untitled 60 ACTA BOT. CROAT. 75 (1), 2016 Acta Bot. Croat. 75 (1), 60–66, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0001 ISSN 0365-0588 eISSN 1847-8476 Resurrection of Ornithogalum brevipedicellatum (Asparagaceae) with morphological and molecular data Candan Aykurt*1, İsmail G. Deniz2, Duygu Sari3, Mecit Vural4, Hüseyin Sümbül1 1 Department of Biology, Faculty of Science, Akdeniz University, Antalya, Turkey 2 Department of Biology Education, Faculty of Education, Akdeniz University, Antalya, Turkey 3 Department of Field Crops, Faculty of Agriculture, Akdeniz University, Antalya, Turkey 4 Department of Biology, Faculty of Science, Gazi University, Ankara, Turkey Abstract – This study evaluates Ornithogalum brevipedicellatum, which was previously accepted as a syn- onym of O. oligophyllum, as a separate distinct species and discusses the similarities and differences between O. brevipedicellatum and its related species (O. oligophyllum and O. pamphylicum). Similarities and differ- ences among these species were identifi ed by morphological and molecular studies. The leaf morphology and infl orescence of O. brevipedicellatum and O. pamphylicum are similar to each other, and in terms of these features, they show differences from O. oligophyllum. Some diagnostic characteristics are quite different in O. brevipedicellatum and O. pamphylicum, such as the size of tepals, length of fruiting pedicels and style. Morphological data were supported by the results obtained from molecular studies. According to a dendro- gram obtained by molecular studies, O. brevipedicellatum and O. pamphylicum are similar. O. oligophyllum is more closely related to O. pyrenaicum used as an out-group. Additionally, the seeds of O. brevipedicella- tum were examined with the use of scanning electron microscopy. Key words: Asparagaceae, endemic, molecular, morphology, Ornithogalum, scanning electron microscopy, synonym, Turkey. * Corresponding author, e-mail: candan@akdeniz.edu.tr Introduction Asparagaceae Juss. (1789) (including Agavaceae Du- mort., Aphyllanthaceae Burnett, Hesperocallidaceae Traub, Hyacinthaceae Batsch ex Borkh., Laxmanniaceae Bubani, Ruscaceae M.Roem., Themidaceae Salisb.) include 143 plant genera (APG III 2009). Hyacinthaceae can be treated as subfamily Scilloideae of Asparagaceae, and the subfami- lies Hyacinthoideae, Ornithogaloideae, Urgineoideae and Oziroëoideae of Hyacinthaceae are then treated as tribes Hyacintheae, Ornithogaleae, Oziroëeae and Urgineeae (e.g. APG III 2009). Ornithogaloideae treated as one of the sub- families in Hyacinthaceae show a distribution through Eu- rope, south-west Asia and Africa, and include about 280 species (Speta 1998). Nowadays, as a result of molecular phylogenetic studies on this subfamily, 19 monophyletic genera are accepted within Ornithogaloideae: Albuca, Avon- sera, Battandiera, Cathissa, Coilonox, Dipcadi, Eliokarmos, Elsiea, Ethesia, Galtonia, Honorius, Loncomelos, Me lom- phis, Neopatersonia, Nicipe, Ornithogalum, Pseudo gal to- nia, Stellarioides and Trimelopter (Martínez-Azorín et al. 2011). Recently, molecular tools have gained importance in identifying taxonomic relations. In Ornithogaloideae, matK, trnL intron, trnL-F spacer, and rbcL plastid DNA sequences have been used for phylogenetic analysis (Manning et al. 2009, Martínez-Azorín et al. 2011) because plastid se- quences comprise an important source for