## 3 Discussion  
Out of the many _Iris_ species that are known today, around 30 species have been reported for their usage in traditional medicine. Three of them, namely _I. pallida_ (Dalmatian iris or sweet iris), _I. germanica_ (German iris or blue German bearded iris), and _I. Florentina_ (Florentine iris or white German bearded iris), are extensively used by various industries, including the pharmacy, perfumery, cosmetic, and food industries [34; 35], although other bearded irises could most likely be processed for the same purpose [36]. Solely in Italy, about 1000 tons of fresh iris rhizomes is used annually to produce iris EO (orris oil) [34]. According to the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) assessment, Orris Root Extract (FEMA 2830) and Orris Concrete Liquid Oil (FEMA 2829), which are obtained from the same three species, have an annual usage greater than or up to 1000 kg [37]. The abovementioned species belong to the group of bearded irises, which have multicellular caterpillar-like hairs on the external petals of their flowers and are popular ornamental plants worldwide [25; 35; 38], as well as in Croatia, where the iris is not only a beloved garden flower but is also the Croatian national flower [39]. The main goal of the present study was to analyze the VOCs profiles obtained from rhizomes of three endemic bearded taxa from Croatia, _I. adriatica, I. illyrica_, and _I. pseudopallida_. A careful selection of extraction methods is key to target a particular class of VOCs. Degradation or polymerization of certain compounds and/or artifact formation are known to occur during conventional methods such as HD [40]. To gain a better insight into the VOCs that could be obtained from the studied species, their GC-MS profiles were compared after isolation by three different methods, two based on extraction of volatiles by HS-SPME using bipolar fibers comprising either two (PDMS/DVB) or three materials (DVB/CAR/PDMS), and the third one being the conventionally used HD. HD and steam distillation are, in general, the primary methods of choice for EO isolation [40]. As such, they have also been the most utilized methods in analyses of _Iris_ rhizomes' EO constituents [21; 22; 23; 41; 42; 43; 44]. Figure 6: Biplot obtained by principal component analysis of EOs composition of the six investigated _Iris_ samples, based on their major components (average content \(\geq\)2.0% in at least one sample), detected using HD; for compound abbreviation, cf. Supplementary Table S1. In the present study, _Iris_ EOs obtained by HD contained several long-chain fatty acids that were detected in greater abundance. As was expected, HD isolated more fatty acids in comparison to HS-SPME. The latter method extracted only short- and medium-chain fatty acids, such as acetic, caproic, and caprylic, as well as methyl and ethyl esters of caprylic acid. All EOs analyzed in the present study contained tetradecanoic (myristic) acid as (one of) the major constituent(s), while palmitic and linoleic acids were the major constituents found exclusively in _I. adriatica_ EO. The latter taxon contained the least amount of myristic acid (4.20%). Myristic acid was especially high in _I. pseudopallida_ B, _I. pseudopallida_ T, and _I. illvica_ V and Z EO (31.92-97.01%). Myristic acid, followed by other long-chain fatty acids (e.g., lauric, capric, and palmitic acid) and/or their esters (e.g., palmitic, octadecanoic, or elaidic acid methyl ester), was found as the major constituent of several Syrian _Iris_ species [22]. Lauric (1.18-3.90%) and capric acids (0.34-2.82%) were also detected in all studied samples, except for the irone-rich _I. pseudopallida_ D, a result that is in accordance with the results obtained for the dried rhizomes of _I. florentina_ EO [41]. In fact, the relative amounts of irones, which were found only in _I. pseudopallida_ D and _I. pseudopallida_ B, were inversely proportional to the amounts of myristic acid. Similarly, [PERSON] et al.