Karyotype analyses of ten sections of Trigonella (Fabaceae)

Abstract Karyotypes of ten sections of genus Trigonella Linnaeus, 1753 (Fabaceae) from Turkey were investigated. Somatic chromosome numbers of examined species were determined as 2n=14 and 16. The karyotype analyses of the species were carried out and somatic chromosome numbers of Trigonella plicata Boiss., 1872, Trigonella brachycarpa (Fisch.) Moris, 1833, Trigonella rostrata Boiss., 1872, Trigonella lunata Boiss., 1843, Trigonella isthmocarpa Boiss. et Balansa 1856, Trigonella rhytidocarpa Boiss. et Balansa, 1859, Trigonella spicata Sibth. et Sm., 1813, Trigonella cephalotes Boiss. et Balansa 1856, Trigonella capitata Boiss., 1843 and Trigonella gladiata Steven, 1808 were reported for the first time. Two pairs of satellite metaphase chromosomes were observed in Trigonella cariensis Boiss., 1843 and one pair in Trigonella lunata.Moreover, 2 B-chromosomes were found only in Trigonella procumbens Rchb., 1830 among all studied species. The aims of this study are to provide karyological data for a significant pool of the taxa, to show differences among them in the number, size and morphology of somatic chromosomes, to verify previous reports or represent numbers which are different from those cited previously.


Introduction
Trigonella L., 1753 (Fabaceae) includes about 135 species worldwide, and most of the species are distributed in dry regions around Mediterranean, West Asia, Europe, North and South Africa, North America, and with only two species being present in South Australia (Mabberly 1997). The genus Trigonella has 13 sections and 50 species in Turkey (Huber-Morath 1970). Trigonella species are localized in different phytogeographical regions in Turkey with 21 endemic species showing 42% endemism rate (Huber-Morath 1970, Martin et al. 2008.

Material and methods
Seedlings were collected between the years of 2002 and 2005 from natural habitats in different localities (Table 1). For karyotype analyses, root tips were obtained from seeds germinated in humidified Petri dishes at room temperature. Root tips were pretreated with α-monobromonaphthalene at 4 °C for 16 h and fixed in Carnoy's fixative for 24 h at 4 °C. Before staining, the material was hydrolyzed with 1N HCl for 13-15 minutes at room temperature. The chromosomes were stained with 2% aceto orcein and mounted in 45% acetic acid. Permanent slides were made by using the standard liquid nitrogen method and then examined under Olympus BX50 Photomicroscope using an oil immersion objective (100 X). Photographs were taken with the same microscope. Karyotype analyses were made by the use of an Image Analysis System (Bs200Pro).

Section: Falcatulae
Trigonella corniculata L., 1759 from the specimen collected from İzmir province; 2n=16 ( Fig. 1, F) and from the specimen collected from Muğla province; 2n=16 ( Fig.  1, G). At somatic metaphase, seven pairs of metacentric and one pair of submetacentric chromosomes were observed in the former while four pairs of metacentric and four pairs of submetacenric chromosomes were detected in the latter. The specimen collected from İzmir province has chromosomes ranging between 0.61 and 0.88 μm, and the total haploid chromosome length for this specimen was measured as 5.71 μm. In the other specimen which was collected from Muğla province, the chromosome length ranges between 1.54 and 2.57 μm with the total haploid chromosome length of 17.58 μm. T. spinosa L., 1753, 2n=16 ( Fig. 1, H). At somatic metaphase, two pairs of metacentric and six pairs of submetacentric chromosomes were observed.
For this species, the chromosome lengths range from 1.49 to 2.76 μm with the total haploid chromosome length of 16.76 μm.

Section: Reflexae
Trigonella monspeliaca L. 1753 from the specimen collected from Muğla province; 2n=16 ( Fig. 1I) while the specimen collected from Antalya province; 2n=16 ( Fig. 1, J). In the former, all chromosome pairs at the somatic metaphase were observed to be metacentric, and the chromosome length ranges between 0.66 and 1.18 μm with the total haploid chromosome length of 6.81 μm. In the latter specimen, five pairs of metacentric and three pairs of metacentric chromosomes were observed at somatic metaphase. The chromosome length ranges between 0.74 and 1.00 μm. The total haploid chromosome length was measured as 7.06 μm.

