Turkish Journal of Botany Turk J Bot (2018) 42: 623-635 http://journals.tubitak.gov.tr/botany/ © TÜBİTAK Research Article doi:10.3906/bot-1712-52 Resolving the position of Astragalus borysthenicus Klokov within the Astragalus L. species 1, 2 2 3 Nataliia KARPENKO *, Viktoriia MARTYNIUK , Oksana TYSHCHENKO , Andrii TARIEIEV , 4 2 2 Ayten TEKPINAR DİZKIRICI , Vitaliia DIDENKO , Igor KOSTIKOV 1 Research Laboratory of Biochemistry, Educational and Scientific Centre “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine 2 Department of Plant Biology, Educational and Scientific Centre “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine 3 Ukrainian Botanical Society, Kyiv, Ukraine 4 Department of Molecular Biology and Genetics, Faculty of Science, Van Yüzüncü Yıl University, Van, Turkey Received: 27.12.2017 Accepted/Published Online: 06.05.2018 Final Version: 26.09.2018 Abstract: The present study is focused on several species from the genus Astragalus L. with the aim to clarify the taxonomic status of Ukrainian local endemic species Astragalus borysthenicus Klokov, which is sometimes considered a synonym to A. onobrychis L. In this study, the morphological features, current taxonomy, taxonomical history, and phylogenetic analysis based on rDNA Bayesian inference, as well as comparative analysis of ITS1 and ITS2 secondary structures, were investigated. It was found that A. borysthenicus is distant from A. onobrychis according to phylogenetic analysis. Moreover, A. borysthenicus has differences from the investigated taxa in its secondary structures of ITS1 and ITS2 transcripts. These data suggest that A. borysthenicus should be treated as a separate species rather than a synonym to A. onobrychis. Key words: Astragalus, Bayesian inference, ITS secondary structure, phylogeny, taxonomy 1. Introduction Threatened Animals and Plants (United Nations, 1991), Astragalus L. (Fabaceae) can be considered one of the and regional red lists (Korzhenevskiy et al., 2012). It ranges largest genera of flowering plants. The exact number of across the littoral zones of the Black Sea and the Sea of species remains unknown and varies from about 2500 Azov, but the populations are damaged by recreational (Ekici et al., 2015) to 3000 species (Hardion et al., 2010) and commercial construction, as well as military conflicts that are mainly distributed in the northern hemisphere. on the coast. It occurs mostly solitary or in small groups; Many Astragalus species are European only (Hardion et sometimes it is the predominant component of plant al., 2010). communities (A. borysthenicus coverage varies from The flora of vascular plants of Ukraine includes 5187 solitary individuals up to 65%). Under anthropogenic species and infraspecific taxa from 160 families, among influences, populations are transformed into insignificant them 380 species of the family Fabaceae. Fifty-three localities with a constantly decreasing number of species belong to Astragalus (Mosyakin and Fedoronchuk, individuals. Complete populations that include plants 1999), 15 of which are endemic (28.3%). In comparison, at different stages of growth are distributed mostly in the endemism rates of Astragalus in different geographical protected areas of the coast of the Sea of Azov (total area regions of East Europe and the Caucasus are as follows: East is approximately 15 ha). The density of these populations European plain – 55.2%, Crimea – 44.8%, Urals – 56.8%, is 1–3 individuals/m2; mature individuals prevail Pre-Caucasus – 17.6%, Caucasus – 64.5%, Transcaucasia – (Korzhenevskiy et al., 2012). 65.7% (Sytin, 2009). A total of 425 Astragalus species are The taxonomic status of A. borysthenicus is interpreted listed for Turkey, and their endemism rate is about 51% ambiguously and remains elusive in various scientific (Podlech and Zarre, 2013; Ekici et al., 2015). studies. Sometimes it is considered a separate species Astragalus borysthenicus Klokov is a Ukrainian local (Visyulina, 1954; Vasilieva, 1987; United Nations, 1991; endemic species listed in the Red Data Book of Ukraine Mosyakin and Fedoronchuk, 1999). However, in many (Krytska, 2009), the European Red List of Globally other taxonomic and floristic publications it is cited as a * Correspondence: [email protected] 623 This work is licensed under a Creative Commons Attribution 4.0 International License. KARPENKO et al. / Turk J Bot junior synonym of nonthreatened multiregional (Europe– protologues and other special sources (Linnaeus, 1753; Asia–Africa) Astragalus onobrychis L. (Borisova, 1946; Bunge, 1868; Grossheim, 1930; Borisova, 1946; Klokov, Chater, 1968; Cherepanov, 1995; Ekici et al., 2015). Both 1946; Visyulina, 1954; Vasilieva, 1987; Ekici et al., 2015) taxa belong