Metropolises of Platanthera chlorantha (PS1 and you can PS2, PB1–PB4, circles) and you can Cephalanthera rubra (CK1 and you will CK2, CB1–CB7, triangles) communities during the north-eastern Poland.
Investigation city and you may sampling
We examined six P. chlorantha and you may nine C. rubra communities in the northern-eastern Poland (Bialowieza and you can Knyszynska Primeval Tree, Szeszupa river valley) inside pure, semi-natural and you can anthropogenic teams away from national and you can surroundings parks, supplies and protected section, eg Natura 2000 web sites ( Fig. 1). Despite the fact that he is situated in secure areas, of a lot exist with the rail embankments, collectively tracks and you can paths into the forest or even in clearings.
New sampling procedure depended toward inhabitants dimensions. Leaf trials regarding nearly all ramets contained in this populations of every kinds were pulled (except inhabitants PS2; Dining table step 1); no samples had been obtained from broken otherwise most younger someone. 100 and you can ninety-eight trials from P. chlorantha and you can 95 samples regarding C. rubra was in fact gathered. Leaf muscle is continued ice until it may be stored from the ?80 °C, pending allozyme investigation. All collected samples were used getting allozyme study.
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; FTry, 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 was in fact prepared by grinding the will leave in a barrier having 2-mercaptoethanol (1%, v/v). Electrophoresis was carried out on the 10% starch gels and you can Titan III cellulose acetate plates (Helena Labs, Beaumont, Tx, USA) following fundamental electrophoretic measures. Ten loci (Adh, Gdh, Got-step one, Got-2, Idh-step 1, Idh-2, Mdh-step 1, Mdh-2, Me personally, Pgi, Pgm, 6Pgd, Skd, Sod, Tpi) from inside the P. chlorantha and you can 16 loci for the C. rubra (Adh, Got-1, Got-dos, Gdh, Idh-step 1, Idh-2, Mdh-1, Mdh-2, Me personally, 6Pgd, Pgi, Pgm, Skd, Sod, Tpi-step one, Tpi-2) had been investigated. Several electrode/solution buffer expertise were used to answer chemical options: GDH and you will Got (10% lithium-borate lateral starch serum on pH 8.2/8.3) and you can MDH, SKD and you will TPI (10% histidine-citrate shield in the pH seven.0/eight.0). Chemical craft staining accompanied Soltis Soltis ( 1989). One other chemical expertise (ADH, IDH, Myself, 6PGD, PGI, PGM, SOD) was in fact screened having fun with Titan III cellulose acetate dishes, which have been fixed using Tris-glycine barrier at pH 8.six and Tris-citrate shield on pH eight.six (Richardson, Adams Baverstock, 1986). The latest chemical staining pattern have been based on Soltis Soltis ( 1989) and you can Richardson et al. ( 1986), which have adjustment.
Statistical 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, Strapon dating review 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 FTry) and population size were tested with Spearman’s pairwise rank correlations (StatSoft, 1995).