PROCEEDINGS 47Th International Conference of the Slovak Society of Chemical Engineering

Slovak Society of Chemical Engineering Institute of Chemical and Environmental Engineering Slovak University of Technology in Bratislava

PROCEEDINGS 47th International Conference of the Slovak Society of Chemical Engineering

Institute of Chemical and Environmental Engineering Bratislava, Slovakia May 18 - 19, 2021

Editors: Prof. Jozef Markoš, Dr. Mário Mihaľ

ISBN: 978-80-8208-057-8, EAN: 9788082080578

Guzowska, M., Wawrzyniak, R., Wasiak, W., Buczkowska-Chmielewska, K.: Composition of volatile compounds in Calypogeia integristipula (Calypogeiaceae, Marchantiophyta), Editors: Markoš, J., Mihaľ, M., In 47th International Conference of the Slovak Society of Chemical Engineering, Bratislava, Slovakia, 2021. 47th International Conference of SSCHE - online conference May 18 - 19, 2021, Bratislava, Slovakia

COMPOSITION OF VOLATILE COMPOUNDS IN CALYPOGEIA INTEGRISTIPULA (CALYPOGEIACEAE, MARCHANTIOPHYTA)

, M. Guzowska1*, R. Wawrzyniak1W. Wasiak1, B. Jasiewicz1, P. Gonera2, A. Bączkiewicz2, K. Buczkowska2

1Faculty of Chemistry, Adam Mickiewicz University in Poznań, Umultowska 89b, 61-614 Poznań, Poland, e-mail: [email protected]

2Faculty of Biology, Adam Mickiewicz University in Poznań, Umultowska 89, 61-614 Poznań, Poland

Keywords: liverworts, Calypogeia integristipula, HS-SPME, gas chromatography, mass spectrometry

ABSTRACT

Calypogeia is a genus of liverworts in the family Calypogeiaceae. It contains the following species of the genus occurring in Europe: Calypogeia azurea, Calypogeia fissa, Calypogeia muelleriana, Calypogeia integritipula. The subject of the research was Calypogeia integristipula. The plant material for analysis came from Tatry, Pieniny, Beskid Sądecki and Bieszczady Mts., from Wielkopolska and Lubusz provinces and from north-eastern Poland. In general, 23 samples determined as C. integristipula based on morphological characters and oil bodies were examined. The samples were determined genetically based on DNA sequences. The volatile compounds from biological materials were extracted by headspace solid-phase microextraction (HS-SPME) technique. The samples were then analysed on the GC/MS. 93 compounds were detected in total of which 38 compounds were identified. The remaining compounds are described using the MS fragmentation spectrum. In terms of quantity among the identified volatile compounds, compounds belonging to the group of sesquiterpenes (up to 61.53%) and sesquiterpenoids (up to 10.87%) prevail. A smaller amount are compounds belonging to aliphatic compounds (up to 1.58%) and aromatic compounds (up to 4.44%). Unfortunately, monoterpenes and monoterpenoids weren’t identified in the samples tested.

The main aim of this study was to select characteristic compounds which enable to differentiate the liverwort species Calypogeia integristipula from the morphologically very similar species Calypogeia neesiana. By comparing the composition of the two species, it was found that they could be distinguished on the basis of their anastreptene content. Calypogeia integristipula contains 4-5 times more of it than Calypogeia neesiana.

Acknowledgements: This work was partly supported by a grant National Science Centre no. 2017/27/B/NZ8/01091. We thank the Directors of the Tatra National Parks, Bieszczady National Parks and Pieniny National Parks for their support provided during the fieldwork and sampling of plant material for research.The work was supported by grant no. POWR.03.02.00-00-I020/17 co-financed by the European Union through the European Social Fund under the Operational Program Knowledge Education Development.

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COMPOSITION OF VOLATILE COMPOUNDS IN CALYPOGEIA INTEGRISTIPULA (CALYPOGEIACEAE, MARCHANTIOPHYTA)

M. Guzowska1, R. Wawrzyniak1 , W. Wasiak1, B. Jasiewicz1, P. Gonera2, A. Bączkiewicz2, K. Buczkowska2

1Faculty of Chemistry, Adam Mickiewicz University in Poznań, Umultowska 89b, 61-614 Poznań, Poland, e-mail: [email protected]

2Faculty of Biology, Adam Mickiewicz University in Poznań, Umultowska 89, 61-614 Poznań, Poland

Keywords: liverworts, Calypogeia integristipula, HS-SPME, gas chromatography, mass spectrometry

ABSTRACT Calypogeia is a genus of liverworts in the family Calypogeiaceae. It contains the following species of the genus occurring in Europe: Calypogeia azurea, Calypogeia fissa, Calypogeia muelleriana, Calypogeia integritipula [1]. The subject of the research was Calypogeia integristipula. Samples of the liverwort Calypogeia integristipula for chemotaxonomic studies were collected from different geographical areas in Poland: Tatras Mts, Pieniny, Beskid Sądecki,The plant material for analysis came from Tatry, Pieniny, Beskid Sądecki and Bieszczady Mts., from Wielkopolska and Lubusz provinces and from north-eastern Poland. In general, 23 samples determined as C. integristipula based on morphological characters and oil bodies were examined. The samples were determined genetically based on DNA sequences. The volatile compounds from biological materials were extracted by headspace solid-phase microextraction (HS-SPME) technique. The samples were then analyzed on the GC/MS. 88 compounds were detected in total of which 38 compounds were identified. The remaining compounds are described using the MS fragmentation spectrum. In terms of quantity among the identified volatile compounds, compounds belonging to the group of sesquiterpenes (up to 61.53%) and sesquiterpenoids (up to 10.87%) prevail. A smaller amount are compounds belonging to aliphatic compounds (up to 1.58%) and aromatic compounds (up to 4.44%). Unfortunately, monoterpenes and monoterpenoids weren’t identified in the samples tested . INTRODUCTION A serious problem in the taxonomy of liverworts, including Calypogeia species, which is considered one of the most difficult genera among liverworts, is the limited number of diagnostically good quality traits due to the low morphological complexity of the gametophyte, as well as the high phenotypic plasticity [1,2]. A unique feature of most liverworts is the presence of oil bodies. Their shape, distribution in leaf and sub leaf cells, and color are important taxonomic features of the genus Calypogeia. Oil bodies are intracellular organelles surrounded by a single unit membrane, in which a wide range of terpenoids and other aromatic compounds are synthesized and accumulated [3-6]. Analysis of the

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composition of secondary metabolites that fall under the scope of chemotaxonomy can be one of the methods to help identify taxonomically difficult species [7]. The genus Calypogeia (Raddi) comprise about 90 species and represents leafy liverworts from the suborder Jungermanniineae. C. intergristipula is one of 9 species of the genus Calypogeia occurring in Europe. Figure 1 shows the occurrence of liverworts of the genus Calypogeia, which are widespread across almost the entire globe. So far, 65 species belonging to this genus have been identified, seven of which can be found in Poland [8,9] These are Calypogeia azurea, Calypogeia fissa, Calypogeia neesiana, Calypogeia sphagnicola, Calypogeia suecica, Calypogeia muelleriana, and Calypogeia integristipula, the subject of this paper. They are most easily found in mountainous areas, and in lake-rich areas of Pomerania [10].

Fig.1. Locations of liverworts [11].

