Seasonal Variability and Effect of Sample Storage on Volatile Components in Calypogeia azurea

A change in the composition of specialized metabolites is often observed in stressed plants. Phytochemicals play an important role in adapting plants to the environment, particularly overcoming stress conditions such as temperature, humidity, and light intensity. In this study, seasonal variations in the concentrations of volatile organic compounds (VOCs) were analysed in species of Calypogeia azurea. The article presents the effect of sample storage on volatile organic compounds present in Calypogeia liverwort cells and whether the collection habitats of the sample affect the content of phytochemicals. The VOCs of the species within the liverwort Calypogeia azurea were analysed by GC-MS. Compounds were isolated from plant material using the HS-SPME technique. The samples were collected over several years (2019–2021). Of the several dozen samples collected, 79 compounds were isolated, of which 47 were identified.


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, sesquiterpenes, 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 of biotics 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 raddi 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 only fridge-3 months, freezer-1 month, freezer-3 months.
In the case of other metabolites in the initial storage period, an increase in content was observed, followed by a slight decrease, as exemplified, among others, by selina-5,11diene (41) and germacrene D (47). These volatile compounds are present in Calypogeia azurea during harvest and decline rapidly in cold stores. The content of limonene (14), βcyclocitral (19), and (-)-arystolene (36) decreases with the time the sample is stored in the refrigerator and then increases after being placed in the freezer. Temperature and humidity change during storage of Calypogeia azurea samples. Mechanical damage may occur during harvesting, transport, and reloading to a storage site; these stresses alter metabolic pathways of plants, which ultimately form the composition of specialized metabolites in stored liverworts. Browning of Calypogeia azurea was noticed during sample storage, which may suggest low-temperature injuries, i.e., chilling injury. The defence mechanism of stored plant material depends entirely on types of stress. All of these changes result in certain phenotypic expressions, i.e., colour change, deterioration of compounds, and emission of certain VOCs. The observed changes in the composition of selected VOCs caused by the stress associated with storage in a refrigerator and a freezer are presented in Figure  1.
In the case of other metabolites in the initial storage period, an increase in content was observed, followed by a slight decrease, as exemplified, among others, by selina-5,11diene (41) and germacrene D (47). These volatile compounds are present in Calypogeia azurea during harvest and decline rapidly in cold stores. The content of limonene (14), βcyclocitral (19), and (-)-arystolene (36) decreases with the time the sample is stored in the refrigerator and then increases after being placed in the freezer. Temperature and humidity change during storage of Calypogeia azurea samples. Mechanical damage may occur during harvesting, transport, and reloading to a storage site; these stresses alter metabolic pathways of plants, which ultimately form the composition of specialized metabolites in stored liverworts. Browning of Calypogeia azurea was noticed during sample storage, which may suggest low-temperature injuries, i.e., chilling injury. The defence mechanism of stored plant material depends entirely on types of stress. All of these changes result in certain phenotypic expressions, i.e., colour change, deterioration of compounds, and emission of certain VOCs. The observed changes in the composition of selected VOCs caused by the stress associated with storage in a refrigerator and a freezer are presented in Figure  1.
In the case of other metabolites in the initial storage period, an increase in content was observed, followed by a slight decrease, as exemplified, among others, by selina-5,11diene (41) and germacrene D (47). These volatile compounds are present in Calypogeia azurea during harvest and decline rapidly in cold stores. The content of limonene (14), βcyclocitral (19), and (-)-arystolene (36) decreases with the time the sample is stored in the refrigerator and then increases after being placed in the freezer. Temperature and humidity change during storage of Calypogeia azurea samples. Mechanical damage may occur during harvesting, transport, and reloading to a storage site; these stresses alter metabolic pathways of plants, which ultimately form the composition of specialized metabolites in stored liverworts. Browning of Calypogeia azurea was noticed during sample storage, which may suggest low-temperature injuries, i.e., chilling injury. The defence mechanism of stored plant material depends entirely on types of stress. All of these changes result in certain phenotypic expressions, i.e., colour change, deterioration of compounds, and emission of certain VOCs. The observed changes in the composition of selected VOCs caused by the stress associated with storage in a refrigerator and a freezer are presented in Figure  1.

fridge-3 months,
Molecules 2022, 27, x FOR PEER REVIEW enyl)benzoic acid methyl ester (74) from 13.61% increase in content was recorded for anastrepte macrene (49) from 4.08% to 15.39%, and alloaro the case of other compounds, the gains were n not frozen samples, the samples after freezing as: isospathulenol (67)(1.85%), germacra-4(15) methyl-2-methylazulene-1-carboxylate (79) (2. (59)(0.25-1.52%) was also detected, which was In the case of other metabolites in the initial s observed, followed by a slight decrease, as ex diene (41) and germacrene D (47). These volatil urea during harvest and decline rapidly in col cyclocitral (19), and (-)-arystolene (36) decrease refrigerator and then increases after being place ity change during storage of Calypogeia azurea during harvesting, transport, and reloading to a pathways of plants, which ultimately form the stored liverworts. Browning of Calypogeia azu which may suggest low-temperature injuries, i. of stored plant material depends entirely on ty certain phenotypic expressions, i.e., colour chan sion of certain VOCs. The observed changes in by the stress associated with storage in a refrige 1.

