Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(4): 129-134, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1354 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Aykut Yilmaz, Yudum Yeltekin (2021) Karyomorphology of some Cro- cus L. taxa from Uşak province in Turkey. Caryologia 74(4): 129-134. doi: 10.36253/caryologia-1354 Received: June 29, 2021 Accepted: November 30, 2021 Published: March 08, 2022 Copyright: © 2021 Aykut Yilmaz, Yudum Yeltekin. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits 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. Karyomorphology of some Crocus L. taxa from Uşak province in Turkey Aykut Yilmaz*, Yudum Yeltekin Department of Molecular Biology and Genetics, Faculty of Science and Arts, Uşak Uni- versity, 64200, Uşak, Turkey *Corresponding author. E-mail: aykut.yilmaz@usak.edu.tr Abstract. The increasing number of new taxa for each day and the presence of the samples exhibiting variable characters depend on this situation make very problemat- ic the genus Crocus as taxonomic and phylogenetic. For this reason, the many studies based on PCR, DNA barcoding and cytogenetics are applied to provide contribution for taxonomic problems and phylogenetic relationships of the genus Crocus. In this study, detailed karyotypic investigation of four taxa (C. pallasii Goldb. subsp. pallasii, C. olivieri J.Gay subsp. olivieri, C. fleischeri J.Gay and C. uschakensis Rukśans) belong- ing to Uşak province in Turkey was carried out and compared with the studies made previously. The somatic chromosome numbers of studied taxa were found to be 2n=14 for C. pallasii subsp. pallasii, 2n=8 for C. olivieri subsp. olivieri, 2n=20 for C. fleischeri and 2n=20 for C. uschakensis. C. uschakensis has only satellite on the short arm of chromosome 7. Some differences with previous studies in aspect of chromosome num- ber and morphology were determined in this study. Furthermore, there is no enough literature information on Crocus uschakensis and it was provided with this study based on detailed chromosomal investigation. Keywords: Crocus, cytogenetics, C. pallasii subsp. pallasii, C. olivieri subsp. olivieri, C. fleischeri and C. uschakensis. INTRODUCTION The genus Crocus L. belonging to the family Iridaceae is represented by about 200 species and show distribution from western Europe and north west Africa to western China (Mathew 1982; Harpke et al. 2016; Saxena 2016; Roma-Marzio et al. 2018). Especially, the Mediterranean region extending east- ward into the Irano-Turanian region is the place containing the majority of the species in the genus Crocus (Saxena 2016). Turkey is one of the most important countries with species number and endemism rate for the Crocus taxa. The genus Crocus is systematically very problematic. The variable char- acters caused by environmental factors due to extensive variety of habitats is the one of the most important reasons for taxonomic problems. Further- more, intermediate characters caused by introgression as a result of hybrid- ization is observed frequently in closely related species (Harrison and Lar- 130 Aykut Yilmaz, Yudum Yeltekin son 2014; Kerndorff et al. 2016; Yılmaz 2021b). The number of the taxa belonging to the genus Crocus have recently doubled with the detailed field studies particu- larly in Turkey (Addam et al. 2019). While Uslu et al. (2012) states that there are almost 70 Crocus taxa which is their 31 endemic to Turkey, Gedik et al. (2017) states that Turkey is represented by 132 taxa which is their 108 endemic. However this caused increase the taxonomic problems at the infraspecific level. As a result, it is pro- posed that subspecies status can not be maintained and anymore must be categorized as species (Harpke et al. 2016; Addam et al. 2019). Another important situation which increase taxo- nomic problems in the genus Crocus is the changes observed in the chromosome number. Studies on the karyotypes of Crocus taxa show chromosome number changes from 2n=6 to 2n=70 within the genus (Brighton et al. 1973; Uslu et al. 2012; Harpke et al. 2013). Further- more, it was observed that some species from different localities show variation in chromosome