C. integristipula is a small liverwort that grows in mats or mixed with other bryophytes. Its shoots are at most 3 mm wide and 30 mm long. The leaves are oblong-oval, in two opposite ranks, and usually pale green and rather dull and opaque. The smaller under leaves which occur as a third row on the underside of the stem are almost round (Fig 2) [12]. Calypogeia integristipula morphologically strongly resembles another species close to it - Calypogeia neesiana. Therefore, for a very long time these species were mistakenly considered as one, and only a few years ago their distinctness was recognized [13].

Fig.2. Calypogeia integristipula [12]

ANALYTICAL PART

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Plant material Samples of the liverwort Calypogeia integristipula for chemotaxonomic analysis were collected from different geographical areas in Poland. Table 1 shows the habitats from which the material for the study was collected.

Table 1. List of studied samples of C. intergristipula Date of Place on Symbol Collection place sample the map collection CI-107 S Poland, Tatry Mts, Bialka Valley 2006 A CI-110 S Poland, Tatry Mts, Bialka Valley 2006 A CI-217 S Poland, Tatry Mts, The valley of Sucha Woda and Panszczycki Potok 2015 A CI-219 S Poland, Tatry Mts, The valley of Sucha Woda and Panszczycki Potok 2016 A S Poland, Tatry Mts - Dolina Kościeliska, Wąwóz Kraków, Smreczyński Staw, CI-306 2006 A Jaferowy Żleb CI-805 S Poland, Tatry Mts - Sichlanski Stream 2006 A CI-1002 S Poland, Tatry Mts - Capovski Forest 2006 A CI-1006 S Poland, Tatry Mts - Capovski Forest 2006 A CI-1301 S Poland, Tatry Mts - Zolta Turnia, Gąsienicowa Valley 2007 A CI-1401 S Poland, Tatry Mts - Psia Trawka 2010 A CI-1405 S Poland, Tatry Mts - Psia Trawka 2017 A CI-603 S Poland, Beskid Sądecki 2008 B CI-3102 S Poland, Beskid Sądecki - Right tributary of the Kotlovy Stream 2015 B CI-2702 SE Poland, Bieszczady Mts - Rozsypaniec Wołosacki 2008 C CI-2704 SE Poland, Bieszczady Mts - Rozsypaniec Wołosacki 2008 C CI-2801 W Poland, Lubuskie Province - Forestry of Nabłoto, section 84i 2010 D CI-2103 W Poland, Greater Poland Voivodship - Antonin 2009 E NW Poland, Western Pomerania - the lake between the lakes of Czarny and CI-1702 2008 F Kulkówko CI-1501 NW Poland, Western Pomerania - Czapliniec reserve, the shore of Książe Lake 2008 G CI-1604 NW Poland, Western Pomerania - Staniszewskie Mudota Reserve (Kartuzy) 2008 G CI-1605 NW Poland, Western Pomerania - Staniszewskie Mudota Reserve (Kartuzy) 2008 G CI-3301 NW Poland, Western Pomerania - Lake Orle, tributary from the side of the road 2016 G CI-2001* NW Poland, Western Pomerania - Lubygost Lake (Kartuzy) 2008 G

The location of the sites is shown graphically in Figure 3.

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Fig. 3. Location of researched populations of Calypogeia integristipula (A - Tatras; B - Beskid Sądecki Mts.; C – Bieszczady Mts.; D - Lubuskie Province; E - Wielkopolskie Province; F,G - West Pomerania)

All collected samples grew on moist decorticated logs. In general, 23 samples determined as C. integristipula based on morphological characters and oil bodies were examined. Figure 4 shows the prepared samples ready for analysis.

Fig. 4. Vials with sample weights

HS-SPME analysis The volatile compounds from biological materials were extracted by headspace solid-phase microextraction (HS-SPME) technique. The fused silica fibers coated with divinylbenzene/ carboxen/polydimethylsiloxane DVB/CAR/PDMS (2cm length, 50/30µm film thickness) were used. Before analysis the fibers were conditioned 1 h at 270oC, according to the

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supplier`s prescriptions. Ten mg of frozen plant material was placed in a 1.7 ml vial hermetically closed with a Teflon/silicone septum, and heated at 50oC. The extraction of the compounds was followed at 50°C for 60 minutes. Desorption of the analytes from fiber was carried out in the injection port of the gas chromatograph at 250oC for 10 min. Sorption and desorption operations were performed using the autosampler TriPlus RSH (Thermo Scientific, USA).

GC/MS Analysis GC/MS analyses using a silphenylene phase were carried out on a Trace 1310 (Thermo Scientific, USA) equipped with a Quadrex 007-5MS column (30m, 0.25mm, 0.25 μm). The mass detector ISQ QD (Thermo Scientific, USA) was operated at 70 eV in the EI mode over the m/z range 30–550. This was used as the carrier gas at a flow rate of 1.0 ml/min. The oven temp. was programmed from 60 to 230oC at 4oC/min and then held isothermal at 230oC for 40 min; injector temp., 250oC; transfer line, 250oC; injection mode, splitless with dedicated liner for SPME technique. The identification of components was confirmed by comparison of the mass spectral fragmentation patterns with those stored in the MS data base (NIST 2011, NIST Chemistry WebBook, Adams 4 Library, MassFinder 4 and Pherobase)[12, 13] and those reported in the literature. In addition, retention indices on the non-polar columns, determined relative to a homologous series of n-alkanes (C8–C26), were compared with published index data. Quantitative data of the components were obtained by integrating the TIC chromatogram and calculating the relative percentage of the peak areas. Each sample of cryptic species was analysed three times.

RESULTS AND DISCUSSION

The results of the studies on the composition of volatile compounds present in the Calypogeia integristipula hepatic cells are presented in Table 2 and chromatogram (Figures 6-7). 93 volatile compounds were detected in the examined plant material, of which 38 compounds were identified. The remaining compounds are described using the MS fragmentation spectrum. After analysis of the collected results, anastraptene (20) (17.03-39.39%) and bicyclogermacrene (41) (6.35-30.09%) were found to be the dominant components in all samples. Variation in the amount of bicyclogermacrene was observed depending on the harvest date of the material. In general, older samples contain lower amounts of this compound. The analyzed biological material also contained other sesquiterpenes commonly found in liverworts such as β-elemene (15), bicycloelemene (16), aromadendrene (31), selin- 5,11-diene (32), ledene (39), γ-dehydro-ar-himachalene (54), and germacrene D (38). The mentioned compounds are already present in much smaller amounts, i.e., at the level of only a few percent. Other compounds from the sesquiterpenoid group were already present in amounts below one percent. The analyzed samples of liverworts of Calypogeia integristipula species were also characterized by the presence of sesquiterpenoid compounds with hydroxyl groups such as: maaliol (56) spathulenol (58), ledol (60), globulol (61), isospathulenol (68), ent-germacra- 4(15),5,10(14)-trien-1β-ol (70) and α-isonootcatol (83). From the sesquiterpenioid group, other oxygen derivatives were also found: 6S,10R-dimethyl-bicyclo[4.4.0]decane-1-en3-one (55), leden oxid-(II) (66), zizanal (78). It is observed that the older samples i.e. collected between 2006 and 2010 contain more spathulenol (58)(3.11-6.42%) than samples collected between 2015 and 2017 (0.43-3.11%). This is because this compound is a product of oxidation of anastreptene.

