Available online: www.notulaebotanicae.ro Print ISSN 0255-965X; Electronic 1842-4309 Not Bot Horti Agrobo, 2017, 45(1):316-322. DOI:10.15835/nbha45110799 Original Article Evidence of Low Chloroplast Genetic Diversity in Two Carpinus Species in the Northern Balkans Mihaela Cristina CĂRĂBUŞ 1, Alexandru Lucian CURTU 1, Dragoş POSTOLACHE 2, Elena CIOCÎRLAN 1, Neculae ŞOFLETEA 1* 1Transilvania University of Brasov, Department of Forest Sciences, 1 Şirul Beethoven, 500123, Braşov, Romania; [email protected] ; [email protected] ; [email protected] ; [email protected] (*corresponding author) 2National Research and Development Institute in Forestry “Marin Dracea”, 65 Horea Str., 400275 Cluj-Napoca, Romania; [email protected] Abstract Genetic diversity and differentiation in two Carpinus species ( C. betulus and C. orientalis ) occurring in Romania was investigated by using three chloroplast Simple Sequence Repeat markers (cpSSRs). A total of 96 and 32 individuals were sampled in eighteen C. betulus and six C. orientalis populations, respectively. A total of four chloroplast haplotypes were observed. Two haplotypes were specific for C. betulus and two for C. orientalis . Most of C. betulus populations were fixed for the predominant haplotype (H1), which was observed in 82% of the individuals. All C. orientalis populations were fixed for one haplotype or the other. Populations with haplotype (H3) are spread in southern Romania and the haplotype (H4) was observed at the northern limit of C. orientalis natural distribution range. Genetic differentiation among populations was moderate in C. betulus (GST = 0.422), compared to the high value observed in C. orientalis (GST = 1.000), which can be explained by the occurrence of a distinct haplotype in the peripheral population. RST values for both species suggest low levels of recurrent gene flow through seeds among populations. Our data on geographic distribution of chloroplast DNA haplotypes may be useful for the identification and conservation of distinct genetic resources of the two Carpinus species. Keywords: Carpinus betulus , C. orientalis , cpSSR , genetic diversity, phylogeography cover (IFN, 2012). This species is spread between 100 and Introduction 450 (500) m altitude, being rare in mixed beech-fir-spruce mountain forests (Horeanu, 1996). C. betulus is considered Genetic diversity is essential for species to evolve and to as a valuable species due to its strong ecological adaptability adapt to changing environmental conditions. Patterns of and increased tolerance to environmental stress, especially in genetic diversity are influenced by population contractions extreme conditions (Șofletea and Curtu, 2007). C. orientalis and expansions during glacial-interglacial fluctuations and is a major pioneer species in arid regions where it c an by post-glacial migration history (Petit et al ., 2005). colonise open and degraded areas (Bergmeier and Phylogeographic studies have provided important evidence Dimopoulos, 2008; Čarni et al., 2009). Both species co- on species demographic history and population structure occur in southeastern Europe and no natural hybrids (Petit et al ., 2005; White et al ., 2007; Xu et al ., 2015) between C. betulus and C. orientalis have been reported; revealing that species-specific life history traits and however, controlled crosses indicated tha t hybrids had geographic barriers may have a strong influence on the inferior survival and growth characteristics, and that adult current patterns of population genetic structure (Ortiz - hybrids were sexually immature (Santamour, 1995; Grivet Ramírez et al ., 2016). and Petit, 2003). The northern edge of C. orientalis The genus Carpinus (Betulaceae family) is r epresented distribution range is the southern part of Romania, but by two species in Europe ( C. betulus L. and C. orientalis isolated small p opulations are also present in the northern Mill). European hornbeam ( C. betulus L.) is a temperate part of the Moldova region (Şofletea and Curtu, 2007 ). hardwood species especially in forests dominated by oak Most of C. orientalis populations in Romania can be species, where it demonstrates a great ability to compete considered as marginal populations and their genetic with oaks at the juvenile stage (Petre et al ., 2012). In diversity assessment is of great importance, due to ongoing Romania C. betulus occupies around 4.8% of the total forest global warming. Received: 23 Mar 2017. Received in revised form: 01 Jun 2017. Accepted: 06 Jun 2017. Published online: 07 Jun 2017. Cărăbuş MC et al / Not Bot Horti Agrobo, 2017, 45(1):316-322 317 Genetic variation of C. betulus and C. orientali s have Materials and Methods been investigated with different DNA markers targeting different polymorphic