number (Uslu et al. 2012; Karamplianis et al. 2013). All of these makes problematic the genus and doubt- ful the species identification within the genus. In addi- tion to cytogenetic studies, many molecular studies based on different PCR methods and DNA barcoding containing nuclear and cpDNA sequences show reality of this situation (Petersen et al. 2008; Harpke et al. 2013; Erol et al. 2014; Yılmaz 2021 a,b). In this study, the Crocus taxa (C. pallasii subsp. pallasii, C. olivieri subsp. olivieri, C. fleischeri and C. uschakensis) from Uşak province were detailed exam- ined based on their somatic chromosome numbers, karyotypic descriptions, length ranges, haploid comple- ments and other morphometric parameters such as IC (Centromeric index), A1 (Intrachromosomic asymmetric index), A2 (Interchromosomic asymmetric index). One of the most important reasons for the choos- ing this region in the study is that there is not enough information about Crocus taxa in Uşak which is one of the regions with the highest species diversity, in addition to cytological data. MATERIALS AND METHODS Plant samples examined in this study were col- lected from Uşak province in Turkey. There are four taxa containing C. pallasii subsp. pallasii, C. olivieri subsp. olivieri, C. fleischeri and C. uschakensis in this study (Table 1). Root tips for plant samples belonging to each taxa were used to provide somatic metaphase chromosomes. Firstly, root tips that are convenient for working were put into small glass bottles and then pre- treated in α-monobromobromonaphthalene for 14-16 h at 4°C. After the first treatment, root tips were fixed with Carnoy solution for overnight. Fixed root tips were transferred to bottles with 70% alcohol and stored at 4°C until use. After the all treatments, hydrolysis with 1 N HCl solution was done at 60°C between 14-16 min. Prior to staining, root tips were washed with distilled water. They were stained with 2% aceto-orcein for two hours and then squashed with 45% acetic acid to obtain metaphase chromosomes. Preparations containing the best metaphase chromosomes were photographed using LEICA DM LB2 microscope with camera. The meas- urements detailed based on small-long arm length and arm ratio were made for each taxa represented by the least five plates. Chromosomes for each taxa examined were classified according to the nomenclature of Levan et al. (1964) and Stebbins (1971). In addition to somatic chromosome number, karyotypic description and length ranges, karyotype asymmetry parameters including cen- tromeric index (IC), intrachromosomic asymmetry index (A1) and interchromosomic asymmetry index (A2) were calculated according to Romero Zarco (1986). RESULTS AND DISCUSSION All samples examined were provided from Uşak province in Turkey (Table 1). This study aims to ana- lyze the karyotypes of four Crocus taxa and to deter- mine the relationships among the taxa studied in addi- tion to Crocus taxa examined previously according to chromosome number and other morphometric param- eters. At the same time, an important Crocus species: C. uschakensis which is not well known and not have sufficient literature information was examined for the first time in detail. The following cytological features belonging to four Crocus taxa examined were observed in this study. Table 1. Species names, localities and chromosome numbers of studied species. Species Locations Somatic chromosome number C. pallasii subsp. pallasii 5-10 km after Kaşbelen/Uşak 2n=14 C. olivieri subsp. olivieri Kent forest/Uşak 2n=8 C. fleischeri 5-10 km after Kaşbelen/Uşak 2n=20 C. uschakensis 5-10 km after Kaşbelen/Uşak 2n=20 131Karyomorphology of some Crocus L. taxa from Uşak province in Turkey C. pallasii subsp. pallasii Plant samples for C. pallasii subsp. pallasii were collected from Kaşbelen around in Uşak province. The chromosome number of C. pallasii subsp. pallasii was determined as 2n=14 (Table 1, Figure 1-2). Karyotypic description consists of 10 metacentric and 4 submetacen- tric chromosomes (4sm+10m) (Table 2). C. pallasii subsp. pallasii evaluated within the series Crocus show wide distribution from Serbia and Mac- edonija to Turkey (Karamplianis et al. 2013). Chromo- some number have been reported in previous studies as 2n=14 and 2n=16 for this taxon (Šopova 1972; Brighton et al. 1973; Brighton 1977; Randelovic et al. 2007; Can- dan et al. 2009). Furthermore, it was determined the both chromosome numbers in the study based on three different populations of C. pallasii subsp. pallasii by Karamplianis et al. (2013). The results provided from the population belonging to Samos Island show similar- Figure 1. Somatic chromosomes of (a) C. pallasii subsp. pallasii; (b) C. olivieri subsp. olivieri; (c) C. fleischeri; (d) C. uschakensis. 