Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(3): 13-19, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/10.13128/caryologia-197 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: İ. Genç, Ş. Kültür (2020) Karyo- logical analysis of twelve Euphorbia species from Turkey. Caryologia 73(3): 13-19. doi: 10.13128/10.13128/caryo- logia-197 Received: March 26, 2019 Accepted: April 13, 2020 Published: December 31, 2020 Copyright: © 2020 İ. Genç, Ş. Kültür. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Karyological analysis of twelve Euphorbia species from Turkey İlker Genç*, Şükran Kültür Department of Pharmaceutical Botany, Faculty of Pharmacy, İstanbul University, İstanbul, Turkey *Corresponding author. E-mail: gencilker1@gmail.com Abstract. Karyotypes of 12 Euphorbia species were studied and described for the first time; Euphorbia cheiradenia, E. pannonica, E. pestalozzae, E. petrophila, E. pisidica, E. thessala and E. yildirimli. Karyological analyses indicate relationships among the spe- cies with respect to their asymmetry indices. Most of the investigated taxa are diploids with 2n = 2x = 18. E. macroclada and E. smirnovii showed tetraploid cytotypes 2n = 4x = 36. All karyotypes are symmetrical, consisting of metacentric and submetacen- tric chromosomes. The chromosomes range in size from 0.79 µm to 2.20 µm. The total haploid chromosome length (THL) ranges from 8.75 μm (E. terracina) to 16.78 μm (E. petrophila). Principal Coordinate Analysis with five uncorrelated parameters was per- formed to determine the karyological relationships among the taxa. Keywords: chromosome number, karyotype asymmetry, Pithyusa, Esula, tetraploid, karyosystematics. INTRODUCTION Genus Euphorbia (Euphorbiaceae), with over 2000 species, is the second- largest genus of flowering plants in the world (Bruyns et al. 2006; Horn et al. 2012). The use of Euphorbia species as medicine and poison are of greatest biocultural importance and the most-valued medicinal use of Euphorbia spe- cies is in the treatment of digestive and respiratory complaints, inflammation and injuries (Ernst et al. 2015). Additionally, Euphorbia taxa have been used in Turkish folk medicine to treat rheumatism, swelling and especially as a wart remover (Baytop 1984). There are also various biological effects as anti- oxidant, antimicrobial and wound healing activities (Barla et al. 2006, 2007; Özbilgin et al. 2018, 2019). The genus is represented in Turkey by two subgenera, E. subg. Chamaesyce Raf. and E. subg. Esula Pers., with a total of 120 taxa (Öztekin 2012; Genç and Kültür 2016). Euphorbia subg. Esula comprises about 480 species, most of which are annual or perennial herbs (Riina et al. 2013). According to the recent classification of Riina et al. (2013), the subgenus is represented by 14 sections in the Turkey (Genç and Kültür 2018). The chromosome numbers in these two subgenera are very variable. Var- ious chromosome numbers have been reported for the subg. Esula (2n = 10, 14 İlker Genç, Şükran Kültür 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 36, 40, 42, 44, 56, 60, 64, 72) and sect. Pithyusa (2n = 16, 18, 28, 36, 40, 72) worldwide (Riina et al. 2013). The chromosome numbers of 64 taxa, which are distributed in Turkey naturally have not been deter- mined yet. Up to now, the chromosome numbers of 12 Euphorbia taxa were reported from Turkey (Radcliffe- Smith 1976; Strid 1987; Kesercioglu et al. 1990; Iyer 1991; Vicens et al. 1991; Rice et al. 2015; Genç and Kültür 2017). Considering that the number of chromosomes of the vast majority of these species is not yet clear, there is a remarkable gap in this area. This study aimed to determine the chromosome numbers and karyomorphology of 11 perennial Euphor- bia sect. Pithyusa species