Metropolises out-of Platanthera chlorantha (PS1 and you can PS2, PB1–PB4, circles) and Cephalanthera rubra (CK1 and you can CK2, CB1–CB7, triangles) populations inside northern-eastern Poland.
Study area and sampling
We examined half a dozen P. chlorantha and you will 9 C. rubra communities within the north-east Poland (Bialowieza and you may Knyszynska Primeval Forest, Szeszupa lake valley) in natural, semi-natural and you may anthropogenic communities away from national and you can surroundings areas, reserves and you may protected parts, like Natura 2000 websites ( Fig. 1). While he’s situated in safe portion, of numerous are present into rail embankments, together routes and you can pathways from inside the woods or perhaps in clearings.
The new sampling techniques relied towards the population dimensions. Leaf examples from nearly all ramets in this populations of every variety was basically drawn (but populace PS2; Desk step one); zero trials was in fact obtained from damaged otherwise really more youthful somebody. 100 and you will ninety-7 products of P. chlorantha and you may 95 products off C. rubra was built-up. Leaf tissues try maintained frost up until it may be stored from the ?80 °C, pending allozyme study. All collected examples were used for allozyme studies.
N, population size; NS, number of samples analysed; NGrams/NW, number of generative ramets/number of vegetative ramets; PPOL, percentage of polymorphic loci; A, mean number of alleles per locus; HO, observed heterozygosity; HE, expected heterozygosity; FIs actually, inbreeding coefficient; G, number of genotypes, G/NS, clonal diversity; GU, number of unique genotypes; GU %, percentage of unique genotypes (Fischer’s exact test: P < 0.05, **P < 0.01, ***P < 0.001); #, sum of parameters.
N, population size; NS, number of samples analysed; NG/NW, number of generative ramets/number of vegetative ramets; PPOL, percentage of polymorphic loci; A, mean number of alleles tips for dating a Spanking Sites per locus; HO, observed heterozygosity; HE, expected heterozygosity; FIs actually, inbreeding coefficient; G, number of genotypes, G/NS, clonal diversity; GU, number of unique genotypes; GU %, percentage of unique genotypes (Fischer’s exact test: P < 0.05, **P < 0.01, ***P < 0.001); #, sum of parameters.
Allozyme polymorphism
Homogenates were served by grinding this new actually leaves within the a boundary with 2-mercaptoethanol (1%, v/v). Electrophoresis is actually achieved into the ten% starch ties in and you can Titan III cellulose acetate plates (Helena Labs, Beaumont, Colorado, USA) pursuing the simple electrophoretic measures. Fifteen loci (Adh, Gdh, Got-step 1, Got-dos, Idh-step 1, Idh-2, Mdh-step one, Mdh-2, Me, Pgi, Pgm, 6Pgd, Skd, Sod, Tpi) inside P. chlorantha and you may sixteen loci inside the C. rubra (Adh, Got-step 1, Got-dos, Gdh, Idh-step 1, Idh-2, Mdh-step 1, Mdh-dos, Myself, 6Pgd, Pgi, Pgm, Skd, Sod, Tpi-step one, Tpi-2) were investigated. Several electrode/gel barrier solutions were used to answer enzyme solutions: GDH and you may Had (10% lithium-borate lateral starch gel during the pH 8.2/8.3) and MDH, SKD and you will TPI (10% histidine-citrate shield within pH 7.0/seven.0). Enzyme activity staining observed Soltis Soltis ( 1989). One other enzyme solutions (ADH, IDH, Myself, 6PGD, PGI, PGM, SOD) was indeed screened playing with Titan III cellulose acetate dishes, that happen to be fixed using Tris-glycine buffer from the pH 8.6 and you can Tris-citrate shield at the pH eight.six (Richardson, Adams Baverstock, 1986). The fresh chemical staining recipes had been based on Soltis Soltis ( 1989) and Richardson mais aussi al. ( 1986), which have changes.
Mathematical study
The data matrix of individuals was analysed using the TFPGA package (Miller, 1997), FSTAT 2.9.3 (Goudet, 2001) and GENEPOP 3.2 (Raymond Rousset, 1995) for calculation of standard measures of allozyme diversity: allelic frequencies, percentage of polymorphic loci (PPOL), number of alleles per locus (A), genetic diversity (i.e. observed HO and expected heterozygosity HE) and inbreeding coefficient (FIs). The occurrence of unique alleles was used to describe population distinctiveness (Slatkin, 1985). Deviations from Hardy–Weinberg expectations were tested for the population by the Markov chain method (GENEPOP).
Parameters of within-population genotypic diversity were also estimated. Three different measures of clonal diversity were used: number of observed genotypes (G), number of genotypes unique to a single population (GU) and the probability that the next ramet sampled would be a different genotype (G/NS; where NS is the number of ramets sampled). POL, A, HO and FAre) and population size were tested with Spearman’s pairwise rank correlations (StatSoft, 1995).