The Effect of Seasonality on the Content of Vola
The liverworts of the Calypogeia azurea spec in the composition of specialized metabolites r plant. Cyclical changes in the VOC compositio autumn, repeated in 2019-2021. It is most visib ing in the composition of the VOC. The observe with the highest content in summer. These incl loaromadendrene (40), selina-5,11-diene (41), g cuparene (51), compound IR = 1637 (63), and ge freezer-1 month, Molecules 2022, 27, x FOR PEER REVIEW enyl)benzoic acid methyl ester (74) from 13.61% increase in content was recorded for anastrepten macrene (49) from 4.08% to 15.39%, and alloarom the case of other compounds, the gains were no not frozen samples, the samples after freezing ha as: isospathulenol (67)(1.85%), germacra-4(15),5, methyl-2-methylazulene-1-carboxylate (79)(2.87 (59)(0.25-1.52%) was also detected, which was no In the case of other metabolites in the initial sto observed, followed by a slight decrease, as exem diene (41) and germacrene D (47). These volatile urea during harvest and decline rapidly in cold cyclocitral (19), and (-)-arystolene (36) decreases w refrigerator and then increases after being placed ity change during storage of Calypogeia azurea sa during harvesting, transport, and reloading to a s pathways of plants, which ultimately form the co stored liverworts. Browning of Calypogeia azure which may suggest low-temperature injuries, i.e., of stored plant material depends entirely on type certain phenotypic expressions, i.e., colour change sion of certain VOCs. The observed changes in th by the stress associated with storage in a refrigera 1.

The Effect of Seasonality on the Content of Volati
The liverworts of the Calypogeia azurea specie in the composition of specialized metabolites res plant. Cyclical changes in the VOC composition autumn, repeated in 2019-2021. It is most visible ing in the composition of the VOC. The observed with the highest content in summer. These includ loaromadendrene (40), selina-5,11-diene (41), ger cuparene (51), compound IR = 1637 (63), and germ freezer-3 months.

The Effect of Seasonality on the Content of Volatile Organic Compounds
The liverworts of the Calypogeia azurea species are characterized by a visible variation in the composition of specialized metabolites resulting from the vegetation period of the plant. Cyclical changes in the VOC composition were observed in spring, summer, and autumn, repeated in 2019-2021. It is most visible in the example of compounds dominating in the composition of the VOC. The observed changes can be divided into compounds with the highest content in summer. These include: δ-elemene (22), anastreptene (25), alloaromadendrene (40), selina-5,11-diene (41), germacrene D (47), bicyclogermacrene (49), cuparene (51), compound IR = 1637 (63), and germacra-4(15),5,10(14)-trien-1-alpha-ol (77). The second group consists of compounds with the lowest content in summer. They include: compound IR = 902 (8), β-pinene (12), limonene (14), β-cyclocitral (19), 1,4-dimethyl-azulene (60), and 4-(cyclopent-1-enyl)benzoic acid methyl ester (74). A small group of compounds showed a continuous increase or decrease in content from spring to autumn; examples include methyl 2-methylazulene-1-carboxylate (79), compound IR = 1224 (18), and compound IR = 1392 (28). Plants secrete a variety of volatile organic compounds that provide protection against mechanical damage, environmental changes, and pathogens. As a result, these tiny plants developed a unique variety of bioactive compounds as part of their survival strategies. Most sesquiterpenoids increase during spring and peak in summer. These compounds are likely to be elevated during the summer to allow liverworts to cope with abiotic stresses such as high temperatures and droughts. Table 2. Volatile compounds detected in the samples C-1-C-5.

