also exhibit a wide range, ranging from 5.87% to 57.70%. Carvacrol values range from 0.01% to 22.27%, Sabinene values within the population range from 0.01% to 10.96%, γ−Terpinen values range from 0.01% to 12.69%, and γ−Cadinene values range from 0.01% to 8.35%. In population number two, the percentage range for Germacrene D is 0.03–31.76%, and for E−Caryophyllene, it is 0.01–36.18%. Germacrene D ranges from 0.01 to 52.56% in population number three, while E−Caryophyllene ranges from 0.01 to 22.23%. For population number four, the values for Germacrene D are between 8.93% and 42.24%, and for E−Caryophyllene, they are between 0.01% and 36.05%. Germacrene D ranges from 7.56% to 26.77% in population number five, with E−Caryophyllene ranging from 0.01% to 26.61%. Population number six ranges from 3.98–40.61% for Germacrene D and 0.01–35.86% for E−Caryophyllene. Lastly, Germacrene D varies from 15.51% to 38.30% in population number seven, and that of E−Caryophyllene varies from 6.31% to 26.08%. The tabulated limits of variation in the material quantities across populations and their respective averages provide valuable insights for the informed selection of populations in breeding programs. This comprehensive dataset aids in identifying populations with desirable traits, thus contributing to the enhancement of breeding strategies and the development of improved plant varieties.

Germacrene D, E−Caryophyllene, Z−β−Ocimene, and Sabinene were reported as the primary components of the essential oil of red oregano in Lithuania [7]. However, the concentrations of Germacrene D (15.4–27.9%) and β−Caryophyllene (7.7–14.6%) reported by another author [21] were considerably less than those observed in the essential oil of our Origanum vulgare ssp. vulgare populations. Sabinene was also identified as the primary component in the essential oil of common oregano [34]. However, contrasting findings from other researchers [35] indicated elevated concentrations of Trans−Sabinene hydrate, α−Caryophyllene, and Germacrene D. Different authors have identified these major compounds in red oregano [13,20,36,37]. Numerous studies have examined the essential oil composition of Origanum vulgare, revealing significant variation in the concentration of crucial compounds like carvacrol, thymol, p−cymene, and γ−terpinene based on environmental factors. The existing research on red and common oregano provides valuable insights into how environmental conditions influence essential oil composition. For instance, oregano grown in the Mediterranean region tends to have higher levels of carvacrol and thymol due to the favorable climate conditions. Red oregano is less extensively studied than common oregano. However, available studies indicate that environmental factors similarly affect its essential oil composition. Altitude, soil type, and climate variations significantly impact thymol, carvacrol, and other essential oil components. Understanding the environmental effects on crucial oil composition can aid in developing optimized cultivation practices to enhance the quality and yield of essential oils in red and white oregano [6,38,39,40,41].

3.2. Correlations between the 15 Major Compounds of Red Oregano Collected in Seven Different Locations in Albania

Table 2. Correlation coefficients between the 15 major essential oil red oregano compounds collected in seven different accessions cultivated in the experimental Agricultural University of Tirana field.

	Sabinene	Z−β−Ocimene	γ−Terpinen	Bornylacetate	Thymol	Carvacrol	E−Caryoph	γ−Muurolene	Germacr.D	E−E−α−Farne	γ−Cadinene	Elemol	Cary.oxide	α−Muurolol	α−Cadinol
Sabinene	1	0.9 **	0.4	−0.4	0.2	0.9 **	0.2	0.4	−0.5	−0.4	0.2	0.2	−0.5	0.1	0
Z−β−Ocimene		1	0.7 *	−0.1	−0.2	0.7 *	0.1	0	−0.3	0	0.4	0.4	−0.2	0.4	0.3
γ−Terpinen				0.2	−0.6 *	0.4	0.3	−0.5	0.4	0.3	0.8 **	0.7 *	0.2	0.5	0.3
Bornylacetate					0.1	−0.4	−0.6 *	−0.4	0.5	0.3	0.1	0.1	0.3	0.2	0.3
Thymol						0.3	−0.4	0.5	−0.6 *	−0.7 *	−0.7 *	−0.7 *	−0.7 *	−0.6 *	−0.5
Carvacrol							0.4	0.3	−0.4	−0.7 *	0.2	0	−0.7 *	−0.2	−0.4
E−Caryophyllene								−0.2	−0.1	−0.4	0.3	0.4	−0.3	0.3	−0.2
γ−Muurolene									−0.5	−0.4	−0.2	−0.3	−0.4	−0.4	−0.2
Germacrene.D										0.5	0.6	0.4	0.7 *	0.1	0.1
E−E−α−Farnesene											0.3	0.4	0.9 **	0.5	0.7
γ−Cadinene												0.8 **	0.4	0.4	0.2
Elemol													0.2	0.9 **	0.7 *
Caryophylleneoxide														0.1	0.3
α−Muurolol															0.9 **
α−Cadinol															1

