Metropolitan areas regarding Platanthera chlorantha (PS1 and you may PS2, PB1–PB4, circles) and Cephalanthera rubra (CK1 and you will CK2, CB1–CB7, triangles) communities in the northern-east Poland.

Research town and you will sampling

We examined half a dozen P. chlorantha and you may nine C. rubra communities into the north-eastern Poland (Bialowieza and you may Knyszynska Primeval Tree, Szeszupa lake valley) inside absolute, semi-natural and you may anthropogenic teams regarding national and you will landscaping parks, supplies and you will protected section, such as for instance Natura 2000 internet ( Fig. 1). Though he’s based in protected elements, of many can be found into the train embankments, with each other tracks and paths in the woods or perhaps in clearings.

The newest sampling process depended to the inhabitants dimensions. Leaf products regarding nearly all ramets within this communities of each and every species were taken (but society PS2; Desk step 1); no samples was basically taken from busted or most younger anybody. A hundred and 90-eight samples from P. chlorantha and you may 95 samples of C. rubra was indeed accumulated. Leaf structure try continued ice until it may be kept at ?80 °C, pending allozyme investigation. All-collected trials were used to possess allozyme studies.

N, population size; N_S, number of samples analysed; N_Grams/N_W, number of generative ramets/number of vegetative ramets; P_POL, percentage of polymorphic loci; A, mean number of alleles per locus; H_O, observed heterozygosity; H_E, expected heterozygosity; F_Is, inbreeding coefficient; G, number of genotypes, G/N_S, clonal diversity; G_U, number of unique genotypes; G_U %, percentage of unique genotypes (Fischer’s exact test: P < 0.05, **P < 0.01, ***P < 0.001); #, sum of parameters.

N, population size; N_S, number of samples analysed; N_G/N_W, number of generative ramets/number of vegetative ramets; P_POL, percentage of polymorphic loci; A, mean number of alleles per locus; H_O, observed heterozygosity; H_E, expected heterozygosity; F_Try, inbreeding coefficient; G, number of genotypes, G/N_S, clonal diversity; G_U, number of unique genotypes; G_U %, percentage of unique genotypes (Fischer’s exact test: P < 0.05, **P < 0.01, ***P < 0.001); #, sum of parameters.

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Allozyme polymorphism

Homogenates was indeed served by grinding brand new will leave in the a barrier that have 2-mercaptoethanol (1%, v/v). Electrophoresis is carried out on the 10% starch fits in and you will Titan III cellulose acetate plates (Helena Labs, Beaumont, Tx, USA) after the standard electrophoretic tips. Fifteen loci (Adh, Gdh, Got-step one, Got-2, Idh-step 1, Idh-dos, Mdh-1, Mdh-2, Myself, Pgi, Pgm, 6Pgd, Skd, Sod, Tpi) for the P. chlorantha and 16 loci for the C. rubra (Adh, Got-step 1, Got-dos, Gdh, Idh-step 1, Idh-2, Mdh-1, Mdh-dos, Myself, 6Pgd, Pgi, Pgm, Skd, Sod, Tpi-step one, Tpi-2) have been examined. A couple of electrode/solution shield expertise were utilized to resolve enzyme systems: GDH and Had (10% lithium-borate lateral starch serum during the pH 8.2/8.3) and you will MDH, SKD and TPI (10% histidine-citrate shield within pH 7.0/seven.0). Enzyme interest staining implemented Soltis Soltis ( 1989). One other chemical expertise (ADH, IDH, Me personally, 6PGD, PGI, PGM, SOD) had been processed playing with Titan III cellulose acetate plates, which were solved using Tris-glycine barrier on pH 8.6 and you can Tris-citrate buffer at the pH seven.6 (Richardson, Adams Baverstock, 1986). The brand new chemical staining recipes have been centered on Soltis Soltis ( 1989) and you may Richardson ainsi que al. ( 1986), having improvement.

Mathematical investigation

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 (P_POL), number of alleles per locus (A), genetic diversity (i.e. observed H_O and expected heterozygosity H_E) and inbreeding coefficient (F_Is). 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 (G_U) and the probability that the next ramet sampled would be a different genotype (G/N_S; where N_S is the number of ramets sampled). _POL, A, H_O and F_{Is actually}) and population size were tested with Spearman’s pairwise rank correlations (StatSoft, 1995).