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In Calypogeia integristipula samples, slight amounts of 1,4-dimethyl-7- (1-methylethyl) azulene were identified (82), which is a derivative of a compound characteristic of the species Calypogeia azurea. In the case of a few samples, deviations from the above-described relationships were noted. Thus, sample CI-107 collected in the Tatra mountains in 2006 - contains 1.13% spathulenol (58), while other samples stored longer contained this compound at much higher levels. It was noticed that the sample labeled CI-110 collected in the Tatra Mountains differs from other samples in its high content of maaliol (56) at the level of 17.76%.

Fig. 5. Structure of characteristic compounds identified in studied plant material: Sesquiterpenoids identifying the liverworts of Calypogeia integristipula (1) - maaliol (56), (2) - spathulenol (58), (3) - ledol (60), (4) - globulol (61), (5) - isospathulenol (68), 6 - ent- germacra-4(15),5,10(14)-trien-1β-ol (70).

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RT: 0.00 - 45.18 20.94 NL: 100 4.31E9 95 TIC MS calypogeia 90 _integristip 25.50 ula_CI1002 85 _1 80 75 70 65

60 55 50 45 23.52

RelativeAbundance 40 35 28.25 30 25 27.70

20 15 28.45 19.72 28.75 10 30.64 34.16 5 23.37 31.99 35.74 1.89 2.51 5.22 6.63 8.39 11.03 13.23 17.04 36.29 38.81 41.43 44.03 0 0 5 10 15 20 25 30 35 40 45 Time (min)

Fig.6. Chromatogram of volatile compounds included in Calypogeia integristipula collected in Tatras Mts.(sample CI-1002).

RT: 0.00 - 45.39

20.90 NL: 100 4.21E9 95 TIC MS calypogeia 90 _integristip ula_CI1604 85 _1 80 25.45 75 70 65 60 55 50 45

40 RelativeAbundance 35 23.49 27.69 30 28.22 25 17.05 26.45 20 10.50 21.74 28.43 15

10 19.70 30.62 34.14 5 11.01 1.87 13.22 31.97 34.41 35.72 2.49 5.22 6.01 8.38 16.70 17.88 38.79 39.67 43.62 0 0 5 10 15 20 25 30 35 40 45 Time (min)

Fig.7. Chromatogram of volatile compounds included in Calypogeia integristipula collected in Western Pomerania (sample CI-1604).

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Table 2. Volatile compounds detected in the analyzed samples of the Calypogeia integristipula collected in Tatra Mountains.

CI CI CI CI CI No. RI Compounds CI 805 107 110 217 219 306 1 486 2-propanol 0,63 0,36 0,32 0,12 0,66 0,45 2 706 pentanal 0,04 0,24 0,16 0,11 0,17 0,14 3 782 hexanal 0,03 0,40 0,01 0,10 0,06 0,02 4 867 1-hexanol 0,23 1,05 0,37 0,67 0,21 0,56 5 987 benzaldehyde 0,01 0,26 0,18 0,09 0,06 0,14 6 1030 2-ethyl-1-hexanol 0,01 0,01 0,03 0,02 0,10 0,03 7 1033 benzyl alcohol 0,02 0,06 0,05 0,04 0,27 1,80 8 1043 benzeneacetaldehyde 0,03 0,14 0,02 0,03 0,10 0,07 9 1111 n-nonanal 0,01 0,03 0,01 0,02 0,03 0,01 10 1116 benzeneethanol 0,08 0,45 0,04 0,05 0,09 0,65 11 1167 152[M+](3) 91(100) 41(98) 0,01 0,05 0,04 0,03 0,03 0,01 12 1204 decanal 0,02 0,01 0,02 0,02 0,03 0,02 13 1224 2-phenoxyethanol 0,08 0,09 0,06 0,09 0,22 0,12 14 1279 184[M+](4) 57(100) 43(82) 0,02 0,01 0,03 0,04 0,05 0,03 15 1320 δ-elemene 0,06 0,10 0,10 0,19 0,10 0,14 16 1341 bicycloelemene 1,15 1,83 1,96 3,62 1,72 2,24 17 1343 204[M+](5) 121(100) 93(95) 0,01 0,01 0,01 0,01 0,26 0,10 18 1345 200[M+](38) 159(100) 117(87) 0,03 0,02 0,88 0,02 0,48 0,28 19 1350 202[M+](15) 81(100) 96(74) 0,19 0,17 0,15 0,24 0,14 0,15 20 1370 anastreptene 33,73 26,54 17,03 25,56 25,05 22,81 21 1384 202[M+](29) 105(100) 91(89) 0,04 0,08 0,05 0,07 0,09 0,28 22 1394 β-elemene 0,58 1,07 2,35 1,02 1,34 1,47 23 1398 204[M+](29) 93(100) 107(89) 0,11 0,08 0,18 0,18 0,16 0,20 24 1408 α-gurjunene 0,32 0,29 0,03 0,01 0,27 0,43 25 1423 204[M+](52) 161(100) 105(96) 0,45 0,55 0,38 0,46 0,35 0,65 26 1427 202[M+](23) 91(100) 105(96) 0,11 0,43 0,46 0,08 0,08 0,08 27 1430 9-aristolene 0,31 0,26 0,08 0,28 0,63 0,34 28 1432 204[M+](7) 107(100) 91(42) 0,41 0,81 1,17 0,27 0,18 0,85 29 1435 γ-maaliene 0,54 0,83 0,33 0,73 0,69 0,70 30 1442 α-panasinsene 0,65 0,33 0,66 0,62 0,48 0,94 31 1445 aromandendrene 5,78 5,10 5,21 10,05 8,05 9,19 32 1447 selina-5,11-diene 1,86 0,79 1,04 1,03 0,84 0,41 33 1452 202[M+](18) 105(100) 159(72) 1,10 1,22 1,56 2,61 1,35 1,48 34 1453 202[M+](25) 159(100) 131(84) 0,24 0,73 0,29 0,72 0,87 0,22 35 1457 204[M+](15) 91(100) 105(88) 0,47 0,55 1,32 0,37 0,39 0,67 36 1468 204[M+](24) 128(100) 143(97) 0,19 0,13 0,06 0,03 0,05 0,17 37 1472 γ-muurolene 0,26 0,35 0,26 0,25 0,27 0,67 38 1475 germacrene D 0,93 1,11 1,40 1,25 0,90 1,17 39 1476 ledene 2,17 2,98 3,49 3,52 3,35 4,97 40 1476 204[M+](32) 105(100) 93(93) 0,05 0,15 0,09 0,01 0,13 0,17 41 1479 bicyclogermacrene 14,05 13,82 23,79 29,38 16,99 21,50 42 1505 204[M+](17) 91(100) 105(87) 0,42 0,42 0,42 1,04 0,30 0,84 43 1509 204[M+](11) 91(100) 105(82) 0,28 0,10 0,10 0,10 0,13 0,08 44 1514 202[M+](35) 131(100) 145(53) 0,06 0,10 0,10 0,10 0,28 0,27 45 1518 204[M+](25) 105(100) 161(98) 0,30 0,22 0,20 1,92 0,35 0,12 46 1521 204[M+](8) 93(100) 91(79) 0,13 0,12 1,11 0,18 0,16 0,02 47 1525 200[M+](52) 185(100) 143(92) 0,09 0,03 0,03 0,01 0,07 0,03 48 1529 200[M+](38) 157(100) 142(57) 1,04 1,28 2,64 0,86 2,93 0,53 49 1531 200[M+](78) 129(100) 157(92) 0,07 0,01 0,73 0,03 0,30 0,03 50 1535 200[M+](23) 171(100) 129(86) 0,02 0,06 0,01 0,01 0,11 0,07 51 1545 200[M+](93) 129(100) 157(98) 0,05 0,03 0,04 0,01 0,03 0,02 52 1549 204[M+](13) 91(100) 157(98) 0,33 0,76 0,04 0,03 0,05 0,04 53 1552 204[M+](87) 189(100) 133(92) 0,18 0,28 0,13 0,42 1,59 0,93 54 1555 γ-dehydro-ar-himachalene 5,98 0,26 2,57 0,53 2,82 1,58 55 1559 6S,10R-dimethyl-bicyclo[4.4.0]decan-1-en3-on 0,12 0,02 0,12 0,04 0,55 0,34 56 1564 maaliol 7,92 17,76 1,98 0,02 1,83 0,65 57 1565 220[M+](12) 43(100) 93(85) 0,12 0,43 0,20 0,01 0,01 0,08 58 1568 spathulenol 1,13 5,36 0,43 0,78 5,88 6,30 59 1579 200[M+](58) 185(100) 143(71) 0,24 0,59 0,05 0,48 1,77 0,85 60 1582 ledol 0,01 0,01 0,74 0,36 0,71 0,01 61 1587 glogulol 1,29 1,85 1,07 0,85 1,36 2,45 62 1588 204[M+](2) 198(100) 183(83) 0,60 0,22 0,50 0,21 0,18 0,29 63 1589 220[M+](3) 145(100) 143(89) 0,43 0,80 1,34 0,47 0,97 0,88 64 1595 220[M+](11) 94(100) 43(77) 0,83 0,10 0,80 0,03 0,20 0,19 65 1599 220[M+](1) 187(100) 91(89) 0,31 0,17 0,09 0,21 0,26 0,20 66 1631 ledene oxid-(II) 1,89 1,69 0,27 0,19 0,54 1,86 67 1633 200[M+](22) 117(100) 95(57) 0,03 0,07 0,30 0,21 0,28 0,13 68 1639 isospathulenol 2,39 0,10 3,96 0,88 1,51 1,19 69 1674 220[M+](32) 105(100) 91(98) 0,36 0,12 0,06 0,05 0,10 0,14 70 1689 ent-germacra-4(15),5,10(14)-trien-1β-ol 0,26 0,11 0,75 1,88 1,93 0,65

