Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 155-161, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-184 Citation: H. Sharghi, M. Azizi, H. Moazzeni (2020) A karyological study of some endemic Trigonella species (Fabaceae) in Iran. Caryologia 73(1): 155-161. doi: 10.13128/caryologia-184 Received: March 8, 2019 Accepted: February 23, 2020 Published: May 8, 2020 Copyright: © 2020 H. Sharghi, M. Azizi, H. Moazzeni. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, 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. A karyological study of some endemic Trigonella species (Fabaceae) in Iran Hamidreza Sharghi1,2, Majid Azizi1,*, Hamid Moazzeni2 1 Department of Horticultural Sciences, Faculty of Agriculture, Ferdowsi University of Mashhad, P.O.Box: 917751163, Mashhad, Iran 2 Department of Botany, Research Center for Plant Sciences, Ferdowsi University of Mash- had, P.O.Box 91779-48974, Mashhad, Iran *Corresponding author. E-mail: azizi@um.ac.ir Abstract. Karyotypes of nine populations belonging to six endemic species of the genus Trigonella (Trifolieae/Fabaceae) studied in this survey. All studied species are perennial and recorded only from Northeast of Iran. Excluding the karyotype of Trigo- nella subenervis, which was previously reported, all of the other species studied here (six species) for the first time. Our results present that all assessed genotypes are dip- loid with 2n=2x=16 and the chromosomal basis of x=8. In addition to the chromo- some counts, length of long and short arms of the chromosome and their ratios ana- lyzed and presented in this study. Keywords. Chromosome number, Cytogenetic, Karyotype, Khorassan, Trifolieae, Trigonella. INTRODUCTION The genus Trigonella L. is a member of the tribe Trifolieae of the fam- ily Fabaceae, with about 135 species worldwide, most of which distributed in the dry regions around the Mediterranean region extended to West Asia, and naturalized in North America. Only two species being present in South Aus- tralia (Mabberley 1997). Trigonella consists of annual or perennial herbs with pinnately trifo- liate leaves, a campanulate or tubular calyx with two large and three small equal lobes, diadelphous stamens, uniform anthers, terminal stigma and ovary with numerous ovules (Širjaev 1928-1932; Hutchinson 1964; Dangi et al. 2016). According to Rechinger (1984), the genus represented by 58 species in to Flora Iranica area. This number has increased to about 66 species as a result of recent researches (Hamzeh’ee 2000; Janighorban 2004; Badrzadeh and Ghafarzadeh-Namazi 2009; Ranjbar, Karamian, and Hajmoradi 2012; Ranjbar and Hajmoradi 2012, 2015, 2016). Of which, 48 taxa (15 endemics (32%)) accommodated in 12 sections growing in Iran; 14 of those are peren- nial species of Trigonella sect. Ellipticae (Boiss.) Sirj. In Flora Iranica account (Rechinger 1984), section Ellipticae is repre- sented with seven perennial species in Iran. The characteristics of the section 156 Hamidreza Sharghi, Majid Azizi, Hamid Moazzeni Ellipticae are: perennial species with entire or dentate stipules, calyx campanulate, petals yellow or rarely white with violet veins, sometimes completely dark violet, standard without interlocking projection, fruit a legume which is different in shape and size, elliptic or lanceolate to oblong, beakless, generally transversely veined, wing- less or with thin wing and smooth seeds. Several cytological investigations have been conducted on approximately a hundred Trigonella species (Agarwal and Gupta 1983; Ahmad et al. 1999; Astanova 1981; Aykut et al. 2009; Bal 1990; Bidak and Amin 1996; Darlington and Wylie 1955; Dundas et al. 2006; Ghosh 1980; Kumari and Bir 1990; Ladizinsky and Vosa 1986; Martin et al. 2006, 2008, 2010, 2011a, 2011b; Pavlova 1996; Ranjbar et al. 2011a, 2011b, 2015; Singh and Roy 1970; Singh and Singh 1976; Tutin and Heywood 1964; Yilmaz et al. 2009). The reported chromosome numbers of the genus Trigonella are 2n=14, 16, 18, 24, 28, 30, 32, 42, 44, 46 and 48. To contribute to the karyological study of the genus, we carried out a karyological study on some peren- nial endemic species collected from different regions in East and Northeast of Iran. This study aimed to verify the chromosome numbers of some endemic Trigonella species recently reported in Iran. In this contribution, we report the somatic chromosome numbers of six taxa (nine populations), that five species are determined for the first time. MATERIAL AND METHODS The chromosome number were analyzed in nine population of Trigonella. The nomenclature of taxa, collection data, and vouchers are given in Table 1. The mitotic chromosome numbers were studied in three populations of T. subenervis Rech. f., two