phylogenetic re- construction, mostly at interspecifi c or higher taxonomic levels (Clegg and Zurawski 1992, Cameron 2004, Kress and Erikson 2007). In Flora of Turkey (Cullen 1984), the species of the ge- nus Ornithogalum L. were evaluated under 4 subgenera, subg. Beryllis (Salisb.) Baker, subg. Ornithogalum, subg. Myogalum (Link) Baker and subg. Caruelia (Parl.) Baker. The recent arrangements recombined Beryllis subg. as Lan- comelos Raf., Myogalum subg. as Honorius Gray and Ca- ruelia subg. as Melomphis Raf. (Martínez-Azorín et al. 2011). Anatolia is an important area for the distribution of the genus Ornithogalum in Asia (Uysal et al. 2005). Since the last revision by Cullen (1984) for the Flora of Turkey, nu- TAXONOMIC NOTES ON ORNITHOGALUM BREVIPEDICELLATUM ACTA BOT. CROAT. 75 (1), 2016 61 merous new taxa, records and combinations of Ornithoga- lum have been introduced from Turkey (e.g. Davis et al. 1988, Özhatay 2000, Düşen and Sümbül 2002, 2003, Düşen and Deniz 2005, Uysal et al. 2005, Özhatay and Kültür 2006, Bağcı et al. 2009, 2011, Özhatay et al. 2009, Yıl dı- rımlı 2009, Koca and Yıldırımlı 2010, Özhatay et al. 2011, Mutlu and Karakuş 2012). A total of 61 species of the genus Ornithogalum are distributed in Turkey, 31 of them endem- ic to the country (Uysal 2012). The fi rst specimens of Ornithogalum brevipedicellatum in Turkey were collected by Bourgeau in 1860 in the higher mountain steppes of the Elmalı (Antalya) district, the re- gion known as ‘Lycia’ in antiquity. These specimens were evaluated under the name of “O. brevipedicellatum” by Boissier, and subsequently were introduced to science in 1873 by Baker (Boissier 1884, Cullen 1984). O. brevipedi- cellatum was reported as the synonym of the O. oligophyl- lum species in the Flora of Turkey (Cullen 1984), the Plant List of Turkey (Uysal 2012) and in “The Plant List” data- base. O. oligophyllum has a substantially wide area of oc- cupancy in Turkey. It was previously reported in The Flora of Turkey that the specimens of the species O. oligophyl- lum, which were collected in Antalya, Isparta and Konya, would be classifi ed as O. brevipedicellatum provided that the distinguishing determinant characteristics were support- ed by a suffi cient number of samples (Cullen 1984). Materials and methods Plant samples and morphological studies The plant specimens of the genus Ornithogalum were collected by fi eld studies between 2012 and 2014 in the Muğla and Antalya Provinces. During the fi eld studies, GPS locations of O. brevipedicellatum were taken, and the individuals of its populations were numbered. This data was used to determine the threat category of O. brevipedi- cellatum according to the Categories and criteria of IUCN (Version 11, 2014). The extent of occurrence (EOO) and area of occupancy (AOO) values were calculated. In the present study, Ornithogalum brevipedicellatum known as a synonym of O. oligophyllum is morphological- ly described in detail and compared with its related species, O. oligophyllum and O. pamphylicum O.D.Düşen & Süm- bül, with the data obtained from morphological and molec- ular studies. The individuals of these species were observed during fi eld studies and the morphological evaluations were done both in the fi eld and in laboratory. Specimens collect- ed and used in molecular studies within the scope of this study, are shown in Tab. 1. The digital isotype photographs