Raca, I., Ljubisavljević, I., Jušković, M., Ranđelović, N., Ranđelović, V.: Comparative anatomical study of the taxa from series Verni Mathew (Crocus L.) in Serbia. Biologica Nyssana, 8 (1), September 2017 BIOLOGICA NYSSANA 8 (1)  September 2017: 15-22 Raca, I. et al.  Comparative anatomical study of the taxa from… 15 Original Article Received: 27 June 2017 Revised: 25 July 2017 Accepted: 12 August 2017 Comparative anatomical study of the taxa from series Verni Mathew (Crocus L.) in Serbia Irena Raca*, Irena Ljubisavljević, Marina Jušković, Novica Ranđelović, Vladimir Ranđelović University of Niš, Faculty of Science and Mathematics, Department of Biology and Ecology, Višegradska 33, Niš, Serbia * E-mail: raca.irena@gmail.com Abstract: Raca, I., Ljubisavljević, I., Jušković, M., Ranđelović, N., Ranđelović, V.: Comparative anatomical study of the taxa from series Verni Mathew (Crocus L.) in Serbia. Biologica Nyssana, 8 (1), September 2017: 15-22. The comparative leaf anatomy of three Crocus L. taxa from series Verni Mathew (Crocus heuffelianus Herb., Crocus tommasinianus Herbert and Crocus kosaninii Pulević) in Serbia is introduced. The general outlook of the cross sections of the leaves was defined with microphotographs. More precisely, leaf shape, leaf surface, mesophyll parenchyma and vascular bundle features were examined. The biggest differences were found at the level of vascular bundles (xylem area, phloem area, sclerenchyma area) and leaf blade features (section height, section length, arm length, white stripe width, lacuna area). In general, listed parameters had the lowest values in C. tommasinianus population, while the highest values could be found in population of C. heuffelianus. Further investigations should be focused on discovering more localities followed by surveying the ecological factors of the habitats. Key words: Crocus, series Verni, cross section, leaf Apstrakt: Raca, I., Ljubisavljević, I., Jušković, M., Ranđelović, N., Ranđelović, V.: Uporedna anatomska studija taksona serije Verni Mathew (Crocus L.) u Srbiji. Biologica Nyssana, 8 (1), Septembar 2017: 15-22. Komparativna anatomska analiza listova tri taksona roda Crocus L. a serije Verni Mathew (Crocus heuffelianus Herb., Crocus tommasinianus Herbert i Crocus kosaninii Pulević) iz Srbije prikazana je na mikrofotografijama. Konkretnije, razmatrani su oblik poprečnog preseka lista, površinske strukture listova, lisni mezofil i karakteristike provodnih snopića. Najveće razlike zabeležene su na nivou provodnih snopića (površina ksilema, floema i sklerenhima) i parametara koji se odnose na oblik preseka (visina i dužina preseka, dužina ručice, širina bele pruge, površina centralnog parenhima). Generalno, navedeni parametri imaju najniže izmerene vrednosti u populaciji vrste C. tommasinianus, dok su najviše izmerene vrednosti zabeležene u populaciji vrste C. heuffelianus. Komparativna anatomska analiza obezbeđuje dodatne informacije o karakterima značajnim za diferencijaciju vrsta. Kako god, buduća istraživanja bi trebalo da budu fokusirana na pronalaženje novih lokaliteta populacija od interesa, kao i na praćenje ekoloških uslova njihovih habitata. Ključne reči: Crocus, serija Verni, poprečni presek, list 8 (1) • September 2017: 15-22 DOI: 10.5281/zenodo.963053 BIOLOGICA NYSSANA 8 (1)  September 2017: 15-22 Raca, I. et al.  