Cyflow sl

To assess genome size homogeneity, the DNA weight per nucleus (genome size; expressed in picograms [pg] ) was determined for each study population (see Online Resource table) using ow cytometry, based on a two-step procedure using Otto I and II buffers (Otto 1990 ). As its genome size (2C DNA = 3.38 pg) is close to that of the species studied, the common daisy Bellis perennis was used as an internal reference standard (Schönswetter et al. 2007 (link)). In each case, 1 cm 2 of fresh leaf tissue from the study species and from B. perennis were macerated together with a sharp blade and placed in a Petri dish containing 0.1 ml of ice-cold Otto I buffer (0.1 M citric acid monohydrate, 0.5% v/v Tween 20) for approx. 90 s, after which the suspension was ltered through a 42 µm nylon mesh. Nuclei within the ltered suspension were then stained with 1 ml of Otto II buffer (0.4M Na 2 HPO 4 .12H 2 O) supplemented with 1 ml of DAPI stock solution (DAPI 10 mg dissolved in 100 ml H 2 O) + 50 µl ß-mercaptoethanol (2 µL/mL). Each sample was then incubated at room temperature for 10 min and analysed using a Sysmex-Partec CyFlow SL equipped with a green solid-state laser ow cytometer (532 nm, 100 mW output power; Sysmex Partec GmbH, Görlitz, Germany).