obtained from MNHN (Museum National d’Histoire Na- turelle) of O. brevipedicellatum were also examined. Addi- tionally, a number of herbarium specimens of O. oligophyl- lum collected from different parts of Turkey in the ISTE (Istanbul University, Herbarium of the Faculty of Pharma- cy) and GAZI (Gazi University Herbarium) were morpho- logically examined (see Appendix). Seed micromorphology of Ornithogalum brevipedicel- latum was investigated using scanning electron microscopy (SEM) techniques. For SEM study, the seeds were treated with gold conjugate on stub. The microphotographs were taken with a Zeiss LEO-1430 scanning electron micro- scope. Molecular study: DNA isolation, PCR amplifi cation and sequencing Complete DNA of Ornithogalum brevipedicellatum and O. pamphylicum was extracted from the leaves of herbari- um specimens using the cetyl trimethyl ammonium bro- mide (CTAB) method of Doyle and Doyle (1990). DNA concentration and quality were tested with 1% agarose gel against a DNA standard. Two different plastid regions were used for molecular analysis to identify the phylogenetic similarities of the species. Amplifi cation of these regions was conducted with the universal primers used in previous Tab. 1. Locality data of collected specimens of Ornithogalum for the current study. Species Locality Collection data O. brevipedi- cellatum C2 Muğla, Fethiye, Seki, 6 km from Seki to Yuva, Ak Mountain, steppe, 1980 m, 27.iv.2012. C. Aykurt 3071(AKDU) Muğla, Fethiye, Seki, 4–6 km from Seki to Yuva, Ak Mountain, 1900–1980 m, steppe, 4.v.2012. C. Aykurt 3084 (AKDU) Muğla: Fethiye, Seki, 4–6 km from Seki to Yuva, Ak Mountain, 1900–1980 m, steppe, 22.v.2012. C. Aykurt 3137* (AKDU) Antalya: Kaş, Gömbe, between Gömbe-İkizgöller, Subaşı environs, plateau, 2080 m, 25.05.2012. C. Aykurt 3156 (AKDU) Muğla, Fethiye, Seki, 6 km from Seki to Yuva, Ak Mountain, steppe, 1975 m, 30.04.2013. C. Aykurt 3741* (AKDU) Muğla, Fethiye, Seki, 6 km from Seki to Yuva, Ak Mountain, steppe, 1827 m, 30.04.2013. C. Aykurt 3742* (AKDU) Muğla, Fethiye, Seki, 6 km from Seki to Yuva, Ak Mountain, steppe, 1825 m, 23.05.2013. C. Aykurt 3868 (AKDU) Muğla, Fethiye, Seki, 6 km from Seki to Yuva, Ak Mountain, steppe, 1975 m, 23.05.2013. C. Aykurt 3869 (AKDU) O. pamphy- licum Antalya, above Fesleğen Plateau, Sakarpınar, 1850–1900 m, calcerous slopes, 4.v.2012. C. Aykurt 3087 (AKDU) Antalya, Elmalı, İmecik Plateau, Bey Mountains, 1800 m, 29.04.2012. C. Aykurt 3089* (AKDU) Antalya, Elmalı, Kızlarsivrisi Mountain, Karakuyu district, 1900 m, openings in Cedrus libani forest, calcareous slopes, 09.v.2012. İ.G. Deniz 4528 (AKDU) Antalya: Çalbalı Mountain, 1700 m, calcareous slopes, 15.v.2014. İ.G. Deniz 5548 (AKDU) O. oligo- phyllum C3 Antalya: Sarıçınar, 36 S 274566, 4096808, 1380 m, 14.iv.2014. C. Aykurt 3882 (AKDU) C2 Antalya: Finike, West side of Finike, under Cedrus libani, 1200 m, 24.iv.2014. C. Aykurt 3911 (AKDU) AYKURT C., DENIZ I. G., SARI D., VURAL M., SÜMBÜL H. 62 ACTA BOT. CROAT. 