Cities out-of Platanthera chlorantha (PS1 and you will PS2, PB1–PB4, circles) and you may Cephalanthera rubra (CK1 and you may CK2, CB1–CB7, triangles) communities during the northern-eastern Poland.
Research urban area and you may testing
We investigated half a dozen P. chlorantha and you can nine C. rubra communities in northern-eastern Poland (Bialowieza and you may Knyszynska Primeval Forest, Szeszupa lake valley) in absolute, semi-absolute and you may anthropogenic groups of national and you will landscape parks, reserves and you may protected portion, such as Natura 2000 internet sites ( Fig. 1). Despite the reality he or she is based in secure components, of several exist towards the train embankments, together ways and you can routes during the forest or even in clearings.
This new sampling procedure relied into population size. Leaf examples out-of almost all ramets inside communities each and every types was removed (but populace PS2; Desk 1); zero trials was basically taken from busted otherwise extremely more youthful some one. 100 and you may ninety-seven examples out of P. chlorantha and you can 95 trials from C. rubra was indeed amassed. Leaf muscle are kept on ice until it can be kept from the ?80 °C, pending allozyme analysis. All-collected examples were used having allozyme study.
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 per locus; HO, observed heterozygosity; HE, expected heterozygosity; FWas, 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 per locus; HO, observed heterozygosity; HE, expected heterozygosity; FAre, 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.
Homogenates were prepared by grinding the fresh will leave for the a buffer having 2-mercaptoethanol (1%, v/v). Electrophoresis is carried out into the ten% starch fits in and Titan III cellulose acetate plates (Helena Laboratories, Beaumont, Colorado, USA) after the practical electrophoretic tips. Fifteen loci (Adh, Gdh, Got-step 1, Got-dos, Idh-step one, Idh-dos, Mdh-step one, Mdh-2, Me, Pgi, Pgm, 6Pgd, Skd, Sod, Tpi) inside P. chlorantha and sixteen loci within the C. rubra (Adh, Got-step 1, Got-dos, Gdh, Idh-step one, Idh-2, Mdh-1, Mdh-dos, Me personally, 6Pgd, Pgi, Pgm, Skd, Sod, Tpi-step 1, Tpi-2) had been investigated. A couple of electrode/serum boundary assistance were used to answer enzyme expertise: GDH and Had (10% lithium-borate lateral starch serum at the pH 8.2/8.3) and MDH, SKD and TPI (10% histidine-citrate boundary at the pH eight.0/7.0). Enzyme interest staining implemented Soltis Soltis ( 1989). The other enzyme options (ADH, IDH, Me, 6PGD, PGI, PGM, SOD) were screened having fun with Titan III cellulose acetate plates, that happen to be fixed having fun Wiccan singles dating sites with Tris-glycine boundary within pH 8.six and Tris-citrate buffer at the pH eight.6 (Richardson, Adams Baverstock, 1986). This new chemical staining treatments was in fact predicated on Soltis Soltis ( 1989) and you will Richardson ainsi que al. ( 1986), having modifications.
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 (FTry). 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 FWas) and population size were tested with Spearman’s pairwise rank correlations (StatSoft, 1995).