131 Diagnostic micromorphological features of leaf surface of selected species of the genus Artemisia L. (Asteraceae) Abstract The leaves of different species of the genus Artemisia show considerable inner and external structural diversity. This paper presents a comparative study of the micromorphological features of leaf surface for seven species from different regions of Ukraine: A. absinthium L., A. annua L., A. dracunculus L., A. marschalliana Spreng., A. vulgaris L., A. argyi H.Lév. & Vaniot, and A. verlotiorum Lamotte. The vegetative organs of these plants have potentially significant value as medicinal raw materials. This study was carried out using both light and scanning electron microscopy. The main diagnostic characteristics for each species – in terms of form and structure of epidermal cells, stomata, and trichomes (glandular and non-glandular) – were described and illustrated. Using combinations of selected qualitative micromorphological characteristics of the investigated species of Artemisia, it is possible to detect the species identity of these raw materials. Iz vleček Listi različnih vrst iz rodu Artemisia so glede na notranjo in zunanjo zgradbo precej raznoliki. V članku je predstavljena primerjalna raziskava mikromorfoloških značilnosti listnih površin pri sedmih vrstah, ki rastejo v različnih regijah v Ukrajini: A. absinthium L., A. annua L., A. dracunculus L., A. marschalliana Spreng., A. vulgaris L., A. argyi H.Lév. & Vaniot in A. verlotiorum Lamotte. Vegetativni organi teh rastlin so potencialno pomembni kot surovina za uporabo v medicini. Raziskavo smo izvedli s pomočjo tako svetlobne kot elektronske mikroskopije. Predstavili smo poglavitne diagnostične značilnosti vsake od vrst – z vidika oblike in strukture celic povrhnjice, listnih rež ter trihomov (žleznih in nežleznih). Z uporabo kombinacije izbranih kvalitativnih mikromorfoloških značilnosti preiskovanih vrst iz rodu Artemisia je v teh surovinah za uporabo v medicini mogoče določiti, katere vrste so prisotne. Key words: Artemisia, micromorphological features, leaves, light microscopy, scanning electron microscopy. Ključne besede: Artemisia, mikromorfološke značilnosti, listi, svetlobna mikroskopija, vrstična elektronska mikroskopija. Corresponding author: Uliana Karpiuk E-mail: uliana.karpiuk@gmail.com Received: 3. 3. 2022 Accepted: 7. 7. 2022 1 Department of Pharmacognosy and Botany, Pharmaceutical Faculty, Bogomolets National Medical University, Kyiv, Ukraine. 2 Department of Systematics and Floristics of Vascular Plants, M.G. Kholodny Institute of Botany, NAS of Ukraine, Kyiv, Ukraine. Valentyna Minarchenko1 , 2  , Uliana Karpiuk1  , Nadiia Kovalska1  , Iryna Tymchenko1 , 2  , Tetiana Dvirna1 , 2  & Iryna Сholak1  DOI: 10.2478/hacq-2022-0012 22/1 • 2023, 131–141 22/1 • 2023, 131–141 132 Minarchenko et al. Diagnostic micromorphological features of leaf surface of Artemisia species Introduction The genus Artemisia L. (wormwood) is one of the larg- est genera in the family Asteraceae (Compositae). It has about 500 species and is an important object of study for taxonomists and florists in almost all countries. There are 30 species of Artemisia in Ukraine; 9 of them have been studied as medicinal plants (Mosyakin & Fedoronchuk, 1999; Minarchenko, 2005). The State Pharmacopoeia of Ukraine (SPhU) has an entry only on Artemisia ab­ sinthium L. (Naukovo-ekspertnyi farmakopeynyit sentr, 2016). The large content of biologically active substances, including essential oils, suggests a wide range of applica- tions of species of Artemisia genus in medical and vet- erinary practice and in the food industry. Artemisia is a taxonomically complicated genus because some species have similar morphology and even very similar micro- scopic structures. It can be difficult to identify related spe- cies, especially as dry raw materials (Hayat et al., 2009b). The species of this genus are also characterized by phyto- chemical variability. Different chemotypes and cytotypes synthesize different compounds with different biological activity (Ivănescu et al., 2015). Taxonomists use a variety of morphological characters to determine Artemisia species, including the life form, shape, and dissection of the