Two New Spiny Species of Dianthus (Caryophyllaceae) from Turkey
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KSÜ Tarım ve Doğa Derg 21(4):545-554, 2018 KSU J. Agric Nat 21(4):545-554, 2018 Two New Spiny Species of Dianthus (Caryophyllaceae) from Turkey Ergin HAMZAOĞLU1 , Murat KOÇ2 , İlker BÜYÜK3 1Gazi University, Gazi Faculty of Education, Department of Mathematics and Science Education, 06500, Ankara, 2Bozok University, Boğazlıyan Vacotional School, 66400, Boğazlıyan, Yozgat, 3Ankara University, Faculty of Science, Department of Biology, 06100, Ankara : [email protected] ABSTRACT DOI :10.18016/ksudobil.347445 Some Dianthus L. (Caryophyllaceae) specimens which have spiny epicalyx scales and spiny leaves have been collected from the Hanönü Article History (Kastamonu) and the Mount Murat (Kütahya/Uşak). As a result of the Received : 28.10.2017 investigations, these specimens have been decided to be new species Accepted : 05.02. 2018 for the scientific world and they have been named as Dianthus varankii Hamzaoğlu & Koç and Dianthus halisdemirii Hamzaoğlu & Keywords Koç. The descriptions, ecological preferences, distribution information Dianthus, and conservation status of the new species was given. Also, they were new taxa, compared with closely related species, D. aculeatus Hamzaoğlu, based Turkey on its general morphology and seed micromorphology. The relationships between new species and their close relatives were Research Article determined based on the ITS sequences. Türkiye’den İki Yeni Dikenli Dianthus Türü (Caryophyllaceae) ÖZET Makale Tarihçesi Hanönü (Kastamonu) ve Murat Dağı’ndan (Kütahya/Uşak) epikaliks Geliş : 28.10.2017 pulları ve yaprakları dikenli olan bazı Dianthus L. (Caryophyllaceae) Kabul : 05.02.2018 örnekleri toplandı. Yapılan incelemeler sonucu bu örneklerin bilim dünyası için yeni oldukları anlaşıldı ve bunlar Dianthus varankii Anahtar Kelimeler Hamzaoğlu & Koç ve Dianthus halisdemirii Hamzaoğlu & Koç olarak Dianthus, adlandırıldı. Yeni türlerin betimlemeleri, ekolojik tercihleri, yayılış yeni taksonlar, bilgileri ve mevcut koruma durumları verildi. Ayrıca, yakın akraba Türkiye oldukları D. aculeatus Hamzaoğlu ile genel morfoloji ve tohum mikromorfolojisi bakımından karşılaştırıldılar. Yeni türler ile yakın Araştırma Makalesi akrabaları arasındaki moleküler benzerlikler ITS sekans analizine bağlı olarak belirlendi. To Cite : Hamzaoğlu E, Koç M, Büyük İ 2018. Two New Spiny Species of Dianthus (Caryophyllaceae) from Turkey. KSU J. Agric Nat 21(4):545-554, DOI :10.18016/ksudobil.347445. INTRODUCTION The development of PCR technology has increased our The genus Dianthus L. has approximately 600 species knowledge about the phylogeny of the Caryophyllaceae that mostly grows in Asia and Europe; there are 78 (White et al., 1990; Rettig et al., 1992; Gardes and species in Turkey (Dequan and Turland, 2001; Gemici Bruns, 1993). In recent years, p16-p25 primer pairs and Leblebici, 1995; Vural, 2008; Yılmaz et al., 2011; (surrounding ITS region) have been frequently used for İlçim et al., 2013; Hamzaoğlu et al., 2015; 2017). molecular phylogenetic studies in Caryophyllaceae Dianthus is the second largest genus after Silene L. in (Popp and Oxelman, 2001; Popp et al., 2005). Caryophyllaceae in Turkey (Reeve, 1967). The genus The aim of this study was to contribute to Dianthus has been divided into five sections in “Flora taxonomy by describe and adding two new species and Orientalis”. The most imported characters of section determine the genetic relationships among other spiny Dentati Boiss. are having a barbulate and dentate species of Dianthus collected from Turkey. petal limbs (Reeve, 1967; Boissier, 1867). On the other hand, some of the taxa of section Dentati have spiny MATERIAL and METHODS epicalyx leaves. Recently new species had been added Plant Materials into this spiny group (Yılmaz et al., 2011; Hamzaoğlu Dianthus specimens which were identified as new et al., 2014). In addition, studies have shown that seed species were collected from Hanönü (Kastamonu) and surface patterns in genus Dianthus are sufficiently Murat Mountain (Kütahya/Uşak) in Turkey. Besides, distinctive (Hamzaoğlu et al., 2014; 2015; Hamzaoğlu the specimens included the materials needed for the and Koç, 2015). seed micromorphology studies. The specimens KSÜ Tarım ve Doğa Derg 21(4):545-554, 2018 Araştırma Makalesi/Research Article collected were comprehensively evaluated by the use of Oxelman, 2001; Popp et al., 2005). The PCR the literature along with the specimens present in amplification was performed in 50 µl volume GAZI, ANK, HUB, KNYA, E, K and P herbaria (Reeve, containing 10 µl of 10 × buffer, 1 unit Taq DNA 1967; Bojňanský & Fargašová, 2007; Yılmaz et al., polymerase (Promega, Madison, Wisconsin), 200 µM 2011; Hamzaoğlu et al., 2014). Pictures were taken dNTPs, 2 