DOI: http://dx.doi.org/10.22092/cbj.2012.100423 Phylogenetic relationships of Onobrychis Mill. (Fabaceae: Papilionoideae) based on ITS sequences of nuclear ribosomal DNA and morphological traits S. Safaei Chaei Kara*, F. Ghanavatib, J. Mozafaric, M. R. Naghavid, H. Amirabadizadehe, and F. Darvishf a and f Department of plant breeding, Science and Research Branch, Islamic Azad University, Tehran, Iran. b and c Seed and Plant Improvement Institute, Karaj, Iran. d Agronomy and Plant Breeding Department, Agricultural College, The University of Tehran, Karaj, Iran. e Agricultural and Natural Resources Research Center of Khorasan-e-Razavi Province, Mashhad, Iran. * Corresponding author's E-mail address: [email protected] Received: August 2012 Accepted: December 2012 ABSTRACT Safaei Chaei Kar, S., F. Ghanavati, J. Mozafari, M. R. Naghavi, H. Amirabadizadeh, and F. Darvish. 2012. Phylogenetic relationships of Onobrychis Mill. (Fabaceae: Papilionoideae) based on ITS sequences of nuclear ribosomal DNA and morphological traits. Crop Breeding Journal 2(2): 91-99. The genus Onobrychis is subdivided into two subgenera: Onobrychis and Sisyrosema. Phylogenetic relationships of 19 species of Onobrychis (Fabaceae: tribe Hedysareae) and one representative each of genera Eversmannia and Ebenus were estimated from DNA sequences of the internal transcribed spacer (ITS) region. Parsimony analysis of the ITS region formed a dendrogram with strong bootstrap support from two groups: Onobrychis subgen. Onobrychis (except O. laxiflora) is in one group and Onobrychis subgen. Sisyrosema is in the other. Within group I, species of the Onobrychis section and species of the Lophobrychis and Dendrobrychis sections form well supported branches A and B, respectively (BP=99% and 91%). The close association between the Onobrychis section and Lophobrychis and Dendrobrychis indicated there is strong sequence homology among them, suggesting that these species are closely related in terms of phylogeny. Also there is strong sequence homology among sections of Sisyrosema subgen. (group II). Species of Heliobrychis, Hymenobrychis and Afghanicae form a branch (branch C) with 77% bootstrap support. Species of the Hymenobrychis section form a clade (clade F) with 82% bootstrap support, which indicates these species are closely related. The present nrDNA ITS data showed that Onobrychis subgen. Sisyrosema appears to be a well-supported monophyletic group (BP=77%), whereas the Onobrychis subgen. Onobrychis is not monophyletic due to the sister group relationship of one species of Onobrychis subgen. (O. laxifolra) to the subgen. Sisyrosema. Cluster analysis of morphological characters showed two major groups separating Onobrychis subgen. Onobrychis (except O. laxiflora) from Onobrychis subgen. Sisyrosema, which are in accordance with molecular phylogenetic groups. Keywords: cluster analysis, Fabaceae, Hedysareae, nrDNA ITS, Onobrychis, phylogenetic analysis, Sisyrosema INTRODUCTION The genus Onobrychis, represented by 170 he Leguminosae are one of the largest families perennial and annual species in the world, is densely Tof flowering plants, with 18,000 species distributed in the Anatolia-Iran-Caucasian triangle classified into around 650 genera. The family is (Nixon, 2006; Emin and Kuddisi, 2010). usually divided into three sub-families: Phylogenetic studies indicated that the primary Papilionoideae, Caesalpinoideae and Mimosoideae center of genetic diversity of Onobrychis is in the (Polhill and Raven, 1981). The tribe Hedysareae was Mediterranean region and that the ecological originally described as one of the tribes comprising separation of this region into western and eastern the subfamily Papilionoideae (Bentham, 1865). Lock sectors represents a main event in the evolution of (2005) included in the Hedysareae many genera, the genus (Ashurmetov and Normatov, 1998). such as: Alhagi, Caragana, Corethrodendron, Onobrychis is a perennial and economically Ebenus, Eversmannia, Calophaca, Halimodendron, important genus used to improve the quality of the Hedysarum, Onobrychis, Sartoria, Sulla and soil. It is also harvested as dried, fresh and purebred Taverniera. Onobrychis Mill. constitutes the second fodder (Emin and Kuddisi, 2010). largest genus (after Hedysarum) of tribe Hedysareae Yildiz et al. (1999) suggested that, based on fruit in the sense adopted by Polhill (1981) (Mabberley, morphology, genus Onobrychis is subdivided into 1990; Lock, 2005). two subgenera, namely, Onobrychis with four 91 Crop Breeding Journal, 2012, 2(2) sections (Dendrobrychis, Lophobrychis, Onobrychis Onobrychis genetic variation, for there are more and Laxiflorae), and Sisyrosema with five sections than 69 annual and perennial species and subspecies (Anthyllium, Afghanicae, Heliobrychis, of