132 Aykut Yilmaz, Yudum Yeltekin ity with this study according to chromosome number (2n=14) and karyotypic description (4sm+10m). C. pallasii subsp. pallasii had the smallest chromo- somes set (1.93–5.17 μm) in addition to lowest haploid com- plement value (22.01 µm) among the studied taxa (Table 2). A1 had the lowest value (0.27) among all the studied taxa, while A2 had the second highest value (0.36) after C. uschakensis. The highest centromeric index value with 41.97 was observed in C. pallasii subsp. pallasii (Table 2). C. olivieri subsp. olivieri Crocus olivieri subsp. olivieri is distributed in Tur- key, Macedonia, Southeast Romania, South Bulgaria, Albania and Greece (Yüzbaşıoğlu 2012). C. olivieri subsp. olivieri chromosome number was found to be 2n=8 (Table 1, Figure 1-2). All chromosomes belonging to this taxon examined were submetacentic. Chromosome counts for C. olivieri subsp. olivieri from different locations were done previously and reported as 2n=6 (Mather 1932; Brighton et al. 1973; Uslu et al. 2012). In this aspect, chromosome number of this taxon from Uşak location show differences from other study results. Chromosome length range was between 7.30–12.80 µm (Table 2). In other words, the longest chromosomes set was observed in C. olivieri subsp. olivieri, although it has the least chromosome number in comparison to oth- er taxa examined. Furthermore the second highest hap- loid complement value with 39.51 µm was found in this taxon (Table 2). The lowest centromeric index value with 26.43 and highest A1 value (0.65) were determined in C. olivieri subsp. olivieri (Table 2). C. fleischeri Chromosome number of C. fleischeri was found to be 2n=20 (Table 1, Figure 1-2). Crocus fleischeri is distrib- uted South and West Anatolia regions of Turkey. Chro- mosome number of this taxon which is endemic was pre- viously reported as 2n=20 (Mathew 1984; Candan et al. 2009). Furthermore, it is stated by Candan et al. (2009) that all of the chromosomes are submetacentric except 3 chromosomes being metacentric. In this study, karyotyp- ic description of this taxon consist of 8 metacentric and 12 submetacentric chromosomes (12sm+8m) (Table 2). Chromosome length range and haploid complement value for this taxon have the lowest value after C. pallasii subsp. pallasii with 2.42–4.22 µm and 34.35 µm respec- tively (Table 2). Chromosomal asymmetry index, A1 and A2 were determined as 0.36 and 0.17, respectively. The lowest A2 Figure 2. Idiograms of (a) C. pallasii subsp. pallasii; (b) C. olivieri subsp. olivieri; (c) C. fleischeri; (d) C. uschakensis. (Bar : 2 µm). 133Karyomorphology of some Crocus L. taxa from Uşak province in Turkey value was observed in C. fleischeri with 0.17 (Table 2). C. uschakensis C. uschakensis is an endemic species and there is not enough information about this taxon. This work repre- sents the first detailed chromosomal study on C. uschak- ensis. Rukśans (2014) states that they observed this taxon on low mountains belonging to North of Uşak. Similar- ly, we observed and collected this taxon on North parts of Uşak province. Chromosome number of C. uschakensis was found to be 2n=20 (Table 1, Figure 1-2). which consist of 6 metacentric, 12 submetacentric and 2 acrocentric chro- mosomes (2a+12sm+6m) (Table 2). Furthermore, satellite was observed on the short arm of chromosome 7. Chro- mosome length range was between 2.87–7.82 µm (Table 2). In other words, the longest chromosomes set was observed in C. uschakensis after C. olivieri subsp. olivieri which has the least chromosome number among taxa examined. The