Pearson’s correlation coefficient is indicated with significance levels (* for p ≤ 0.05 and ** for p ≤ 0.01 are indicated in bold). Negative and positive correlations between factors are shown by minus and plus signs.

illustrates the correlations among the primary components of essential oils. Positive correlations were observed within the essential oil components of red oregano, specifically in the hydrocarbon sesquiterpene (HS) and oxygenated sesquiterpene (OS) classes. Noteworthy positive correlations were identified between pairs of E−E−α−Farnesene (HS) and Caryophyllene oxide (OS) (0.9 **), Germacrene D (HS), and γ−Cadinene (HS) (0.6 *), as well as Germacrene D (HS) and Caryophyllene oxide (OS) (0.7 *). These findings suggest that an increase in the concentration of Germacrene D correlates with elevated concentrations of both γ−Cadinene and Caryophyllene oxide. Similarly, a strong correlation was observed between γ−Cadinene (HS) and Elemol (OS) (0.8 **), indicating that an increase in γ−Cadinene concentration is associated with higher concentrations of Elemol (refer to Table 2).

A robust positive association was also noted between hydrocarbon monoterpenes (HM) and oxygenated monoterpenes (OH). Sabinene (HM) exhibited strong positive correlations with Z−β−Ocimene (HM) and Carvacrol (OH) (0.9), indicating that an increase in Sabinene concentration correlates with elevated concentrations of both Z−β−Ocimene and Carvacrol. Similarly, Z−β−Ocimene (HM) displayed positive correlations with γ−Terpinene (HM) (0.7 *) and Carvacrol (OH) (0.9 **), suggesting that an increase in Z−β−Ocimene concentration is associated with higher concentrations of γ−Terpinene and Carvacrol. The correlations among Thymol, Carvacrol, p−Cymene, and γ−Terpinene are also discussed in the work of other authors [42]. Additionally, a robust positive correlation of 0.8 was observed between γ−Terpinene (HM) and γ−Cadinene (HS), implying that these components fluctuate. Furthermore, positive relationships within the same class were observed, such as Elemol (OS) with α−Muurolol (OS) (0.9 **) and α−Cadinol (OS) (0.7 *). The strong positive correlations among these components suggest that they vary in tandem. Previous studies have demonstrated positive correlations between these compounds [43].

3.3. Principal Component Analysis (PCA)

Principal component analysis (PCA) was undertaken to unravel the variance distribution among the variables. Our statistical analyses were directed explicitly toward oil components whose concentrations exceeded 0.5% of the total oil content, providing insights into essential oil composition variations. The contents of the 15 selected essential oil compounds differed significantly (p < 0.05) between the populations collected at different sites (Figure 2). The horizontal axis of the PCA accounted for 14.6% of the total variance, while the vertical axis contributed an additional 20.7 (refer to Figure 2). PC1 and PC2, the first two principal components, collectively elucidated 35.3% of the total variance. Figure 2 visually depicts the relationships among the variables on the PCA plot, with PC1 represented along the x−axis and PC2 represented along the y−axis. A principal component analysis (PCA) of data on the 13 significant compounds of essential oils in five oregano populations was previously conducted [21]. The analysis identified three principal components with eigenvalues exceeding 1, collectively explaining 96.5% of the total variation.

In our study, PC1 characterized by positive influences from Thymol, γ−Muurolene, Carvacrol, γ−Terpinene, Sabinene, Z−β−Ocimene, Bornyl acetate, Caryophyllene oxide and E,E−α−Farnesene, α −Cadinol, α−Muurolol, and Elemol but a negative contribution from E−Caryophyllene and Germacrene D to γ−Cadinene.