No.
Compounds                    This percentage drops in autumn, which may be due to Calypogeia azurea's reaction to fewer hours of sunshine per day and the amount of water in the soil. In nature, light plays an irreplaceable role in plant growth and inducing or regulating plant metabolism. In response to light radiation, plants can adapt to changing conditions by releasing and accumulating various specialized metabolites. During experiments, it was shown that plants exposed to drought stress accumulate higher concentrations of specialized metabolites.
The changes in the composition of the specialized metabolites described above are caused by differences in the exposure of the plant to the sun, to water, and nutrients in various stages of vegetation, which in turn affects the metabolic processes of the plant. The described seasonal variability (spring-summer-autumn) for selected compounds is presented in Figure 2. However, no significant differences were observed in the composition of the specialized metabolites resulting from the location of the sites. Samples collected in the Beskid Sądecki or Karkonosze Mountains have a similar composition. The second group consists of compounds with the lowest content in summer. They include: compound IR = 902 (8), β-pinene (12), limonene (14), β-cyclocitral (19), 1,4-dimethyl-azulene (60), and 4-(cyclopent-1-enyl)benzoic acid methyl ester (74). A small group of compounds showed a continuous increase or decrease in content from spring to autumn; examples include methyl 2-methylazulene-1-carboxylate (79), compound IR = 1224 (18), and compound IR = 1392 (28). Plants secrete a variety of volatile organic compounds that provide protection against mechanical damage, environmental changes, and pathogens. As a result, these tiny plants developed a unique variety of bioactive compounds as part of their survival strategies. Most sesquiterpenoids increase during spring and peak in summer. These compounds are likely to be elevated during the summer to allow liverworts to cope with abiotic stresses such as high temperatures and droughts. This percentage drops in autumn, which may be due to Calypogeia azurea's reaction to fewer hours of sunshine per day and the amount of water in the soil. In nature, light plays an irreplaceable role in plant growth and inducing or regulating plant metabolism. In response to light radiation, plants can adapt to changing conditions by releasing and accumulating various specialized metabolites. During experiments, it was shown that plants exposed to drought stress accumulate higher concentrations of specialized metabolites.
The changes in the composition of the specialized metabolites described above are caused by differences in the exposure of the plant to the sun, to water, and nutrients in various stages of vegetation, which in turn affects the metabolic processes of the plant. The described seasonal variability (spring-summer-autumn) for selected compounds is presented in Figure 2. However, no significant differences were observed in the composition of the specialized metabolites resulting from the location of the sites. Samples collected in the Beskid Sądecki or Karkonosze Mountains have a similar composition. To date, it has been reported in the literature that the accumulation of VOC depends on various environmental factors such as light, temperature, soil water, soil fertility, and salinity, and for most plants, a change in one factor can change the VOC content even if other factors remain constant. External factors can significantly affect some processes re- The second group consists of compounds with the lowest content in summer. They include: compound IR = 902 (8), β-pinene (12), limonene (14), β-cyclocitral (19), 1,4-dimethyl-azulene (60), and 4-(cyclopent-1-enyl)benzoic acid methyl ester (74). A small group of compounds showed a continuous increase or decrease in content from spring to autumn; examples include methyl 2-methylazulene-1-carboxylate (79), compound IR = 1224 (18), and compound IR = 1392 (28). Plants secrete a variety of volatile organic compounds that provide protection against mechanical damage, environmental changes, and pathogens. As a result, these tiny plants developed a unique variety of bioactive compounds as part of their survival strategies. Most sesquiterpenoids increase during spring and peak in summer. These compounds are likely to be elevated during the summer to allow liverworts to cope with abiotic stresses such as high temperatures and droughts. This percentage drops in autumn, which may be due to Calypogeia azurea's reaction to fewer hours of sunshine per day and the amount of water in the soil. In nature, light plays an irreplaceable role in plant growth and inducing or regulating plant metabolism. In response to light radiation, plants can adapt to changing conditions by releasing and accumulating various specialized metabolites. During experiments, it was shown that plants exposed to drought stress accumulate higher concentrations of specialized metabolites.
The changes in the composition of the specialized metabolites described above are caused by differences in the exposure of the plant to the sun, to water, and nutrients in various stages of vegetation, which in turn affects the metabolic processes of the plant. The described seasonal variability (spring-summer-autumn) for selected compounds is presented in Figure 2. However, no significant differences were observed in the composition of the specialized metabolites resulting from the location of the sites. Samples collected in the Beskid Sądecki or Karkonosze Mountains have a similar composition. To date, it has been reported in the literature that the accumulation of VOC depends on various environmental factors such as light, temperature, soil water, soil fertility, and salinity, and for most plants, a change in one factor can change the VOC content even if other factors remain constant. External factors can significantly affect some processes re- The second group consists of compounds with the lowest content in summer. They include: compound IR = 902 (8), β-pinene (12), limonene (14), β-cyclocitral (19), 1,4-dimethyl-azulene (60), and 4-(cyclopent-1-enyl)benzoic acid methyl ester (74). A small group of compounds showed a continuous increase or decrease in content from spring to autumn; examples include methyl 2-methylazulene-1-carboxylate (79), compound IR = 1224 (18), and compound IR = 1392 (28). Plants secrete a variety of volatile organic compounds that provide protection against mechanical damage, environmental changes, and pathogens. As a result, these tiny plants developed a unique variety of bioactive compounds as part of their survival strategies. Most sesquiterpenoids increase during spring and peak in summer. These compounds are likely to be elevated during the summer to allow liverworts to cope with abiotic stresses such as high temperatures and droughts. This percentage drops in autumn, which may be due to Calypogeia azurea's reaction to fewer hours of sunshine per day and the amount of water in the soil. In nature, light plays an irreplaceable role in plant growth and inducing or regulating plant metabolism. In response to light radiation, plants can adapt to changing conditions by releasing and accumulating various specialized metabolites. During experiments, it was shown that plants exposed to drought stress accumulate higher concentrations of specialized metabolites.
The changes in the composition of the specialized metabolites described above are caused by differences in the exposure of the plant to the sun, to water, and nutrients in various stages of vegetation, which in turn affects the metabolic processes of the plant. The described seasonal variability (spring-summer-autumn) for selected compounds is presented in Figure 2. However, no significant differences were observed in the composition of the specialized metabolites resulting from the location of the sites. Samples collected in the Beskid Sądecki or Karkonosze Mountains have a similar composition. The second group consists of compounds with the lowest content in summer. They include: compound IR = 902 (8), β-pinene (12), limonene (14), β-cyclocitral (19), 1,4-dimethyl-azulene (60), and 4-(cyclopent-1-enyl)benzoic acid methyl ester (74). A small group of compounds showed a continuous increase or decrease in content from spring to autumn; examples include methyl 2-methylazulene-1-carboxylate (79), compound IR = 1224 (18), and compound IR = 1392 (28). Plants secrete a variety of volatile organic compounds that provide protection against mechanical damage, environmental changes, and pathogens. As a result, these tiny plants developed a unique variety of bioactive compounds as part of their survival strategies. Most sesquiterpenoids increase during spring and peak in summer. These compounds are likely to be elevated during the summer to allow liverworts to cope with abiotic stresses such as high temperatures and droughts. This percentage drops in autumn, which may be due to Calypogeia azurea's reaction to fewer hours of sunshine per day and the amount of water in the soil. In nature, light plays an irreplaceable role in plant growth and inducing or regulating plant metabolism. In response to light radiation, plants can adapt to changing conditions by releasing and accumulating various specialized metabolites. During experiments, it was shown that plants exposed to drought stress accumulate higher concentrations of specialized metabolites.
The changes in the composition of the specialized metabolites described above are caused by differences in the exposure of the plant to the sun, to water, and nutrients in various stages of vegetation, which in turn affects the metabolic processes of the plant. The described seasonal variability (spring-summer-autumn) for selected compounds is presented in Figure 2. However, no significant differences were observed in the composition of the specialized metabolites resulting from the location of the sites. Samples collected in the Beskid Sądecki or Karkonosze Mountains have a similar composition. To date, it has been reported in the literature that the accumulation of VOC depends on various environmental factors such as light, temperature, soil water, soil fertility, and salinity, and for most plants, a change in one factor can change the VOC content even if other factors remain constant. External factors can significantly affect some processes re-autumn.
To date, it has been reported in the literature that the accumulation of VOC depends on various environmental factors such as light, temperature, soil water, soil fertility, and salinity, and for most plants, a change in one factor can change the VOC content even if other factors remain constant. External factors can significantly affect some processes related to the growth and development of plants, and even their ability to synthesize specialized metabolites, ultimately leading to changes in general phytochemical profiles that play a strategic role in the production of bioactive substances [26][27][28][29].

