Towns from Platanthera chlorantha (PS1 and PS2, PB1–PB4, circles) and you may Cephalanthera rubra (CK1 and CK2, CB1–CB7, triangles) populations inside the north-east Poland.
Study town and testing
We examined half dozen P. chlorantha and you may 9 C. rubra communities when you look at the north-east Poland (Bialowieza and Knyszynska Primeval Tree, Szeszupa lake area) in sheer, semi-pure and you can anthropogenic groups regarding federal and you can landscape areas, reserves and you will secure elements, such as for instance Natura 2000 sites ( Fig. 1). Although he is located in safe areas, of a lot exist into the railway embankments, collectively tracks and you will pathways in woods or perhaps in clearings.
Brand new sampling procedure depended towards the people size. Leaf products regarding almost all ramets inside communities each and every varieties had been removed (except populace PS2; Table 1); zero trials was indeed extracted from damaged otherwise extremely more youthful people. A hundred and you may ninety-7 products away from P. chlorantha and you can 95 examples of C. rubra were amassed. Leaf dating services Social Media Sites structure was kept on frost up until it can be stored in the ?80 °C, pending allozyme study. All collected samples were utilized to own 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, 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; FIs, 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 have been made by grinding this new renders within the a shield having 2-mercaptoethanol (1%, v/v). Electrophoresis is actually accomplished to your 10% starch gels and you may Titan III cellulose acetate dishes (Helena Labs, Beaumont, Texas, USA) adopting the practical electrophoretic measures. Fifteen loci (Adh, Gdh, Got-1, Got-dos, Idh-step 1, Idh-dos, Mdh-step one, Mdh-dos, Me personally, Pgi, Pgm, 6Pgd, Skd, Sod, Tpi) in P. chlorantha and 16 loci from inside the C. rubra (Adh, Got-step 1, Got-2, Gdh, Idh-1, Idh-2, Mdh-1, Mdh-dos, Me, 6Pgd, Pgi, Pgm, Skd, Sod, Tpi-step one, Tpi-2) had been investigated. One or two electrode/solution barrier systems were used to respond to enzyme expertise: GDH and you will Had (10% lithium-borate horizontal starch gel from the pH 8.2/8.3) and you may MDH, SKD and you can TPI (10% histidine-citrate buffer from the pH seven.0/seven.0). Enzyme activity staining implemented Soltis Soltis ( 1989). The other chemical possibilities (ADH, IDH, Myself, 6PGD, PGI, PGM, SOD) was basically processed having fun with Titan III cellulose acetate plates, which were resolved using Tris-glycine boundary at the pH 8.six and Tris-citrate shield at the pH seven.6 (Richardson, Adams Baverstock, 1986). Brand new enzyme staining recipes had been based on Soltis Soltis ( 1989) and you may Richardson et al. ( 1986), having variations.
Analytical 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 (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).
