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A New Variety of Plocama Calabrica (Rubiaceae) from Denizli (Turkey) Confirmed by Morphological and Molecular ISSR Markers

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A New Variety of Plocama Calabrica (Rubiaceae) from Denizli (Turkey) Confirmed by Morphological and Molecular ISSR Markers Acta Bot. Croat. 78 (2), 142–146, 2019 CODEN: ABCRA 25 DOI: 10.2478/botcro-2019-0016 ISSN 0365-0588 eISSN 1847-8476 A new variety of Plocama calabrica (Rubiaceae) from Denizli (Turkey) confirmed by morphological and molecular ISSR markers Ramazan Süleyman Göktürk1, Olcay Düşen2*, Ergun Kaya3, Betül Gürcan2, Uygar Sarpkaya2 1 Akdeniz University, Faculty of Science, Department of Biology, 07058 Antalya, Turkey. 2 Pamukkale University, Faculty of Arts and Science, Department of Biology, 20160, Kinikli, Denizli, Turkey. 3 Muğla Sıtkı Koçman University, Faculty of Science, Molecular Biology and Genetic Department, 48000, Kötekli, Muğla, Turkey. Abstract – Plocama calabrica (L.f.) M.Backlund & Thulin var. alba Göktürk, O.D.Düşen, B.Gürcan & U. Sarp- kaya variety nova is described from South-West Anatolia. The new variety grows on limestone slopes between Akpınar and Yaylapınar villages in the Çameli district in Denizli. It is closely related to P. calabrica var. calabrica, and can be readily distinguished by morphological and molecular characters from the related variety. Taxonom- ic comments such as descriptive and diagnostic characters, distribution and ecology, phenology and proposed conservation status for this new variety are given in the current study. Morphological affinities and the -in ter-simple-sequence repeat (ISSR)-PCR based phylogenetic relationships between the new and the related vari- ety are also discussed Keywords: ISSR-PCR, molecular marker, new variety, Plocama, systematics, Turkey Introduction The family Rubiaceae comprises 650 genera and ca. True and rapid determination of degrees of genetic re- 11000 species, distributed throughout almost all the regions lationship and genetic diversity levels are required for con- of the world, although they are found mainly in tropical and servation and/or utilization of plant breeding programs. subtropical regions (Ortiz et al. 2000). Putoria is a mono- During the last twenty years, usage of DNA-based molec- typic genus which belongs to Rubiacaeae in Turkey, and was ular marker systems has increased faster than chemical or revised by Ehrendorfer in the Flora of Turkey and the East other morphological characterization systems (Collard et al. Aegean Islands (Ehrendorfer 1982). The genus of Putoria 2005, Bernardo 2008). Formerly, these markers were quite was treated as a synonym of Plocama genus by Backlund et expensive and laborious, but technological improvements al. 2007. Plocama was represented by 34 species and 1 sub- have made them cheaper, faster and relatively easy (Yang et species until 2009 (Backlund et al. 2007, IPNI 2015). Since al. 2015). The inter-simple-sequence repeat (ISSR) marker then, one new species has been described from Uzbekistan system is based on amplification of DNA fragments using a (Khassanov et al. 2014). With the description here of P. cal - single microsatellite sequence primer designed with six tri- abrica var. alba there are now two taxa in Turkey and world- nucleotide or eight dinucleotide repeats and one more an- wide there are in all 37 Plocama taxa. In the flora of Turkey, chor nucleotide (Zietkiewicz et al. 1994). This marker tech- the genus Plocama is represented only by P. calabrica (L. fil) nique has been verified as a simple, fast and low-cost way to DC. P. calabrica is known from Spain, Italy (incl. Sicily), Mal- determine genetic diversity (Sarla et al. 2003), to investigate ta, Montenegro, Bosnia and Herzegovina, Croatia, Albania, relationships between cultivars (Martins et al. 2003, Kaya Greece (incl. Crete), Cyprus, Turkey, Israel, Lebanon, Iraq, 2015), to use evolutionary studies such as gene flow (Wolfe Syria, Morocco, Algeria, Tunisia, and Libya (Backlund and et al. 1998), and to detect genetic stability (Kaya et al. 2017). Thulin 2007). The practicality and utility of ISSR primers has been evalu- * Corresponding author e-mail: [email protected] 142 ACTA BOT. CROAT. 78 (2), 2019 A NEW VARIETY OF PLOCAMA CALABRICA (RUBIACEAE) ated in this work for identifying the differences between two Tab. 1. ISSR primer sequences and GenBank accessions numbers taxa of Plocama calabrica. (Martins-Lopes et al. 2009, Smykal et al. 2011) used in molecular phylogenetic analysis of Plocama calabrica var. calabrica and P. ca- labrica var. alba. Materials and methods GenBank Primer Sequence Morphological study accesion number ISSR1 (AG)8T UBC 807 In June 2017, during a project named “Biodiversity and ISSR2 (AG)8G UBC 809 Monitoring studies of Terrestrial and Inland Water Ecosys- ISSR3 (GA)8T UBC 810 tems in Denizli Province”, the authors collected some inter- ISSR4 (GA)8C UBC 811 esting Plocama specimens (Fig. 1, 2). While the normal color ISSR5 (CA)8A UBC 817 of P. calabrica var. calabrica individuals is pink, individuals of ISSR6 (TC)8C UBC 823 this new variety were interesting because of their white flow- ers. In total, 