Karyological studies of the genus Onobrychis Mill. and the related genera Hedysarum L. and Sartoria Boiss. & Heldr. (Fabaceae, Hedysareae) from Turkey

Abstract

Nine chromosome counts in several genera of the tribe Hedysareae from Turkey, six species of Onobrychis, two species of Hedysarum and one species of Sartoria, were reported and all karyotypes were prepared. Six of them are new. One of the other three reports is a confirmation of polyploidy, another report is a confirmation of chromosome count and the last report is a confirmation of both chromosome count and karyotype morphology. Except for karyotype of O. altissima, all karyotypes were presented in this study first.

Keywords:

• chromosome number
• Hedysarum
• karyotype
• Onobrychis
• Sartoria

Introduction

Fabaceae, represented by 650 genera and 18000 species in the world, is one of the well-known families in terms of containing the most number of species and of having an economic value (Polhill 1981a). The tribe Hedysareae is represented by four genera in Turkey; Ebenus L., Hedysarum L., Onobrychis Mill. and Sartoria Boiss. & Heldr. The genus Ebenus has its centre in Anatolia, where all species are endemic. Only six species are known outside Turkey (Davis et al. 1988). The genera Onobrychis and Hedysarum constitute the main part of the tribe Hedysareae in the sense adopted by Polhill (1981b). In Turkey, the genera Onobrychis and Hedysarum are represented by 52 species and 22 species respectively (Davis et al. 1988).

Sartoria is a monotypic endemic genus of Turkey. Hedge (1970) indicated that, although Sartoria allied to Onobrychis and Hedysarum, it is distinct from both. According to Hedge, Sartoria differs from Onobrychis in the three ovulate ovary and 2-3- seeded, large, ovate-oblong unarmed fruit; from Hedysarum it is distinguished by the non-lomentoid fruit, small corollas and dwarf habit. According to phylogenetic analysis of the tribe Hedysareae using chloroplast trnL-F sequences, Amirahmadi et al. (2009) declared that the genus Sartoria, endemic to Turkey, was nested within Eurasian Hedysarum. Also, the seed storage protein profiles results showed that Sartoria and Hedysarum are closer to each other than they are to Onobrychis (Arslan and Ertuğrul 2010).

In recent years, karyological studies have played an important role in solving taxonomic problems. Variations of chromosome number besides the karyological analyses represent fundamental steps toward understanding of plant evolution and diversification, especially the relationship between each of the ploidy level, biogeography and origin of the basic chromosome number (Badr et al. 1997; Abou-El-Enain 2002).

Cytological studies on genera Onobrychis and Hedysarum are very limited and little is known about the genus Hedysarum, about the nature of genetic variability in diploid species and the taxonomic relationships of the different taxa (Baatout et al. 1991). Some researchers have contributed to this subject with a few species (Löve 1972; Kuzmanov 1993; Abou-El-Enain 2002; Hatami and Nasirzadeh 2006; Hoşgören 2006; Issolah et al. 2006; Yıldız and Gücel 2006; Hesamzadeh Hejazi et al. 2010; Ranjbar et al.2010). Furthermore there is no karyological data in the monotypic genus Sartoria.

Most of the cytological studies in the genus Onobrychis, have centered on the chromosome count (Baltisberge 1991; Karshibaev 1992; Slavivk et al. 1993; Akalın and Alpınar 1994), with little focus on detailed karyological criteria for taxonomic purposes (Khatoon et al. 1991; Mesicek and Sojak 1992). It is evident that in these and other reports, the chromosome count is known for just over 25% of the species (Fedorov 1969; Goldblatt 1981a; 1984; 1985; 1988; Romano et al. 1987; Goldblatt and Johnson 1991; Diaz-Lifante et al. 1992; Gomurgen 1996).

In this paper, karyotypes of selected species of the three genera particularly those with the unknown morphology and/or chromosome counts have been analyzed in order to examine their patterns of chromosomal variations in relation to current taxonomic delimitation, in order to compare the data obtained with those gathered from previous studies.