Effect of the Type of Substratum on the Content of Secondary Metabolites
On the basis of samples of C. azurea grown on decayed wood, it was found that soil influenced the VOC composition. Samples of these plants were istinguished by a much higher content of anastreptene (25) and a much lower content of 1,4-dimethyl azulene (60) and 4-(cyclopent-1-enyl)benzoic acid methyl ester (74) in the spring season. On the other hand, in summer and autumn, the VOC composition was similar to that of C. azurea samples growing in forest litter. In samples of C. azurea grown in this medium, the presence of compounds not present in liverworts grown in soil was found. They are: 2-methyl-1propanol (1), (Z)-3-hexen-1-ol (6), tricyclene (9), camphene (11), compound IR = 1407 (30), (+)-spathulenol (61), and compound IR = 1664 (66). On the other hand, plants growing on soil had 1,2-dihydro-6-methylnaphthalene (20), which was not detected in plants growing on rotting wood. The presented results show that environmental factors related to the substrate, such as temperature, pH, and humidity of the substrate, influence the composition of specialized metabolites produced by liverworts.
The observed differences in the VOC composition resulting from the substrate, based on the example of selected compounds, are shown in Figure 3. lated to the growth and development of plants, and even their ability to synthesize specialized metabolites, ultimately leading to changes in general phytochemical profiles that play a strategic role in the production of bioactive substances [26][27][28][29].

Effect of the Type of Substratum on the Content of Secondary Metabolites
On the basis of samples of C. azurea grown on decayed wood, it was found that soil influenced the VOC composition. Samples of these plants were istinguished by a much higher content of anastreptene (25) and a much lower content of 1,4-dimethyl azulene (60) and 4-(cyclopent-1-enyl)benzoic acid methyl ester (74) in the spring season. On the other hand, in summer and autumn, the VOC composition was similar to that of C. azurea samples growing in forest litter. In samples of C. azurea grown in this medium, the presence of compounds not present in liverworts grown in soil was found. They are: 2-methyl-1propanol (1), (Z)-3-hexen-1-ol (6), tricyclene (9), camphene (11), compound IR = 1407 (30), (+)-spathulenol (61), and compound IR = 1664 (66). On the other hand, plants growing on soil had 1,2-dihydro-6-methylnaphthalene (20), which was not detected in plants growing on rotting wood. The presented results show that environmental factors related to the substrate, such as temperature, pH, and humidity of the substrate, influence the composition of specialized metabolites produced by liverworts.
The observed differences in the VOC composition resulting from the substrate, based on the example of selected compounds, are shown in Figure 3.