Comparative anatomical study of the taxa from… 16 Introduction Saffrons are known as “golden spice” and ornamentals (C a n d a n , 2007). Moreover Crocus species have antitumor, antimutagenic, cytotoxic activities and inhibitory effect on nucleic acid synthesis in human malignant cells (F a t e h i et al., 2003). As a matter of fact, the genus Crocus contains more than 200 taxa (R u k š ā n s , 2017) from the West Mediterranean to Central Asia with Poland as Northern limit of distribution. In the latest detailed revision of the genus (M a t h e w et al., 2009), the genus was divided into: section Crocus Mathew and section Nudiscapus Mathew. The Balkan Peninsula is known as one center of diversity (M a t h e w , 1982). In Serbia, there are 17 noticed species (R a n đ e l o v i ć et al., 1990). They belong to section Crocus Mathew (series Verni Mathew, Scardici Mathew, Crocus Mathew) and section Nudiscapus Mathew (series Reticulati Mathew, Biflori Mathew, Flavi Mathew) (R a n đ e l o v i ć et al., 1990). However, there are many contentious questions related to systematic positions of Crocus taxa. One of them certainly refers to taxa from series Verni. Series Verni is represented by three species in Serbia: Crocus heuffelianus Herb.(Fig. 1a), Crocus kosaninii Pulević (Fig.1b) and Crocus tommasinianus Herb. (Fig. 1c) (R a n đ e l o v i ć et al., 1990). Since good differential markers can be found on the level of micromorphological aspects of the leaves, there have been several investigations about leaf anatomy of Crocus taxa from different parts of its distribution range (R u d a l l & M a t h e w , 1990; A k a n , 2007; E r o l & K u ç u k e r , 2007; S a t i l , 2007; C a n d a n , 2007, 2015; K a n d e m i r , 2009, 2010, 2012; C o ş k u n , 2010; Ö z d e m i r , 2010; Y e t i ş e n , 2013). As can be seen in the literature, no detailed comparative study of series Verni has been available, yet. Therefore, the initial objective of this paper is to introduce the comparison of leaf anatomy in cross sections of series Verni taxa in Serbia and to identify their possible differences. Material and methods Plant samples were collected from natural populations in the flowering time. The sampling localities (Fig. 2) with the associated information are given below (Tab. 1). Taxonomic description of the species was made according to R a n đ e l o v i ć et al. Fig. 1. Verni taxa in Serbia: 1a. Crocus heuffelianus, 1b. C. kosaninii, 1c. C. tommasinianus Table 1. Localities with associated information Species Crocus heuffelianus Crocus kosaninii Crocus tommasinianus Locality Velika Ivanča Strojkovce Mađere N lat/E long 44º 42' 33" 20º 59' 20" 42º 54' 00" 21º 55' 00" 43º 41' 29" 21º 30' 34" Altitude 173 m 305 m 207 m https://www.google.rs/url?sa=t&rct=j&q=&esrc=s&source=web&cd=7&cad=rja&uact=8&ved=0ahUKEwjtw_rjv_PMAhVIExoKHaUUA0cQFghIMAY&url=https%3A%2F%2Fwww.facebook.com%2Foznur.kucuker&usg=AFQjCNGod6h_3lbA3ebSio7vj928hXSVog&sig2=hL-gJBxDRy4czPUO-BJRWg BIOLOGICA NYSSANA 8 (1)  September 2017: 15-22 Raca, I. et al.  Comparative anatomical study of the taxa from… 17 (1990). Specimens were deposited in Herbarium Moesiacum Niš. Five individuals per population were fixed in 50% alcohol for anatomical study, which was done in the laboratory for Plant Systematics and Ecology, Faculty of Science and Mathematics, University of Niš. Manual microtome (G l i g o r i j e v i ć & P e j č i n o v i ć , 1983) was utilized in order to make cross sections of the leaves. 30 transverse sections per population were stained with Safranin - Alcian Blue and examined by Leica DM 1000 microscope afterwards. Following parameters were considered in leaf cross sections: section height, section length, arm length, white stripe width, lacuna area, adaxial epidermis cells height and width, palisade cells height and width, palisade tissue height, spongy cells height and width, spongy tissue height, abaxial epidermis cells height and width, number of big vascular bundles, xylem area, phloem area and sclerenchyma area. Listed anatomical features were measured with ImageJ software. Anatomy characteristics were also