distributed in Turkey. In addi- tion, E. terracina (sect. Pachycladae) was also included in the present study as an external group. MATERIALS AND METHODS Twelve Euphorbia species were analysed in this study. Seeds of 12 species were collected from mature capsules in their natural habitats as given in Table 1. Voucher specimens are deposited in İstanbul University, Faculty of Pharmacy Herbarium (ISTE). For each popu- lation, 9-27 mature seeds were selected from at least 5 individuals (inflorescence). The seeds germinated on wet filter paper in petri dishes at room temperature. The root tips meristems were excised at 9-9.30 am. Mitotic chromosomes were prepared from root tips and pre-treated with 8-Hydroxyquinoline at +4 °C for 24 h. Roots were fixed for a minimum of 2 h in abso- lute ethanol:glacial acetic acid, (3:1,v/v), hydrolysed at 60 °C in 1 N HCl for 15 min and stained with Feulgen reagent. Finally, root tips were squashed in a drop of 1% aceto-orcein. Permanent microscopic slides were pre- pared with Entellan (Rapid Mounting Medium / Merck Darmstadt Germany). Microphotographs of good qual- ity metaphase plates were taken using an Olympus BX53 (Tokyo, Japan) microscope equipped with a high-resolu- tion digital camera. Metaphase observations and chro- mosome measures were made using the image analysis systems Kameram (Argenit Microsystems, İstanbul, Tur- key). The chromosome number and karyotype details were studied in five to fourteen well-spread metaphase plates from different individuals; mean values were used for the analysis. Chromosome pairs were identified and arranged on the basis of chromosome/chromatid length and any other evident karyomorphological features. The nomen- clature used for describing karyotype followed Levan et Table 1. Localities and voucher data of Euphorbia taxa examined in the present study. Species Voucher specimens Previous counts (Rice et al. 2014) E. cheiradenia Boiss. & Hohen. Şanlıurfa, Tektek Mountain National Park, 460m, 18.6.2015, İ.Genç 2398, Ş.Kültür. -- E. macroclada Boiss. Eskişehir-Antalya road, roadsides, 10.8.2015, İ.Genç 2472. 18 E. niciciana Borbás ex Novák Karabük, Bolkuş village, 220 m, 03.vi.2015, İ.Genç 2290, G. Ecevit Genç. 18, 18+1 E. pannonica Host İstanbul, Dağyenice-Kalfaköy, roadsides, 110m, 18.vii.2016, İ.Genç 2477, G. Ecevit Genç. -- *E. pestalozzae Boiss. Antalya, Saklıkent, 1780m, 30.vii.2016, İ.Genç 2499. -- E. petrophila C.A.Meyer Kastamonu, Kastamonu-Sinop roadsides, 720m, 14.vii.2015, İ.Genç 2419, G. Ecevit Genç. -- *E. pisidica Hub.-Mor. & M.S.Khan Burdur, Altınyayla-Gölhisar road, Dirmil pass, 1585m, 23.vi.2014, İ.Genç 2114, G. Ecevit Genç. -- E. seguieriana Necker Ağrı, between Doğubeyazıt-Çaldıran, roadsides, 2600m, 24.vii.2015, İ.Genç 2453. 16, 18, 40 E. smirnovii Geltman Erzincan, Ergan mountain, 1510m, 25.vii.2015, İ.Genç 2468, A. Kandemir. 18 (Genç & Kültür 2017) E. thessala (Form.) Degen & Dörf. Kırklareli, Kırklareli-Pınarhisar roadside, 190m, 5.vi.2015, İ.Genç 2304, G. Ecevit Genç. -- *E. yildirimlii Dinç Eskişehir, Sivrihisar, Aşağıkepen village, gypsum slopes, 900m, 26.viii.2014, İ.Genç 2267, G. Ecevit Genç. -- E. terracina L. Bursa, Mudanya, Söğütlüpınar village, s.l., 7 vıı 2014, İ.Genç 2192, S. Yüzbaşıoğlu 18, 20, 36 * The species endemic to Turkey. 