75 (1), 2016 studies. The trnL intron and trnL spacers were amplifi ed with c (5’-CGAAATCGGTAGACGCTACG) and f (5’-AT T- TGAACTG-GTGACACGAG) primers described in Ta ber let et al. (1991). Amplifi cation of the rbcL gene was performed using a forward primer (427F), 5’-GCTTATTCAAAAA- C T TTCCAAGGCCCGCC, and a reverse primer (724R), 5’-TCGCATGTACCTGCAGTTGC (Lledó et al. 1998). Polymerase chain reaction (PCR) for both trnL and rbcL regions contained 2 mM MgCl2, 0.2 mM of each dNTP, 10 pM μL–1 of each primer, 1 U of Taq DNA polymerase (Fer- mentas Life Sciences, Burlington, Canada) with supplied reaction buffer at 10× concentration, and 40 ng of template DNA. The amplifi cations were performed on a program- mable thermocycler (BIONEER, MyGenie™) with the fol- lowing program: one cycle of 4 min at 94 °C, 28 cycles of 1 min at 94 ºC, 30 s at 48 °C for rbcL, or 1 min at 50 °C for trnL, 1 min at 72 °C, and for fi nal extension one cycle of 7 min at 72 °C. PCR products were cleaned up using the GeneJET gel extraction kit (Thermo Scientifi c Fermentas, Vilnius, Lithuania). Sequencing process was carried out at Iontek Laboratory in Istanbul, Turkey as direct sequencing from PCR products. The sequences of other species (O. oli- gophyllum and O. pyrenaicum) were retrieved from Gen- Bank databases at the National Center for Biotechnology Information (NCBI) and compared with O. brevipedicella- tum trnL and rbcL sequences. A phylogenetic tree was constructed using the software MEGA 5. Bootstrap values are displayed at tree nodes (Tamura et al. 2011). Results Morphological studies Ornithogalum brevipedicellatum Boiss. ex Baker, J. Linn. Soc., Bot. 13: 263 (1873) (Figs. 1–3) Geophyte, 5–10(–15) cm high; bulb 1.3–1.8(–2) × 1.3– 1.8(–2) cm, ovoid-spherical, without bulblets; outer tunics cream to pale-brown. Leaf synanthous, 4–6(–7), 9–37 × 2.3–0.5 cm, linear-lanceolate, canaliculate, with a whitish median line, gradually narrowing towards the base, longer than scape, margins entire, glabrous. Scape below the ground level, 3–12 cm, slender. Infl orescence congested racemose, racem borne at ground level, dense, 2–6 × 3–4.5 cm, erect, with (1–)5–15(–18) fl owers. Bracts lanceolate, long acuminate, (12–)15–23 × 3–5 mm, membranous, lon- ger than pedicels. Flowers sessile or subpedicellate up to 3 mm at anthesis and fruiting period. Tepals eliptic to ovate- eliptic, obtuse to acute at apex; outers 12–18 × 4–5 mm; inners 13–18 × 4.5–5 mm, white inside, green with narrow- ly white margins outside. Filaments white, conspicuously tapering towards the apex, 4–6 × 1–2 mm; anthers medifi xed, milky white, 2–2.2 mm length. Ovary ovoid, 3.5–5 × 2–3.5 mm, pale green, longer than style; style 1.5–2 mm length; stigma capitate. Capsule 10–15 × 10–15 mm, ovoid, erect, distinctly winged, pale brown. Seeds numerous, 1.8–2 × 1.2–1.8 mm, elliptic-subglobose to globose, black, strongly apiculate; testa reticulate, with reticules formed by promi- nent crests, testa cells irregular. Hitherto Ornithogalum brevipedicellatum was evaluat- ed as the synonym of O. oligophyllum; however the species is morphologically more related to O. pamphylicum, de- scribed in 2002. The infl orescence type of O. brevipedicel- latum is similar to O. pamphylicum. The raceme of O. bre- vipedicellatum is very condensed in fl owering and fruiting period and borne at ground level. Its fl owers are sessile or the fl oral or fruiting pedicels are up to 3 mm long. The in- fl orescence of O. pamphylicum resembles O. brevipedicel- latum, but the internodes are more elongated and the pedi- cels are fl accid and longer at fruiting time. On the other hand, the lower pedicels of O. oligophyllum are longer then the fl owers; fruiting pedicels are 10–30 mm long. There- fore, the infl orescence of O. oligophyllum can be evaluated as corymbose or pseudocorymbose. Moreover, the infl ores- cence of O. brevipedicellatum is different due to its sessile or subpedicellate fl owers. Fig. 1. Ornithogalum brevipedicellatum (from C. Aykurt 3071): a) habit, b): fl ower, c) stamen, d) pistil, e) capsule. Scale bars: 1 cm (a,b,e), and 1 mm (c,d). Fig. 2. SEM photographs of the seeds of Ornithogalum brevipedi- cellatum (from C. Aykurt 3137): a) seed, b) seed surface. TAXONOMIC NOTES ON ORNITHOGALUM BREVIPEDICELLATUM ACTA BOT. CROAT. 