Materials and methods

Chromosome counts were made on somatic metaphases using the squash technique. Root meristems from germinating seeds collected in the wild were used. Root-tips were pre-treated with α-monobromo naphthalene solution at 4˚ C for 16 h, washed and fixed in 3:1 absolute ethanol/glacial acetic acid overnight. Samples were hydrolyzed with 1N HCl at 60˚ C, washed for 12 min and stained in Aceto-orcein’s solution for 1-2h. Stained tips were squashed with a drop of 1% aceto-orcein and mounted in 45% acetic acid (Gray 1964). Cells with a good spread of chromosomes were photographed using an Olympus Photomicroscope. Chromosomes were counted from well-spread metaphases in intact cells, through direct observation and photomicrographs. The chromosome counts of the studied species were confirmed in at least five cells. As Levan et al. (1964) defined, long and short arm measurements of the chromosomes were made. Idiograms were drawn for each species.

Results and discussion

In this study, new chromosome numbers are given for six taxa, karyotype morphology is given for the first time in eight taxa.

Onobrychis Mill. Section Onobrychis Miller.

Onobrychis altissima Grossh.

Turkey, C6 ¹ Hatay: between Iskenderun and Arsuz 15^th km, 2m, 36°31’524”N-36°02’035”E, Ertuğrul 3456 & Tugay, 21.v.2005 (KNYA). 2n = 14 (Figure. 1-1). According to our data the chromosome number and morphology are the second report for this species. Hesamzadeh Hejazi et al. (2010) and Ranjbar et al. (2010) analysed a tetraploid individual of species (2n = 4x = 28) and karyotype morphology of the species. Most of the chromosomes are metacentric, with two pairs of acrocentric (Figures. 2-1). Our results of chromosome counts and karyotype morphology agree with the previous counts and karyotype reported by Hesamzadeh Hejazi et al. (2010).

Fig. 1 Somatic metaphases and karyotypes 1. O. altissima (2n = 14), 2. O. oxyodonta (2n = 14), 3. O. hajastana (2n = 14), 4. O. subacaulis (2n = 16), 5. O. galegifolia (2n = 16), 6. O. tournefortii (2n = 14), 7. S. hedysaroides (2n = 16), 8. H. syriacum (2n = 16), 9. H. pannosum (2n = 16).

Fig. 2 Idiograms: 1. O. altissima (2n = 14), 2. O. oxyodonta (2n = 14), 3. O. hajastana (2n = 14), 4. O. subacaulis (2n = 16), 5. O. galegifolia (2n = 16), 6. O. tournefortii (2n = 14), 7. S. hedysaroides (2n = 16), 8. H. syriacum (2n = 16), 9. H. pannosum (2n = 16).

Onobrychis oxyodonta Boiss.

Turkey, C4 Konya: between Hadim and Taşkent steppe. 1600 m. Ertuğrul 3166, Bağcı & Dural, 14.vii.2004 (KNYA). 2n = 14 (Figures. 1-2). Our counts disagree with 2n = 28 reported by Akalın and Alpınar (1994) under the name O. oxyodonta, without further details, we suppose that they analysed a tetraploid individual of species. However, the karyotype morphology of O. oxyodonta is presented here for the first time. All of the chromosomes are metacentric (Figures. 2-2).

Onobrychis hajastana Grossh.

Turkey, C4 Karaman: Taşkale Avdan Mountain, northern stony slopes, 1880 m, 37° 02’459”N-33°40’859”E., Ertuğrul 3588 & Uysal, 25.vi.2005 (KNYA). 2n = 14 (Figures. 1-3). The chromosome number and morphology are presented here for the first time. All of the chromosomes are metacentric. Secondary constriction (Satellite) is observed on a chromosome pair (Figures. 2-3).

Our results and most of previous counts; 2n = 14 in O. elata (Cartier 1976). 2n = 14 in O. kotschyana and O. carduchorum (Hoşgören 2006) 2n = 22, 27, 28, 29 in O. viciifolia, i.e. 2n = 3x+1, 4x-1, 4x, 4x+1, respectively (Corti 1930; SacristÁn 1966; Zi and Jing 1996), support x = 7 as the basic number for this section. However, there is another incongruous count in section Onobrychis: x = 16 in O. megataphros (Hoşgören 2006).

