Locations away from Platanthera chlorantha (PS1 and PS2, PB1–PB4, circles) and you can Cephalanthera rubra (CK1 and you will CK2, CB1–CB7, triangles) populations for the northern-east Poland.

Data urban area and you can sampling

I investigated six P. chlorantha and nine C. rubra communities into the northern-eastern Poland (Bialowieza and you will Knyszynska Primeval Tree, Szeszupa lake area) inside the pure, semi-absolute and you will anthropogenic communities out of federal and you may surroundings parks, supplies and you will safe components, such as for example Natura 2000 sites ( Fig. 1). Despite the reality he is located in secure section, of numerous can be found towards train embankments, together routes and you may routes into the woods or perhaps in clearings.

The latest testing processes depended on populace proportions. Leaf trials off almost all ramets within this populations each and every types was in fact taken (but people PS2; Dining table step 1); no examples were taken from damaged or extremely more youthful some body. A hundred and ninety-seven samples regarding P. chlorantha and you can 95 products away from C. rubra were accumulated. Leaf cells try continued ice until it can be held from the ?80 °C, pending allozyme investigation. All-collected trials were used having allozyme studies.

N, population size; N_S, number Bisexual dating online 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_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.

Allozyme polymorphism

Homogenates was in fact made by milling brand new leaves when you look at the a shield having 2-mercaptoethanol (1%, v/v). Electrophoresis try accomplished into 10% starch fits in and you may Titan III cellulose acetate plates (Helena Labs, Beaumont, Colorado, USA) adopting the standard electrophoretic measures. Fifteen loci (Adh, Gdh, Got-step 1, Got-2, Idh-step one, Idh-dos, Mdh-step one, Mdh-dos, Myself, Pgi, Pgm, 6Pgd, Skd, Sod, Tpi) during the P. chlorantha and you can 16 loci into the C. rubra (Adh, Got-step one, Got-2, Gdh, Idh-1, Idh-2, Mdh-step one, Mdh-dos, Me, 6Pgd, Pgi, Pgm, Skd, Sod, Tpi-step one, Tpi-2) was basically examined. A few electrode/gel shield expertise were utilized to resolve enzyme options: GDH and you can Got (10% lithium-borate horizontal starch gel on pH 8.2/8.3) and you may MDH, SKD and you can TPI (10% histidine-citrate buffer within pH 7.0/eight.0). Chemical activity staining implemented Soltis Soltis ( 1989). Others chemical solutions (ADH, IDH, Myself, 6PGD, PGI, PGM, SOD) have been processed using Titan III cellulose acetate plates, which were solved playing with Tris-glycine boundary within pH 8.six and you will Tris-citrate buffer on pH eight.six (Richardson, Adams Baverstock, 1986). The brand new enzyme staining pattern was indeed considering Soltis Soltis ( 1989) and Richardson mais aussi al. ( 1986), having modifications.

Mathematical studies

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_Try). 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_Was) and population size were tested with Spearman’s pairwise rank correlations (StatSoft, 1995).