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PCR products were cleaned with the Illustra DNA Purification Kit (GE Healthcare), and standardized aliquots were then submitted to Eurofins MWG Operon ().
Optimal multiple alignments were obtained with CLUSTALW 1.81 (Thompson et al. The phylogenetic investigation was performed on both the cp DNA (joined mat K trn H-psb A sequences) and cp DNA ITS datasets.
Radiation occurred during the Middle Miocene, with a later extension into southern Europe, Iberia and North Africa, as documented by samples collected from the late Miocene-early Pliocene. student of our working group, for helping translate Croatian into Italian.
The causes of extinction in the east, however, should be investigated further and may lead to investigations about other species that might have experienced range shifts similar to that of cork oak. Maura Fracassini for the revision and support of our Ancient Greek translation of Theophrastus. Most of the ancient books we consulted were retrieved freely form Google Books, the Biodiversity Heritage Library ( – Plants were all sampled in the wild.
Leaves were dried in silica gel, preserved as herbarium specimens and liophilised.