Section: Capitatae
Trigonella procumbens (Besser) Reichp., 1830, 2n=16 (Fig. 1O) and T. capitata Boiss., 1843, 2n=16 ( Fig. 1, P). Both species have metacentric chromosome pairs at the somatic metaphase. Endemic to Turkey, T. procumbens have chromosomes ranging from 0.82 to 1.32 μm with the total haploid chromosome length of 7.75 μm.  Moreover, two B chromosomes are observed in this species. The other species of the section examined, T. capitata, have chromosomes ranging from 0.96 to 1.30 μm. The total haploid chromosome length was measured as 9.33 μm. This species is also endemic to Turkey.

Section: Biebersteinianae
Trigonella coerulescens (Bieb.) Hal., 1901, in the specimen collected from Konya province; 2n=16 (Fig. 1, R) and in the specimen collected from Kayseri province; 2n=16 (Fig. 1, S). All chromosome pairs at the somatic metaphase phase were observed to be metacentric for both specimens. The chromosome length ranges between 1.38 and 1.91 μm for the former while that of the latter ranges between 0.91 and 1.32 μm. The total haploid chromosome length was measured as 12.91 μm in the specimen collected from Konya while that of the other specimen was measured as 9.06 μm.

Section: Foenum-graecum
Trigonella gladiata Stev. Fischer, 1808, 2n=16 (Fig. 1T). All chromosome pairs at the somatic metaphase phase were observed to be metacentric. The chromosome length ranges between 2.03 and 4.72 μm. The total haploid chromosome length was measured as 27.70 μm. T. cariensis Boiss. In the specimen collected from İzmir province; 2n=16 (Fig. 1, U), and in the specimen collected from Antalya province; 2n=16 (Fig. 1, V). Both specimens have four pairs of metacentric and four pairs of submetacentric chromosomes at somatic metaphase. Of the two specimens, the former has chromosomes whose lengths vary from 2.66 to 3.94 μm with the total haploid chromosome length of 25.28 μm. For the latter, the chromosome length ranges between 3.93 and 6.28 μm, and the total haploid chromosome length was measured as 39.78 μm. In addition, one pair of satellite metaphase chromosomes has been detected in the specimen from Antalya province. T. foenum-graecum L., 1753, 2n=16 (Fig. 1, W), and T. macrorrhyncha Boiss., 1843, 2n=16 (Fig. 1, X). At somatic metaphase, two pairs of metacentric and six pairs of submetacentric chromosomes were observed for both species. For T. foenum-graecum, the chromosome length ranges between 3.03 and 4.84 μm, and the total haploid chromosome length was measured as 30.23 μm. Endemic to Turkey, T. macrorrhyncha has chromosomes varying from 2.23 to 4.40 μm. In this species, the total haploid chromosome length is 25.67 μm. Idiograms of each species were arranged in order of decreasing length (Fig. 2). The total haploid chromosome lengths were given in Table 2, and the information of the 19 species studied were presented in Table 3.

Basic chromosome number variations
In this karyological study, two different basic chromosome numbers of x=7 and x=8 were observed in the species belonging to ten sections of Trigonella. Bidak and Amin (1996) reported two different basic chromosome numbers of x=8 and x=9 for Trigonella species studied. Among studied species, only two species, T. plicata and T. rostrata, from the sections of Pectinatae and Lunatae, respectively, have the basic chromosome number of x=7.