described by microphotograps. Results and discussion Typical microphotographs of leaf cross sections for all of three Verni species from Serbia are presented in Fig. 3. The first part of collage refers to the general outline of leaf cross sections. Lateral arm details are given in the middle. The last part of collage represents the structure of big bundles positioned at the end of the arms. Leaf blade: Crocus leaves cross sections have a unique shape - with a square and almost rectangular central keel and two lateral arms recurved towards the keel (R u d a l l & M a t h e w , 1990). However, there are some exceptions where the keel and arms are of the same length (C. scharojanii, C. carpetanus, C. scardicus). The leaf type was declared as morphologicaly and physiologicaly bifacial (E r o l , 2007). The keel is squared with wide base and acute corners in all Verni cross sections. The length of arms and their curving degree differ amongst the species (Fig. 3A) (Tab. 2). The papillae are detected on the adaxial side at the end of the arms. Also, the abaxial side of the corner of keel might have hair. The mentioned hair abundance pattern is characteristic of each of three species. Leaf surface: Adaxial epidermis cells are rectangular, while abaxial cells are elliptical in shape. The cuticle is thicker on the adaxial side of the leaf. Stomata occurs on the abaxial surface in the region of arms and lateral sides of the keel. The stomata type is anomocytic (E r o l , 2007). Mesophyll: Large, almost round parenchyma cells are located in the central part of the keel. Because of their thin walls, they might break down, forming an air space called lacuna. Disintegration of parenchyma cell walls varies between species (Fig. 3A, Tab. 2). Since parenchyma cells of the keel lack chloroplasts, lacuna area can be noticed as typical white stripe all along the central part of the Crocus leaves (E r o l , 2007; Y e t i ş e n , 2013). There are two types of parenchyma cells in the mesophyll of arms – palisade and spongy cells. Palisade is oriented to the adaxial surface and it is made of two layers of polygonal cells. Spongy parenchyma is three – four layered and consists elliptical - irregular shaped cells sometimes with intercellular space within (Fig. 4). Two layers of palisade cells are noticed at the base of the keel as well. Vascular bundles: Collateral vascular bundles are located between the palisade and spongy cells in one row. There are four big vascular bundles (two of them are placed at the ends of the lateral arms, while two others are positioned in the base of the central keel) (Fig. 3A). The number of small and the smallest vascular bundles depends on the leaf dimensions. Additionally, they are located all along Fig. 2. The geographical position of the investigated populations in Serbia (1-Crocus heuffelianus; 2-C. tommasinianus; 3-C. kosaninii) BIOLOGICA NYSSANA 8 (1)  September 2017: 15-22 Raca, I. et al.  Comparative anatomical study of the taxa from… 18 the arms and lateral and basal side of the keel. The xylem is oriented towards the adaxial side with the phloem beneath. Sclerenchyma “caps” below the phloem are well developed (Fig. 5). The anatomy features measurements expressed through the average values and standard deviation are given in the table below (Tab. 3). Apparently, the highest values of leaf blade features (section height and length, arm length and lacuna area) are noticed in C. heuffelianus population. In the other two species values of those parameters are much lower. The white stripe is narrow in C. kosaninii cross sections (Fig. 6). Both adaxial and abaxial epidermis cells were found to be Fig. 3. Anatomic characters of leaves of Crocus ser. Verni species from Serbia: