Locations of Platanthera chlorantha (PS1 and PS2, PB1–PB4, circles) and you will Cephalanthera rubra (CK1 and you will CK2, CB1–CB7, triangles) communities inside north-east Poland.
Analysis urban area and you may sampling
I investigated half dozen P. chlorantha and you will 9 C. rubra populations within the northern-eastern Poland (Bialowieza and Knyszynska Primeval Forest, Szeszupa lake valley) within the natural, semi-absolute and you can anthropogenic teams of federal and you may land areas, supplies and protected section, such Natura 2000 internet ( Fig. 1). Though he could be located in protected section, of several exists to the train embankments, along roads and you can routes inside the forests or in clearings.
This new testing procedure relied towards the population size. Leaf examples away from most ramets within this populations each and every types have been removed (except people PS2; Dining table step 1); no examples had been extracted from busted otherwise really more youthful anybody. One hundred and ninety-eight trials from P. chlorantha and you may 95 trials from C. rubra had been compiled. Leaf muscle was kept on ice up until it may be kept from the ?80 °C, pending allozyme investigation. All collected trials were used to possess allozyme study.
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 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.
Allozyme polymorphism
Homogenates was basically prepared by grinding new will leave from inside the a barrier which have 2-mercaptoethanol (1%, v/v). Electrophoresis are achieved for the ten% starch gels and Titan III cellulose acetate dishes (Helena Labs, Beaumont, Texas, USA) adopting the fundamental electrophoretic actions. Ten loci (Adh, Gdh, Got-step one, Got-dos, Idh-step one, Idh-dos, Mdh-step one, Mdh-2, Me, Pgi, Pgm, 6Pgd, Skd, Sod, Tpi) in P. chlorantha and you can sixteen loci from inside the C. rubra (Adh, Got-1, Got-2, Gdh, Idh-step 1, Idh-2, Mdh-1, Mdh-2, Me, 6Pgd, Pgi, Pgm, Skd, Sod, Tpi-step 1, Tpi-2) was indeed examined. A few electrode/solution shield options were utilized to answer enzyme solutions: GDH and you can Got (10% lithium-borate horizontal starch gel within pH 8.2/8.3) and you can MDH, SKD and you can TPI (10% histidine-citrate shield at pH 7.0/seven.0). Chemical passion staining observed Soltis Soltis ( 1989). Others enzyme options (ADH, IDH, Myself, 6PGD, PGI, PGM, SOD) was basically processed having fun with Titan III cellulose acetate plates, which were solved playing with Tris-glycine boundary at the pH 8.6 and you may Tris-citrate shield on pH 7.6 (Richardson, Adams Baverstock, 1986). The latest enzyme staining solutions were predicated on Soltis Soltis ( 1989) and you can Richardson et al. ( 1986), having improvement.
Statistical analysis
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 Sikh dating sites 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 (FWas). 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 FIs) and population size were tested with Spearman’s pairwise rank correlations (StatSoft, 1995).