Chromosome number and morphology variations
Two different somatic chromosome numbers (2n=14 and 2n=16) were observed in studied sections. The smallest chromosome length is 0.43 μm measured in T. cephalotes (section Uncinatae) while the biggest of that is 6.28 μm measured in T. cariensis (section Foenum-graecum). The smallest total haploid chromosome length was measured as 4.49 μm from the species of T. cephalotes (section Uncinatae). T. cariensis (section Foenum-graecum) has the biggest total haploid chromosome length of 39.78 μm. T. rostrata (section Lunatae) has the smallest arm ratio (1.17), and T. foenum-graecum (section Foenum-graecum) has the biggest (2.17). The smallest centromeric index (4.18) was measured in T. foenum-graecum (section Foenum-graecum) while the biggest of that (6.56) was observed in T. rostrata (section Lunatae). In this study, there is a marked difference in somatic chromosome lengths compared to other species of the section Foenum-graecum. Chromosome numbers are rather close to each other excluding several species (Lunatae, T. corniculata and T. spinosa) in other sections. Besides, 2B chromosome was observed in T. procumbens in the section Capitatae. Karyotype formulae of the sections of Samaroideae, Pectinatae, Lunatae, Isthmocarpae, Capitatae and Biebersteinianae are completely composed of metacentric chromosome pairs. Karyotype formulae of species in other sections are composed of metacentric and submetacentric chromosome pairs. While there is one pair of satellite metaphase chromosomes in T. lunata (section Lunatae), T.cariensis (section Foenum-graecum) has two pairs of satellite metaphase chromosomes. Section Samaroideae is represented by a single species (T. cretica) in Turkey. The karyotype of this species has been analysed by Yılmaz (2006) reporting the karyotype formulae 2n=16 as in our study. Our results agree with one of the reports of somatic chromosome number of 2n=16 from the same locality (Yılmaz 2006). Section Pectinatae is represented by a single species (T. plicata) in Turkey. Karyotype analysis of this species has been performed by us for the first time. Section Luna-   tae is represented by four species (T. brachycarpa, T. rostrata, T. lunata and T. sırjaevii Hub. -Mor., 1939) in Turkey. Making a general evaluation of the section, diploid chromosome numbers were found to be different although the localities of the two types (T. brachycarpa and T. rostrata) in the section were similar. The diploid chromosome number is 2n=16 in T. brachycarpa whereas it is 2n=14 in T. rostrata. The smallest chromosome length of 0.81 μm was measured in T. rostrata in this section while the biggest  Bal, 1990;Tutin, Heywood, 1964 " T. macrorrhyncha 16 16 Ladizinsky, Vosa, 1986 of that was observed in T. lunata with a length of 3.92 μm. T. rostrata is the species with the smallest arm length of 1.17; however, it is also the species with the biggest centromeric index (6.56). T. lunata is the one with the biggest total haploid chromosome length of 26.44 μm. This length is rather different compared to the species in other sections. Besides, one pair of satellite chromosome has been observed in T. lunata. Karyotype formulae of all species in the section are composed of metacentric chromosome pairs. T. sırjaevii could not been studied due to its inability to be germinated. Section Falcatulae is represented by two species (T. corniculata and T. spinosa) in Turkey. T. corniculata was studied in two different localities (İzmir and Muğla). In Muğla case, tetraploidy was observed differing from the other. Total haploid chromosome length was measured as 5.71 μm in İzmir province while that was 17.58 μm in Muğla province. It can be stated that the distinction between the karyological values obtained from these two localities resulted from locality differences. In Izmir province, the karyotype formula was 7m+1sm whereas it was set as 4m+4sm in Muğla province. Our diploid counts are in agreement with the literature such as one of the reports from Turkish material and many others from different territories (Tutin and Heywood 1964). From a karyological point of view, to obtain same results from the same species confirms the previous studies. The species of T. spinosa is also placed in the section Falcatulae. Compared with the other species (T. corniculata) of the section, the chromosome number is the same and the chromosome size measured is very close to each other. However, karyotype formulae are different. Karyotype formulae of T. spinosa is 2m+6sm. Bidak et Amin (1996) reported the somatic chromosome numbers as 2n=16 and 18 in T. gladiata, 2n=16 in T. ornithopodiodes, 2n=16 in T. spinosa and 2n=18 in T. stellata.
