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ŠUMARSKI LIST 5-6/2018 str. 35     <-- 35 -->        PDF

cited authors recommended the use of molecular data and DNA analysis to definitely assess its taxonomic status.
In this study, we carried out for the first time a phylogenetic analysis of the “green oak” in comparison to other closely related oak species from the surrounding region, including all putative parental species: Q. cerris, Q. ilex and Q. suber. A further oak tree from Croatia displaying similar morphology to the “green oak” individual, as well as members of Q. crenata Lam., i.e. the supposed stable hybrid between Q. cerris and Q. suber (Bellarosa et. al., 2005; Cristofolini et al., 2005; Conte et. al., 2007) from Italy and Slovenia, were included in the dataset. To finally resolve the origin of the green oak tree, we combined both plastid and nuclear DNA markers widely used for DNA barcoding in plants and which have proven to be helpful in taxonomic identification and delimitation of some closely related Euro-Mediterranean oak taxa (Simeone et. al., 2013).
Plant material – Biljni materijal
We collected leaf samples from the “green oak” individual (Q. × viridis) growing in the village of Islam Latinski near Zadar, as well as from another putative green oak growing near Rijeka (Donje Jelenje) where unusual evergreen oak individuals of unknown origin have been observed (J. Franjić, personal observation) (Table 1). In addition, leaf samples from 1-3 individuals were collected from all closely related oak taxa (Q. cerris, Q. suber, Q. ilex, Q. coccifera L. and Q. crenata) from Croatia, Slovenia, Italy and Greece to maximally cover the geographic area surrounding the target “green oak”. Finally, samples from single individuals of three more distant oak species belonging to the infrageneric group Quercus (Q. robur L., Q. petraea /Matt./ Liebl. and Q. pubescens Willd.) were also investigated and used as outgroups. In total, nine Quercus taxa were included in the analyses (Table 1).
DNA extraction and amplification – Izolacija i umnažanje DNK
DNA extractions from silica-gel dried leaves were performed with the DNeasy Plant Minikit (QIAGEN), following the manufacturer’s instructions. In a search for maximum variability in our taxon-based study, we used two highly variable plastid regions (trnK-matK and trnH-psbA), and the nuclear ribosomal region 5.8S + ITS2 (Simeone et al., 2013). Plastid primers were the same as in Piredda et. al. (2011); ribosomal oligonucleotides were designed as follows: 5’-ACG ACT CTC GGC AAC GGA TA-3’ (5.8S_Fw), 5’-CAG CGG GTA GTC CCG CCT GA-3’ (25S_Rev). DNAs (ca. 40 ng) were amplified with RTG PCR beads (GE Healthcare) in 25 μl final volume according to the manufacturer’s protocol. Thermocycling conditions were: 94° for 3 min, followed by 35 cycles of 94° for 30 sec, 53° for 40 sec and 72° for 40 sec, with a final extension step of 10 min at 72° (plastid loci), and 98° for 3 min, followed by 35 cycles of 98° for 30 sec, 60° for 30 sec and 72° for 30 sec, with a final extension step of 5 min at 72° (ITS region). PCR products were cleaned with Illustra DNA and Gel Band Purification Kit (GE Healthcare), and eluted in 30 µl type 6 elution buffer. Standardized aliquots were then submitted to Eurofins MWG Operon ( for sequencing. Electropherograms were edited with CHROMAS 2.3 ( and checked visually.
Phylogenetic analyses – Filogenetske analize
All sequences were first aligned with MUSCLE (Edgar, 2004) and checked by eye. We calculated summary statistics of the aligned sequences (number of variable sites – V,