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71 1695 220[M+](18) 159(100) 91(97) 0,20 0,28 2,79 0,09 0,03 0,09 72 1708 220[M+](9) 107(100) 91(86) 0,08 0,09 0,07 0,05 0,12 0,18 73 1720 220[M+](8) 109(100) 43(84) 0,46 0,12 0,42 0,53 0,53 0,15 74 1722 218[M+](17) 120(100) 105(82) 0,09 0,12 0,02 0,03 0,08 0,15 75 1725 236[M+](49) 43(100) 150(92) 0,02 0,06 0,06 0,01 0,12 0,24 76 1729 220[M+](11) 159(100) 91(98) 0,18 0,22 1,48 0,48 0,47 0,44 77 1751 220[M+](4) 157(100) 200(96) 0,43 0,06 0,10 0,19 0,12 0,31 78 1690 zizanal 0,23 0,05 0,41 0,01 0,18 0,04 79 1761 218[M+](18) 145(100) 173(74) 0,24 0,11 0,18 0,02 0,14 0,22 80 1766 218[M+](76) 41(100) 55(97) 0,04 0,04 0,24 0,05 0,17 0,03 81 1782 218[M+](7) 143(100) 198(83) 0,12 0,08 0,09 0,02 0,08 0,03 82 1801 1,4-dimethl-7-(1-methlethyl)-azulene 0,26 0,23 1,15 0,44 1,25 0,17 83 1815 α-isonootkatol 0,35 0,04 0,78 0,13 0,13 0,05 84 1828 220[M+](13) 189(100) 91(27) 0,11 0,11 0,30 0,05 0,41 0,03 85 1857 218[M+](1) 196(100) 165(88) 0,01 0,08 0,03 0,11 0,36 0,01 86 1868 218[M+](4) 196(100) 165(81) 0,01 0,01 0,18 0,01 0,02 0,02 87 1880 223[M+](7) 149(100) 57(29) 0,02 0,00 0,01 0,02 0,03 0,03 88 1984 223[M+](4) 149(100) 44(11) 0,07 0,02 0,02 0,02 0,04 0,01 % Identification 85,21 85,97 73,48 85,13 80,79 86,14 Aliphatic 0,95 2,09 0,90 1,04 1,23 1,21 Aromatics 0,22 1,00 0,35 0,30 0,74 2,78 Monoterpene hydrocarbons 0,00 0,00 0,00 0,00 0,00 0,00 Oxygen-containing monoterpenes 0,00 0,00 0,00 0,00 0,00 0,00 Sesquiterpene hydrocarbons 68,45 55,89 61,72 78,65 64,20 68,61 Oxygen-containing sesquiterpenes 15,59 26,99 10,51 5,14 14,62 13,54 Total 96,81 98,28 94,78 98,18 98,77 99,30

Table 4. Volatile compounds detected in the analyzed samples of the Calypogeia intergristipula collected in Tatra Mountains

CI CI CI CI CI No. RI Compounds 1002 1006 1301 1401 1405 1 486 2-propanol 0,32 0,26 0,38 0,32 0,11 2 706 pentanal 0,26 0,16 0,19 0,37 0,03 3 782 hexanal 0,07 0,07 0,13 0,17 0,37 4 867 1-hexanol 0,13 0,17 0,45 0,35 1,25 5 987 benzaldehyde 0,09 0,20 0,81 0,76 0,05 6 1030 2-ethyl-1-hexanol 0,04 0,20 0,04 0,25 0,02 7 1033 benzyl alcohol 0,04 0,07 6,34 0,04 0,42 8 1043 benzeneacetaldehyde 0,24 0,12 0,51 0,59 0,03 9 1111 n-nonanal 0,05 0,07 0,10 0,05 0,01 10 1116 benzeneethanol 0,30 1,18 1,28 1,25 0,07 11 1167 152[M+](3) 91(100) 41(98) 0,04 0,05 0,09 0,09 0,01 12 1204 decanal 0,03 0,02 0,06 0,03 0,02 13 1224 2-phenoxyethanol 0,61 1,79 1,86 5,17 0,03 14 1279 184[M+](4) 57(100) 43(82) 0,07 0,04 0,03 0,04 0,05 15 1320 δ-elemene 0,12 0,09 0,07 0,11 0,19 16 1341 bicycloelemene 1,99 1,43 0,95 2,41 3,47 17 1343 204[M+](5) 121(100) 93(95) 0,01 0,10 0,30 0,11 0,10 18 1345 200[M+](38) 159(100) 117(87) 0,42 0,57 1,48 0,03 0,04 19 1350 202[M+](15) 81(100) 96(74) 0,16 0,14 0,12 0,12 0,18 20 1370 anastreptene 25,48 27,24 21,41 28,21 25,68 21 1384 202[M+](29) 105(100) 91(89) 0,04 0,11 0,14 0,14 0,03 22 1394 β-elemene 0,28 0,25 1,46 2,01 0,70 23 1398 204[M+](29) 93(100) 107(89) 0,20 0,26 0,33 0,07 0,21 24 1408 α-gurjunene 0,02 0,34 0,28 0,33 0,18 25 1423 204[M+](52) 161(100) 105(96) 0,34 0,31 0,27 0,35 0,44 26 1427 202[M+](23) 91(100) 105(96) 0,08 0,08 0,08 0,09 0,09 27 1430 9-aristolene 0,19 0,10 0,92 1,04 0,29 28 1432 204[M+](7) 107(100) 91(42) 0,69 0,23 0,30 0,31 0,25 29 1435 γ-maaliene 0,56 0,41 0,29 0,50 0,89 30 1442 α-panasinsene 0,56 0,31 0,18 0,35 0,76 31 1445 aromandendrene 8,61 5,49 3,64 6,13 11,10 32 1447 selina-5,11-diene 0,89 0,44 0,39 0,65 1,16 33 1452 202[M+](18) 105(100) 159(72) 1,58 1,33 1,10 1,29 1,75 34 1453 202[M+](25) 159(100) 131(84) 0,72 0,62 0,73 1,59 0,52 35 1457 204[M+](15) 91(100) 105(88) 0,36 0,29 0,30 0,26 1,08 36 1468 204[M+](24) 128(100) 143(97) 0,04 0,05 0,21 0,45 0,34 37 1472 γ-muurolene 0,20 0,08 0,10 0,09 0,35 38 1475 germacrene D 1,07 1,07 0,66 1,23 1,18