Volatile Organic Compounds in In Vitro Culture
A separate group of samples was the liverworts collected from the in vitro cultures. Samples were grown from material collected in spring in Beskid Sądecki. The composition of the VOCs was tested after 6 months of cultivation. Each time, the in vitro samples had a composition similar to that of the samples collected in summer in the natural environment. This indicates that in vitro culture was conducted under optimal growth conditions. lated to the growth and development of plants, and even their ability to synthesize specialized metabolites, ultimately leading to changes in general phytochemical profiles that play a strategic role in the production of bioactive substances [26][27][28][29].

Effect of the Type of Substratum on the Content of Secondary Metabolites
On the basis of samples of C. azurea grown on decayed wood, it was found that soil influenced the VOC composition. Samples of these plants were istinguished by a much higher content of anastreptene (25) and a much lower content of 1,4-dimethyl azulene (60) and 4-(cyclopent-1-enyl)benzoic acid methyl ester (74) in the spring season. On the other hand, in summer and autumn, the VOC composition was similar to that of C. azurea samples growing in forest litter. In samples of C. azurea grown in this medium, the presence of compounds not present in liverworts grown in soil was found. They are: 2-methyl-1propanol (1), (Z)-3-hexen-1-ol (6), tricyclene (9), camphene (11), compound IR = 1407 (30), (+)-spathulenol (61), and compound IR = 1664 (66). On the other hand, plants growing on soil had 1,2-dihydro-6-methylnaphthalene (20), which was not detected in plants growing on rotting wood. The presented results show that environmental factors related to the substrate, such as temperature, pH, and humidity of the substrate, influence the composition of specialized metabolites produced by liverworts.
The observed differences in the VOC composition resulting from the substrate, based on the example of selected compounds, are shown in Figure 3.

Volatile Organic Compounds in In Vitro Culture
A separate group of samples was the liverworts collected from the in vitro cultures. Samples were grown from material collected in spring in Beskid Sądecki. The composition of the VOCs was tested after 6 months of cultivation. Each time, the in vitro samples had a composition similar to that of the samples collected in summer in the natural environment. This indicates that in vitro culture was conducted under optimal growth conditions. lated to the growth and development of plants, and even their ability to synthesize specialized metabolites, ultimately leading to changes in general phytochemical profiles that play a strategic role in the production of bioactive substances [26][27][28][29].

Effect of the Type of Substratum on the Content of Secondary Metabolites
On the basis of samples of C. azurea grown on decayed wood, it was found that soil influenced the VOC composition. Samples of these plants were istinguished by a much higher content of anastreptene (25) and a much lower content of 1,4-dimethyl azulene (60) and 4-(cyclopent-1-enyl)benzoic acid methyl ester (74) in the spring season. On the other hand, in summer and autumn, the VOC composition was similar to that of C. azurea samples growing in forest litter. In samples of C. azurea grown in this medium, the presence of compounds not present in liverworts grown in soil was found. They are: 2-methyl-1propanol (1), (Z)-3-hexen-1-ol (6), tricyclene (9), camphene (11), compound IR = 1407 (30), (+)-spathulenol (61), and compound IR = 1664 (66). On the other hand, plants growing on soil had 1,2-dihydro-6-methylnaphthalene (20), which was not detected in plants growing on rotting wood. The presented results show that environmental factors related to the substrate, such as temperature, pH, and humidity of the substrate, influence the composition of specialized metabolites produced by liverworts.
The observed differences in the VOC composition resulting from the substrate, based on the example of selected compounds, are shown in Figure 3.

Volatile Organic Compounds in In Vitro Culture
A separate group of samples was the liverworts collected from the in vitro cultures. Samples were grown from material collected in spring in Beskid Sądecki. The composition of the VOCs was tested after 6 months of cultivation. Each time, the in vitro samples had a composition similar to that of the samples collected in summer in the natural environment. This indicates that in vitro culture was conducted under optimal growth conditions. liverworts growing on a rotten tree under the forest litter.

Volatile Organic Compounds in In Vitro Culture
A separate group of samples was the liverworts collected from the in vitro cultures. Samples were grown from material collected in spring in Beskid Sądecki. The composition of the VOCs was tested after 6 months of cultivation. Each time, the in vitro samples had a composition similar to that of the samples collected in summer in the natural environment. This indicates that in vitro culture was conducted under optimal growth conditions.