75 (1), 2016 63 The other morphological differences of O. brevipedicel- latum, O. oligophyllum and O. pamphylicum are shown in Tab. 2. Habitat, distribution and conservation status Ornithogalum brevipedicellatum shares its habitats with several herbaceous plants such as Cerastium macranthum Boiss. Ceratocephala falcata (L.) Pers., Corydalis erdelii Zucc., Crocus bifl orus Mill. subsp. isauricus (Siehe ex Bowles) B. Mathew, Fumaria asepala Boiss., Gagea villo- sa (M. Bieb.) Sweet subsp. hermonis Dafni & Heyn, Scilla bifolia L., Tussilago farfara L., Viola heldreichiana Boiss. An old specimen collection record was documented in Flora Orientalis indicating that specimens of the Ornithog- alum brevipedicellatum species were collected on Mount Trodos in southern Cyprus (Boissier 1884). However, re- cent data supporting these dubious records was not docu- mented and for that reason, the species O. brevipedicella- tum was evaluated as endemic to Turkey. The conservation status of Ornithogalum brevipedicel- latum is described below according to the rules of citation. Considering the IUCN (2014) categories and criteria (Ver- sion 11), the species should be placed in the EN (endan- gered) category, according to the criteria for critically en- dangered, endangered and vulnerable. The subpopulations of the species were identifi ed in Seki (Muğla) and Gömbe (Kas, Antalya) (Fig. 4). No artifi - cial factors endangering the development of the species in its area of occupancy were present. Therefore, it was pro- jected that the population of the species in terms of percent- age did not decrease through time as a result of anthropo- logical effects. Item A cannot therefore be evaluated. The distance between the two locations of the species is 25 km. The EOO value of the species was determined as 130 km2 (EN B1b(iii)) taking both locations of occupancy and the area contained within the shortest continuous imaginary boundary. The AOO value in this area, where the species was identifi ed was calculated as 13 km2 (EN B2b(ii)). The number of individuals identifi ed in the subpopulations of the species was determined as 400 at the Seki location and 200 at the Gömbe location (EN C). Molecular studies Individuals of Ornithogalum brevipedicellatum and O. pamphylicum were collected from different localities for molecular analysis (Tab. 1). The sequences of these speci- mens were compared and evaluated with O. oligoliphylum Fig. 3. Ornithogalum brevipedicellatum on Ak Mountain (Elmalı, Antalya). Tab. 2. Comparison of the morphological characters of Ornithogalum brevipedicellatum, O. oligophyllum and O. pamphylicum. Features O. brevipedicellatum O. oligophyllum O. pamphylicum Scape length (cm) 3–12 (borne at ground level) 4–15 3–15 Number of leaves 4–6(–7) 2–3 (rarely 4) (3–)4–11(–13) Leaves with a whitish median line without a whitish median line with a whitish median line Leaves width 2–5 mm 5–20 mm 1–4 mm Infl orescence