10 47th International Conference of SSCHE - online conference May 18 - 19, 2021, Bratislava, Slovakia

39 1476 ledene 3,08 1,35 1,35 2,59 4,20 40 1476 204[M+](32) 105(100) 93(93) 0,05 0,07 0,13 0,25 0,11 41 1479 bicyclogermacrene 20,73 15,26 8,63 14,01 30,09 42 1505 204[M+](17) 91(100) 105(87) 0,84 0,53 0,10 0,29 1,08 43 1509 204[M+](11) 91(100) 105(82) 0,01 0,03 0,15 0,08 0,03 44 1514 202[M+](35) 131(100) 145(53) 0,01 0,02 0,36 0,09 0,06 45 1518 204[M+](25) 105(100) 161(98) 0,18 0,05 0,28 0,38 1,43 46 1521 204[M+](8) 93(100) 91(79) 0,13 0,21 0,11 0,12 0,24 47 1525 200[M+](52) 185(100) 143(92) 0,01 0,10 0,26 0,04 0,01 48 1529 200[M+](38) 157(100) 142(57) 4,10 5,91 9,28 1,72 0,90 49 1531 200[M+](78) 129(100) 157(92) 0,59 0,61 1,24 0,18 0,08 50 1535 200[M+](23) 171(100) 129(86) 0,04 0,06 0,15 0,06 0,05 51 1545 200[M+](93) 129(100) 157(98) 0,04 0,08 0,08 0,10 0,01 52 1549 204[M+](13) 91(100) 157(98) 0,05 0,16 0,09 0,07 0,03 53 1552 204[M+](87) 189(100) 133(92) 0,27 0,18 1,09 2,59 0,25 54 1555 γ-dehydro-ar-himachalene 3,90 5,59 8,89 1,54 0,56 55 1559 6S,10R-dimethyl-bicyclo[4.4.0]decan-1-en3-on 0,10 0,07 0,26 0,56 0,03 56 1564 maaliol 0,71 2,95 0,37 2,74 0,03 57 1565 220[M+](12) 43(100) 93(85) 0,04 0,02 0,05 0,03 0,01 58 1568 spathulenol 5,38 3,11 6,03 3,80 0,74 59 1579 200[M+](58) 185(100) 143(71) 2,15 0,55 1,47 0,94 0,40 60 1582 ledol 0,40 0,17 0,24 0,12 0,17 61 1587 glogulol 1,49 2,75 0,27 0,92 0,51 62 1588 204[M+](2) 198(100) 183(83) 0,15 0,26 1,07 0,11 0,15 63 1589 220[M+](3) 145(100) 143(89) 0,89 1,17 0,54 0,59 0,43 64 1595 220[M+](11) 94(100) 43(77) 0,17 0,07 0,15 0,06 0,06 65 1599 220[M+](1) 187(100) 91(89) 0,31 0,20 0,19 0,54 0,13 66 1631 ledene oxid-(II) 0,35 0,36 0,81 0,20 0,04 67 1633 200[M+](22) 117(100) 95(57) 0,19 0,18 0,31 0,09 0,01 68 1639 isospathulenol 1,17 0,86 0,59 1,32 1,46 69 1674 220[M+](32) 105(100) 91(98) 0,08 0,05 0,03 0,04 0,03 70 1689 ent-germacra-4(15),5,10(14)-trien-1β-ol 1,49 2,00 1,19 1,24 0,47 71 1695 220[M+](18) 159(100) 91(97) 0,09 0,03 0,30 0,07 0,43 72 1708 220[M+](9) 107(100) 91(86) 0,11 0,05 0,08 0,07 0,01 73 1720 220[M+](8) 109(100) 43(84) 0,33 0,02 0,16 0,39 0,06 74 1722 218[M+](17) 120(100) 105(82) 0,36 1,38 0,17 0,15 0,06 75 1725 236[M+](49) 43(100) 150(92) 0,20 0,01 0,05 0,12 0,01 76 1729 220[M+](11) 159(100) 91(98) 0,36 0,44 0,30 0,38 0,12 77 1751 220[M+](4) 157(100) 200(96) 0,20 0,13 0,07 0,09 0,16 78 1690 zizanal 0,20 0,04 0,05 0,14 0,01 79 1761 218[M+](18) 145(100) 173(74) 0,08 0,07 0,06 0,19 0,02 80 1766 218[M+](76) 41(100) 55(97) 0,05 0,04 0,04 0,08 0,03 81 1782 218[M+](7) 143(100) 198(83) 0,05 0,07 0,04 0,05 0,16 82 1801 1,4-dimethl-7-(1-methlethyl)-azulene 1,42 1,34 2,07 0,71 0,28 83 1815 α-isonootkatol 0,07 0,07 0,10 0,29 0,44 84 1828 220[M+](13) 189(100) 91(27) 0,06 0,07 0,28 0,47 0,02 85 1857 218[M+](1) 196(100) 165(88) 0,28 0,34 0,23 0,24 0,07 86 1868 218[M+](4) 196(100) 165(81) 0,02 0,01 0,02 0,01 0,02 87 1880 223[M+](7) 149(100) 57(29) 0,03 0,16 0,02 0,02 0,02 88 1984 223[M+](4) 149(100) 44(11) 0,02 0,05 0,05 0,09 0,01 % Identification 82,74 77,33 72,36 81,54 87,34 Aliphatic 0,87 0,93 1,29 1,51 1,79 Aromatics 1,28 3,36 10,80 7,81 0,60 Monoterpene hydrocarbons 0,00 0,00 0,00 0,00 0,00 Oxygen-containing monoterpenes 0,00 0,00 0,00 0,00 0,00 Sesquiterpene hydrocarbons 69,23 60,66 50,36 60,89 81,05 Oxygen-containing sesquiterpenes 11,36 12,38 9,91 11,33 3,90 Total 99,93 95,04 97,83 98,32 99,18

11 47th International Conference of SSCHE - online conference May 18 - 19, 2021, Bratislava, Slovakia