Observation of Changes Depending on the Season
Analyses carried out within individual years of the samples taken showed that in 2019 there were no significant differences between seasons in terms of volatile compounds aliphatics (p = 0.420), aromatics (p = 0.062), monoterpenes (p = 0.097), monoterpenoids (p = 0.368), and sesquiterpenoids (p = 0.717). However, a statistically significant effect of the difference between seasons in terms of undefined relationships was confirmed, F = 7.87; df = 2; p = 0.020, and sesquiterpenes, F = 11.31; df = 2; p = 0.003.
As it turned out, the level of volatile compounds not identified was significantly higher in condition C-1 (p = 0.040) and was biased higher in condition C-9 (p = 0.058) compared to condition C-13. Furthermore, condition C-13 for sesquiterpene compounds showed higher levels of volatile compounds compared to C-9 (p = 0.003). No statistically significant differences were found in the other conditions in 2019 (Figure 4).
Similarly to 2019, samples collected in 2020 showed statistically significant differences only for volatile sesquiterpene compounds, F = 9.64; df = 2; p = 0.008, and non-identified compounds, F = 10.87; df = 2; p = 0.004. No significant differences were found between seasons in 2020 for the remaining compounds: aliphatics (p = 0.779), aromatics (p = 0.449), monoterpenes (p = 0.368), monoterpenoids (p = 0.368), and sesquiterpenoids (p = 0.717). Analysis of pairwise comparisons between seasons in terms of sesquiterpenes and differences not identified showed that the level of undefined compound in sample C-22 was significantly higher than in sample C-26 (p = 0.004). In contrast, the intensity of sesquiterpene compounds in sample C-22 was significantly lower than in sample C-26 (p = 0.006). No significant differences were found in the other conditions.
Furthermore, an analysis of differences was performed using the Kruskal-Wallis test for the severity of individual volatile compounds in each section from C-9 to C-39 separately to confirm the possibility that individual compounds were more prevalent. As it turned out, statistically significant differences were found for C-9 in 2019, H = 13.45; df = 6; p = 0.036, also C-22 in 2020, H = 14.09; df = 6; p = 0.029, and C-35 in 2021, H = 13.45; df = 6; p = 0.036. Analysis of pairwise comparisons showed that the severity of sesquiterpene compounds was always significantly higher compared to aliphatic compounds, and these differences were significant at the level of p < 0.05 level. No significant differences were found for the other volatile compounds. Figure 5 shows the average content of volatile compounds depending on the storage method. Analysis of differences between all individual storage methods, taking into account the duration of use of a given method, showed statistically significant differences only between sesquiterpene compounds, F = 20.58; df = 3; p < 0.001.

Observation of Changes Depending on Storage
Pairwise comparisons showed that the severity of the relationship was higher in condition C-5 compared to condition C-2 (p = 0.005) and C-3 (p = 0.030), and also lower in condition C-4 compared to condition C-2 (p = 0.003) and C-3 (p = 0.017). There were no significant differences between conditions C-2 and C-3, as well as conditions C-4 and C-5. Furthermore, it was confirmed that there were no differences in the storage of volatile compounds such as aliphatics (p = 0.801), aromatics (p = 0.266), monoterpenes (p = 0.072), monoterpenoids (p = 0.392), and sesquiterpenoids (p = 0.122). Additional analysis of the differences between the volatile compounds themselves under particular conditions showed significant differences in the case of condition C-2, H = 14, 62; df = 6; p = 0.023, and for condition C-4, H = 19.08; df = 6; p = 0.004. As it turned out, volatile sesquiterpene compounds under both conditions showed a higher intensity compared to aliphatic compounds, and these were the only significant differences between all volatile compounds at p < 0.05.