leaf blade, the general shape of the inflorescence, the aggregation of the heads, the length of the stem of the head, the nature of pubescence, and so on. The parameters of these structures in worm- wood, for example, are variable. Therefore, the issue of clear demarcation of all species in this group still remains controversial (Hayat et al., 2009a). In addition, the iden- tification of whole plants and raw materials of species in- volves a number of variables. The raw materials usually contain only parts of plant organs, and one of the require- ments for the quality of raw materials is the absence of coarser parts of plants (stems, roots, etc.). The raw ma- terial of wormwood contains mostly leaves with a small proportion of inflorescences and stems, and it is impor- tant to identify the main species-specific morphological or anatomical features of these organs, which would allow a clear determination of their species affiliation. There are no summary data of such characteristics for different spe- cies of wormwood in Ukraine. This paper presents for the first time a comparative characterization of diagnostic features of the leaf epi- dermis of 7 species of genus Artemisia that are used as sources of valuable biologically active compounds: A. ab­ sinthium L., A. annua L., A. dracunculus L., A. marschal­ liana Spreng., A. vulgaris L., A. argyi H.Lév. & Vaniot, and A. verlotiorum Lamotte. The two last invasive species, A. argyi and A. verlotiorum, are of particular interest. They have appeared relatively recently in Ukraine and in recent decades have shown a tendency to expand into natural phytocenoses (Mosyakin et al., 2019 and the references therein). Moreover, the dry raw material of A. argyi is morphologically similar to A. absinthium and so can po- tentially occur as an impurity in the raw material of the latter species. Likewise, the raw material of A. verlotiorum is similar to A. vulgaris. The raw materials of these two species contain many important biologically active com- pounds that are widely studied and used in medicine in Asia and Europe. Moreover, the raw material of A. ver­ lotiorum is more valuable than A. vulgaris (Carnat et al., 2001), and there are cases of hybridization between these two species (James et al., 2000). Materials and methods This study is based on materials collected during many years of field research by the authors in different regions of Ukraine, with the additional use of herbarium speci- mens that were deposited in the National Herbarium of Ukraine (KW) (Table 1). Species Locality Collecting Date Collectors, Herbarium code Artemisia absinthium Odesa region, Ovidiopol district, the village of Sukhyi Lyman, the roadsides 10.6.2005 Bondarenko O.J., KW № 120226 Sumy region, near the town of Hlukhiv, the dry slopes 21.8.2010 Koval L.V., KW № 154497 Kyiv region, Obukhiv district, the village of Kozyn, the roadsides 2.7.2019 Tymchenko I.A., Dvirna T.S., KW № 155601 Poltava region, Poltava district, near the village of Novi Sanzhary, the steppe meadows 6.8.2019 Dvirna T .S., KW № 155600 Kyiv region, Makariv district, near the village of Mykolaivka, the abandoned fields 15.7.2020 Minarchenko V .M., KW № 155599 Table 1: Specimens of the Artemisia species analyzed in the study. Tabela 1: Primerki vrst rodu Artemisia, proučevani v raziskavi. 22/1 • 2023,131–141 133 Minarchenko et al. Diagnostic micromorphological features of leaf surface of Artemisia species A. annua Kharkiv region, near the village of Kehychivka, the forest strip along the railway 19.9.2015 Davydov D.A., KW № 123866 Vinnytsya region, Chechelnyk district, western suburbs of the village of Bondurivka, the forest road 28.9.2016 Kolomiychuk V. P ., KW № 123463 Poltava region, Poltava district, near the village of Lelyukhivka, the vegetable gardens 26.7.2019 Dvirna T .S., KW № 155594 Kyiv region, Obukhiv district, near village Kozyn, floodplain meadow of Kozynka river, the roadsides 7.8.2020 Tymchenko I.A., KW № 155595 Poltava region, Lubny district, near the village of Berezotocha, the roadsides 10.8.2020 Glushchenko L.A., KW № 155610 A. argyi The city of Kyiv, the railway station of Darnytsya 18.9.1989 Мосякін С.