Table 4. Volatile compounds detected in the analyzed samples of the Calypogeia intergristipula collected in Bieszczady Mountains and Beskid Sądecki Mountains and Western Poland

CI CI CI CI CI CI No. RI Compounds 603 3102 2702 2704 2801 2103 1 486 2-propanol 0,23 0,41 0,44 0,09 0,11 0,44 2 706 pentanal 0,06 0,16 0,14 0,01 0,21 0,47 3 782 hexanal 0,20 0,23 0,12 0,03 0,36 1,83 4 867 1-hexanol 0,06 0,03 0,19 0,06 0,89 0,01 5 987 benzaldehyde 0,09 0,04 0,19 0,01 1,93 0,03 6 1030 2-ethyl-1-hexanol 0,06 0,01 0,04 0,04 0,06 0,03 7 1033 benzyl alcohol 0,61 0,45 2,96 0,10 4,20 3,24 8 1043 benzeneacetaldehyde 0,28 0,23 0,42 0,38 0,27 0,61 9 1111 n-nonanal 0,08 0,02 0,07 0,01 0,11 0,15 10 1116 benzeneethanol 0,08 0,07 0,80 0,01 0,14 0,05 11 1167 152[M+](3) 91(100) 41(98) 0,04 0,01 0,12 0,05 0,11 0,05 12 1204 decanal 0,04 0,01 0,09 0,04 0,08 0,06 13 1224 2-phenoxyethanol 2,15 0,79 1,30 1,09 6,63 2,43 14 1279 184[M+](4) 57(100) 43(82) 0,05 0,03 0,08 0,05 0,04 0,02 15 1320 δ-elemene 0,06 0,09 0,13 0,11 0,08 0,06 16 1341 bicycloelemene 1,10 1,66 1,98 1,89 0,78 0,58 17 1343 204[M+](5) 121(100) 93(95) 0,11 0,05 0,36 0,63 0,03 0,06 18 1345 200[M+](38) 159(100) 117(87) 1,10 0,01 0,75 0,45 0,91 0,20 19 1350 202[M+](15) 81(100) 96(74) 0,06 0,09 0,19 0,13 0,16 0,06 20 1370 anastreptene 22,19 22,55 21,53 24,13 25,42 39,39 21 1384 202[M+](29) 105(100) 91(89) 0,05 0,05 0,13 0,17 0,12 0,07 22 1394 β-elemene 0,23 0,09 1,46 1,86 1,33 0,65 23 1398 204[M+](29) 93(100) 107(89) 0,27 0,01 0,25 0,18 0,17 0,05 24 1408 α-gurjunene 0,16 0,21 0,17 0,04 0,25 0,07 25 1423 204[M+](52) 161(100) 105(96) 0,24 0,47 0,32 0,37 0,28 0,28 26 1427 202[M+](23) 91(100) 105(96) 0,05 0,05 0,09 0,80 0,09 0,17 27 1430 9-aristolene 0,18 0,23 0,57 0,21 0,67 0,20 28 1432 204[M+](7) 107(100) 91(42) 0,11 0,33 0,15 0,18 0,31 0,39 29 1435 γ-maaliene 0,44 0,53 0,65 0,65 0,20 0,22 30 1442 α-panasinsene 0,34 0,65 0,60 0,47 0,30 0,18 31 1445 aromandendrene 5,41 7,47 8,29 9,03 3,45 4,27 32 1447 selina-5,11-diene 0,44 0,67 0,91 0,96 1,43 0,34 33 1452 202[M+](18) 105(100) 159(72) 0,76 2,21 1,04 1,50 1,02 2,57 34 1453 202[M+](25) 159(100) 131(84) 0,33 0,49 0,84 1,96 0,13 1,56 35 1457 204[M+](15) 91(100) 105(88) 0,28 2,21 0,36 0,20 0,13 0,82 36 1468 204[M+](24) 128(100) 143(97) 0,13 0,09 0,17 0,57 0,20 0,31 37 1472 γ-muurolene 0,20 0,24 0,31 0,25 0,45 0,23 38 1475 germacrene D 1,05 1,17 1,15 1,25 0,69 1,00 39 1476 ledene 2,11 3,31 4,29 3,48 1,51 1,60 40 1476 204[M+](32) 105(100) 93(93) 0,07 0,06 0,17 0,15 0,10 0,27 41 1479 bicyclogermacrene 13,00 18,42 15,65 22,37 8,11 6,35 42 1505 204[M+](17) 91(100) 105(87) 0,24 0,37 0,43 0,56 0,04 0,33 43 1509 204[M+](11) 91(100) 105(82) 0,18 0,39 0,21 0,05 0,06 0,02 44 1514 202[M+](35) 131(100) 145(53) 0,02 0,73 0,14 0,17 0,35 0,44 45 1518 204[M+](25) 105(100) 161(98) 0,24 0,30 0,23 1,09 0,08 0,36 46 1521 204[M+](8) 93(100) 91(79) 0,08 0,36 0,13 0,13 0,14 0,06 47 1525 200[M+](52) 185(100) 143(92) 0,21 0,11 0,10 0,12 0,01 0,01 48 1529 200[M+](38) 157(100) 142(57) 11,67 0,61 4,07 3,97 7,58 1,66 49 1531 200[M+](78) 129(100) 157(92) 0,04 0,02 0,64 0,77 1,13 0,02 50 1535 200[M+](23) 171(100) 129(86) 0,09 0,04 0,08 0,10 0,16 0,21 51 1545 200[M+](93) 129(100) 157(98) 0,09 0,01 0,03 0,06 0,12 0,52 52 1549 204[M+](13) 91(100) 157(98) 0,11 0,01 0,04 0,01 0,01 0,07 53 1552 204[M+](87) 189(100) 133(92) 0,17 0,27 1,57 1,57 7,94 1,64 54 1555 γ-dehydro-ar-himachalene 6,65 0,46 3,97 3,87 0,36 0,43 55 1559 6S,10R-dimethyl-bicyclo[4.4.0]decan-1-en3-on 0,13 0,11 0,39 0,10 0,06 0,16 56 1564 maaliol 0,71 0,69 1,05 0,30 0,04 0,21 57 1565 220[M+](12) 43(100) 93(85) 0,08 0,15 0,06 0,06 0,12 0,17 58 1568 spathulenol 6,50 1,01 4,30 1,45 2,47 4,35 59 1579 200[M+](58) 185(100) 143(71) 0,94 0,40 2,94 2,24 4,27 1,46 60 1582 ledol 0,13 0,15 0,14 0,13 0,39 0,60 61 1587 glogulol 1,86 1,05 0,80 0,23 0,32 2,48 62 1588 204[M+](2) 198(100) 183(83) 0,06 0,11 0,12 0,01 0,03 0,08 63 1589 220[M+](3) 145(100) 143(89) 2,27 0,26 1,01 0,63 1,73 1,64 64 1595 220[M+](11) 94(100) 43(77) 0,20 0,11 0,08 0,12 0,39 0,33 65 1599 220[M+](1) 187(100) 91(89) 0,35 0,39 0,42 0,22 0,32 1,37 66 1631 ledene oxid-(II) 0,96 0,25 0,39 0,13 0,54 0,91 67 1633 200[M+](22) 117(100) 95(57) 0,33 0,09 0,13 0,27 0,12 0,17 68 1639 isospathulenol 1,41 15,87 1,01 0,27 1,73 2,49