to sect. Onobrychoidei DC., whose main in order to create a list of morphological characters that diagnostic characteristic is pubescence with mostly potentially differentiate the studied taxa. A comparative medifixed or asymmetrically bifurcate hairs (Podlech list of A. borysthenicus and A. onobrychis morphological and Zarre, 2013). Ambiguity in the taxonomical status characters was completed on the basis of mentioned of A. borysthenicus may cause the loss of its endemic and protologues and other special sources, field experience, protected status. and the study of herbarium material from KW and KWU When morphological characteristics are not enough, (approximately 100 specimens). molecular analysis can help resolve the problem with Morphological analysis of the Astragalus species from separation of close taxa. Nuclear ribosomal spacers (ITS1 Turkey and adjacent areas (A. bachmarensis, A. akmanii, A. and ITS2) are widely used as molecular phylogenetic dasycarpus, A. ansinii, and A. viridissimus) was also used to markers for plants because of their universality, simplicity verify the presence of morphologically similar parameters in amplification, intragenomic uniformity, and variability between A. borysthenicus and related species endemic to at the specific, generic, and family levels (Baldwin et Turkey or the Western Caucasus. Morphological features al., 1995; Álvarez and Wendel, 2003). Moreover, ITS2 were analyzed according to protologues and other special secondary structure analysis allows to assess the level of sources (Freyn, 1892; Grossheim, 1930; Chamberlain reproductive isolation and to distinguish taxa (Coleman and Matthews, 1969, 1970; Duman et al., 1995; Güner et and Mai, 1997; Coleman, 2000, 2007; Ruhl et al., 2009). al., 2000; Uzun et al., 2009). Only those characteristics ITS sequences obtained without cloning may contain that were comprehensively represented in the available ambiguous sites. This can happen due to PCR and literature for all comparable species were taken in order sequencing errors (Clarke, 2001; Kunin et al., 2011), or to create a list of morphological characters that potentially can be the signature of intragenomic polymorphism. differentiate these taxa. The names of Astragalus sections Considering that from 30% to 70% of all flowering plant are given according to Podlech and Zarre (2013). species had hybridization events in their phylogenetic 2.2. Molecular analysis histories (Wendt et al., 2001), another reason for the 2.2.1. Plant material presence of ambiguous sites is hybridization (Hřibová et Three herbarium specimens of A. borysthenicus and al., 2011). However, a number of plant sequences have no two specimens of A. onobrychis were used for molecular ambiguous positions and it is reasonable to suggest that analysis. Data on vouchers and origins of plants are they most likely did not have hybridization events in the presented in Table 1. past. Therefore, such organisms can probably be treated as relative analogs of “pure homozygous lines” or true 2.2.2. DNA extraction, amplification, sequencing, and breeding organisms (TBOs) (King et al., 2007). annotation Thus, the aim of our study was to assess the taxonomical Total genomic DNA was extracted from herbarium status of A. borysthenicus using morphological feature material using a modification (Tarieiev et al., 2011) of comparisons, phylogenetic reconstructions, and analyses the method developed by Doyle and Doyle (1990). PCR of secondary structures of ITS1 and ITS2 transcripts. reactions of ITS1-5.8S-ITS2 regions were performed as Moreover, an additional aim was to find a “pure” described by White (1990) using ITS1 and ITS4 universal nonhybrid A. borysthenicus (TBO) or to ascertain the primers. Sequencing was carried out by Macrogen Inc. and probability of the hybridogenic origin of analyzed obtained sequences were deposited in GenBank (see Table specimens of A. borysthenicus. We also used the same 1). type of data for genetically related species (A. dasycarpus Sequences were assembled and edited manually using Chamberlain, A. akmanii Aytaç & H.Duman, A. ansinii BioEdit software (Hall, 1999). Ambiguous positions were Uzun, Terzioğlu & Pal.-Uzun, A. viridissimus Freyn & coded using the NC-IUPAC ambiguity codes if two peaks Sint., and A. bachmarensis Grossh.) to assess the level of were present at the same position and the lower peak was separation of A. borysthenicus. more than 1/2 of the higher peak in the chromatograms. Additionally, we analyzed the presence of ambiguous 2. Materials and methods positions in sequences of A. bachmarensis (JQ685625), A. akmanii (JQ685622), and A. dasycarpus (JQ685634) 2.1. Morphological analysis from previous investigations (Dizkirici et al., 2013)