populations of T. binaloudensis and one population from each of T. lasi- ocarpa, T. stipitata, T .heratensis and T. Torbatejamensis. Seed materials were collected between the years of 2016 and 2018 from natural habitats. Voucher specimens were deposited at the Ferdowsi University of Mashhad Her- barium (FUMH), Iran. For karyological observation, to accelerate germina- tion, the seed surfaces were abraded with emery paper. Seeds were sown on wet filter paper in Petri dishes at room temperature. The seeds germinated after 2-3 days. One cm fresh root tip cells were used to study the somatic chromosomes. The root tips pretreated in an ice-water mixture for 24 hours. Afterward, they fixed in Carnoy’s fixative (ethanol: acetic acid 3:1) for 24 h at 4 °C (Fukui and Nakayama 1996). The root tips were washed in distilled water to remove the fixative, then hydrolyzed in 1N HCl for 13–15 minutes at room tem- perature, and finally stained with 2% aceto-orcein for two hours. The slides were created using the squash method. The prepared slides were slightly heated under an alco- hol frame for 1–2 s before observation. Photographs of chromosomes were taken using a Nikon Eclipse Ni-u (Tokyo, Japan) photomicroscope equipped with Nikon Ds-Fi3 digital camera. Chromosome counts were made from well-spread metaphases in intact cells, by direct observation, and from photomicrographs. To ensure for chromosome number, a minimum of five cells, at somat- ic metaphase, observed (Figure 1). Karyotypic analyses were conducted on IdeoKar software (version 1.2) to cal- culate karyotypic parameters and generate ideograms (Mirzaghaderi and Marzangi 2015). Karyotype formu- lae and nomenclature followed Levan et al. (1964), and karyotype asymmetry followed Stebbins (1971). Table 1. Iranian endemic species of Trigonella analyzed in this study, their locations, and voucher specimens data. Taxa Location Collection Date Elevation (m) Herbarium number T. binaloudensis* population 1 SW Chenaran, Ferizi towards Binaloud mountains 2016/05/30 1730 46443 population 2 NW Sabzevar, W Jalambadan, in Mnts. near Chromite mine 2018/05/29 1770 46323 T .heratensis S Fariman, between Torbate-Heydariyeh & Fariman 2016/06/06 1685 45959 T. lasiocarpa* NE Birjand, Now-Qand towards Bidesk 2016/05/24 2320 46442 T. stipitata* N Mashhad, E Chenaran, Mian-Marq 2016/05/25 1420 25771 T. subenervis* population 1 N Torbate-Heydariyeh, Khomari pass 2016/05/23 1851 46441 population 2 N Kashmar, NE hills of Chalpu village 2017/06/06 1879 46446 population 3 N Shirvan, 12 km from Lowjalli toward Namanlu village 2017/06/24 1820 45844 T. torbatejamensis* NE Torbate-Jam, between Saleh-Abad & Gush-Laqar, 2016/05/04 750 45958 157A karyological study of some endemic Trigonella species (Fabaceae) in Iran RESULTS Our investigations comprised nine populations belonging to 6 species of the genus Trigonella, of which data for five species reported here for the first time. The chromosomes of these taxa at mitotic metaphase shown in Figure 1. Detailed karyotypic parameters, formu- lae, and asymmetry are summarized in Table 2. In this study, the basic chromosome number of all taxa is x=8, and all of them are diploid. Trigonella binaloudensis Ranjbar & Karamian Population 1 (SW Chenaran): The chromosome number was 2n=2x=16 (Figure 1e1). Haploid chromosome length was 27.73 μm. Chro- mosome length varied from 2.75 to 4.83 μm, and arm ratio from 1.06 to 1.88. The chromosome complement at mitotic metaphase consisted of 14 median region and two submedian region chromosomes. Karyotypic asym- metry was 1A. Population 2 (NW Sabzevar): The chromosome number was 2n=2x=16 (Figure 1e2). Haploid chromosome length was 24.03 μm. Chro- mosome length varied from 2.15 to 3.72 μm, and arm ratio from 1.030 to 1.82. The chromosome complement at mitotic metaphase consisted of 14 median region and two submedian region chromosomes. Karyotypic asym- metry was 1A. Trigonella heratensis Rech.f The chromosome number of T. heratensis was 2n=2x=16 (Figure 1c). Haploid chromosome length was 22.98 μm. Chromosome length varied from 2.46 to 3.40 Figure 1. Photographs of somatic metaphase chromosomes of nine populations belonging to six species of the genus Trigonella collected from northeast of Iran. (a, b) T. binaloudensis from two locality; (c) T. heratensis; (d) T. lasiocarpa; (e) T. stipitata; (f, g, h) T. subenervis from three locality; (i) T. torbatejamensis. Scale bars = 10 μm. 158 Hamidreza Sharghi, Majid Azizi, Hamid Moazzeni μm, and arm ratio from 1.02 to 1.69. The chromosome complement at mitotic metaphase consisted of 16 medi- an region chromosomes, and karyotypic asymmetry was 1A. Trigonella lasiocarpa Ranjbar & Z.Hajmoradi The chromosome