Observation of Changes Depending on the Season
Analyses carried out within individual years of the samples taken showed that in 2019 there were no significant differences between seasons in terms of volatile compounds aliphatics (p = 0.420), aromatics (p = 0.062), monoterpenes (p = 0.097), monoterpenoids (p = 0.368), and sesquiterpenoids (p = 0.717). However, a statistically significant effect of the difference between seasons in terms of undefined relationships was confirmed, F = 7.87; df = 2; p = 0.020, and sesquiterpenes, F = 11.31; df = 2; p = 0.003.
As it turned out, the level of volatile compounds not identified was significantly higher in condition C-1 (p = 0.040) and was biased higher in condition C-9 (p = 0.058) compared to condition C-13. Furthermore, condition C-13 for sesquiterpene compounds showed higher levels of volatile compounds compared to C-9 (p = 0.003). No statistically significant differences were found in the other conditions in 2019 (Figure 4).  In 2020, they showed statistically significant differences in terms of volatile sesquiterpene compounds, F = 29.03; df = 5; p < 0.001, and non-identified compounds, F = 11.46; df = 5; p = 0.043. There were no significant differences between seasons in 2020 for the remaining compounds: aliphatics (p = 0.207), aromatics (p = 0.350), monoterpenes (p = 0.221), monoterpenoids (p = 0.416), and sesquiterpenoids (p = 0.982). Analysis of pairwise comparisons between different substrates in terms of differences between sesquiterpenes and non-identified compounds showed that the level of undefined compound in the C-22 segment was significantly higher than in the C-26 segment (p = 0.018). However, the intensity of sesquiterpene compounds in the C-19 segment was significantly higher than in C-18 (p = 0.013), C-26 (p < 0.001), and C-27 (p = 0.002), and, in addition, the compound level in C-26 was lower than in C-23 (p = 0.035). No significant differences were found in the remaining conditions. In 2021, the comparisons showed statistically significant differences only for volatile sesquiterpene compounds, F = 26.27; df = 5; p < 0.001. The results of the analyses confirmed that the sesquiterpene compounds had a significantly lower intensity in the C-31 (p = 0.005) and C-39 (p < 0.001) segment compared to C-32; moreover, in the C-39 condition, a lower level of sesquiterpene was found compared to C-36 (p = 0.026). There were also no significant differences in 2021 for the remaining compounds: aliphatics (p = 0.609), aromatics (p = 0.152), monoterpenes (p = 0.371), monoterpenoids (p = 0.416), not identified (p = 0.118), and sesquiterpenoid (p = 0.885).
Furthermore, an analysis of differences was performed using the Kruskal-Wallis test for the severity of individual volatile compounds in each section from C-9 to C-39 separately to confirm the possibility that individual compounds were more prevalent. As it turned out, statistically significant differences were found for C-9 in 2019, H = 13.45; df = 6; p = 0.036, also C-22 in 2020, H = 14.09; df = 6; p = 0.029, and C-35 in 2021, H = 13.45; df = 6; p = 0.036. Analysis of pairwise comparisons showed that the severity of sesquiterpene compounds was always significantly higher compared to aliphatic compounds, and these differences were significant at the level of p < 0.05 level. No significant differences were found for the other volatile compounds.
2.6.2. Observation of Changes Depending on Storage Figure 5 shows the average content of volatile compounds depending on the storage method. Analysis of differences between all individual storage methods, taking into account the duration of use of a given method, showed statistically significant differences only between sesquiterpene compounds, F = 20.58; df = 3; p < 0.001. Pairwise comparisons showed that the severity of the relationship was higher in condition C-5 compared to condition C-2 (p = 0.005) and C-3 (p = 0.030), and also lower in condition C-4 compared to condition C-2 (p = 0.003) and C-3 (p = 0.017). There were no significant differences between conditions C-2 and C-3, as well as conditions C-4 and C-5. Furthermore, it was confirmed that there were no differences in the storage of volatile compounds such as aliphatics (p = 0.801), aromatics (p = 0.266), monoterpenes (p = 0.072), monoterpenoids (p = 0.392), and sesquiterpenoids (p = 0.122). Additional analysis of the differences between the volatile compounds themselves under particular conditions showed significant differences in the case of condition C-2, H = 14, 62; df = 6; p = 0.023, and Analyses conducted within each sampling year showed that in 2019, there were no significant differences between the substrates for volatile compounds: aliphatics (p = 0.178), aromatics (p = 0.142), monoterpenes (p = 0.119), monoterpenoids (p = 0.416), non-identified compounds (p = 0.126), and sesquiterpenoids (p = 0.885). However, sesquiterpenes were confirmed, F = 29.62; df = 5; p < 0.001. As it turned out, the measurement in period C-6 was statistically significantly higher compared to C-1 (p = 0.004), C-13 (p < 0.001), and C-14 (p = 0.008); in addition, the measurement of C-13 was higher compared to C-10 (p = 0.020). No statistically significant differences were found in the other conditions ( Figure 6). In 2020, they showed statistically significant differences in terms of volatile sesquiterpene compounds, F = 29.03; df = 5; p < 0.001, and non-identified compounds, F = 11.46; df = 5; p = 0.043. There were no significant differences between seasons in 2020 for the remaining compounds: aliphatics (p = 0.207), aromatics (p = 0.350), monoterpenes (p = 0.221), monoterpenoids (p = 0.416), and sesquiterpenoids (p = 0.982). Analysis of pairwise comparisons between different substrates in terms of differences between sesquiterpenes and non-identified compounds showed that the level of undefined compound in the C-22 segment was significantly higher than in the C-26 segment (p = 0.018). However, the intensity of sesquiterpene compounds in the C-19 segment was significantly higher than in C-18 (p = 0.013), C-26 (p < 0.001), and C-27 (p = 0.002), and, in addition, the compound level in C-26 was lower than in C-23 (p = 0.035). No significant differences were found in the remaining conditions. In 2021, the comparisons showed statistically significant differences only for volatile sesquiterpene compounds, F = 26.27; df = 5; p < 0.001. The results of the analyses confirmed that the sesquiterpene compounds had a significantly lower intensity in the C-31 (p = 0.005) and C-39 (p < 0.001) segment compared to C-32; moreover, in the C-