Л., KW № 155609 Zhytomyr region, Ovruch district, the railway station of Ihnatpil 7.8.2010 Orlov O.O., KW № 092379 The city of Kyiv, Pushcha-Vodytsya, near the lake of Synye, the forest margin 22.8.2018 Bagatska T .S., KW № 134301 The city of Kyiv, the roadsides of ring road 14.8.2020 Minarchenko V .M., KW № 155602 A. dracunculus Kyiv region, Makariv district, near the village of Mykolaivka, the forest margin 24.7.2019 Minarchenko V.M., Makhynia L.M., KW № 155604 Kyiv region, Obukhiv district, near the village of Kozyn, the floodplain of Kozynka river, the abandoned vegetable gardens 20.7.2020 Tymchenko I.A., KW № 155603 Zhytomyr region, the town of Berdychiv, the railway station, ruderal places 25.7.2021 Orlov O.O., Shynder O.I., KW № 153832 A. marschalliana Chernihiv region, Borzna district, near the village of Hryshivka, the floodplain meadow of lake T rubyn 6.8.2004 Shyian N.M., KW № 00112743 Volyn region, Ratne district. Near the town Ratne, the roadsides 21.8.2008 Minarchenko V.M., Tymchenko I.A., KW № 155596 Zhytomyr region, Narodychi district, near the village of Velyki Klishchi, Drevlyanskyi nature reserve, the forest glades 18.8.2015 Kolomiychuk V. P ., KW № 116426 The city of Kyiv, Syretsky Park, the roadsides 11.9.2020 Minarchenko V .M., KW № 155598 Kyiv region, Makariv district, near the village of Khmilna, the wastelands 26.9.2020 Minarchenko V .M., KW № 155597 A. verlotiorum Crimea, the city of Simferopil, the mountain of Salhirka 14.7.2004 Shevera M.V., KW № 090866 Crimea, near the town of Hurzuf, Ay-Danil, the weed in the vineyard 11.10.2013 Ryff L.E., KW № 148035 Zakarpattya region, the city of Uzhhorod, Hrushevskoho street, ruderal areas 12.9.2019 Shevera M.V., KW № 145936 The city of Kyiv, Syretsky Park, the roadsides 11.9.2020 Minarchenko V.M., Boyko G.V., KW № 155605 A. vulgaris Chernihiv region, Bobrovytsya district, the village of Nova Basan, the roadsides of the forest margin 26.7.2002 Ilyinska A.P ., Futorna O.A., Dyachenko I.I., Nitsenko L.M., KW № 007317 Khmelnytskyi region, the town of Slavuta, Suvorova street 21.8.2002 Gubar L.M., KW № 060927 Kyiv region, Makariv district, near the village of Mykolaivka, the abandoned fields 24.7.2019 Minarchenko V.M., Makhynia L.M., KW № 155606 Poltava region, Poltava district, near the village of Novi Sanzhary, the roadsides 6.8.2019 Dvirna T .S., KW № 155608 Kyiv region, Obukhiv district, the village of Kozyn, the roadsides 8.8.2020 Tymchenko I.A., KW № 155607 Light microscopy (LM) Fragments of dried leaves (basal stem and from the middle of the inflorescence) of 10 samples (individuals) of each species were obtained for morphological and anatomical analysis. The raw materials were boiled in water or 5% so- dium hydrochloride solution for 2–5 minutes. The speci- mens were then prepared for light microscopy (LM). Mi- cropreparations were produced from fresh plants in some cases. The dense layer of trichomes on the leaf blade was removed with adhesive tape in order to analyze the epi- dermal surfaces. Microscopic examination was performed 10 times for each organ from the different parts of the sample. Observations and photomicrographs were per- formed using an Olympus CX23 light microscope and a Philip Harris stereomicroscope, using Levenhuk M1000 PLUS camera software. 22/1 • 2023, 131–141 134 Minarchenko et al. Diagnostic micromorphological features of leaf surface of Artemisia species Scanning electron microscopy (SEM) Investigations of diagnostic features of model species were also performed using a scanning electron microscope (SEM) JSM-6060LA according to standard methods. The main terminology used in describing the diagnostic features of raw materials is based on classical works in this field (Hayat et al., 2009a; Hussain, 2019). The paper also uses the classic terms of characterization of plant organs micromorphology. Results and discussion Features of the epidermis of leaves play a decisive role in the identification of raw materials, as the share of leaves in raw materials of Artemisia species is 60–80%. The main microdiagnostic characteristics of wormwood leaves in raw materials are the shape of epidermal cells, as well as the presence/absence and structure of non-glandular and glandular (or glands) trichomes; slightly less important