Study areas and sampling
Plant material was collected from both heavy metalliferous (HM) and non-metalliferous (NM) sites in the summer of 2013. The polluted sites (heaps of waste Triassic dolomite rocks left by historical Zn-Pb ore mining) are located in western Olkusz Upland (Southern Poland), in the vicinity of the former mining village of Galman (G), approx. 5 km north-east of the town of Trzebinia. Detailed characteristics of the geology and climate of the region, the history of mining, the mine waste heaps as well as the vegetation covering them are described in Woch (2015) and Woch et al. (2017). Plants at the metalliferous site were collected from five heaps, scattered over approx. 2 km2. Three fern specimens were taken from each heap and pooled to obtain one sample per heap (n = 6). Additionally, three soil samples (0–15 cm in depth) were collected from the same heaps, and pooled into one composite sample per heap. Control ferns were taken from five non-metalliferous sites located in Southern Poland: 1) the Czupel (C) mountain (n = 4), located in the Beskidy Małe mountains, 6 km south-east of the city of Bielsko-Biała, 480 m ASL, the Carpathian flysch; 2) the Cisowe Skały (CS) mountain (n = 2) located in western Olkusz Upland, 5 km north-east from the town of Trzebinia, 400 m ASL, the Triassic dolomite; 3) the Murońka (Mu) mountain (n = 2) located in the Beskid Śląski mountains, 10 km south-west from the town of Żywiec, 950 m ASL, the Carpathian flysch; 4) the Kletno (K) village (n = 2) located in the Masyw Śnieżnika mountains, 7 km south-west from the town of Stronie Śląskie, 830 m ASL, the Devonian marble; 5) the Marzysz (M) village (n = 5) located in the Świętokrzyskie Mountains, 9 km south-east from the city of Kielce, 240 m ASL, the Devonian limestone.
Analysis of element concentrations in the soil and plant tissues
Soil samples were sieved using a 2 mm mesh and dried at 105°C. Total As, Cd, Pb, Tl and Zn were extracted by digestion of the ground soil with hot concentrated HClO4 (Foss Tecator Digestor Auto). Asplenium viride specimens from the metalliferous site were washed carefully in running tap water, followed by double-distilled and deionized water. The plants were divided into shoots and roots and dried at 80°C for 48 h. To analyze As, Cd, Pb, Tl and Zn content, the plant material was ground and digested in a hot concentrated mixture of HNO3 and HClO4 (4:1; Foss Tecator Digestor Auto). The elemental contents of the soil and plant extracts were analyzed using flame or graphite furnace atomic absorption spectrometry (Varian AA280FS; Varian AA280Z, GTA 120). Certified reference materials were used to estimate the quality of the metal analyses of soil (CRM048-050; RTC) and plants (Oriental Basma Tobacco Leaves, INCT-OBTL-5; The Institute of Nuclear Chemistry and Technology and moss Pleurozium schreberi M2; The Finnish Forest Research Institute).
Plant material
Ploidy level and genome size were estimated in fresh and young leaves of A. viride, collected from metalliferous (HM) and non-metalliferous (NM) sites. Ploidy was determined using the diploid plants of A. viride as a reference standard. Leaves of Vicia faba ‘Inovec’ (2C=26.90 pg; Doležel et al. 1992) were used as an internal standard for nuclear DNA content estimation. Molecular analyses were performed on 24 specimens originating from one metalliferous and five non-metalliferous sites.
Flow cytometry measurements
Samples for cytometry analysis were prepared according to Galbraith et al. (1983), with some modifications. Plant tissues of each sample were chopped simultaneously with a sharp razor blade in a plastic Petri dish, with 1 ml of TrisMgCl2 (200 mM Tris, 4mM MgCl2∙6H2O, 0.5% (v/v) Triton X-100, pH 7.5) nucleus-isolation buffer, supplemented with fluorochrome 4,6'-diamidino-2-phenylindole (DAPI, 2μg/mL) and 2 % (v/v) polyvinylpyrrolidone (PVP-10) for ploidy level, or propidium iodide (PI, 50 μg/mL), ribonuclease A (50 μg/mL) and 2 % PVP-10 for genome size estimation. Ploidy level was estimated by comparison of the position of the G0/G1 peak of the target sample on a histogram with that of the diploid reference standard. Analyses were performed for 5000-7000 nuclei using a Partec CCA flow cytometer (Partec GmbH, Münster, Germany), equipped with a mercury UV lamp. The obtained histograms were analyzed using DPAC v.2.2 software (Partec, GmbH). For nuclear DNA content, 7000 – 10 000 nuclei were measured using a Partec CyFlow SL Green flow cytometer (Partec GmbH, Münster, Germany), equipped with a high-grade solid-state laser with green light emission at 532 nm and side (SSC) and forward (FSC) scatters. Analyses were performed on 15 individual samples from the HM and NM sites. The obtained histograms were evaluated manually using a FloMax software (Partec, GmbH). Genome size was estimated using the linear relationship between the ratios of target species and the internal standard 2C peak positions on the histograms. The coefficient of variation (CV) of the G0/G1 peak of Asplenium samples ranged between 4.45 and 4.49%. The 2C DNA contents (pg) were transformed to megabase pairs of nucleotides using the following conversion: 1 pg = 978 Mbp (Doležel and Bartoš 2005).
