Locations of Platanthera chlorantha (PS1 and you may PS2, PB1–PB4, circles) and you can Cephalanthera rubra (CK1 and CK2, CB1–CB7, triangles) populations for the north-east Poland.
Data town and you can testing
We investigated half a dozen P. chlorantha and nine C. rubra populations inside northern-eastern Poland (Bialowieza and you will Knyszynska Primeval Forest, Szeszupa lake area) from inside the pure, semi-absolute and you can anthropogenic communities from national and you will surroundings parks, reserves and you may safe components, such as for example Natura 2000 internet sites ( Fig. 1). Ageven though he is located in secure elements, of a lot are present for the railway embankments, with each other courses and you can paths in woods or even in clearings.
The fresh new testing procedure depended for the population proportions. Leaf samples of most ramets in this populations of every varieties was in fact removed (except populace PS2; Dining table step 1); no examples were taken from damaged or most young anyone. 100 and you can 90-eight samples out of P. chlorantha and 95 samples of C. rubra was basically compiled. Leaf tissue try maintained frost up to it can be kept in the ?80 °C, pending allozyme studies. All collected products were utilized to own allozyme research.
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; 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.
Allozyme polymorphism
Homogenates had been served by milling the brand new renders from inside the a shield that have 2-mercaptoethanol (1%, v/v). Electrophoresis is achieved on ten% starch gels and you may Titan III cellulose acetate plates (Helena Laboratories, Beaumont, Texas, USA) adopting the standard electrophoretic strategies. Fifteen loci (Adh, Gdh, Got-1, Got-2, Idh-step 1, Idh-2, Mdh-1, Mdh-dos, Myself, Pgi, Pgm, 6Pgd, Skd, Sod, Tpi) in P. chlorantha and you will sixteen loci inside the C. rubra (Adh, Got-step 1, Got-dos, Gdh, Idh-step 1, Idh-2, Mdh-1, Mdh-dos, Me personally, 6Pgd, Pgi, Pgm, Skd, Sod, Tpi-step one, Tpi-2) was in fact investigated. Two electrode/serum barrier solutions were utilized to resolve chemical possibilities: GDH and you will Had (10% lithium-borate lateral starch serum on pH 8.2/8.3) and you will MDH, SKD and you can TPI (10% histidine-citrate buffer during the pH eight.0/eight.0). Enzyme interest staining accompanied Soltis Soltis ( 1989). Others chemical solutions (ADH, IDH, Me personally, 6PGD, PGI, PGM, SOD) have been screened playing with Titan III cellulose acetate dishes, that have been solved using Tris-glycine boundary from the pH 8.6 and you can Tris-citrate boundary within pH 7.6 (Richardson, Adams Baverstock, 1986). The new enzyme staining treatments had been centered on Soltis Soltis ( 1989) and you will Richardson ainsi que al. ( 1986), with adjustment.
Analytical 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 (PPOL), number of alleles per locus (A), genetic diversity (i.e. observed HO and expected heterozygosity HE) and inbreeding coefficient (FAre). 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 Sports Sites dating apps. 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 FAre) and population size were tested with Spearman’s pairwise rank correlations (StatSoft, 1995).