15Karyological analysis of twelve Euphorbia species from Turkey al. (1964). To determine the karyological relationships among taxa, we performed Principal Coordinate Analy- sis (PCoA) with fi ve uncorrelated parameters as sug- gested by Peruzzi and Altınordu (2014). Th ese param- eters are basic chromosome number (x), total haploid length (THL), mean centromeric asymmetry (MCA) it is calculated as the mean (L-S)/(L+S)×100 where, for each chromosome, L is the length of long arm and S is the length of short arm; coeffi cient of variation of chromo- some length (CVCL=(L+S)×100) and coeffi cient of varia- tion of centromeric index (CVCI =S/(L+S) ×100) (Paszko 2006; Peruzzi et al. 2009; Zuo and Yuan 2011; Peruzzi and Eroğlu 2013). Th e soft ware Past 3.03 (Hammer et al. 2001; Hammer 2018) was used to perform this analysis. Seed and gland morphology in the classifi cation of the genus Euphorbia is one of the most important tax- onomic characters. Th erefore, in order to evaluate the resulting PCoA graphic in terms of the seed and gland morphological characters, some symbols are given to the studied species (Table 2). Table 2. PCoA graphic symbols of some important taxonomic char- acteristics for Euphorbia. Seed surface smooth Seed surface not smooth Cyathial glands horned Cyathial glands hornless Figure 1. Somatic chromosomes of the studied taxa. (a) E. cheiradenia; (b) E. macroclada; (c) E. niciciana; (d) E. pannonica; (e) E. pestaloz- zae; (f ) E. petrophila; (g) E. pisidica; (h) E. seguieriana; (i) E. smirnovii; (j) E. thessala; (k) E. yildirimli; (l) E. terracina. (Scale bars 2 μm). 16 İlker Genç, Şükran Kültür Mitotic metaphase chromosomes are given in Fig- ure 1. Ideograms of these taxa are arranged in order of centromere position and then decreasing the length of homologue chromosome pairs (Figure 2). RESULTS Karyomorphological details (shortest chromosome length; longest chromosome length; mean long arm length; mean short arm length; karyotype formula) of 12 species of Euphorbia are listed in Table 3. The chro- mosome numbers, total haploid length, mean centro- meric asymmetry, coefficient of variation of chromo- some length and the coefficient of variation of centro- meric index of the species are also summarized in Table 4. Metaphase plates and their related ideograms of the studied species are presented in Figures 1 and 2. Karyotype analysis revealed the basic chromosome number x=9 for all species. Most of the species showed diploid cytotypes 2n = 18 but E. macroclada and E. smirnovii showed tetraploid cytotypes 2n = 4x = 36 (Fig- ure 1). The chromosomes were mainly metacentric with centromeres localized in the median position. The kar- yotypes of four species (E. cheiradenia, E. macroclada, E. smirnovii, E. yildirimli) included one submetacentric chromosome. Only karyotype of E. petrophila included two submetacentric chromosomes. The ratio of the shortest and the longest chromo- some lengths ranged from 0.79 μm (E. smirnovii) to 2.20 μm (E. petrophila). The ratio of the shortest and the longest total haploid chromosome length (THL) ranged from 8.75 μm to 16.78 μm in E. terracina and E. petroph- ila, respectively. The smallest mean short arm length (q) was observed in E. terracina (0.44 μm) and E. seguieri- ana (0.51 μm), the largest mean long arm length (p) was observed in E. petrophila (1.07 μm) (Table 3). Figure 2. Haploid ideograms of the studied taxa. (a) E. cheirad- enia; (b) E. macroclada; (c) E. niciciana; (d) E. pannonica; (e) E. pestalozzae; (f ) E. petrophila; (g) E. pisidica; (h) E. seguieriana; (i) E. smirnovii; (j) E. thessala; (k) E. yildirimli; (l) E. terracina. Table 3. Karyotype analysis of the investigated Euphorbia taxa. SC–LC q (μm) Mean (±SD) p (μm) Mean (±SD) p+q Mean (±SD) Karyotype formula E. cheiradenia 1.35–2.12 0.76(±0.10) 0.98(±0.14) 1.75(±0.22) 16 m + 2 sm E. macroclada 1.21–1.89 0.67(±0.09) 0.85(±0.13) 1.53(±0.20) 32 m + 4 sm E. niciciana 0.94–1.53 0.55(±0.08) 0.65(±0.10) 1.20(±0.17) 18 m E. pannonica 1.05–1.60 0.63(±0.08) 0.68(±0.08) 1.31(±0.16) 18 m E. pestalozzae 0.98–1.44 0.54(±0.04) 0.67(±0.09) 1.21(±0.13) 18 m E. petrophila 1.59–2.20 0.79(±0.12) 1.07(±0.12) 1.86(±0.18) 14 m + 4 sm E. pisidica 1.10–1.64 0.63(±0.06) 0.75(±0.10) 1.38(±0.16) 18 m E. seguieriana 0.88–1.42 0.51(±0.06) 0.61(±0.11) 1.11(±0.16) 18 m E. smirnovii 0.79–1.52 0.52(±0.10) 0.62(±0.14) 1.14(±0.22) 32m + 4 sm E. thessala 1.15–1.78 0.65(±0.06) 0.79(±0.13) 1.44(±0.19) 18 m E. yildirimli 1.13–1.74 0.66(±0.08) 0.80(±0.13) 1.47(±0.18) 16 m + 2 sm E. terracina 0.80–1.14 0.44(±0.03) 0.54(±0.07) 0.97(±0.09) 18 m Abbreviations:SC: the shortest chromosome length; LC: the longest chromosome length; p: mean long arm length; q: mean short arm length; SD: standard deviation; m: metacentric; sm: submetacentric. 