12 47th International Conference of SSCHE - online conference May 18 - 19, 2021, Bratislava, Slovakia

69 1674 220[M+](32) 105(100) 91(98) 0,09 0,29 0,05 0,05 0,04 0,37 70 1689 ent-germacra-4(15),5,10(14)-trien-1β-ol 1,83 2,50 1,23 1,08 0,50 0,30 71 1695 220[M+](18) 159(100) 91(97) 0,13 0,46 0,16 0,06 0,07 0,34 72 1708 220[M+](9) 107(100) 91(86) 0,14 0,18 0,08 0,04 0,09 0,18 73 1720 220[M+](8) 109(100) 43(84) 0,51 0,15 0,13 0,37 0,33 0,18 74 1722 218[M+](17) 120(100) 105(82) 0,44 0,07 0,16 0,09 0,08 0,57 75 1725 236[M+](49) 43(100) 150(92) 0,04 0,12 0,05 0,05 0,22 0,66 76 1729 220[M+](11) 159(100) 91(98) 0,53 0,30 0,32 0,42 0,07 0,06 77 1751 220[M+](4) 157(100) 200(96) 0,20 0,06 0,08 0,90 0,18 0,19 78 1690 zizanal 0,06 0,06 0,11 0,14 0,09 0,09 79 1761 218[M+](18) 145(100) 173(74) 0,04 0,23 0,05 0,07 0,09 0,25 80 1766 218[M+](76) 41(100) 55(97) 0,05 0,05 0,05 0,03 0,05 0,10 81 1782 218[M+](7) 143(100) 198(83) 0,07 0,06 0,07 0,03 0,05 0,08 82 1801 1,4-dimethl-7-(1-methlethyl)-azulene 3,12 0,37 1,21 0,96 2,32 1,07 83 1815 α-isonootkatol 0,09 0,06 0,12 0,07 0,08 0,12 84 1828 220[M+](13) 189(100) 91(27) 0,15 0,10 0,30 0,25 0,08 0,09 85 1857 218[M+](1) 196(100) 165(88) 0,45 0,10 0,26 0,13 0,40 0,33 86 1868 218[M+](4) 196(100) 165(81) 0,02 0,00 0,02 0,01 0,02 0,03 87 1880 223[M+](7) 149(100) 57(29) 0,05 0,02 0,01 0,01 0,02 0,04 88 1984 223[M+](4) 149(100) 44(11) 0,17 0,01 0,02 0,01 0,06 0,09 % Identification 74,16 82,34 78,66 77,38 67,84 77,65 Aliphatic 0,69 0,86 1,00 0,24 1,74 2,93 Aromatics 3,21 1,58 5,67 1,59 13,17 6,36 Monoterpene hydrocarbons 0,00 0,00 0,00 0,00 0,00 0,00 Oxygen-containing monoterpenes 0,00 0,00 0,00 0,00 0,00 0,00 Sesquiterpene hydrocarbons 56,58 58,15 62,45 71,65 46,71 56,65 Oxygen-containing sesquiterpenes 13,68 21,75 9,54 3,90 6,22 11,71 Total 98,40 95,41 98,43 99,36 98,81 98,70

Table 5. Volatile compounds detected in the analyzed samples of the Calypogeia intergristipula collected in Pomerania and Western Pomerania

CI CI CI CI CI CI No. RI Compounds 1702 1501 1604 1605 3301 2001 1 486 2-propanol 0,60 0,10 0,66 0,36 4,10 0,39 2 706 pentanal 0,35 0,34 0,34 0,03 0,18 0,70 3 782 hexanal 0,07 0,29 0,23 0,38 0,28 3,47 4 867 1-hexanol 0,29 0,11 0,21 0,19 0,03 0,32 5 987 benzaldehyde 0,09 0,48 0,80 0,22 0,06 0,26 6 1030 2-ethyl-1-hexanol 0,06 0,05 0,11 0,08 0,08 0,15 7 1033 benzyl alcohol 0,92 5,18 7,46 5,17 0,01 4,11 8 1043 benzeneacetaldehyde 0,26 0,45 1,00 0,27 0,02 0,58 9 1111 n-nonanal 0,04 0,07 0,08 0,10 0,01 0,27 10 1116 benzeneethanol 0,64 0,78 0,60 0,17 0,02 0,49 11 1167 152[M+](3) 91(100) 41(98) 0,03 0,03 0,06 0,04 0,01 0,07 12 1204 decanal 0,02 0,04 0,08 0,03 0,01 0,11 13 1224 2-phenoxyethanol 0,19 1,60 5,10 1,82 0,06 2,40 14 1279 184[M+](4) 57(100) 43(82) 0,01 0,01 0,03 0,02 0,04 0,05 15 1320 δ-elemene 0,04 0,09 0,10 0,10 0,10 0,07 16 1341 bicycloelemene 1,13 1,18 1,45 0,94 1,98 0,62 17 1343 204[M+](5) 121(100) 93(95) 0,31 0,23 0,49 0,10 0,11 0,02 18 1345 200[M+](38) 159(100) 117(87) 1,10 1,40 0,73 0,88 0,23 0,31 19 1350 202[M+](15) 81(100) 96(74) 0,16 0,09 0,12 0,09 0,01 0,05 20 1370 anastreptene 24,84 18,34 18,67 19,89 33,39 35,71 21 1384 202[M+](29) 105(100) 91(89) 0,19 0,14 0,22 0,07 0,36 0,10 22 1394 β-elemene 2,50 0,25 2,18 1,06 1,26 0,39 23 1398 204[M+](29) 93(100) 107(89) 0,19 0,37 0,20 0,20 0,21 0,11 24 1408 α-gurjunene 0,23 0,46 0,02 0,02 0,30 0,25 25 1423 204[M+](52) 161(100) 105(96) 0,28 0,23 0,39 0,25 0,46 0,51 26 1427 202[M+](23) 91(100) 105(96) 0,05 0,06 0,25 0,07 0,34 0,13 27 1430 9-aristolene 0,21 0,14 0,22 0,52 0,35 0,69 28 1432 204[M+](7) 107(100) 91(42) 2,21 0,34 1,01 0,18 0,74 0,33 29 1435 γ-maaliene 0,42 0,29 0,39 0,36 0,61 0,22 30 1442 α-panasinsene 0,31 0,29 0,26 0,02 0,05 0,25 31 1445 aromandendrene 5,32 4,64 4,96 5,07 9,05 3,98 32 1447 selina-5,11-diene 1,53 0,47 0,45 0,55 0,96 0,04 33 1452 202[M+](18) 105(100) 159(72) 1,45 1,05 1,15 1,33 2,07 2,43 34 1453 202[M+](25) 159(100) 131(84) 0,06 0,54 0,17 0,83 1,60 1,20 35 1457 204[M+](15) 91(100) 105(88) 0,08 0,27 0,52 0,38 0,51 0,59

13 47th International Conference of SSCHE - online conference May 18 - 19, 2021, Bratislava, Slovakia