congested racemose corymbose or pseudocorymbose laxly racemose (internodes more elongated) Number of fl owers (1–)5–15(–18) 3–10(–20) 3–25 Fruiting pedicels Capsules sessile or subpedicellate (up to 3 mm long), erect 10–30 mm, become fl accid at base Capsules pedicettale (6–12 mm), erect Flowers odour odourless odourless Tepals (mm) 12–18 × 4–5 – 20–30 × 5–9 Style length (mm) 1.5–2 2–3.5 4–5 Fig. 4. Distribution areas of Ornithogalum brevipedicellatum (), Ornithogalum oligophyllum (●) and Ornithogalum pamphylicum (◼) in Turkey. AYKURT C., DENIZ I. G., SARI D., VURAL M., SÜMBÜL H. 64 ACTA BOT. CROAT. 75 (1), 2016 and O. pyrenaicum retrieved from the GenBank database. To amplify the trnL and rbcL region of O. brevipedicella- tum and O. pamphylicum species, c/f and 427F/724R prim- er pairs were used, respectively, as described in methodolo- gy section. About 1200 bp amplicons for trnL and 300 bp amplicons for rbcL were yielded in PCR assays. These am- plicons were sequenced and compared with those of O. oli- gophyllum species (Fig. 5) available in the GenBank using the BLAST similarity search tool. In the trnL tree, two groups were identifi ed. All of the individuals of O. brevi- pedicellatum were aligned in the fi rst group. O. pamphyli- cum was the closest species to O. brevipedicellatum while O. oligophyllum and O. pyrenaicum were more distant from this group. Similarly, three individuals of O. brevipedicel- latum were located together in the rbcL tree. Although O. pamphylicum was placed in a different branch, it was the closest species to O. brevipedicellatum while O. oligophyl- lum and O. pyrenaicum species were positioned in another group. Discussion The Ornithogalum genus, previously classifi ed into nu- merous subcategories by several different researchers, was rearranged recently in the light of the novel molecular phy- logenetic studies conducted on the Ornithogaloideae sub- family (Martínez-Azorín et al. 2011). Moret and Galland (1992) indicated that many taxa have been described in subg. Ornithogalum which is a taxonomically problematic subgenus on the basis of subtle morphological variations, usually with little biological signifi cance. The complex tax- onomy of subg. Ornithogalum may be due to intraspecifi c variations, the plasticity of individuals, their habits being strongly dependent on environmental factors and poor con- servation of the types (Moret and Galland 1992). Martínez-Azorín et al. (2010) reported that some au- thors have used the infl orescence structure and/or the length of the fl oral bracts relative to their pedicels as diagnostic characteristics. When Ornithogalum brevipedicellatum, O. pamphylicum and O. oligohyllum species are evaluated with respect to the infl orescence type and length of the ped- icels, it is seen that the specimens of O. brevipedicellatum are distinct; distinguished from the other species by the lack of pedicels, or the presence of very short pedicels- either during fl owering or fruiting periods. The presence of very short internodes between the fl owers in the subpedicellate or the sessile fl owers allowed the infl orescence of the spe- cies to be evaluated as congested racemose, rather than cor- ymb or pseudocorymb. Furthermore, the habitus of these three species show differences, while the fi rst fl owers of O. brevipedicellatum and O. pamphylicum are at ground level but the infl orescence of O. oligophyllum