Conversely, the second principal axis showcased positive contributions from Caryophyllene oxide, counteracted by negative influences from γ−Muurolene, E,E−α−Farnesene, Elemol, α−Cadinol, and α−Muurolol to E−Caryophyllene, Bornyl acetate, Z−β−Ocimene, Sabinene, γ−Terpinene, Thymol, γ−Muurolene, and Carvacrol, thereby illustrating the distinct contributions of individual components to the overall chemical variance.

The two−dimensional representation of PCA facilitated the delineation of four distinct clusters within the essential oil dataset, each defined by principal components making significant contributions.

The first cluster was notably characterized by Germacrene D, emerging as its predominant contributing component in many individuals for most of the population. The corresponding data revealed the presence of E−Caryophyllene and Germacrene D [44,45]. Sesquiterpenes, such as β−caryophyllene and germacrene D, were also identified in this study, consistent with findings from other studies on the essential oils of oregano [6,46,47]. It is crucial to highlight the biological activities of the most abundant sesquiterpenes, particularly E−Caryophyllene and Germacrene D, found in red oregano. E−caryophyllene has been widely studied for its anti−inflammatory, antimicrobial, and antioxidant properties, contributing to its potential therapeutic benefits [6,44,46].

In the second cluster, E−Caryophyllene was a principal component in most individuals in most populations. The third cluster exhibited pronounced influences from α−Muurolol, α−Cadinol, E−α−Farnesene, and Elemol, collectively shaping its composition. The fourth cluster was distinguished by the influence of Thymol, Carvacrol γ−Muurolene, γ−Terpinenene, Sabinene, Z−β−Ocimene, and Bornyl acetate, collectively defining its unique chemical. Stešević et al. [21] found that the average Euclidean distance among populations, calculated based on the leading essential oil constituents, was 41.6, ranging from 8.9 to 79.1. Another study identified the primary chemotypes of essential oils as β−Ocimene–Germacrene D–β−Caryophyllene [7]. Conversely, Stešević et al. [21] emphasized that in Montenegrin populations of red oregano, the prevalent chemotype is primarily monoterpene.

3.4. Hierarchical Cluster Analysis (HCA)

The dendrogram from the hierarchical cluster analysis (HCA) offers valuable insights for identifying similarities and differences in essential oil compositions across seven populations of red oregano in various geographical locations. Furthermore, the findings revealed variations in the composition’s quantitative and qualitative aspects, a resemblance noted in the diversity described by [48].

The hierarchical cluster analysis (HCA), based on the Euclidean distance between groups, delineated two distinct population groups (refer to Figure 3), each identified by unique essential oil chemotypes. The first group, characterized by a dissimilarity of four populations, was split into two sub−groups (C1, C5, C2, and C6). Meanwhile, the second group, exhibiting a dissimilarity of three populations, underwent further subdivision into two sub−groups (C3, C4, and C7). Notably, the populations in the sub−groups (C1 and C5), (C2 and C6), and (C3 and C4) emerged prominently, forming distinct groups in the principal component analysis (PCA) and establishing a significant dichotomy in the hierarchical cluster analysis (HCA) (refer to Figure 3)

Findings from the cluster analysis revealed considerable inter−production variability in the essential oil of red oregano [49]. Through multivariate analysis of the six production samples, cluster analysis distinctly identified two well−defined groups based on the essential oil composition.

4. Conclusions

In conclusion, the essential oils derived from seven distinct red oregano populations displayed noteworthy chemical diversity. The findings also highlight discrepancies in the compositions’ quantity and quality. Utilizing the chemical profiles of these essential oils, the hierarchical cluster analysis (HCA) effectively categorized them into two clusters and four primary subclusters. This categorization offers valuable insights for organizing and understanding similarities and distinctions in essential oil compositions across different geographical locations. The cluster analysis and principal component analysis (PCA) emphasized Germacrene D, E−Caryophyllene, α−Muurolol, and Thymol as the key clusters for red oregano. In the Albanian population of O. vulgare subsp. vulgare, all groups exhibit the hydrocarbon sesquiterpenes chemotype, comprising 49.02% to 71.09% of the essential oil content. Germacrene D and E−Caryophyllene were identified as the primary constituents, underscoring the pharmaceutical benefits of these compounds in chemotypes where they are more prevalent. The observed disparities in the essential oil compositions of red oregano at the intraspecific level are attributed to genetic diversity, underscoring the imperative for additional studies to discern optimal clones for breeding programs within red oregano populations. Moreover, genotypic analyses will enhance our understanding of the impact of environmental factors and plant genotype on plant diversity and adaptation.