in identification are the structure, size, and location of the stomata. Epidermal cells According to our research, the cells of the leaves’ abaxial epidermis, in the most studied species of Artemisia genus, are characterized by an irregular shape with wavy walls (Figure 1a–f) and mostly smooth cuticles. The abaxial epidermis of A. marschalliana leaves is characterized by a polygonal shape of cells with slightly wavy or smooth walls and an elongated-comb cuticle in some samples (Figure 1g). The tortuosity of the anticline walls of the adaxial epidermis is well expressed only in A. annua and A. vulgaris, somewhat weaker in A. verlotiorum (Figure 1i, l–n); in A. absinthium, A. dracunculus, A. argyi, and A. marschalliana, the shape of the cells is polygonal (Figure 1h, j, o). In A. verlotiorum and A. vulgaris, the shapes of the cells of upper and lower leaf epidermis are very similar, but in the latter species, the tortuosity of the anticline walls of epidermal cells is much higher on both sides. Probably, this characteristic has considerable variability, as in some works we find opposite data (Bini Maleci & Bagni Marchi, 1983). Stomata The leaf blade of A. absinthium, A. annua, A. dracunculus and A. marschalliana is amphistomatic with anomocytic- type stomata, although A. marschalliana has a predomi- nance of tetracytic stomata (Figure 1g, o). Stomata occur much less frequently on the adaxial epidermis of A. ab­ sinthium, A. annua (Figure 3b, d), while in A. dracuncu­ lus (Figure 1c, j) and A. marschalliana (Figure 1g, o) the density of stomata is approximately the same on the both epidermises. We have found that leaves of A. argyi, A. ver­ lotiorum and A. vulgaris have hypostomatic type with nu- merous stomata in the lower epidermis, but according to some data (Kondratieva & Kondratieva, 2010), stomata on the leaves A. argyi can be on both sides. The shape of the stomata and the length of the stomatal slit are characterized by moderate variability. Thus, a common feature for the stomata of the leaves of the analyzed species is their rounded or elongated shape with 3–6 side cells; they are slightly sunken, raised, or located at the level of epidermal cells. In general, the stomata are larger in size on the adaxial epidermis in all analyzed species with amphistomatic leaf type. The range of ostiole lengths for some species is species-specific. It is the smallest in A. absinthium (17–20 μm), and the largest in A. argyi (13–23 μm) and A. marschalliana (18–28 μm). The size of the ostiole in A. annua was 10–17 μm, in A. dracunculus 12.7–18.5 μm, in A. verlotiorum and A. vulgaris, this figure ranged from 13–20 μm. In the last two species, the stomata clearly protrude above the epidermis and have a rounded shape (Figure 4d–e). The size of individual structures (epidermal cells, stomata, glandular and non-glandular trichomes) is different in samples of wormwood collected from different places, and often overlap in a certain range, so a greater diagnostic role in raw materials plays a combination of qualitative (shape, location, presence/absence) and quantitative characteristics In A. absinthium, A. annua, A. argyi, A. verlotiorum and A. vulgaris, the stomata are divergent, often convex or elongated. The stomata of A. marschalliana are predomi- nantly unidirectional along the leaf axis; they are located at the level of the epidermis or slightly sunken with pro- truding anticline walls of the closing cells (Figure 3a, 4f). On the abaxial epidermis of A. annua, the stomata are multidirectional, and on the adaxial epidermis they are Figure 1: Diagnostic features of the leaf epidermal cells of different Artemisia species as seen under LM. Slika 1: Diagnostične značilnosti epidermalnih celic listov različnih vrst iz rodu Artemisia kot so vidne s pomočjo svetlobnega mikroskopa. Legend: abaxial epidermis (abaksialna povrhnjica): a – A. absinthium, ×400; b – A. annua, ×400; c – A. dracunculus, ×100; d – A. argyi, ×400; e – A. v er lotior um, ×400; f – A. vulga ris, ×400; g – A. marschalliana, ×100; adaxial epidermis (adaksialna povrhnjica): h – A. absinthium, ×400; i – A. annua, ×400; j – A. dracunculus, ×100; k – A. argyi, ×400; l – A. verlotiorum, ×400; m–n – A. vulgaris, ×400; o – A. marschalliana, ×100. → 22/1 • 2023,131–141 135 Minarchenko et al. Diagnostic micromorphological features of leaf surface of Artemisia species a c g e f j k l m n o d h i b 50 μ 50 μ 50 μ 100 μ 50 μ 50 μ 50 μ 100 μ 50 μ 50 μ 50 μ 50 μ 50 μ 50 μ 100 μ 22/1 • 2023, 131–141 136 Minarchenko et al. Diagnostic micromorphological features of leaf surface of Artemisia species mainly oriented along the leaf axis (Figure 3b, 4b). In A. dracunculus, the stomata are oriented mainly along the leaf axis on both sides and are located at the level of epidermal cells (Figure 3g, 4h). The density of stomata on the leaf surface exhibits a very significant variability within each species, which is confirmed by the correlation of this indicator with environmental conditions (Ochi- rova et al., 2015). Glandular and non-glandular trichomes One of the most important characteristics of the epidermis of wormwood leaves is the presence/absence and struc- ture of glandular (glands) and non-glandular trichomes. In most members of the genus Artemisia, indicators of density of glandular trichomes on the leaf surface are characterized by significant variability in plants according to different environmental conditions and different stages of development. Therefore, it is likely that quantitative indicators of the density of glandular trichomes cannot play a decisive role in the identification of raw materials. Multicellular peltate or biseriate trichomes, differentiat- ed into basal, basic, and apical cells, are dominant among the glandular trichomes in the studied species (Figure 2a). They are the site of synthesis and storage of various types of secondary metabolites, the most noted of which are terpenoids and flavonoids (Nigam et al., 2019). The most valuable compound in wormwood species is artemisinin (Xiao et al., 2016), which is synthesized and concentrated in glandular trichomes – this indicator is important in determining the quality of raw materials of a particular species (Ferreira & Anick, 2009). It is believed that biseriate glandular trichomes of Arte­ misia species consist of 10 pairs of secretory cells arranged in 2 rows (Duke et al. 1994; Appezzato-da-Glória et al., 2008), while Cui et al. (2020) suggest that A. argyi has 8-celled biseriate glands. A. absinthium glands are large, 25–50 μm long in the frontal plane. They are localized in the recesses of the adaxial and abaxial epidermis under a dense layer of T-shaped non-glandular trichomes (Fig- ure 2a–b, 3d–f). In general, their density on the adaxial surface is significantly higher than on the abaxial surface, which is characteristic of most of the studied species. On the epidermis of A. argyi leaves, we found several morphotypes of glandular trichomes. Biseriate trichomes dominate, especially on the adaxial leaf surface (Figure 3h, i). Moreover, their density is much higher than in A. absinthium. The length of these glands in the fron- tal plane is 28–57 μm. They are basically more or less sunken into the epidermis (Figure 3i). There are also 4-celled glandular trichomes with vertical cell placement in A. argyi; in our study, they were found rarely on the adaxial epidermis between biseriate trichomes. There are also small round glands with a diameter of 10–16 μm with secretory content. They are embedded in the epider- mis (Figure 2c). It was noted that the density of glandu- lar and non-glandular trichomes in this species is lower in mature leaves compared to young leaves (Cui et al., 2020). An important feature of the epidermis of A. dra­ cunculus leaves is also the presence of peltate glands, but they are somewhat different from those in other species of the genus. Peltate glands on the leaves of A. dracunculus are large, lyre-shaped, and the longest in the frontal plane among the analyzed species (37–63 μm) (Figure 4g, h). Occasionally, on the epidermis of A. dracunculus leaves, there are also classic peltate (Figure 2e) and unicellular glands embedded in the epidermis, and their diameter varies in the range of 10–16 μm. The surface of A. annua leaves on both sides and on the stem is abundantly covered with peltate glandular tri- chomes. There are unicellular glands among them (Figure 2d, 3c). The size of