Section Heliobrychis Bunge.

Onobrychis subacaulis Boiss.

Turkey, B9 Iğdır: Aralık-Kazım Karabekir Tarım İşletmesi. 1000 m, 37°47’160”N-44°37’200”E, Ertuğrul 2610 & Tugay, 12.vi.2002 (KNYA). 2n = 16 (Figres. 1-4). As far as we know, this is the first count for this species. Karyotype of this species is also presented here for the first time. Except for a pair’s submetacentric chromosomes, all of the chromosomes are metacentric (Figures. 2-4). The same chromosome number, 2n = 16, reported by Cartier (1976) in O. ornate, another species of this section. These counts confirm x = 8 as the basic chromosome number of section Heliobrychis.

Section Hymenobrychis DC.

Onobrychis galegifolia Boiss.

Turkey, C8 Mardin: Bakırkırı Yusufdağ vineyards, 1000 m, 37°18.997’N-40°46.790’E, Ertuğrul 2911 & Tugay, 04.vii.2003 (KNYA). 2n = 16 (Figures. 1-5). According to our information, this is the second report for this species. It agrees with the previous counts reported by Hoşgören and Saya (2006). But, a detailed karyotype analysis is given for the first time. The species has three pairs of metacentric, two pairs of submetacentric and three pairs of acrocentric chromosomes (Figures. 2-5).

Onobrychis tournefortii (Willd.) Desv.

Turkey, C4 Konya; Bozkır, Ördek Bogazı, sandy and, stony place, 1230 m, Tugay 2864, 24.vii.2002, (KNYA). 2n = 14 (Figures. 1-6). According to our data the chromosome number and karyotype morphology of this species are presented here for the first time. A total of five out of the seven chromosome pairs are submetacentric. Others are metacentric (Figures. 2-6). All results show that this section has to different basic number x = 7 and x = 8. Although it is rare that the species of the same section have different chromosome numbers, this difference is considered as normal even in different populations of the same species (Issolah et al. 2006).

Sartoria Boiss. & Heldr.

Sartoria hedysaroides Boiss & Heldr.

Turkey, C4 Konya: Taşkent to Gevne Valley, Çukuryurt, Kızılgedik steppe, 1800 m, Ertuğrul 3713, 30.v.2008 (KNYA). 2n = 16 (Figures. 1-7). To our knowledge, this is the first report of chromosome number and the karyotype morphology in this monotypic endemic genus Sartoria for Turkey. All of the chromosomes belonging to this species are metacentric (Figures. 2-7). Our result suggests x = 8 to be the basic chromosome number of this genus.

Hedysarum L. Section Multicaulia (Boiss.) B. Fedtsch.

Hedysarum syriacum Boiss.

Turkey, C4 Karaman: Karadağ–around Akkaya, debrises 1300-1500 m, Ertuğrul 2979 & Dural 12.vii.2003 (KNYA). 2n = 16 (Figures. 1-8). According to our knowledge, the chromosome number and karyotype morphology of H. syriacum are reported here for the first time. All chromosomes appear to be metacentric (Figures. 2-8). Our results agree with previous counts, 2n = 16, reported by Akpınar and Yıldız (1999) in H. nitidum, H. pestalozzae, and H. varium. These counts support x = 8 as the basic number of section Multicaulia.

Section Crinifera (Boiss.) B. Fedtsch.

Hedysarum pannosum Boiss.

