Species-Based Field Cultivation of Thymus: Essential Oil Yield and Chemotype Differentiation

The genus Thymus L. is characterized by high taxonomic complexity and pronounced phytochemical polymorphism, which underlie its economic and medicinal importance. While a limited number of species (Thymus vulgaris, Thymus pulegioides, Thymus × citriodorus) are traditionally cultivated, the cultivation potential of many Balkan taxa remains poorly explored. The present study aimed to evaluate the field cultivation performance, essential oil yield, and chemotype differentiation of three traditional and three lesser-studied Thymus species (Thymus zygioides Griseb., Thymus longedentatus (Degen & Urum.) Ronniger, and Thymus pannonicus All.). Plants were established through vegetative propagation and cultivated under field conditions, followed by essential oil isolation and GC–MS analysis. The newly introduced species exhibited higher essential oil yields, reaching 2.30% in T. longedentatus, 1.48% in T. pannonicus, and 0.94% in T. zygioides, compared to 0.24–0.60% in traditionally cultivated species. Clear and species-specific chemotypes were identified: a citral (neral/geranial) chemotype in T. longedentatus, a thymol chemotype in T. zygioides, and a sesquiterpene-dominated profile in T. pannonicus. In contrast, traditionally cultivated species displayed overlapping and less differentiated chemical profiles. All species were propagated vegetatively and cultivated in an open-field experimental plantation under temperate continental climatic conditions, following environmentally responsible horticultural practices. Vegetative propagation ensured genetic uniformity and supported consistent chemotype expression of the planting material under the applied cultivation conditions. These results demonstrate that species-based selection represents a robust alternative to conventional thyme cultivation, enabling higher essential oil productivity, clearer chemotypic differentiation, and improved standardization for horticultural and medicinal plant production, while supporting the sustainable use of native Bulgarian biodiversity.