# Seasonal Variability and Effect of Sample Storage on Volatile Components in _Calypogeia azurea_
## 1 Introduction  
The chemistry of liverworts (Hepaticae) has been the subject of intensive research in recent decades [1]. Liverworts are the ancestors of all land plants and abundantly produce specialized metabolites, including monoterpenes, sesquiterpennes, monoterpenoids, sesquiterpenoids, and aromatic compounds, many of which exhibit noteworthy biological activities, such as an inhibitory effect on allergic contact dermatitis, cytotoxicity, antibacterial and antifungal activity, anti-insect activity, and antioxidant properties [2, 3, 4, 5]. Due to their morphology, liverworts (Hepaticae) are difficult to classify and identify. Since they are rich in volatile organic compounds, the latter one can be used to access their chemosystematicity [6, 7]. Phytochemicals produced by liverworts are unique sources of pharmaceuticals, food additives, flavours, and other industrial materials. The accumulation of these metabolites occurs often in plants subjected to stress, including various stimulators or signal molecules [8]. Modern theories state that the specialized metabolites produced by plants are widely dependent on the environmental condition, duration and intensity of stress, genetic plasticity, and composition of plants. Currently, liverworts are of great biological interest because they contain a wide range of secondary metabolites that are produced to combat a variety ofbiotics and abiotic stress (microorganisms, insects, UV radiation, and various environments conditions). Most stress factors are seasonal, which means that the total content and relative proportions of specialized metabolites in plants fluctuate with seasonal environmental changes [9]. Consequently, these changes can affect the therapeutic effectiveness of the plant in question. The genus _Calypogeia radial_ comprises approximately 90 described species distributed throughout the world, with the highest diversity of species observed in the tropics [10, 11]. In Holarctis, the species richness of _Calypogeia_ is much lower and is represented by only9-13 species in its different parts [10; 12]. In Europe, _Calypogeia_ is represented by only eight species: _C. azurea_, _C. integristipula_, _C. neesiana_, _C. suecica_, _C. muelleriana_, _C. sphagnicola_, _C. fissa_, and _C. arguta_[13]. All occur in Poland with the exception of _C. arguta_ of the genus _Calypogeia_ and are quite common in the flora of the Polish hypophytes, except for _C. fissa_[14]. Most species in the genus _Calypogeia_ have colourless and pellucid oil bodies, while coloured oil bodies, including gray to pale brown, purple to blue, are rare [15]. _Calypogeia azurea_ is a species with blue oil bodies that has been considered a species of the Holarctic range, green or bluish when alive, yellowish green when dried, from 1.6 to 1.8 mm wide \(\times\) 7-16 mm long to 2-2.5 mm wide \(\times\) 7-20 mm long. Leaves are imbricate, ovate to cordate, with maximal width near the base. Blue or deep blue oil bodies under fresh conditions, discernible with a hand lens as dark or blue minute spots, are present in all cells on the top and underside of the leaf [16; 17]. The intense blue colour indicates the presence of azulenes, which are not uncommon constituents of essential oils, although in some cases they are presumably formed during sample processing [18; 19]. One of these azulenes exhibits anti-inflammatory and anti-ulcer activity [20]. _Calypogeia azurea_ prefers to grow on steep walls, for example, stream incisions with acidic loam in coniferous or peech forests. The species sometimes also grows on rotten wood and also in peaty places and north of heathand. Perhaps the future will show that _Calypogeia azurea_ is more common than we have to assume now. Searching through relief-rich forests or relief-rich vegetation could lead to new finds. The species can be confused with _C. muelleriana_, which sometimes has a blue tinge. The lower petals of _C. azurea_ are more dissected and almost as sharply serrated as those of _C. fissa_.