longest haploid complement was deter- mined in C. uschakensis with 50.93 µm. The second low- est centromeric index value with 34.49 and highest A2 value with 0.37 was observed in this taxon (Table 2). The variation in chromosome counts for two spe- cies were observed in this study. While the chromosome number for C. pallasii subsp. pallasii have been reported as 2n=14 and 2n=16 in previous studies, it was deter- mined 2n=14 in present study. Similarly, another differ- ent chromosome count was found in C. olivieri subsp. olivieri as previously reported as 2n=6, whereas in the present study it was found as 2n=8. It is observed wide range of variation on the chro- mosomes counts (from 2n=6 to 70) and morphology of the species belonging to the genus Crocus (Brighton et al. 1973; Uslu et al. 2012; Harpke et al. 2013). The most probably reasons of variations in the chromosome num- ber and morphology of the species are geographical dif- ferences, environmental factors caused by locations of taxa, hybridization, polyploidization and aneuploidy. Geographical differences and variations in environmen- tal factors caused by geographical differences could be reason of chromosome count differences in C. olivieri subsp. olivieri. Similarly, Karamplianis et al. (2013) examining the C. pallasii subsp. pallasii in three differ- ent populations states that chromosome numbers for this taxon change as 2n=14 and 2n=16. Other an important taxon, C. uschakensis were examined in detailed according to chromosome number and other morphometric parameters. Furthermore, in addition to contribution for literature based on its kar- yotype informations determined, it was firstly evaluated the relationships of C. uschakensis with other Crocus taxa. Besides the first detailed karyotype analysis, satel- lite chromosome was determined in C. uschakensis. Turkey with 132 taxa which is their 108 endemic is found in very rich region according to species num- ber and diversity in the world (Gedik et al. 2017) and accepted as the center of species diversity for the genus Crocus (Erol et al. 2012; Candan and Özhatay 2013). The genus Crocus in the world in comparison to Tur- key according to their species number and diversity, it can be said that Turkey is the center of genetic variation for the genus. Furthermore, the high endemism rate for the Crocus species make very important the Turkey in aspect of studies on the genus. In this study, four species belonging to Uşak prov- ince located in the Western Anatolia region which is the richest region of Turkey according to species diver- sity were examined caryologically. One of the most important gains of this study is to obtain literature information on Crocus uschakensis which is an endem- ic species, in addition to determining the species diver- sity of the region. ACKNOWLEDGMENTS The authors would like to thank Uşak University Directorate of Scientific Research Projects (BAP-2021/ MF003) for providing financial support and also special thanks to Ahmet Kahraman for helping to identify the plant material. Table 2. Karyotypic descriptions, length ranges and other morphometric parameters of studied Crocus species. Species Karyotypic description Length range (µm) Haploid complement (µm) IC A1 A2 C. pallasii subsp. pallasii 4sm+10m (1.93 – 5.17) 22.01 41.97 0.27 0.36 C. olivieri subsp. olivieri 8sm (7.30 – 12.80) 39.51 26.43 0.65 0.24 C. fleischeri 12sm+8m (2.42 – 4.22) 34.35 38.80 0.36 0.17 C. uschakensis 2a+12sm+6m (2.87 – 7.82) 50.93 34.49 0.47 0.37 134 Aykut Yilmaz, Yudum Yeltekin REFERENCE Addam K, Bou-Hamdan M, Sabbagh N, Takkoush J, Hout K. 2019. Crocus baalbekensis K. Addam and M. Bou Hamdan sp. Nov and its three forms (IRIDACE- AE), new endemic species and forms from Lebanon, joined the Lebanese Flora. MOJ Eco Environ Sci. 4(2): 75–83. Brighton CA, Mathew B, Marchant CJ. 1973. Chromo- some Counts in the Genus Crocus (Iridaceae). Kew Bulletin. 28(3):451–464. Brighton CA. 1977. Cytology of Crocus sativus and its allies (Iridaceae). Plant Syst Evol. 211:149–154. Candan F, Şik L, Kesercioğlu T. 2009. Cytotaxonomical studies on some Crocus L. taxa in Turkey. 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