A-Leaf cross sections (5x), B-Arm details (20x) and C-Big bundles (40x) (a-C. heuffelianus; b-C. kosaninii; c-C. tommasinianus) Table 2. Qualitative anatomic characters of leaves of Crocus ser. Verni species from Serbia Crocus heuffelianus Crocus kosaninii Crocus tommasinianus Leaf shape Arms the longest with the highest curving degree shorter and slightly recurved Keel rectangulared, with ± acute corners squared, with ± acute corners squared, with conspicuo- usly acute corners Epidermis Cell shape adaxial rectangular, abaxial eliptical Papillae finger-like, at the end of the arms and in the corner of the keel Mesophyll parenchyma Lacuna area large round cells disintegration of parenchyma cell walls is more frequent Palisade 2 layers of polygonal cells Spongy 3 - 4 layers of elliptical – irregular shaped cells Big Vascular Bundles 2 of them at the ends of the lateral arms, 2 others at the base of the central keel sometimes one more pair in the middle of the arms BIOLOGICA NYSSANA 8 (1)  September 2017: 15-22 Raca, I. et al.  Comparative anatomical study of the taxa from… 19 the largest in C. kosaninii population. Palisade tissue characters are of a similar value. The thinnest spongy layer was observed in the investigated C. tommasinianus population. The most developed vascular tissue features (xylem, phloem and sclerenchyma cap area) were found in C. heuffelianus cross sections. Crocus tommasinianus vascular bundles are the smallest, while C. kosaninii big bundles are middle-sized (Fig. 6). The objective of this study was to introduce and compare leaf anatomy of the three Crocus species from series Verni present in Serbia. Leaf blade, epidermis, mesophyll and vascular bundle features were analyzed. As might be expected (R u d a l l & M a t h e w , 1990), those species tend to correspond closely in their leaf anatomy. However, major differencies can be found on the level of big vascular bundles and their examined parameters (xylem area, phloem area and sclerenchyma area). Moreover, leaf blade features (section height and length, arm length, white stripe width and lacuna area) are good distinguishing characters as well. The Table 3. Quantitative anatomic characters of leaves of Crocus ser. Verni species from Serbia Species Crocus heuffelianus Crocus kosaninii Crocus tommasinianus Character Mean ± StDev Min - Max Mean ± StDev Min - Max Mean ± StDev Min - Max Section height (µm) 881 ± 173 570 - 1210 654 ± 124 494 - 1017 630 ± 125 382 - 916 Section length (µm) 7003 ± 1095 3611 - 9035 4527 ± 946 2810 - 6298 4152 ± 959 2391 - 5598 Arm length (µm) 3139 ± 492 1684 - 4263 1908 ± 429 1109 - 2725 1701 ± 462 892 - 2467 White stripe width (µm) 752 ± 122 500 - 996 590 ± 93 434 - 792 740 ± 111 584 - 955 Lacuna area (µm2) 371375 ± 105510 193667 - 648276 209992 ± 69767 111999 - 335380 248603 ± 74234 141373 - 395804 Adaxial epidermis cell height (µm) 20 ± 3 16 - 28 22 ± 2 19 - 27 16 ± 2 11 - 20 Adaxial epidermis cell width (µm) 18 ± 3 15 - 30 19 ± 2 16 - 25 17 ± 2 13 - 20 Palisade cell height (µm) 37 ± 6 27 - 55 34 ± 4 27 - 42 34 ± 4 28 - 43 Palisade cell width (µm) 15 ± 2 11 - 21 14 ± 1 12 - 17 16 ± 2 13 - 20 Palisade tissue height (µm) 59 ± 13 35 - 88 59 ± 9 45 - 82 54 ± 7 40 - 66 Spongy cell height (µm) 22 ± 3 16 - 29 22 ± 2 17 - 27 16 ± 2 12 - 20 Spongy cell width (µm) 19 ± 3 15 - 26 20 ± 2 18 - 23 24 ± 3 17 - 30 Spongy tissue height (µm) 68 ± 11 38 - 91 69 ± 16 38 - 107 47 ± 9 27 - 66 Abaxial epidermis cell height (µm) 19 ± 2 14 - 24 22 ± 2 15 - 25 14 ± 2 11 - 18 Abaxial epidermis cell width (µm) 18 ± 2 14 - 21 22 ± 2 17 - 27 16 ± 3 12 - 23 Number of big bundles 4 ± 0 4 - 4 4 ± 0 4 - 4 4 ± 0 4 - 4 Sclerenchyma cap area (µm2) 2614 ± 636 1323 - 3710 2219 ± 375 1596 - 2929 1459 ± 409 895 - 2303 Phloem area (µm2) 1230 ± 245 669 - 1645 720 ± 337 338 - 1965 609 ± 87 442 - 768 Xylem area (µm2) 2477 ± 586 1657 - 3946 1234 ± 491 621 - 2323 1059 ± 249 496 - 1509 BIOLOGICA NYSSANA 8 (1)  September 2017: 15-22 Raca, I. et al.  