DNA extraction
Genomic DNA was extracted from silica gel-dried leaf tissue of HM and NM samples using a CTAB extraction protocol (Rogers and Bendich 1994) modified by Trewick et al. (2002) and Kwiatkowska et al. (2019). Leaves with the sori removed underwent homogenization in 500 μl 2% CTAB isolation buffer (2% CTAB, 1.4 M NaCl, 100 mM Tris, 20 mM EDTA), following the addition of 50 μl 10 % sodium dodecyl sulphate buffer (10% SDS, 100 mM Tris-HCl pH 8.0, 20 mM EDTA) and 5 μl 98% β-mercaptoethanol, after which the whole mixture was incubated at 60° C for 1 h. An equal volume of chloroform:isoamyl alcohol mixture 24:1 (v/v) was added, mixed for 10 minutes and centrifuged for 3 minutes at 13000 rpm at room temperature. The collected aqueous phase was mixed with 12.5 μl sodium acetate (pH 5.2) per 100 μl of the mixture. Precipitation was performed by adding a 2/3 volume of isopropanol and incubating at -20° C overnight. DNA pellets were centrifuged at 13000 rpm for 3 minutes at 4° C and rinsed in 500 µl 70% ethanol, centrifuging at 13000 rpm for 1 minute. Vacuum-dried pellets were dissolved in 50 μl nuclease-free water (EURx, Gdańsk, Poland). The quality and quantity of extracted DNA was established using a UV/Vis Q5000 spectrophotometer (Quawell Technology, Inc, CA, USA) and gel electrophoresis on 1% agarose gel. Only samples found to be high quality were used for ISSR-PCR.
ISSR analysis
After an initial screening of 15 ISSR primers, five were chosen for further analysis (Tab. 1). ISSR-PCR amplifications were performed in 25 µl reaction volumes containing 2.5 µl 10X DreamTaq™ Green Buffer (Thermo Scientific), 2 µl 10 mM dNTPs (Thermo Scientific), 0.5 µl bovine serum albumin (BSA), 0.25 µl of each primer, 1 µl template DNA and 0.25 µl DreamTaq™ DNA polymerase. PCR reactions were performed as follows: initial denaturation at 94° C for 2 minutes and 35 cycles of denaturation at 94° C for 30 seconds, annealing at 44° C for 45 seconds and elongation at 72° C for 90 seconds. Final extension was performed at 72° C for 20 minutes. Obtained ISSR products were separated through gel electrophoresis on 1% (v/v) agarose gel. The bands were visualized using a GelDoc®-it2 imager (UVP, Jena, Germany), sized using GeneRuler 1kb Plus DNA ladder (Thermo Scientific), and scored by the presence/absence of each feature using PyElph v. 1.4 (Pavel and Vasile 2012).
Statistical analysis
Student’s t-test for paired samples was used to compare element concentrations between A. viride shoots and roots. The nuclear DNA content results were analyzed using one-way analysis of variance (ANOVA) and Student’s t-test (STATISTICA v. 10, StatSoft, Poland) to determine possible differences in nuclear DNA content among A. viride plants collected from HM and NM sites. We estimated genetic diversity by calculating parameters both at the population and the whole study area levels. These included: number (P) and proportion (%poly) of polymorphic markers, number of private and discriminating markers (Nprt, Nd; Schlüter 2013), Nei’s gene diversity (Hj; Nei 1973), total gene diversity (HT), mean gene diversity within populations (HS; Nei 1973), gene diversity among populations (GST) and estimated gene flow (Nm; Slatkin and Barton, 1989). All calculations were performed in FAMD v. 1.31 (Schlüter and Harris 2006) and POPGENE v. 1.31 (Yeh et al. 1999).
A dendrogram, based on Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and NeigborNet, was constructed using Treecon v. 1.3b (Van de Peer and De Wachter 1994) and SplitsTree v. 4.6 (Huson and Bryant 2006), based on a matrix of Nei-Li coefficients (Nei and Li 1979). Bootstrap analysis was performed using the Neighbor-Joining method, with 2000 replicates. Population genetic structure analysis was completed using Bayesian clustering inference in STRUCTURE v. 2.3 (Falush et al. 2007; Pritchard et al. 2000), following the protocol described in Migdałek et al. (2017) and Kwiatkowska et al. (2019). For these calculations, a recessive allele model for dominant markers, admixture and independent allele frequencies between clusters were assumed. Ten independent runs were performed for each K value ranging from 2 to 5, with a burn-in of 200000, followed by 1000000 Markov Chain Monte Carlo replicates. The estimated mean logarithmic likelihoods of K values and ∆K values were calculated to determine the optimal K value (Evanno et al. 2005) using Structure Harvester v. 0.6 (Earl and VonHoldt 2012). STRUCTURE results were summarized in CLUMPAK (Kopelman et al. 2015), using the LargeKGreedy search method and 2000 random input repeats. A three-level hierarchical analysis of molecular variance (AMOVA) was performed in ARLEQUIN v. 3.5 (Excoffier et al. 2005), aiming on testing the statistical significance of inferred groups. Calculations were run on all individuals and population groups suggested by STRUCTURE using a pairwise difference distance matrix at P = 0.05.