number of T. lasiocarpa was 2n=2x=16 (Figure 1a). Haploid chromosome length was 28.9 μm. Chromosome length varied from 2.44 to 4.44 μm, and arm ratio from 1.09 to 1.51. The chromosome complement at mitotic metaphase consisted of 16 medi- an region chromosomes, and karyotypic asymmetry was 1A. Trigonella stipitata Ranjbar & Joharchi The chromosome number of T. stipitata was 2n=2x=16 (Figure 1b). Haploid chromosome length was 19.17 μm. Chromosome length varied from 1.92 to 3.13 μm, and arm ratio from 1.02 to 1.44. The chromosome complement at mitotic metaphase consisted of 16 medi- an region chromosomes, and karyotypic asymmetry was 1A. Trigonella subenervis Rech.f Population 1 (N Torbate-Heydariyeh): The chromosome number was 2n=2x=16 (Figure 1f1). Haploid chromosome length was 19.87 μm. Chro- mosome length varied from 1.94 to 3.00 μm, and arm ratio from 1.01 to 1.94. The chromosome complement at mitotic metaphase consisted of 14 median region and two submedian region chromosomes. Karyotypic asym- metry was 1A. Population 2 (N Kashmar): The chromosome number was 2n=2x=16 (Figure 1f3). Haploid chromosome length was 20.68 μm. Chro- mosome length varied from 2.03 to 3.04 μm, and arm ratio from 1.01 to 1.52. The chromosome complement at mitotic metaphase consisted of 16 median region chro- mosomes, and Karyotypic asymmetry was 1A. Population 3 (N Shirvan): The chromosome number was 2n=2x=16 (Figure 1f3). Haploid chromosome length was 28.04 μm. Chro- mosome length varied from 2.67 to 4.18 μm, and arm ratio from 1.01 to 1.44. The chromosome complement at mitotic metaphase consisted of 16 median region chro- mosomes, and Karyotypic asymmetry was 1A. Trigonella torbatejamensis Ranjbar The chromosome number of T. torbatejamensis  was 2n=2x=16 (Figure 1d). Haploid chromosome length was 26.77 μm. Chromosome length varied from 2.42 to 4.50 μm, and arm ratio from 1.05 to 2.21. The chromo- some complement at mitotic metaphase consisted of 14 median region and two submedian region chromosomes. Karyotypic asymmetry was 2A. DISCUSSION All taxa in the present study showed the same basic chromosome number x=8 and same polyploidy level, which is congruent with those previously reported by Ranjbar et al. (2016) in Trigonella subenervis and six other species of the section. Ellipticae. This section comprises most of the perennial species of the genus Trigonella and widely distributed in Iran, Afghanistan and Middle Asia. Table 2. Somatic chromosome numbers and Karyotypes of nine Trigonella taxa. HCl: haploid chromosome length, Cl: chromosome length, AR: arm ratio(L/S), RL%: relative length of the chromosome, CI: centromeric index. Species 2n X HCL (μm) CL μm) AR RL% CI Karyotype formulae Karyotypic asymmetry (Stebbins) T. binaloudensis* (1) 16 8 27.73 2.75-4.83 1.06-1.88 4.96-8.70 0.35-0.49 14m+2sm 1A T. binaloudensis* (2) 16 8 24.03 2.15-3.72 1.03-1.82 4.48-7.74 0.35-0.49 14m+2sm 1A T. heratensis 16 8 22.98 2.46-3.40 1.02-1.69 5.36-7.39 0.37-0.50 16m 1A T. lasiocarpa* 16 8 28.90 2.41-4.44 1.09-1.51 4.17-7.68 0.40-0.48 16m 1A T. stipitata* 16 8 19.17 1.92-3.13 1.02-1.44 5.00-8.15 0.41-0.49 16m 1A T. subenervis* (1) 16 8 19.87 1.94-3.00 1.01-1.94 4.88-7.54 0.34-0.50 14m+2sm 1A T. subenervis* (2) 16 8 20.68 2.03-3.04 1.01-1.52 4.91-7.34 0.40-0.50 16m 1A T. subenervis* (3) 16 8 28.04 2.67-4.18 1.01-1.44 4.75-7.45 0.41-0.50 16m 1A T. torbatejamensis* 16 8 26.77 2.42-4.50 1.05-2.21 4.51-8.41 0.31-0.49 14m+2sm 2A 159A karyological study of some endemic Trigonella species (Fabaceae) in Iran All of the studied taxa in this study, analyzed karyo- typically for the first time, covering chromosome length, karyotype formulae, and asymmetry (Table and Figure 2). In term of karyotypic parameters, Our results sup- port the results reported by Riasat (2015) about Trigo- nella elliptica, a perennial species from section Ellip- ticae. In later study, the karyotype formulae reported as 14m+2sm, 16m, 8m+8sm, and 12m+4sm in different genotypes. We found the same variations in karyotype formulae and asymmetry among different species and populations that are shown in Table 2. Karyotypic asymmetry in most of the studied taxa, was as A1 but in T. torbatejamensis which is A2. Karyo- type formulae in most of the specimens were as 16m and in four specimens (T. torbatejamensis, two populations of T. binaloudensis and one population of T. subenervis) was as 14m+2sm. The incongruences may result from variations among different populations and chromosome preparation treatments. It seems that chromosome varia- tion and evolution of Trigonella species need more com- parative cytological studies based on more collections from different populations. 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