36 1468 204[M+](24) 128(100) 143(97) 0,38 0,06 0,26 0,21 0,12 0,23 37 1472 γ-muurolene 0,44 0,14 0,34 0,31 0,42 0,35 38 1475 germacrene D 1,40 1,08 1,08 0,80 1,19 0,43 39 1476 ledene 1,44 1,16 2,07 2,65 3,38 2,02 40 1476 204[M+](32) 105(100) 93(93) 0,28 0,48 0,27 0,29 0,17 0,14 41 1479 bicyclogermacrene 11,51 11,70 13,09 10,52 20,36 7,27 42 1505 204[M+](17) 91(100) 105(87) 0,25 0,04 0,11 0,18 0,62 0,41 43 1509 204[M+](11) 91(100) 105(82) 0,16 0,03 0,03 0,03 0,34 0,01 44 1514 202[M+](35) 131(100) 145(53) 0,10 0,19 0,06 0,11 0,08 0,01 45 1518 204[M+](25) 105(100) 161(98) 0,48 0,08 0,37 0,12 0,20 0,34 46 1521 204[M+](8) 93(100) 91(79) 0,08 0,14 0,10 0,16 0,15 0,02 47 1525 200[M+](52) 185(100) 143(92) 0,21 0,21 0,12 0,12 0,01 0,01 48 1529 200[M+](38) 157(100) 142(57) 7,05 11,81 5,34 6,02 1,00 1,44 49 1531 200[M+](78) 129(100) 157(92) 1,20 0,03 0,83 0,11 0,05 0,06 50 1535 200[M+](23) 171(100) 129(86) 0,07 0,04 0,04 0,06 0,12 0,06 51 1545 200[M+](93) 129(100) 157(98) 0,06 0,09 0,06 0,09 0,06 0,04 52 1549 204[M+](13) 91(100) 157(98) 0,08 0,09 0,06 0,06 0,73 0,03 53 1552 204[M+](87) 189(100) 133(92) 1,22 1,15 1,23 0,88 0,29 0,68 54 1555 γ-dehydro-ar-himachalene 7,24 8,88 5,86 6,42 0,03 1,16 55 1559 6S,10R-dimethyl-bicyclo[4.4.0]decan-1-en3-on 0,82 0,06 0,35 2,10 1,05 0,14 56 1564 maaliol 0,02 0,02 0,14 0,11 0,01 0,16 57 1565 220[M+](12) 43(100) 93(85) 0,15 0,33 0,02 0,12 2,24 5,63 58 1568 spathulenol 3,17 5,41 3,45 6,42 0,36 0,33 59 1579 200[M+](58) 185(100) 143(71) 4,80 3,30 3,33 3,80 0,86 1,24 60 1582 ledol 0,02 0,02 0,07 0,43 0,46 0,36 61 1587 glogulol 0,36 0,98 0,77 2,03 0,31 2,21 62 1588 204[M+](2) 198(100) 183(83) 0,37 0,43 0,23 0,12 0,09 0,07 63 1589 220[M+](3) 145(100) 143(89) 1,69 1,79 1,20 1,39 0,45 1,44 64 1595 220[M+](11) 94(100) 43(77) 0,11 0,31 0,17 0,01 0,09 0,14 65 1599 220[M+](1) 187(100) 91(89) 0,31 0,20 0,20 0,61 0,45 1,31 66 1631 ledene oxid-(II) 0,74 0,84 0,49 0,64 0,21 2,59 67 1633 200[M+](22) 117(100) 95(57) 0,22 0,26 0,19 0,39 0,12 0,62 68 1639 isospathulenol 0,93 0,79 0,94 1,61 1,11 0,74 69 1674 220[M+](32) 105(100) 91(98) 0,04 0,05 0,04 0,03 0,12 0,22 70 1689 ent-germacra-4(15),5,10(14)-trien-1β-ol 0,87 0,10 0,83 0,96 0,54 0,16 71 1695 220[M+](18) 159(100) 91(97) 0,17 0,01 0,07 0,10 0,17 0,25 72 1708 220[M+](9) 107(100) 91(86) 0,06 0,13 0,06 0,11 0,16 0,21 73 1720 220[M+](8) 109(100) 43(84) 0,09 0,57 0,11 0,16 0,24 0,68 74 1722 218[M+](17) 120(100) 105(82) 0,18 0,31 0,09 0,22 0,13 0,50 75 1725 236[M+](49) 43(100) 150(92) 0,04 0,03 0,10 0,11 0,03 0,02 76 1729 220[M+](11) 159(100) 91(98) 0,28 0,33 0,23 0,30 0,27 0,35 77 1751 220[M+](4) 157(100) 200(96) 0,16 0,18 0,10 0,09 0,13 0,01 78 1690 zizanal 0,07 0,01 0,06 0,06 0,05 0,08 79 1761 218[M+](18) 145(100) 173(74) 0,04 0,04 0,07 0,04 0,20 0,17 80 1766 218[M+](76) 41(100) 55(97) 0,03 0,05 0,06 0,11 0,02 0,18 81 1782 218[M+](7) 143(100) 198(83) 0,11 0,05 0,01 0,08 0,04 0,15 82 1801 1,4-dimethl-7-(1-methlethyl)-azulene 2,24 2,29 1,47 2,15 0,22 1,20 83 1815 α-isonootkatol 0,11 0,11 0,10 0,02 0,02 0,04 84 1828 220[M+](13) 189(100) 91(27) 0,25 0,07 0,25 0,18 0,05 0,08 85 1857 218[M+](1) 196(100) 165(88) 0,39 0,32 0,30 0,36 0,07 0,35 86 1868 218[M+](4) 196(100) 165(81) 0,02 0,02 0,01 0,03 0,01 0,07 87 1880 223[M+](7) 149(100) 57(29) 0,01 0,17 0,04 0,03 0,02 0,06 88 1984 223[M+](4) 149(100) 44(11) 0,02 0,04 0,06 0,07 0,01 0,07 % Identification 71,26 68,82 76,55 74,26 82,43 74,17 Aliphatic 1,41 0,96 1,63 1,14 4,68 5,30 Aromatics 2,10 8,49 14,96 7,65 0,17 7,84 Monoterpene hydrocarbons 0,00 0,00 0,00 0,00 0,00 0,00 Oxygen-containing monoterpenes 0,00 0,00 0,00 0,00 0,00 0,00 Sesquiterpene hydrocarbons 60,64 51,03 52,76 51,09 73,46 54,22 Oxygen-containing sesquiterpenes 7,11 8,34 7,20 14,38 4,12 6,81 Total 98,72 97,42 97,64 95,92 99,23 97,91

14 47th International Conference of SSCHE - online conference May 18 - 19, 2021, Bratislava, Slovakia

CONCLUSIONS The main aim of this study was to select characteristic compounds which enable to distinguish the liverwort species Calypogeia integristipula from the morphologically very similar species Calypogeia neesiana. Comparing the composition of the two species, it was found that they could be distinguished on the basis of their anastreptene content. Calypogeia integristipula contains 4-5 times more of it than Calypogeia neesiana. Another problem considered was the influence of harvest time on the chemical composition of the plant material. It turned out that older samples contain more spathulenol, which is formed as a result of anastraptene oxidation, and smaller amounts of bicyclogermacrene. This proves that in the case of the studied liverwort species, chemotaxonomic studies should be performed immediately after collecting the plant material.

Acknowledgements This work was partly supported by a grant National Science Centre no. 2017/27/B/NZ8/01091. We thank the Directors of the Tatra National Parks and Bieszczady National Parks for their support provided during the fieldwork and sampling of plant material for research.

The work was supported by grant no. POWR.03.02.00-00-I020/17 co-financed by the European Union through the European Social Fund under the Operational Program Knowledge Education Development.

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15 47th International Conference of SSCHE - online conference May 18 - 19, 2021, Bratislava, Slovakia

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