mM MgCl2, and 10 pmol of both primers, p16 with a CANON EOS60D digital camera. The seed and p25. PCR amplification was performed in a surface micromorphology was visualized using the Biometra TProfessional Standard (Biometra, GmbH, FEI Quanta 450 FEG (in 2017) and LEO 440 (in 2013) Germany) as follows: 94°C for 2 min, 35 cycles of 94°C scanning electron microscopes. The vegetative for 30 s, 55°C for 1 min, and 72°C for 1 min, and a final characteristics were measured with a ruler with 0.5 extension step of 8 min at 72°C. The amplification mm accuracy and the floral characteristics were products were analyzed by electrophoresis in 1.2% determined using an ocular micro-meter. agarose gel containing ethidium bromide, and the product sizes were determined on gels by nucleotide DNA Extraction size marker (100 bp ladder; Fermentas, Vilnius, All of the molecular experiments were performed in Lithuania). ‘Plant Molecular Biology Laboratory of Ankara The PCR products were sequenced in both directions University’ by İlker BÜYÜK. Leaf material of six (once using the forward primer, and once using the Dianthus (Caryophyllaceae) specimens was dried in reverse primer) with a BigDye cycle sequencing kit silica gel to preserve the tissue in the field for DNA (Applied Biosystems, Foster City, California) using an extraction. ABI 3130XL genetic analyzer Applied Biosystems, The genomic DNA was extracted according to the Foster City, California). The derived ITS sequences modified CTAB method as described by Aras et al. identity was confirmed by appropriate program Blast (2003). NanoDrop Lite Spectrophotometer (Thermo (NCBI) to search the DNA sequence databases for high Scientific, USA) was used to check DNA quality and similarity with other Dianthus species quantity and also the integrity of each DNA was (http://www.ncbi.nlm. nih.gov/BLAST). The quality checked by running a 1% agarose gel containing check of the nucleotide peaks and the collapsing the ethidium bromide and were visualized using an forward and reverse Sanger reads into consensus ultraviolet transilluminator (Syngene Chemi Genius 2 sequence was done through Geneious version R9 Bio Imaging System, UK). (http://www.geneious.com, Kearse et al., 2012). PCR Amplification and Sequencing Sequence Alignment and Phylogenetic Analysis Sequence comparison of the ITS is frequently used in The amplified fragments were sequenced and the phylogenetic reconstructions at the intraspecific outgroup Petrorhagia saxifraga (L.) Link. was taxonomic level (Chase et al. 2000; White et al., 1990; obtained from GenBank (https://www.ncbi.nlm. Hamzaoğlu et al., 2017). On this basis, ITS sequences nih.gov/genbank/) to compare with Dianthus of six Dianthus specimens were analyzed in the sequences (Table 1). All sequences were first aligned current study. The ITS region was amplified using a using CLC Main Workbench ver. 8.0 primer set P16 (5´- (https://www.qiagenbioinformatics.com/) and the TCACTGAACCTTATCATTTAGAGGA- 3´) and P25 alignment was checked using the MEGA7 software (5´- GGGTAGTCCCGCCTGACCTG - 3´) (Popp and (Kumar et al., 2016). Table 1. GenBank accession numbers of the ITS sequences from six spiny Dianthus sect. Dentati specimens which were obtained in this study. Collector number Species GenBank accession no. Hamzaoğlu 6589 Dianthus erinaceus Boiss. var. erinaceus KY364854 Hamzaoğlu 6590 D. erinaceus Boiss. var. alpinus Boiss. KY364855 Hamzaoğlu 6596 D. goekayi Kaynak, Yılmaz & Daşkın KY364856 Hamzaoğlu 6744 D. aculeatus Hamzaoğlu KY364857 Hamzaoğlu 7241 D. varankii Hamzaoğlu & Koç KY364858 Koç 2127 D. halisdemirii Hamzaoğlu & Koç KY364859 Outgroup Petrorhagia saxifraga (L.) Link KX184019.1 The pairwise sequence divergence between species in Maximum likelihood (ML) method (Felsenstein, 1985; ITS was determined by Kimura-2 parameter model Tamura et al., 2011; Kumar et al., 2016). (Kimura, 1980). The base composition analysis was performed using MEGA7 and the phylogenetic tree RESULTS and DISCUSSION (bootstrap replicates=1000) was constructed using the Dianthus varankii Hamzaoğlu & Koç, sp. nov. 546 KSÜ Tarım ve Doğa Derg 21(4):545-554, 2018 Araştırma Makalesi/Research Article Type: Turkey, A5 Kastamonu, Hanönü, Küreçayı Hamzaoğlu 7241 & Koç (holo. GAZI, iso. GAZI, ANK) village turnout, Pinus brutia Ten. and Quercus (Figure 1). pubescens Willd. openings, 480 m, 13.08.2016, Figure 1. Dianthus varankii sp. nov. (A) habit and habitat, (B) spiny leaves, (C) petals, (D) calyx and epicalyx scales. Diagnosis: Dianthus varankii is related to D. 23.5 mm, veinless below, distinctly 3540-veined aculeatus. It differs from this species mainly by stem above, puberulous, purplish; teeth narrowly oblong- 10‒26 cm long (not 2–6 cm); calyx 24‒30 mm long (not lanceolate, 710 × 11.5 mm, 79-veined, with ciliate 18‒26 mm); petal 30–37 mm long (not 22–26 mm); claw