peltate trichomes of A. annua in the frontal plane varies between 29 and 47 μm. They are usu- ally sunken at the base into the abaxial and adaxial epi- dermis of the leaf. Duke et al. (1994) reported that there are two biotypes of A. annua: with and without secretory trichomes; plants without glandular trichomes lack arte- misinin. Peltate glands are present mainly on the abaxial epi- dermis under a dense cover of non-glandular T-shaped trichomes in the studied samples of A. verlotiorum (Figure 4i, j). On the adaxial epidermis there are occasionally pel- tate and unicellular glands, as well as single flat multicel- lular glands, which rise slightly above the epidermis (Fig- ure 3j). The density of glandular trichomes on the abaxial epidermis of A. verlotiorum leaves was 12–18 pieces/mm 2 , while in other studies this figure reached 30 pieces/mm 2 (Bini Maleci & Bagni Marchi, 1983). We can explain this difference by the limiting environmental conditions in our area, given the southern origin of this species. Peltate glands are sunken at the base into the epidermis, large, up to 63 μm long in the frontal plane, generally divided in half at the apex (Figure 4j). The glands of A. vulgaris are sunken at the base into the epidermis, up to 35 μm long in the frontal plane, generally divided in half at the apex (Figure 2j, 4k). Glandular trichomes of the capitate type are also rarely present here (Hayat et al., 2009b) without constriction in the terminal part (Figure 4l). Note that in A. vulgaris, the peltate glands are almost half the size of those in A. verlotiorum, and their density is also about half lower (Figure 4m). Among the studied samples of A. marschalliana plants, small peltate glands were found mainly on the leaves of pubescent forms (Figure 2f ). Note 22/1 • 2023,131–141 137 Minarchenko et al. Diagnostic micromorphological features of leaf surface of Artemisia species Figure 2: Variations in glandular and non-glandular trichomes on the leaf surfaces of some Artemisia species as seen under LM. Slika 2: Razlike v žleznih in nežleznih trihomih na listnih površinah nekaterih vrst iz rodu Artemisia kot so vidne s pomočjo svetlobnega mikroskopa. a–b – A. absinthium, ×400; c – A. argyi, ×400; d – A. annua, ×400; e – A. dracunculus, ×400; f – A. marschalliana, ×400; g – A. marschalliana, ×1000; h – A. verlotiorum, ×400; i – A. verlotiorum, ×1000; j – A. vulgaris, ×1000; k – A. vulgaris, ×100; l – A. dracunculus, ×400. that in our study, the size of the peltate glands (length in the frontal plane) differed significantly on the leaves from different fragments of the shoot in all species. They also differ from those in some publications (Karbalaei et al., 2021). In addition, the size of the glandular trichomes of- ten overlap in related species. This may confirm the weak diagnostic value of quantitative values of the size of these trichomes. Another diagnostic feature of all analyzed species of the genus Artemisia is also the presence/absence of non- glandular trichomes and their structure. These trichomes are classified according to their morphology and the stud- ied species are dominated by T-shaped (or dolabriform) non-glandular trichomes. They are present on the above- ground parts of all studied species but their localization, 20 μ a c g e f j k l d h i b 50 μ 20 μ 100 μ 50 μ 50 μ 50 μ 50 μ 50 μ 50 μ 20 μ 50 μ 22/1 • 2023, 131–141 138 Minarchenko et al. Diagnostic micromorphological features of leaf surface of Artemisia species Figure 3: Variations in stomata, glandular and non-glandular trichomes of adaxial leaf epidermis of different Artemisia species by means of SEM. Slika 3: Razlike v listnih režah, žleznih in nežleznih trihomih adaksialne povrhnjice listov različnih vrst iz rodu Artemisia kot so vidne s pomočjo elektronskega mikroskopa. a – A. marschalliana, ×500 ; b–c – A. annua, ×500; d – A. absinthium, ×500; e – A. absinthium, ×1000; f – A. absinthium, ×250; g – A. dracunculus, ×100; h – A. argyi, ×100; i – A. argyi, ×1000; j – A. verlotiorum, ×500; k – A. vulgaris, ×150; l – A. argyi, ×500; m – A. verlotiorum, ×100; n – A. marschalliana, ×1000; o – A. marschalliana, ×500. a c g e f j k l m n o d h i b 22/1 • 2023,131–141 139 Minarchenko et al. Diagnostic