Systematic placement of the Turkish endemic genus Ekimia (Apiaceae) based on morphological and molecular data

Turkish Journal of Botany Turk J Bot (2015) 39: 673-680 © TÜBİTAK doi:10.3906/bot-1405-111 http://journals.tubitak.gov.tr/botany/ Research Article Systematic placement of the Turkish endemic genus Ekimia (Apiaceae) based on morphological and molecular data 1, 2 3 2 Dmitry LYSKOV *, Galina DEGTJAREVA , Tahir SAMIGULLIN , Michael PIMENOV 1 Department of Higher Plants, Biological Faculty, Moscow State University, Moscow, Russia 2 Botanical Garden, Biological Faculty, Moscow State University, Moscow, Russia 3 Department of Evolutionary Biochemistry, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Received: 30.05.2014 Accepted/Published Online: 01.02.2015 Printed: 30.07.2015 Abstract: The systematic position of the monotypic genus Ekimia H.Duman & M.F.Watson (Apiaceae), a narrow endemic to Turkey, was evaluated on the basis of morphological data and nrDNA ITS sequences. Ekimia bornmuelleri (Hub.-Mor. & Reese) H.Duman & M.F.Watson was initially described in Prangos Lindl. Due to the unique fruit morphology uncommon for this genus it was later shifted to an independent genus. In the Bayesian and most parsimonious trees, E. bornmuelleri is sister to Laserpitium petrophilum Boiss. & Heldr and Laserpitium glaucum Post within the Daucinae clade. This result is consistent with its morphology: the presence of the primary and secondary ribs of E. bornmuelleri fruits brings the species closer to Laserpitium rather than Prangos. Key words: Apiaceae, carpology, Daucinae, Ekimia, internal transcribed spacer (ITS), Laserpitium, molecular phylogeny, Turkey 1. Introduction Ekimia bornmuelleri (Hub.-Mor. & Reese) H.Duman & M.F.Watson is a narrow endemic to the Turkish province Burdur in Central Anatolia, where it was collected for the first time in 1938. Huber-Morath and Reese described this species in the genus Prangos, due to resemblance of immature fruits to fruits of Prangos lophoptera Boiss. (Huber-Morath, 1945). The authors, however, paid attention to notable distinction of this species from the other members of Prangos. According to Huber-Morath and Reese, it differs from other congeners by fleshy brushlike blue-green leaves, small number of umbel rays, broadly elliptic to orbicular bracteoles, and winged secondary ribs of immature mericarps. The authors of the taxonomic treatment of Prangos, Herrnstadt and Heyn (1972, 1977), adhered to the same opinion; they expressed reasonable doubts about the generic attribution of this species based on the morphology of fruits, umbels, and leaves, which is not typical to Prangos. Although Herrnstadt and Heyn considered that this species should be excluded from Prangos, they indicated that a study of mature fruits was needed to decide on the generic placement of the species. Duman and Watson (1999) collected new material * Correspondence: with mature fruits and well-developed flowers during their fieldwork in Turkey. They examined the cross-sections of mature fruits, presented a detailed description of plant morphology, and placed the species in the independent monotypic genus Ekimia, noting its similarity to Prangos. The difference between genera was supported by the analysis of pollen morphology (Pehlivan et al., 2009). The objectives of the present research were to: (1) reassess in detail the carpological characters of Ekimia; and (2) ascertain its phylogenetic placement using sequences of nrDNA ITS, since it has been shown to be the most suitable instrument to clarify relationships of genera and species in Apiaceae (Spalik and Downie, 2007; Downie et al., 2010). 2. Materials and methods 2.1. Morphology and micromorphology Four species from three genera were chosen for carpological analysis. Prangos ferulacea (L.) Lindl. is the type species of the genus Prangos and has the typical anatomy of mericarps for this genus. Prangos lophoptera is a species with which E. bornmuelleri was initially compared (Huber-Morath, 1945). Laserpitium petrophilum and Laserpitium glaucum 673 LYSKOV et al. / Turk J Bot Post form the clade with Ekimia in molecular analysis and so the choice of L. petrophilum for carpological analysis was justified by the results of our preliminary molecular studies. Close examination of L. glaucum Post would have been more desirable, but we failed to obtain specimens of this species. The material was collected during our expedition to Turkey or taken from herbaria; the origin of the material is indicated in Figure 1. Fruits were examined under a light microscope. Mericarps were crosscut in the middle with a hand razor and cross-sections were then treated with phloroglucinol (Erdmann et al., 1986). We used standard terms to describe the fruit and other parts of the plant (Kljuykov et al., 2004). Microstructure of the fruit surface was studied by scanning electron microscopy (SEM). Fruits were coated with a 25-mm layer of Au-Pd using an Eiko IB-3 sputter coater. Micrographs were taken at 15 kV using a CamScan S-2 microscope at Moscow State University. Micromorphological features were described in the terms used by Ostroumova et al. (2010). 2.2. Molecular phylogenetic analyses For molecular phylogenetic study, nrDNA ITS sequences of Ekimia bornmuelleri and a close relative to Prangos, Bilacunaria microcarpa (M.Bieb.) Pimenov & V.N.Tikhom., were generated and analyzed along with a selection of sequences retrieved from GenBank. Total DNA was extracted from a herbarium specimen using a NucleoSpin Plant II kit (Macherey-Nagel, Germany) according to the protocol. Primers and PCR conditions conformed to those described in Valiejo-Roman et al. (2002). PCR products were purified using the DNA cleaning kit (Evrogen, Moscow, Russia) as indicated by the manufacturer’s instructions. Direct sequencing was performed on an automated DNA sequencer ABI Prism 3100-Avant (Applied Biosystems, Foster City, CA, USA) using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit for cycle sequencing reactions in accordance with the manufacturer’s instructions. The newly obtained sequences were deposited in GenBank (see Appendix; on the journal’s website). The initial set of taxa to be compared with Ekimia was selected using the BLAST option of the GenBank database. This search suggested that taxa belonging to the clade currently known as Daucinae (Downie et al., 2010) were most alike. The coherence in nrDNA ITS sequences was highest between Ekimia and Laserpitium species with 95%–93% values of identity. Other species from the Scandiceae and Cachrys clade were appended to the matrix to demonstrate relationships between groups. After preliminary analysis, a dataset of 99 species (see GenBank numbers in Appendix) including representatives of Laserpitium, Prangos, and allied taxa was compiled. 674 The trees were rooted with Physospermum cornubiense DC. in reliance upon the results obtained previously (Downie et al., 2010). A total set of 100 species, including an outgroup, were analyzed (...truncated)