ACTA BOT. CROAT. 79 (1), 2020 55 Acta Bot. Croat. 79 (1), 55–58, 2020 CODEN: ABCRA 25 DOI: 10.37427/botcro-2020-005 ISSN 0365-0588 eISSN 1847-8476 A comparative anatomical study of the genus Puschkinia Adams in Turkey Kadriye Yetişen1*, Hasan Yıldırım2, Canan Özdemir1 1 Department of Biology, Faculty of Art and Science, Celal Bayar University, Manisa, Turkey 2 Department of Biology, Faculty Science, Ege University, İzmir, Turkey Abstract – In the study, Puschkinia scilloides Adams, P. bilgineri Yıldırım, and P. peshmenii Rix et B. Mathew were compared anatomically. Although the stem vascular bundles were arranged in two rows in P. peshmenii, they can observed in three rows in P. scilloides and P. bilgineri. In addition, some differences were found, such as the metaxylem number in the root, number of vascular bundles in the scape, presence of raphide crystals, and num- ber of protruding epidermis cells at the tip of the leaf. These differences have been found to be taxonomically useful for distinguishing these species. Moreover, similarities and differences between Puschkinia Adams and the taxonomically closely related Scilla L. section Chionodoxa Boiss. and Scilla bifolia L. taxa were also discussed. Keywords: anatomy, Puschkinia, Scilla, taxonomy, Turkey * Corresponding author e-mail: kadriyeyetisen@gmail.com Introduction The genus Puschkinia is represented by three species in Turkey (P. scilloides Adams, P. peshmenii Rix et B. Mathew, and P. bilgineri Yıldırım). The species of this genus show high morphological variations, especially P. scilloides (Edmond- son 1984, Yıldırım 2012, 2013, 2014). These species are dis- tributed naturally in Turkey. Puschkinia scilloides is distrib- uted in north Caucasus, Lebanon, northern Iran, Iraq and Syria (Rix and Mathew 2007, Yıldırım 2013). As for the other species, P. bilgineri is endemic to Turkey, and P. peshmenii is distributed in Turkey and Iran. In the present study, P. scil- loides, P. bilgineri and P. peshmenii are compared anatomi- cally and it is aimed at contributing to the taxonomic sepa- ration of the species. In addition, the anatomical properties of the Puschkinia taxa and the taxonomically closely related Scilla L. section Chionodoxa Boiss. and Scilla bifolia L. taxa are also discussed. Materials and methods Study area The species were collected from natural habitats in east- ern and southeastern Anatolia (Fig. 1). The study area is sit- uated in the Armenio-Iranian subregion of the Irano-Tura- nian phytogeographic region (Rivas-Martínez et al. 2004), and mostly has a continental semiarid climate with hot and dry summers, and cold and snowy winters. Puschkinia bilgi- neri (Fig. 2A) was gathered from Van Province, Kavuşşahap mountain chain, at Karabet Gateway, alpine mountain meadows and mountain slopes, from 2885–3000 m a.s.l. (type locality). Puschkinia peshmenii (Fig. 2B, C) was col- lected from Hakkari Province, near the Yüksekova district, in the gorge leading into the valley from the west, among dwarf shrubs, from 1850–1900 m a.s.l. (type locality). Pusch- kinia scilloides (Fig. 2D) was gathered from Van Province, Fig. 1. Location of sampling sites of the Puschkinia species in Tur- key: P. scilloides Adams (▲), P. bilgineri Yıldırım (■), P. peshmenii Rix et B. Mathew (●). YETIŞEN K, YILDIRIM H, ÖZDEMIR C 56 ACTA BOT. CROAT. 