regions of the chloroplast genome by Sampling and DNA isolation PCR-RFLP (Restriction Fragment Length Polymorphism) Shoots and leaves were collected from 18 populations of (Postolache et al ., 2017) and also by using chloroplast C. betulus and six populations of C. orientalis covering the microsatellites (Simple Sequence Repeats; cpSSRs ) (Grivet species distribution range in Romania (Table 1, Fig. 1). The and Petit, 2003; Fărcaş et al ., 2006). The chloroplast DNA material was stored i n an ultrafreezer at temperatures of diversity for C. betulus was significantly lower in western -60 °C until used for DNA extraction. Europe compared with southeastern European populations, Genomic DNA was extracted from biological specimens that harbour nearly all genetic variation and consequently a using the CTAB method described by Doyle and Doyle more deta iled analyses in this region is absolutely necessary (1987) with minor modifications. The DNA was to quantify the genetic structure and diversity. Beside this, quantified using NanoDrop 8000 (Thermo Scientific, previously published results Petit and Grivet (2003) Wilmington, USA, 2008) and extracted DNA was stored at indicate for very low introgression between these two -20 °C. species. The C. betulus population s from Romania have a very distinct Holocene postglacial history with direct Chloroplast microsatellites analysis consequences on current genetic pattern that needs to be Due to the lack of intraspecific variation observed in investigated further in order to understand the species Carpinus spp. we initially tested ten universal chloroplast diversity and evolutionary history (Grivet and Petit, 2003; Fărcaş et al ., 2006). Moreover, the phylogeographic data on microsatellites ( ccmp1 , ccmp2 , ccmp3 , ccmp4 , ccmp5 , ccmp6 , C. orientalis are very scarce and these data ar e very ccmp7 , ccmp8 , ccmp9 and ccmp10 ) developed by Weising important in the conservation and management of this and Gardener (1999). Nine out of ten primers showed the drought-tolerant woody species. amplification products, but only three primers ( ccmp4 , The aim of this study was to analyse the chloroplast ccmp7 and ccmp10 ) showed fragment length polym orphism. DNA was diluted (1:30) prior to PCR amplification . PCR DNA variation in two sister species of hornbeam ( C. betulus and C. orientalis ) in Romania (Northern Balkan Pen insula) reactions were performed in a total volume of 15 µl of a using cpSSRs markers, that can bring detailed information reaction mixture consisting of 1 × PCR Buffer (Promega), on species genetic structure, which could be applied in 2.5 mM MgCl 2, 200 µM of each of the four dNTPs, 1 unit developing site-oriented conservation strategy and of Taq DNA Polymerase (P romega), 0.2 µM of each primer sustainable management of Carpinus spp. genetic resources. added to 2 µl of genomic DNA. PCR amplifications were performed in a Corbett Palm-Cycler CG1-96 with the The major objective of t his work is to characterize the genetic structure of Carpinus spp. in Romania and to test following conditions: an initial denaturation for 15 minutes at 95 °C followed by 30/35 cycles of denaturation for 1 weather Carpathian Mountains acted as a major geographic barrier during the Holocene postglacial recolonization minute at 94 °C , annealing for 1 minute at 50 °C, extension history. More specifically we aimed: i) to assess the for 1 minute at 72 °C and a final extension for 20 minutes at chloroplast DNA genetic variation within and among 72 °C (30 cycles for ccmp4 and 35 cycles for ccmp7 and Carpinus spp. populations using cpSSRs; ii) to analyse the ccmp10 ). The amplification of chloroplast SSRs was geographic distribution of chloroplast haplotypes and infer performed using fluorescence dy ed forward primers direct consequences on the management and conservation (Metabion) for genotyping purpose, namely 6 -FAM/Blue of Carpinus spp. genetic resources. (ccmp7 and ccmp10 ) and HEX/Green ( ccmp4 ). Fig. 1. Distribution of C. betulus (A) and C. orientalis (B) chloroplast DNA haplotypes in Romania Cărăbuş MC et al / Not Bot Horti Agrobo, 2017, 45(1):316-322 318 Table 1. Geographic location of populations No Population Abbreviation Latitude Longitude Sample size Species 1 Apa Sărată -Maramure ș MM -CB 47°40’ 23°29’ 5 2 Teaca -Bistri ța -Năsăud BN -CB 46°52’ 24°32’ 5 3 Ro șcani -Ia și IS -CB 47°26’ 27°24’ 5 4 Panciu -Vrancea VN -CB 46°04’ 27°01’ 5 5 Babadag -Tulcea TLB -CB 44°51’ 28°41’ 5 6 Măcin -Tulcea TLM -CB 45°14’ 28 °11’ 5 7 Warthe -Bra șov BVW -CB 45°39’ 25°34’ 5 8 Tâmpa -Bra șov BVT -CB 45°38’ 25°35’ 5 9 Gura Teghii -Buzău BZGT -CB 45°32’ 26°28’ 8 10 Măgura -Buzău BZM -CB 45°16’ 26°33’ 8 11 Col ți -Buzău BZC -CB 45°22’ 26°23’ 5 C.betulus 12 Lotrisor -Vâlcea VL -CB