In 1987, a strong mast year was recorded, followed by three more in 1994, 1998 and 2004. When for the first time abundant regeneration was recorded under shelterwood, small regeneration gaps sized ATM Kinase Inhibitor one to two tree

heights were opened up in the stand. Regeneration centres subsequently extended into the stand and have been later released. Attributes of regeneration centres, measured on five 1 m2 subplots situated in regeneration centres, are presented in Table 1. One of the subplots was always situated in the middle of the regeneration centre. The remaining four subplots were located in a cross; each subplot was situated halfway from the middle of the regeneration centre to its edge in the directions of north, south, east and west. The shape of the regeneration centres was plotted according to coordinates recorded during sampling. Twigs with dormant buds from 35 adult trees and 35 saplings (>1.3 m tall and < 5 cm DBH) per site were collected in spring Ceritinib solubility dmso 2012. Trees >30 m apart from the entire sampling area were sampled and their geographical location was recorded using a Garmin GPSMAP 60CSx (Garmin International, Kansas, U.S.A.). For saplings, the midpoint of the regeneration

centres and their borders were recorded. Differently sized regeneration centres at Osankarica were located in the prevailing horizontal structures (i.e. mature and rejuvenation stages), based on height and DBH of adult trees around the centres (Table 1). In the old growth, smaller regeneration centre was located in a gap while larger one was situated in the part of the old growth where different small gaps were already interconnected

and regeneration was continuously present in the whole area. Regeneration centres where only seedlings were present (<0.5 m tall) were not considered for this analysis as initial phases of high mortality might not have come to an end. From the whole area of the regeneration centres, two (15–20 saplings per centre) and four (5–11 saplings per centre) regeneration centres in the old growth and managed stand, respectively, were randomly sampled. Total DNA was isolated from dormant buds using a DNeasy plant kit (QIAGEN, Germany), as per the manufacturer’s specifications. All adults and saplings were genotyped at 16 highly variable microsatellite loci using primers described by Lefevre et al. (2012). Primers were renamed Pembrolizumab concentration with consecutive numbers to ease reporting; csolfagus_31 became Fs1 and DE576_A_0 became Fs16. Multiplex Kit 1 was split into two separate kits (kit 1a: primer pairs Fs1, Fs2, Fs4 and Fs5; kit 1b: primer pairs Fs3, Fs6, Fs7 and Fs8) to avoid the overlapping of alleles labelled with the same fluorescent dye. Polymerase chain reactions (PCRs) were performed as described by Lefevre et al. (2012) but primer pair concentrations required further optimisation and final concentrations differed from the published ones by a maximum of 0.9 for primer pair Fs16.