10 herbarium specimens of the new variety were ucts were separated on 1.5% agarose gel and visualized under collected from the type locality. Plocama specimens were UV light after being stained with ethidium bromide. PCR dried for morphological and molecular phylogenetic stud- band profiles were recorded as 1 (present) or 0 (absent) and ies according to standard herbarium techniques and pre- cluster analysis was performed to construct dendrograms, served in the Pamukkale University Herbarium (PAMUH). with the unweighted pair-group method by arithmetic aver- After drying process, these specimens were checked using ages (UPGMA) from the similarity data matrices using Jac- the basic floras, Flora of Turkey (Ehrendorfer 1982), Flo- card’s coefficient (D-UPGMA, 2002). ra Europaea (Ball 1976), Flora Italiana (Tanfani 1887) and related papers (Backlund et al. 2007, Backlund and Thulin Results 2007, Karabacak 2012) and also confirmed by comparison with the e-herbarium samples in the B, BGBM, C, BR, E, M, Taxonomy P, SAV, W and WU herbaria. After detailed morphological Plocama calabrica (L.f.) M. Backlund & Thulin var. alba and molecular phylogenetic studies, we decided that P. cal - Göktürk, O. D. Düşen, B. Gürcan & U. Sarpkaya var. nov. abrica var. alba was a variety new to science. (Figs. 1, 3) Fig. 1. A general view of Plocama calabrica var. alba in nature (A) Fig. 2. A general view of Plocama calabrica var. calabrica in nature with detail of flowers (B). (A) with detail of flowers (B). Molecular phylogenetic study Holotype:—TURKEY. C2 Denizli: Çameli, between Genomic DNA was isolated from three individual plants Akpınar and Yaylapınar villages, limestone slopes, 1166 m, of two different characteristic taxa belonging to dryP. cal abr i - 21 June 2017, O. D. Düşen (2584) & R. S. Göktürk (holotype ca herbarium samples using a modified protocol developed by PAMUH!, isotypes Akdeniz Univ. Herb.!). Ferdous et al. (2012). The method based on cetyltrimethylam- Procumbent shrubs. Stem much branched, woody at monium bromide (CTAB) extraction included one more step, base, 5–25 cm, forming mats up to 1.5 m in diameter, pu- the use of chloroform:isoamyl alcohol:phenol (24:1:5, v/v). berulent. Leaves opposite, 10–15(–20) × 2–3.5(–4.5) mm, –1 Polymerase chain reactions were carried out using 40 ng µl lanceolate to oblong, obtuse, narrowed into a short petiole, DNA template, 1×PCR buffer, 2.5 mM MgCl2, 0.4 mM dNTP, revolute, slightly scabrid on margins and midrib, somewhat 1 unit Taq DNA polymerase and ten ISSR primers (Tab. 1) in leathery-succulent, blackening when dry; stipules small, a 25 µL reaction mix (Martins-Lopes et al. 2009, Ozudogru linear-oblong, ± fused. Flowers grouped in laxly contract- et al. 2011, Smykal et al. 2011, Düşen et al. 2018). DNA band ed shortly pedicellate cymes, hermaphrodite, 4-merous. Ca- profiles were amplified at 95 °C for 3 min initial denaturation lyx greenish, tubular, persistent and inflated in fruit; teeth followed by 35 reaction cycles (95 °C 15 s; 55 °C 30 s; 72 °C 4, greenish, unequal triangular. Corolla infundibular with 3 min) and finally extended with 72 °C 10 min. PCR prod- a long tube, 8–15 (–20) mm, white, glabrous outside, hairy ACTA BOT. CROAT. 78 (2), 2019 143 GÖKTÜRK R.S, DÜŞEN O., KAYA E., GÜRCAN B., SARPKAYA U. inside; lobe 4, valvate. Stamen 4, inserted at corolla throat; ed that this new variety should be placed under the IUCN anther white, dorso-basifixed, exserted; filament white. Style threat category “Critically Endangered (CR)” (IUCN 2012), white, filiform, with very short bifid stigma. Ovary bilocular, because the estimated area of occupancy is less than 10 km2 each cell with one basal ovule. Fruit drupe, 4–6 mm, oblong, (criterion B2) and it is known only from one locality (crite- with two pyrenes, glossy. rion B2a). The population size of the new species is estimat- Plocama calabrica includes 2 varieties and the diagnostic ed to be less than 50 mature individuals (criterion C2-ai). In keys are presented below; addition, the distribution area of the new taxon may be de- stroyed by anthropogenic effects such as road construction Calyx teeth reddish; corolla pink; anther and filament or grazing in the near future. pink ..................................................................var. calabrica Etymology: the specific epithet is derived from corolla, Calyx teeth greenish; corolla white; anther and filament anther and filament color. white ........................................................................var. alba Specimens examined of Plocama calabrica var. cal- The new variety is different from P. calabrica var. cal- abrica: TURKEY. C3 Antalya: Çakırlar, south of Çakırlar, abrica (Figs. 1, 2) and the two are compared on the basis of roadside, 25 m, 13 August 1993, R. S. Göktürk (3080) (Ak- morphological characters in Tab. 2. deniz Univ. Herb.!). C3 Antalya: Saklıkent, Pinus clearings, 1600 m, 8 July 1995, O.Dinç (1087) (Akdeniz Univ. Herb.!)). C2 Denizli: Kocabaş, limestone slopes, 604 m, 21 May 2017, Tab. 2. Morphological comparison of Plocama calabrica var. cal- O.D.Düşen (1871) & R. S. Göktürk (PAMUH!). C2 Den- abrica and P. calabrica var.
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