79 (1), 2020 Gevaş district, Artos Mountain, opening slopes and plateau, from 2400–2550 m a.s.l. Sampling For the anatomical studies, the plant specimens were fixed in 70% ethanol. Ten specimens of each species were studied. The paraffin method (Algan 1981) was used for preparing cross-sections of the root, scape, and leaves of the species. Transverse sections of 15–20 μm were made with a sliding microtome and stained with safranin-fast green. Measurements of the root and stem, and the leaf cell sizes of the species were taken using an ocular-micrometer and photographed with a motorized Leica DM 3000 micro- scope. The minimum, maximum, mean, and standard de- viation were determined and are shown in Tab. 1. Fifteen cross-sections from each plant were made for each mea- surement (Tab. 1). Results Root The rhizodermis, which consisted of elongated cells, was single-layered on the outer surface of the root in all of the species. There was no cuticle layer on the hypodermal cells. The cortex consisted of 6–8, 6–9, and 6–10 layered paren- chymatous cells with intercellular spaces in P. scilloides, P. bilgineri, and P. peshmenii, respectively (Fig. 3A, C, F). The endodermis cell wall of all of the species was thick on all four sides. On the inside of the endodermis and adjacent to it was the cylinder of parenchyma cells called the pericycle in all of the studied species. The pericycle was 1 cell wide and consisted of thin-walled cells. Puschkinia scilloides had 1–2 metaxylem-vessels in the center of the root while P. bilgineri and P. peshmenii had just one. There were four xylem strands on the midpoint of the vascular cylinder in all of the spe- Fig. 2. Habitus of the Puschkinia species: P. bilgineri (A), P. pesh- menii (B, C), P. scilloides (D). Scale bar = 1 cm. Ta b. 1 . M ea su re m en ts of an at om ic al p ro pe rt ie s o f t he st ud ie d Pu sc hk in ia sp ec ie s ( n = 10 ). P. bi lg in er i P. pe sh m en ii P. sc ill oi de s W id th Le ng th W id th Le ng th W id th Le ng th M in -M ax (µ m ) M ea n± SD M in -M ax (µ m ) M ea n± SD M in -M ax (µ m ) M ea n± SD M in -M ax (µ m ) M ea n± SD M in -M ax (µ m ) M ea n± SD M in -M ax (µ m ) M ea n± SD R oo t Ep id er m is 10 .0 –2 3. 0 15 .0 ±1 .4 8. 0– 27 .0 14 .5 ±2 .1 10 .0 –2 5. 0 16 .0 ±2 .7 7. 5– 17 .5 15 .0 ±3 .4 17 .0 –2 5. 0 20 .0 ±3 .4 7. 50 –1 7. 0 11 .0 ±1 .2 C or te x (d ia m et er ) 22 .0 –5 5. 0 29 .0 ±2 .8 . . 20 .0 –3 7. 0 28 .0 ±5 .1 . . 17 .0 –3 8. 0 23 .0 ±4 .1 . . M et ax yl em (d ia m et er ) 30 .0 –3 7. 0 34 .0 ±1 .3 . . 30 .0 –4 2. 0 27 .0 ±9 .0 . . 20 .0 –3 7. 0 31 .0 ±2 .3 . . St em Ep id er m is 12 .0 –3 3. 0 25 .0 ±5 .1 12 .0 –3 4. 0 22 .0 ±6 .7 10 .0 –1 9. 0 16 .0 ±3 .7 17 .0 –3 7. 0 30 .0 ±5 .2 15 .0 –2 7. 0 20 .0 ±3 .1 20 .0 –3 2. 0 25 .0 ±5 .4 C or te x (d ia m et er ) 16 .0 –4 3. 0 37 .0 ±7 .5 . . 25 .0 –5 5. 0 46 .0 ±3 .1 . . 20 .0 –5 2. 0 34 .0 ±3 .6 . . Tr ac he a (d ia m et er ) 12 .0 –2 6. 0 16 .1 ±4 .3 . . 13 .0 –3 5. 0 20 .0 ±4 .2 . . 10 .0 –2 0. 0 14 .0 ±1 .7 . . Pi th (d ia m et er ) 25 .0 –9 6. 0 57 .1 ±2 2 . . 52 .0 –8 7. 0 66 .0 ±1 3 . . 30 .0 –9 0. 0 60 .0 ±1 1 . . Le af A da xi al e pi de rm is 12 .0 –2 7. 0 19 .0 ±3 .6 15 .0 –2 5. 0 20 .0 ±2 .7 17 .0 –3 0. 