Turkey, C4 Karaman: Karadağ-above Akkaya stony slopes 37°21.773’N-33°10.090’E 1496 m, Ertuğrul 2973 & Dural,12.vii.2003 (KNYA). 2n = 16 (Figures. 1-9). As far as we know, this is the first report of chromosome number and karyotype morphology for this species. Seven out of the eight chromosome pairs in the total are metacentric. Only one chromosome pair is submetacentric (Figures. 2-9). Our results agree with previous counts, 2n = 16, reported by Akpınar and Yıldız (1999) in H. aucheri, H. pycnostachyum, H. rotundifolium. These counts confirm x = 8 as the basic number of section Crinifera as section Multicaulia. There are many previous counts reported from the rest of the studied taxa of the genus Onobrychis in this study; 2n = 32 in O. pulchella, 2n = 28 in (Aboul-El-Enain 2002), 2n = 16 in O. aucheri subsp. teheranica 2n = 32 in O. aucheri subsp psammophila (Hatami and Nasirzadeh 2006). Controversial chromosome counts were also reported from the some taxa of section Lophobrychis; 2n = 14, 16, 28 in O. aequidentata (De-Montmollin 1984; Romano et al. 1987; Baltisberger 1991; Abou-El-Enain 2002), 2n = 14, 16, 28 in O. caput-galli (Heyn 1962; Slavivk et al. 1993; Abou-El-Enain 2002), 2n = 14, 16, 32 in O. crista-galli (Corti 1931; Diaz-Lifante et al. 1992; Abou-El-Enain 2002) respectively. These reports show that the variation in chromosome number in each of species above can be related to differences in their taxonomic delimitation, at least the subspecies level (Aboul-El-Enain 2002).

The chromosome numbers reported from the rest of the studied taxa of the genus Hedysarum are as follows: 2n = 14 in H. cyprium (Yıldız and Gücel 2006), 2n = 16 and 18 in H. coronarium (Issolah et al. 2006), 2n = 16 and 18 in H. pallidum (Abdelguerfi-Berrekia 1985), 2n = 16 in H. naudinianum (Bousba 1992), 2n = 18 and 32 in H. perrauderianum (Bousba 1992).

The previous studies on these genera are insufficient not only on the chromosome morphologies but on the chromosome counts. The genus Onobrychis have two basic chromosome counts (x = 7 and x = 8) and three ploidy levels (2n = 2x = 14, 2n = 4x = 28, 2n = 8x = 56 and 2n = 2x = 16, 2n = 4x = 32) (Sirjaev 1925; Goldblatt 1981b). Diaz-lifante et al. (1992) and Gömürgen (1996) suggested that the basic chromosome count, x = 8, is associated with the annual species while x = 7 is more frequent in perennial Onobrychis species. This suggestion was confirmed by Abou-El-Enain (2002) in three annual Onobrychis species. Our results disagreed with this suggestion because some perennial Onobrychis species have two basic chromosome count x = 8 and 7 as O. tournefortii. Our new counts confirm that the genus Onobrychis has two basic chromosome numbers x = 7 and x = 8.

According to previous reports, the genus Hedysarum has three basic chromosome counts (x = 7, 8 and 9) (Darlington and Wylie 1955; Löve and Löve 1961; Federov 1974; Löve 1978; Moore 1982). Our new two counts confirm one of basic chromosome counts of Hedysarum species.

Abou-El-Enain (2002) reported that all chromosomes in members of Lophobrychis section were metacentric or submetacentric. While our study is in accordance with the study above in terms of chromosome morphology of all species except for this section including metacentric and submetacentric chromosomes, it contradicts that study since it also includes acrocentric chromosomes.

Genus Ebenus is very close morphologically to genus Hedysarum (Polhill 1981). All the previous studied species of the genus Ebenus; E. longipes, E. argentea E. plumosa var. plumosa, E. plumosa var. speciosa, E. macrophylla and E. barbigera have 2n = 14 chromosomes (Davis et al. 1988; Aytaç 2000; Aksoy et al.2001). Although E. plumosa var. plumosa and E. plumosa var. speciosa have six pairs of metacentric chromosomes and one pair of submetacentric chromosome, all chromosomes of E. longipes, E. argentea, E. macrophylla and E. barbigera are metacentric (Aytaç et al. 2000; Aksoy et al. 2001). All our records of basic chromosome numbers agree with earlier reports on the tribe Hedysareae.

Acknowledgement

We would like to thank Dr. Tuna Uysal and Dr. Yavuz Bağcı for providing plant specimens.

Notes

¹According to Grid system adopted by Davis in Flora of Turkey.