has generally a scape above ground level. The pedicels of O. oligophyllum are distinctly longer than the pedicels of O. brevipedicella- tum and O. pamphylicum in fl owering and fruiting periods. Ornithogalum brevipedicellatum and O. pamphylicum are similar to each other with respect to their leaf morphol- ogy and infl orescence in fl owering time. They have nar- rower leaves from O. oligophyllum. We observed the white line on the leaf surface of O. pamphylicum and the ovary of the species is distinctly winged, contrary to the description of the species by Düşen and Sümbül (2002). Therefore, O. pamphylicum was included in subg. Ornithogalum in the present study but, on the contrary, in subg. Myogalum by Düşen and Sümbül (2002). The closer morphologic prox- imity between O. brevipedicellatum and O. pamphylicum as opposed to O. oligophyllum was supported by the data obtained from molecular studies. Considering to the phylo- genetic tree obtained from trnL and rbcL sequences, the in- dividuals of O. brevipedicellatum were separated from O. oligophyllum in two different dendograms. O. brevipedicel- latum are closer to O. pamphylicum compared with O. oli- gophyllum. The most remarkable morphological differences be- tween O.brevipedicellatum and O. pamphylicum can be de- fi ned as follows: (1) The capsules of O. brevipedicellatum are generally sessile, sometimes subpedicellate but they are pedicellate and pendant in O. pamphylicum; (2) lengths of Fig. 5. Phylogenetic trees of trnL (a) and rbcL (b) sequences from six Ornithogalum specimens. Locations (*) and GenBank accession numbers (**) are given next to the Ornithogalum name. TAXONOMIC NOTES ON ORNITHOGALUM BREVIPEDICELLATUM ACTA BOT. CROAT. 75 (1), 2016 65 the style are quite different; (3) O. brevipedicellatum has more congested infl orescence while the internodes are elon- gated in O. pamphylicum; (4) The tepals are more narrow in O. brevipedicellatum (Tab. 2). The seed size, shape, color, surface ornamentation and shape of the cells, raphe, micropylar and chalazal poles are useful diagnostic characteristics for seed micro-morpholo- gical studies conducted on the genus Ornithogalum (Bed- norz and Czarna 2008, Çitak et al. 2015). Moret et al. (1990) and Martínez-Azorín et al. (2010) reported that subg. Orni- thogalum shows globose and apiculate seeds with reticulate testa. Similarly, the seeds of O. brevipedicellatum are ellip- tic-subglobose to globose, distinctly apiculate with reticu- late testa. The results obtained from the present study clear- ly indicate that O. brevipedicallatum is a distinct species and cannot be claimed to be a synonym of O. oligophyllum. Acknowledgements The authors are indebted to Akdeniz University Scien- tifi c Research Projects Unit (Project number: 2012.01. 0115.004) for fi nancial support to fi eld studies and thanks to the curators of AKDU, ISTE and GAZI. Appendix Additional specimens examined O. oligophyllum: A1(E) Kırklareli: Demirköy, Mahya- dağ, Mareşal road, Fagus gap, 860 m, 22.5.1974, A. Baytop et E. Tuzlacı (28271 ISTE). Kırklareli: Demirköy, Mahya- dağ, 1030 m, 22.5.1974, A. Baytop et E. Tuzlacı (28304 ISTE). Kırklareli: Merkez, between Dereköy and boundary, 1.5.1973, G. Ertem (24281 ISTE). Kırklareli: Centre, wo- odlands in Dereköy, Kocakaynak environs, 26.4.1974, N. Özhatay, E. Özhatay (27642 ISTE), Kırklareli: Demirköy, between Yeniceköy and Velika, 1 km to