0 19 .0 ±3 .8 20 .0 –3 3. 0 27 .0 ±4 .1 25 .0 –4 2. 0 34 .0 ±5 .2 17 .0 –3 0. 0 23 .0 ±1 .9 Pa lis ad e ce ll 12 .0 –3 0. 0 23 .0 ±5 .0 25 .0 –7 5. 0 50 .0 ±1 2 22 .0 –5 0. 0 30 .0 ±4 .1 45 .0 –7 5. 0 55 .0 ±9 .2 21 .0 –3 7. 0 28 .0 ±2 .1 38 .0 –5 9. 0 47 .0 ±7 .1 Sp on gy ce ll 10 .0 –2 5. 0 16 .0 ±4 .7 32 .0 –2 8. 0 19 .0 ±3 .7 12 .0 –2 8. 0 15 .0 ±1 .7 12 .0 –3 5. 0 23 .0 ±6 .2 20 .0 –4 3. 0 31 .0 ±3 .1 . . A ba xi al e pi de rm is 10 .0 –2 2. 0 17 .0 ±2 .9 13 .0 –2 1. 0 18 .0 ±2 .0 20 .0 –3 7. 0 28 .0 ±3 .9 23 .0 –3 0. 0 27 .0 ±3 .5 25 .0 –3 2. 0 28 .0 ±3 .7 22 .0 –3 0. 0 24 .0 ±1 .7 ANATOMY OF PUSCHKINIA SPECIES IN TURKEY ACTA BOT. CROAT. 79 (1), 2020 57 cies root cross-sections (Fig. 3B, D, E, G, H). Needle-shaped raphide crystals were present in the root cortex of P. scilloi- des and P. bilgineri. Scape A very thick cuticle layer covered the scape epidermis in all of the studied species. The cortex was composed of 6–9, 5–8, and 4–7 layered thin-walled, circular parenchyma cells with intercellular spaces in P. scilloides, P. bilgineri and P. peshmenii, respectively, under the epidermis (Fig. 4A, C, E). In P. scilloides, 23–29 collateral vascular bundles with thick- walled cells were arranged in 3 rings, while in P. bilgineri there were 16–20 vascular bundles arranged in 3 rings, and in P. peshmenii, there were 14–17 vascular bundles arranged in two rings. In P. peshmenii, a 1–3 cell lined collenchyma layer was present, whereas a 3–5 cell lined layer was present in the other two species. There were pith cells in the center of the scape in all of the species (Fig. 4). Leaf Leaf anatomy was quite similar in all of the studied spe- cies. The epidermis comprised a single layer of cells cover- ing both the abaxial and adaxial surfaces of the leaf, where there was a thick cuticle layer in all of the species. Outward epidermis cell protrusions were present at the ends of the leaves with, respectively 4–7, 3–5, and 3–6 in P. scilloides, P. bilgineri and P. peshmenii (Fig. 5G, J). Two regions of the mesophyll were distinguishable, and the adaxial and abax- ial palisade parenchyma consisted of a rectangle, and oval- shaped cells were arranged in 2–3 rows in all of the species. The middle region consisted of loosely arranged spongy pa- renchyma cells with abundant air spaces between them in all of the taxa. The raphides present in the mesophyll of P. pesh- menii were different from those in the other species. The vascular bundles were arranged regularly in the cen- ter of the mesophyll in all of the species (Fig. 5). When the anatomical measurements were taken, the cell widths and lengths were generally close to each other. The measured values are detailed in the Tab. 1. Discussion When the anatomical features of the roots were com- pared, it was understood that P. scilloides differed from the other species by its 1–2 metaxylem. When the scape proper- ties were considered, the vascular bundles were arranged in two rings in P. peshmenii, while the other species had three rings and the number of vascular bundles in the species dif- fered. The scape epidermis cells protruded outward in P. peshmenii, while they were almost non-existent in the other Puschkinia species. Scape cortex and pith cell measurements of P. peshmenii