micromorphological features of leaf surface of Artemisia species Figure 4: Variations in stomata, glandular and non-glandular trichomes of abaxial leaf epidermis of different Artemisia species as seen under SEM. Slika 4: Razlike v listnih režah, žleznih in nežleznih trihomih abaksialne listne povrhnjice različnih vrst iz rodu Artemisia kot so vidne s pomočjo elektronskega mikroskopa. a – A. absinthium, ×1000; b – A. annua, ×500; c – A. argyi, ×500; d – A. verlotiorum, ×500; e – A. vulgaris, ×500; f – A. marschalliana, ×500; g – A. dracunculus, ×500; h – A. dracunculus, ×200; i – A. verlotiorum, ×300; j – A. verlotiorum, ×1000; k – A. vulgaris, ×1000; l – A. vulgaris, ×1000; m – A. vulgaris, ×100; n – A. absinthium, ×1000; o – A. argyi, ×300; p–q – A. argyi, ×500; r–s – A. annua, ×500; t – A. verlotiorum, ×1000; u – A. vulgaris, ×500; v – A. dracunculus, ×200; w – A. dracunculus, ×500; x – A. dracunculus, ×220. structure, density and orientation have species-specific features. A common feature of these non-glandular tri- chomes is their placement on the same level with the epi- dermal cells, or with base slightly convex, in contrast to the glandular trichomes, which are usually sunken into the epidermis. T-shaped non-glandular trichomes are a c g e f j k l m n o p q r s t u v w x d h i b 22/1 • 2023, 131–141 140 Minarchenko et al. Diagnostic micromorphological features of leaf surface of Artemisia species elongated generally parallel to the surface of the epidermis terminal cell in the form of a compass needle, with a stem base formed by 1–4 cells. The basal cell in these trichomes is usually enlarged. The site of fastening has the form of rounded rollers. A characteristic feature of T-shaped non-glandular tri- chomes of A. absinthium is the presence of a wide (up to 18 μm in the middle) and long (2–5 mm) tape-shaped terminal cell with a saucer-shaped depression at the site of attachment to the stem (Figure 4n). The stem part of the trichome is usually short (1–2 cells) with an enlarged basal cell, which is attached at the level of the epidermis. In some trichomes, the number of stem cells can reach four. T-shaped trichomes are mostly placed unidirection- ally (Figure 3d, f ), densely covering the leaf blade on both sides with a higher density on the abaxial epidermis. A. argyi is similar to A. absinthium in pubescence. All aboveground organs of plants of this species are also glau- cous from dense pubescence with T-shaped trichomes, but their structure is somewhat different. We found some morphological differences in the T-shaped trichomes of A. argyi. In particular, the felt covering on the abaxial side of the leaf is formed mainly by T-trichomes with long filamentous nodular apical cells, which sometimes grow together and form anastomoses (Figure 4p). They are placed so densely that it is difficult to notice the glands under them. The terminal T-trichome cell on the adaxial epidermis is long, thick, smooth or slightly tor- tuous (Figure 4q). T-trichomes with a ribbon-like apical cell appear here singly. The stem part of both morpho- types is composed of 2–3 cells with a thickened basal cell. On the adaxial epidermis, T-shaped trichomes are less tortuous and less densely spaced, with peltate glands clearly visible between them. A. annua presents a different type of T-shaped tri- chomes than those described above. These are 5-celled trichomes with a ribbon-like apical cell, but much shorter and narrower than in A. absinthium; the stem part is 2–4 celled, and the basal cell is large and barrel-shaped (Fig- ure 4r), as in A. verlotiorum (Figure 4t). Such trichomes are rarely present on both sides of the leaf blade of A. annua (Figure 4s). T-shaped trichomes are present on A. verlotiorum on the abaxial epidermis with a long termi- nal filamentous cell and a saucer-shaped depression in the center (Figure 4i–j). They have a number of similar features to those described above: their base is 2–3 celled, as in A. absinthium, but the basal cell is large, sometimes barrel-shaped, especially along the veins (Figure 4t); on the adaxial surface of the leaf blade, these trichomes are shorter and almost flat without a clear orientation (Figure 3m), as in A. vulgaris (Figure 3k). Radial placement of adjacent epidermal cells is often present around the base of the trichome on the adaxial surface. The abaxial epider- mis of A. vulgaris is dominated by filamentous T-shaped trichomes, which form a less dense cover than in A. ver­ lotiorum, and occasionally there are T-shaped trichomes with a thick, long, slightly tortuous or flat terminal cell (Figure 4u). Their stem base is formed by 2–4 cells. T- trichomes of A. vulgaris on the adaxial epidermis are simi- lar to those in A. verlotiorum, although the terminal cell is longer in A. vulgaris. Of particular note is the pubescence of the epidermis in leaves of A. marschalliana. This species is characterized by huge variations of this feature: from dense pubescence of all aboveground organs of plants to the complete ab- sence of T-shaped trichomes. Moreover, plants that are pubescent, sparsely pubescent, or even glabrous can grow together where there is predominance of one kind in the coenopopulation. The only thing that was established in this study was the predominance of pubescent plants of A. marschalliana in the populations in Southeast Ukraine, whereas in Polissya the plants are weakly pubescent or bare. T-shaped trichomes with a unicellular broad base and a long flat terminal cell (Figure 3n) are usually ori- ented along the axis of the leaf on both sides. Non-glandular trichomes of A. dracunculus are differ- ent from those in other studied species. Specific macro- morphic multibranched trichomes (Figure 2l, 3g, 4v–x), which are also a unique diagnostic feature of A. persica (Hayat et al., 2009b), were found scattered on the leaf in the samples that we studied. These are large branched trichomes with a broad short base and various numbers of branches. They are scattered in A. dracunculus on both sides of the leaf blade. Typical for most species of the ge- nus Artemisia, non-glandular T-shaped trichomes with al- most equally long terminal cells were very rare (Figure 4x) in the samples we studied. Conclusions We found that the leaf surfaces of A. absinthium, A. an­ nua, A. dracunculus, A. marschalliana, A. vulgaris, A. ar­ gyi and A. verlotiorum have some common and species specific features in the structure of epidermis, stomata, glandular and non-glandular trichomes. Comprehensive analysis of these characteristics of Artemisia species allows us to determine their species affiliation clearly. Targeted falsification or incorrect identification of a particular spe- cies of Artemisia during the microscopic analysis of raw materials is unlikely. Among the analyzed structures of leaf surfaces of Artemisia species, the species specificity is more pronounced in the morphology and localization of the glandular and non-glandular trichomes. 22/1 • 2023,131–141 141 Minarchenko et al. Diagnostic micromorphological features of leaf surface of Artemisia species Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We are thankful to M.G. Kholodny Institute of botany, NAS of Ukraine and special thanks to S. L. Mosyakin, Prof., Dr. Biol. Sci., Corr. Member of NAS of Ukraine, for providing research facilities and appreciated techni- cal support throughout the scanning electron microscopy. This research was supported by the Ministry of Health of Ukraine (grant number 012U001306). Valentyna Minarchenko  https://orcid.org/0000-0002- 5049-7620 Uliana Karpiuk  https://orcid.org/0000-0002-8316-4910 Nadiia Kovalska  https://orcid.org/0000-0003-2673-5446 Iryna Tymchenko  https://orcid.org/0000-0001-7505-3164 Tetiana Dvirna  https://orcid.org/0000-0002-9279-9766 Iryna Сholak  https://orcid.org/0000-0003-1833-321Х References Appezzato-da-Glória, B., Hayashi, A. H., Cury, G., Soares, M. K. M., & Rocha, R. (2008). Occurrence of secretory structures in underground systems of seven Asteraceae species. Journal of the Linnean Society. Botany, 157, 789–796. https://doi.org/10.1111/ j.1095-8339.2008.00823.x Bini Maleci, L., & Bagni Marchi, A. (1983). «Artemisia vulgaris» L. ed «A.verlotiorum» Lamotte: studio di alcuni caratteri morfo-anatomici distintivi delle due specie. Webbia, 37(1), 185–196. https://doi.org/10. 1080/00837792.1983.10670272 Carnat, A. 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