The section Reflexae is represented by a single species (T. monspeliaca) in Turkey. This species was studied in two different localities. Diploid chromosome numbers are the same (2n=16) in both localities of T. monspeliaca species. The chromosome sizes, total haploid chromosome lengths, arm lengths and centromeric indices are very close to each other while the karyotype formulae are different, i.e. the karyotype formula of Muğla province is 8m, of Antalya province is 5m+3sm. For Darlington and Wylie (1955), in a cytological study conducted on species belonging to Trigonella, diploid chromosome numbers varied from 2n=16 to 2n=32. For example, they are 2n=16 in T. gladiata, 2n=16 in T. monspeliaca, 2n=28, 30 and 2n=32 in T. polyceratia.
The section Isthmocarpae is represented by two species (T. isthmocarpa and T. rhytidocarpa) in Turkey. Karyology of the two species in this section was studied for the first time. The diploid chromosome number of T. isthmocarpa and T. rhytidocarpa species were found to be 2n=16=8m. Karyological characteristics of these two species are very close to each other.
The section Uncinatae is represented by two species (T. spicata and T. cephalotes) in Turkey. The diploid chromosome number of T. spicata and T. cephalotes were found to be 2n=16=5m+3sm. Karyological characteristics of these two species are very close to each other. There is a marked difference only in terms of total haploid chromosome length. While total haploid chromosome length was 10.36 μm in T. spicata, that is 4.49 μm in T. cephalotes. T. cephalotes is also the species having the smallest haploid chromosome length among studied species.
The section Capitatae is represented by three species (T. procumbens, T. capitata and T. pseudocapitata) in Turkey. The diploid chromosome number of T. procumbens and T. capitata was found as 2n=16=8m. Karyological characteristics of these two species are very close to each other. In addition to A chromosomes, two examples of B chromosomes were observed in T. procumbens differing from the other species of the sections studied. The chromosome number of T. procumbens was reported as 2n=18 by Yılmaz (2006) from the same locality, but he did not mention B chromosomes. The other species of the section, T. pseudocapitata, could not be studied due to its inability to germination.
The section Biebersteinianae is represented by T. coerulescens in Turkey. It was studied from two different localities. Diploid chromosome numbers are the same (2n=16=8m) in both localities of T. coerulescens. Chromosome sizes, total haploid chromosome lengths, arm lengths and centromeric indices are very close to each other.
The chromosome number of Trigonella coerulescens is in agreement with the previous report (2n=16) by Yılmaz (2006).
The section Foenum-graecum is represented by five species (T. gladiata, T. cariensis, T. foenum-graecum, T. macrorrhyncha and T. cassia) in Turkey. Diploid chromosome numbers of the four species studied in this section are the same (2n=16). T. cassia could not be studied due to failure to germinate. The chromosome morphologies of the species are very close to each other. The karyotype formula of Trigonella gladiata is 8m. In a cytological study performed by Bidak & Amin (1996), diploid chromosome number was found to be 2n=16 and 2n=18 in T. gladiata. Researchers reported two different basic chromosome numbers for this species (x=8 and x=9). However, the basic chromosome number in our study was found to be x=8 for this species. In a cytological study conducted on Trigonella species, Darlington and Wylie (1955) reported that the diploid chromosome numbers varied from 2n=16 to 2n=32. For example, 2n=16 in T. gladiata, 2n=16 in T. monspeliaca, 2n=28, 30 and 32 in T. polyceratia. Somatic chromosome numbers found in our study are parallel with that report. Two double satellite chromosome pairs were observed in a sample collected from Antalya. The sample collected from İzmir has the biggest total haploid chromosome length (39.78 μm) among all sections. has diploid chromosome number of 2n=16 as in other species in the section. The karyotype formula is 2m+6sm. T. foenum-graecum is the species with the biggest arm ratio in all sections (2.17). Chromosome numbers of only two Trigonella species were reported in European Flora records, T. corniculata 2n=16 and T. foenum-graecum 2n=16 (Tutin and Heywood 1964). Our results obtained from this study agree with that report. In a study on karyotype analysis of T. foeanum-graecum, somatic chromosome numbers were found to be similar to that of our study (Bal, 1990). The diploid chromosome number of T. macrorrhyncha is 2n=16 as in all other species in the section. The karyotype formula is 3m+5sm. In another study conducted in six different species (T. gladiata, T. cariensis, T. foenum-graecum, T. berythea, T. macrorrhyncha and T. cassia) of the section Foenum-graecum, diploid chromosome numbers were reported as 2n=16 (Ladizinsky and Vosa, 1986). In this case, our counts agree with the previous study. It is considered that the results obtained from this karyological study have contributed to the taxonomical revision of the genus Trigonella.