are bigger than the other two species. Leaf Fig. 3. Root cross-sections of the Puschkinia species: P. bilgineri (A, B), P. peshmenii (C, D, E), P. scilloides (F, G, H). c – cortex, e – epidermis, en – endodermis, mx – metaxylem. Scale bar = 40 μm. Fig. 4. Scape cross-sections of the Puschkinia species: P. bilgineri (A), P. peshmenii (B, C, D), P. scilloides (E, F, G, H). c – cortex, cl – collenchyma, cu – cuticle, e – epidermis, p – pith, s – stoma, v – vascular bundle. Scale bar = 40 μm. KARAHAN F 58 ACTA BOT. CROAT. 79 (1), 2020 Fig. 5. Leaves cross-sections of Puschkinia species: P. bilgineri (A, B, C), P. peshmenii (D, E, F, G), P. scilloides (H, I, J). a – aerenchyma, ab – abaxial epidermis, ad – adaxial epidermis, cu – cuticle, m – mesophyll, s – stoma, v – vascular bundle. Scale bar = 40 μm. spongy cells of P. scilloides are larger than in the other two species (Tab. 1). The location and type of crystals in a plant are taxonomically important (Metcalfe and Chalk 1983, Fahn 1990). In this study, needle-shaped crystals were pres- ent in the root cross-sections of P. scilloides and P. bilgineri, but the presence of raphide crystals in the mesophyll layer of the leaves of P. peshmenii distinguished this species from the others. The differences in the number of vascular bundles in the scape, the presence of raphide crystals, and the num- ber of protruding epidermis cells at the tips of the leaves can be used to differentiate species of the genus Puschkinia. These taxa are known to be taxonomically closely related to Scilla section Chionodoxa and Scilla bifolia taxa in Turkey (Yıldırım et al. 2017). The number of metaxylems in the root of Scilla taxa was higher than that in species of Puschkinia. Disintegration areas in the scape pith of the Scilla taxa were wider than that of the genus Puschkinia. A single-layered epidermis formed projections that formed longitudinal ribs in all of the taxa, except Scilla  allenii in the scape. These protrusions were present in the endemic P. peshmenii scape, but were almost non-existent in other Puschkinia species. Hyacinthoides Heist. ex Fabr. and Scilla taxa generally have aerenchyma cavities in the leaf mesophyll (Almeida da Silva and Rosselló 1990). We also observed aerenchyma cavities in the leaves of studied Scilla and Puschkinia taxa. Needle- shaped raphide crystals were present in all three species of Puschkinia. In the studies carried out previously, such crys- tals were found in the leaves and stems of Scilla taxa (Satil and Akan 2006, Yıldırım et al. 2017). In the three Puschki- nia species, 3–7 epidermis cells protruded outwardly at the tip of the leaves. Such protrusions were not observed in the studied Scilla taxa (Yıldırım et al. 2017). This feature can be used to distinguish between the two genera. References Algan, G., 1981: Microtechnics for plant tissues. PhD Thesis. Firat University, Faculty of Science, Istanbul, Turkey (in Turkish). Almeida da Silva, R., Rosselló, J.A., 1990: Anatomical studies on the Scilla verna (Hyacinthaceae) complex. Israel Journal of Plant Sciences 47, 103–110. Edmondson, J., 1984: Puschkinia Adams. In: Davis, P.H. (ed.), Flora of Turkey and the East Aegean islands 8, 226–227. 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