Haydut Mountain, 24.4.1988, G. Dalgıç, N. Başak (59757 ISTE). Kırklareli: Centre, Dereköy, boundary, under Fagus, 23.4.1988, G. Dalgıç, N. Başak (59758 ISTE). Kırklareli: Demirköy, Veli- ka, Balaban village picnic areas, under woodlands, 24.4.1988, G. Dalgıç, N. Başak (59766 ISTE). A3 Bolu: between Bolu and Mudurnu, under Quercus, 1250–1350 m, 03.05.1993, Z. Aytaç 5789 et al. (GAZI). Bolu: Kale, Tenekeci River, streamside, woodlands, 800 m, İ. Kılınç 1029 (GAZI). A4 Ankara: Kızılcahamam, Soğuksu Nation- al Park, Çakmaklı environs, 1450–1500 m, 12.05.1990, Ö. Eyüboğlu (GAZI). Ankara: Çubuk, between Ovacık and Saraycık villages, Borucağın Hill, shrubs, 1250–1380 m, 20.5.1992, E. Dündar 1193 (GAZI). Çankırı: Atkaracalar, Dumanlı Mountain, Atkaracalar plateau, Abaza River envi- rons, steppe, 1300–1450, 09.05.1992, A. Duran 1516 (GAZI). Ankara: Ayaş, Ayaşbeli, Çal Hill, steppe, 1300– 1380 m, 13.04.1986, M. Vural 4029 (GAZI). Kırıkkale: Koçubaba Town, 1200 m, 31.03.1990, A.A. Dönmez 1696 (GAZI). A9 Kars: Arpaçay, Karakale Village, under Rumex shrubs, 2250 m, 16.06.1984, H. Ocakverdi 1714 (GAZI). Ardahan: Posof, Baykent village, birch woodlands, 1900 m, 25.6.2007, Sezgin Esen (87083 ISTE). Ardahan: Çataldere village birch woodlands, 9.6.2008, Sezgin Esen (87275 ISTE). B4 Kırşehir: Çiçek Mountain, Halaçlı Village, Almanderebaşı environs, Quercus shrubs, slopes, 1550 m, 09.05.1993, F.A. Karavelioğulları 1026 et al. (GAZI). B5 Nevşehir: between Uçhisar and Göreme, erosional dry slopes, 1250 m, 19.04.1989, M. Vural 4497 et al. (GAZI). B6 Kayseri: Sarız, Yalak environs, Binboğa Mountain, steppe, 1800–2200 m, 07.05.1991, Z. Aytaç 3709 et al. (GAZI). Kahramanmaraş: Binboğa Mountain, west of Kaşvut Village, alpine steppe, under Cedrus libani, cliffs, 1750–2250 m, 08.05.1991, Z. Aytaç 3733 et al. (GAZI). Kayseri: Sarız, Binboğa Mountain, Doğankonak, Karagöz, 1450–1900 m, 21.04.1992, Z. Aytaç 4486 et al. (GAZI). Kayseri: Pınarbaşı, south of Aşağıbeyçayırı village, eroded west slopes, 29.4.2004, B. Yıldız, T. Arabacı (89931 ISTE). B7 Malatya: Kubbe Mountain, Kubbe plateau, mountain steppe, 1700 m, 1.6.1992, B.Yıldız 9378 (ISTE). B8 Er- zurum: between Aşkale and Tercan, Kükürtlü environs, slopes and shrubs, 1950 m, 23.5.2004, M. Koyuncu, M. Fı- rat, M. Armağan (88864 ISTE). B9 Bitlis: Adilcevaz, Süphan Mountain hillside, Süte plateau, meadows, 1900 m, 04.06.1993, Y. Altan 4796 (GAZI). Van: Gevaş, west slopes of Çadır Mountain, steppe, 2300 m, 06.06.1993, Y. Altan 5077 (GAZI). C3 Isparta: Barla, Gelincik Mountain, Tahtacıçukuru Hills environs, Pinus nigra, 1800–1900 m, 09.05.2009, H. Duman 9921 (GAZI). C6 Kahramanmaraş: Engizek Mountain, Küçükcerit Village, under Quercus, 1400–1500 m, 21.04.1987, H. Duman 2486 (GAZI). Kahramanmaraş: Engizek Mountain, south of Şaç Rocks, wetlands, 2400–2500 m, 20.05.1987, H. Duman 2680 (GAZI). Kahramanmaraş: Erince Mountain, under Quer- cus, 1300–1400 m, 22.04.1987, H. Duman 2507 (GAZI). Kahramanmaraş: between Göksun and Geben, Kayranlı Mountain, east slopes, 30.4.2014, B. Yıldız, T. Arabacı (89959 ISTE). C7 Şanşıurfa: Karacadağ, 2 km from Karabahça to Dağ, Reme River environs, stony areas, 1400 m, İ. Eker 181-b (GAZI). References APG III., Angiosperm Phylogeny Group, 2009: An update of the angiosperm phylogeny group classifi cation for the orders and families of fl owering plants: APG III. 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