the genus distributed over different climatic Hymenobrychis and Insignes) distinguished by regions. Because these species have high genetic different karyotypes, morphological features and variability, it is possible to use them as a rich and geographical origins. valuable genetic resource for breeding farm species. Typically, one of the areas utilized for The range of the Onobrychis genus is very variable phylogenetic inference at the generic and and the boundary between species is not clear. infrageneric levels in plants is the internal Molecular phylogenetic studies of Onobrychis transcribed spacer (ITS) region of 18S-5.8S-26S species are limited to a small number of taxa nuclear ribosomal DNA (nrDNA). The advantages (Wojciechowski et al., 2000; Ahangarian et al., of this locus for phylogenetic reconstruction include: 2007). Therefore, study of the reconstruction of the biparental inheritance, universal primers, phylogenetic relationships and the boundary among intragenomic consistency and intergenomic species in this genus-using nrDNA ITS sequences variability (Baldwin et al., 1995). The internal are also considered. Determination of the transcribed spacer (ITS) mostly provides enough phylogenetic relationships of these species, beside molecular markers which are acceptable for the introduction of new species and wild relatives of evolutionary studies at the species level (Ganzalo cultivated species of this genus would enhance the and Josep, 2007). It has been proved that ITS is efficiency of Onobrychis breeding program. suitable for studying interspecific relationships in The aim of this study was to investigate the many plant families (reviewed in Baldwin et al., relationships of taxa within two separate subgenera 1995), and particularly within the Fabaceae of Onobrychis, subgen. Onobrychis and subgen. (Wojciechowski et al., 1993; Sanderson et al., 1996; Sisyrosema. To accomplish this goal, the internal Downie et al., 1998). transcribed spacer (ITS) region of the 18S-5.8S-28S The Hedysareae in general and Hedysarum nuclear ribosomal DNA (nrDNA) was sequenced. (Chennaoui et al., 2007) and Onobrychis (Yildiz et Morphological characteristics were studied and the al., 1999; Ahangarian et al., 2007) in particular resulting cluster compared with the molecular remain under-sampled in molecular phylogenetic cluster. analyses. In a study by Yildiz et al. (1999), 40 species belonging to five sections of the Onobrychis MATERIALS AND METHODS genus and six species belonging to four sections of Taxon sampling Hedysarum as an out-group were evaluated based on To conduct molecular phylogenetic studies fruit morphology. Results showed there are no during 2011-2012, leaf materials were sampled from common traits for fruit, which supports the herbarium specimens deposited in the Herbarium monophyly of the Onobrychis genus. The Research Center of Khorasan-e-Razavi Agricultural monophyly of Onobrychis and subgen. Sisyrosema and Natural Resources Center (MRCH), Mashhad, has not been confirmed. Abou-El-Enain (2002) Iran; the Herbarium of National Plant Gene Bank of evaluated 22 taxa of six Onobrychis species Iran (HNPGBI); and the Herbarium of the Research belonging to the Lophobrychis section based on Institute of Forests and Rangelands (TARI). In this chromosome number, chromosome length and study, 21 species including 19 species representing ploidy levels. His evaluation confirmed the seven sections of the Onobrychis genus and two monophyly of the Onobrychis genus. Ahangarian et species of the other genus of tribe Hedysareae as an al. (2007) estimated that there are 11 species of out-group, were selected for molecular phylogenetic Onobrychis representing two subgenera and 8 reconstruction. Details of these species, including sections. Results showed that Onobrychis subgen. accession identities, geographical origins and Onobrychis is not monophyletic, whereas genebank sequence accession numbers, are given in Onobrychis subgen. Sisyrosema forms a strongly Table 1. supported clade. In contrast to Onobrychis section Heliobrychis, sections Dendrobrychis and DNA extraction, PCR and sequencing Onobrychis are not monophyletic. Based on Total genomic DNA was isolated using a cytological evaluation of annual species of the modified CTAB extraction method (Doyle and Onobrychis genus in Iran, Ghanavati et al. (2012) Doyle, 1987). The DNA concentration was reported that the basic chromosome number varied determined with a Gene-Guant spectrophotometer from x=7 to x=8. (Pharmacia) and DNA quality was analyzed by Iran is one of the most important centers of agarose gel electrophoresis. 92 Safaei Chaei Kar et al.: Phylogenetic relationships of Onobrychis … Table 1. Locality, voucher and sequence accession number of Onobrychis species.