Comparative anatomical study of the taxa from… 20 length of arms and their curving degree differ amongst the species (E r o l , 2007). Crocus heuffelianus cross sections have the longest arms with conspicuously high curving degree. This population is also characterized by the most prominent vascular bundles. The features referred to leaf blade are of a higher value as well (section height and length, arm length, white stripe width, lacuna area). While C. tommasinianus can be distinguished from the other two species by the smallest vascular bundles, thinnest spongy layer and wide white stripe, C. kosaninii has middle - sized vascular bundles and the biggest epidermal cells (both adaxial and abaxial). Also, disintegration of parenchyma cell walls in lacuna area is more frequent in C. tommasinianus and C. kosaninii populations in comparision to C. heuffelianus. Further researches should be carried out including more populations, other closely related species and surveying also the ecological conditions in each locality. This would allow us to evaluate which impact habitat factors have on the anatomy of leaves as well as the potential of leaf anatomy features as diagnostic characters. Conclusion As could be seen from the literature, no detailed comparative study of series Verni had been done before. Therefore, the major aim of this paper was to introduce a leaf anatomy comparison of series Verni taxa in Serbia and to highlight the differences between them. This study is a basis for further researches within series Verni. Acknowledgements. The work was funded by the Ministry of Education, Science and Technological Development of Republic of Serbia (project no. 173030). Fig. 4. Arm details on leaves cross sections of Crocus ser. Verni species from Serbia (20x): a-C. heuffelianus; b-C. kosaninii; c-C. tommasinianus (ade-adaxial epidermis, p-palisade parenchyma, s-spongy parenchyma, abe-abaxial epidermis, st-stomata) Fig. 5. Big bundles on leaves cross sections of Crocus ser. Verni species from Serbia (40x): a-C. heuffelianus; b-C. kosaninii; c-C. tommasinianus; (sc-sclerenchyma cap; ph-phloem; xy-xylem) BIOLOGICA NYSSANA 8 (1)  September 2017: 15-22 Raca, I. et al.  Comparative anatomical study of the taxa from… 21 Fig 6. Box plots of quantitative anatomic characters of leaves of Crocus ser. Verni species from Serbia: a-C. heuffelianus; b-C. kosaninii; c-C. tommasinianus (Ad ec-Adaxial epidermal cell; Ab ec-Abaxial epidermal cell) 200 700 1200 c b a Section height (µm) 2000 4000 6000 8000 10000 c b a Section Length (µm) 500 1500 2500 3500 4500 c b a Arm Length (µm) 400 600 800 1000 c b a White Stripe Width (µm) 0,1 0,2 0,3 0,4 0,5 0,6 0,7 c b a Lacuna area (mm2) 10 15 20 25 c b a Ad ec height (µm) 12 17 22 27 32 c b a Ad ec width (µm) 10 15 20 25 30 c b a Ab ec height (µm) 10 15 20 25 30 c b a Ab ec width (µm) 25 35 45 55 c b a Palisade cell height (µm) 10 15 20 c b a Palisade cell width (µm) 30 55 80 c b a Palisade tissue height (µm) 10 20 30 c b a Spongy cells height (µm) 10 20 30 c b a Spongy cells width (µm) 20 45 70 95 c b a Spongy tissue height (µm) 400 1400 2400 3400 c b a Xylem area (µm2) 200 700 1200 1700 c b a Phloem area (µm2) 500 2500 4500 c b a Sclerenchyma cap area (µm2) BIOLOGICA NYSSANA 8 (1)  September 2017: 15-22 Raca, I. et al.  Comparative anatomical study of the taxa from… 22 References Akan, H., Eker, I., Satil, F. 2007: The morphological and anatomical properties of endemic Crocus leichtlinii D. 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