Plant Material
The plant material studied included 43 Calypogeia azurea samples cultured and obtained from habitats in different regions of Poland (Table 1). Natural liverwort samples were collected in the years 2019-2021 in three seasons: spring, summer, and autumn. Research materials were collected at three locations in the Karkonosze and Beskid Sądecki regions near Karpacz, Szklarska Poręba, and Krynica Zdrój at 700-1200 m A.S.L. All samples, except those from Krzyżowa Góra, were taken from the slope near the frequented routes. Plants collected on Góra Krzyżowa grew on fallen, rotten trees and rotting trees under forest litter. Five samples weighing approximately 15 g each were taken from each natural site. Only green plants that did not show signs of drying out and were not affected by visible diseases were eligible for collection and further research. In natural habitats, liverwort samples are initially identified on the basis of their morphological structure. In the laboratory, samples of C. azurea identified as species based on six DNA barcodes. Before analysis, the samples were cleaned from different plant material and soil. Research was conducted on fresh material. For selected samples from Beskid Sądecki, the impact of storage on VOC was analysed. For this purpose, the collected research material was stored for 1 month and 3 months in a refrigerator at a temperature of 5 • C and in a freezer at a temperature of −30 • C.

HS-SPME Extraction
Volatile compounds from Calypogeia azurea were extracted by the headspace solid phase microextraction technique. Fused silica fibres coated with divinylbenzene/carboxen/ polydimethylsiloxane (DVB/CAR/PDMS) were used. A 2-cm long fibre covered with a 50/30 µm thick film was used. Before analysis, the fibres were conditioned for 1 h at 270 • C, according to the supplier's instructions. Then, 5 mg of clean and dried plant material was placed in a 1.7 mL vial hermetically closed with a Teflon/silicone septum and heated at 50 • C. The extraction of the compounds was followed at 50 • C for 60 min. Fibre analyte desorption was carried out in the injection port of the gas chromatograph at 250 • C for 10 min. Sorption and desorption operations were performed using the TriPlus RSH autosampler (Thermo Scientific, Waltham, MA, USA).

GC-MS Analysis
The analysis of volatile compounds was performed using a previously described GC-MS method [30,31]. GC-MS analyses using a silphenylene phase were performed on a Trace 1310 (Thermo Scientific, Waltham, MA, USA) equipped with a Quadrex 007-5MS column (30 m, 0.25 mm, 0.25 µm). The ISQ QD mass detector (Thermo Scientific, Waltham, MA, USA) was operated at 70 eV in the EI mode in the m/z range of 30 to 550. This was used as the carrier gas at a flow rate of 1.0 mL/min. The oven temperature was programmed from 60 to 230 • C at 4 • C/min and then isothermal at 230 • C for 40 min. The injector temperature and transfer line were 250 • C. Injection samples were in splitless mode with a dedicated liner for the SPME technique. The identification of components was confirmed by comparing the mass spectral fragmentation patterns with those stored in the MS database (NIST 2011, NIST Chemistry WebBook, Adams 4 Library, MassFinder 4, and Pherobase) and those reported in the literature [32,33]. Furthermore, retention indices on nonpolar columns, determined relative to a homologous series of n-alkanes (C7-C40), were compared with the data of the published indices. Quantitative data of the components were obtained by integrating the TIC chromatogram and calculating the relative percentage of the peak areas. Each sample of Calypogeia azurea was analysed three times.

Statistical Analysis
The methods used were Friedman's ANOVA and Kurskal-Wallis rank tests because of the high intensity of variance. By transforming the results to a unified scale, rank analyses allowed more accurate comparisons to be made without accounting for measurement error for the means. A threshold of α = 0.05 was used as the significance level. The determination of the statistical analysis conditions is shown in Tables 1-10. The paper discusses the results of the statistical analysis performed on the example of samples for the location of the Krzyżowa Pass, because similar relationships were observed in other locations.

Conclusions
This study examined whether and to what extent Calypogeia liverworts are susceptible to environmental stress. A total of 43 samples collected at different times of the year and samples grown in vitro were analysed. In these samples, 79 volatile organic compounds were detected, 47 of which were identified. Knowing that the content of specialized metabolites varies with the seasons, Calypogeia azurea has been characterized in three different seasons (spring, summer, and autumn). Since winter is considered the dormant period for plants, including liverworts, the winter season has not been included in the present study.
Based on the VOC composition tests performed, it was found that Calypogeia azurea is a liverwort species particularly susceptible to stress and plant defence reactions. The biosynthesis of specialized metabolites is induced by environmental conditions. Studies have shown that abiotic factors such as sample storage and freezing and the type of medium on which Calypogeia azurea grows influence the production of specialized metabolites. Sunlight and humidity increase or decrease the percentage of volatile organic compounds present in plant cells. On the other hand, C. azurea obtained from in vitro culture is similar in terms of VOC composition to plant material obtained in summer from natural sites.
Due to their low morphology, liverworts are difficult to classify and identify. As they are rich in volatile organic compounds, they can be used to evaluate their chemosystematics. The conducted research shows that storing the sample in a refrigerator or a freezer may cause changes in the content of volatile organic compounds, which is why it is so important to test liverworts on fresh samples.