17Karyological analysis of twelve Euphorbia species from Turkey Euphorbia petrophila, with relatively high intrachro- mosomal (MCA= 14.90) and E. pannonica (4.17) with low intrachromosomal asymmetry, also E. smirnovii with high interchromosomal (CVCL=19.65) and E. petrophila (9.49) low interchromosomal asymmetry were observed. Intrachromosomally, seed surface smooth and cyathial glands horned species are more asymmetrical than seed surface smooth and cyathial glands hornless species (Table 4). Twelve Euphorbia species were analysed by PCoA (cumulative variance explained by the first two axes: 88.21%). And, the species with the same seed and cyathi- al gland morphology tend to cluster together except E. smirnovii (Figure 3). The most important characters in recognizing these groups as distinct resulted THL, MCA and CVCL. DISCUSSION The number, size, and asymmetry of chromosomes are important characteristics that help explain the phy- logenetic relationships of species (Eroğlu et al., 2013). Karyotype data for seven taxa (3 endemic) are reported for the first time in the present study. Various chromosome numbers have been reported for the sect. Pithyusa (2n=16, 18, 28, 36, 40, 72) (Riina et al 2013). However, most of the investigated taxa in this study have the same chromosome number (2n=18), only E. macroclada and E. smirnovii showed tetra- ploid cytotypes 2n = 4x = 36. According to Riina et al. (2013) chromosome number of E. terracina is 18 and our results supported this report. However, even though E. terracina has 18 chromosomes, it differs from the other investigated taxa in smaller chromosomes (THL=8.75). Table 4. Chromosome numbers and karyo-morphometric param- eters, symmetry indices for investigated taxa. 2n x THL MCA CVCL CVCI E. cheiradenia 18 9 15.71 12.56 12.51 6.82 E. macroclada 36 9 13.73 11.37 12.89 7.73 E. niciciana 18 9 10.78 8.43 14.61 3.34 E. pannonica 18 9 11.77 4.17 12.40 1.53 E. pestalozzae 18 9 10.86 10.52 10.87 5.16 E. petrophila 18 9 16.78 14.90 9.49 10.44 E. pisidica 18 9 12.41 8.06 11.25 2.99 E. seguieriana 18 9 10.01 8.49 14.33 4.73 E. smirnovii 36 9 10.30 7.71 19.65 7.96 E. thessala 18 9 12.93 9.56 13.16 4.64 E. yildirimli 18 9 13.21 9.37 12.35 7.54 E. terracina 18 9 8.75 9.85 9.76 3.93 Abbreviations: THL: total haploid length; MCA: mean centromeric asymmetry; CVCL: coefficient of variation of chromosome length; CVCI: coefficient of variation of centromeric index. Figure 3. PCoA analysis based on five quantitative karyological parameters of investigated taxa. 18 İlker Genç, Şükran Kültür The chromosome number of E. macroclada was reported 2n = 2x = 18 by Lessani and Chariat-panahi (1979) and Chariat-Panahi et al. (1982). Tetraploid cyto- type is reported for the first time for this species. Differ- ent chromosome numbers were reported for E. seguieri- ana subsp. seguieriana (2n= 16, 18, 40) (Rice et al. 2015). In this study, the chromosome number of this species is reported to be 2n = 2x = 18. Euphorbia terracina is a bit separated from other taxa according to the PCoA analysis. So, our results sup- port the position of E. terracina in a distinct section (E. sect. Pachycladae), as suggested by Riina et al. (2013). 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