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Viola tricolor (Violaceae) is a karyologically unstablespeciesA. SŁomkaa, E. Wolnyb & E. Kutaa

a Department of Plant Cytology and Embryology, Jagiellonian University, Polandb Department of Plant Anatomy and Cytology, University of Silesia, PolandAccepted author version posted online: 21 Mar 2013.Published online: 19 Apr 2013.

To cite this article: A. SŁomka, E. Wolny & E. Kuta (2014) Viola tricolor (Violaceae) is a karyologically unstable species,Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa BotanicaItaliana, 148:4, 602-608, DOI: 10.1080/11263504.2013.788576

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ORIGINAL ARTICLE

Viola tricolor (Violaceae) is a karyologically unstable species

A. SŁOMKA1, E. WOLNY2, & E. KUTA1

1Department of Plant Cytology and Embryology, Jagiellonian University, Poland and 2Department of Plant Anatomy and

Cytology, University of Silesia, Poland

AbstractViola tricolor is a pseudometallophyte covering heavy-metal-polluted and non-polluted areas. The species is a member of theevolutionarily young sect. Melanium of Viola. In this study, we sought to determine whether the karyotype of V. tricolor isstable with respect to chromosome structure or is altered depending on environmental conditions (heavy-metal-polluted vs.non-polluted areas). We established the karyotypes of plant material originating from a Zakopane meadow (non-metallicolous population) and from the Bukowno mine waste heap (metallicolous population), showing evidentinterpopulation differentiation in chromosome type (2M þ 20m þ 2sm þ 2st vs. 18m þ 8sm), in the number, size, anddistribution of rDNA loci (25S and 5S), and also in chromosome mutations, mainly fission of chromosomes into acentricfragments and translocation of the fragments. Variable numbers of both 25S and 5S rDNA loci were distributed at differentpositions of the chromosomes and not on specific pairs of chromosomes. The results clearly indicate that the karyotype ofV. tricolor results from the unstable genetic structure of the species. This character, typical for relatively young evolutionarygroups, proves its membership to the Melanium section considered to be young within the genus Viola.

Keywords: Karyotype repatterning, Melanium section, pseudometallophyte, rDNA loci, Viola tricolor

Introduction

Viola tricolor L. (wild pansy) belongs to sect.

Melanium of the genus Viola, which forms a derived

and monophyletic clade. Polyploidy and hybridiz-

ation have played a key role in the evolution of this

group, resulting in a wide range of chromosome

numbers. Its low level of genetic differentiation and

its complex cytological evolution indicate explosive

and quite recent radiation of this section (Erben

1996; Ballard et al. 1999; Yockteng et al. 2003).

Karyotype studies ofV. tricolor plants fromdifferent

parts of its area of distribution, including an

ornamental variety, have shown interpopulation

variability of karyotype structure using standard

staining techniques (Lausi & Cusma Verali 1986;

Krahulcova et al. 1996; He & Zhou 2007). The

variation involves chromosome structure and also

chromosome number. Intra- and interpopulation and

also intra- and interindividual variability of chromo-

some number were found in plants from the same

populations as analyzed in this study. Aneuploid cells

with lower (2n ¼ 18–25), higher (2n ¼ 27, 28), and

also polyploid (2n ¼ 42) chromosome numbers were

counted, in addition to the standard chromosome

number (2n ¼ 26), and the frequency of non-standard

numbers was higher in plants from the site polluted

with heavy metals than in those from the non-polluted

site (43% vs. 28%) (Słomka et al. 2011a). Continuing

our research on the V. tricolor karyotype, in this paper

we focus on karyotype alteration: changes in chromo-

somemorphology (type of chromosome), size, and the

distribution of selected rDNA sequences, using the

fluorescence in situhybridization (FISH) technique. In

view of previously reported intraspecific variability of

chromosome number in V. tricolor (Słomka et al.

2011a) and chromosomal aberrations in other plant

species, attributable to the effects of heavy metals

(Coulaud et al. 1999; Nkongolo et al. 2001; Prus-

Głowacki et al. 2006; Sedel’nikova & Pimenov 2007),

we expected soils pollutedwith heavymetals to have an

evident impact on karyotype alteration inViola tricolor.

Changes in chromosome number, chromosome

structure, and nuclear size (DNA content), generating

interpopulation, intrapopulation, and interindividual

karyological variability, are the crucial mechanisms of

q 2013 Societa Botanica Italiana

Correspondence: A. Słomka, Department of Plant Cytology and Embryology, Jagiellonian University, 52 Grodzka St., 31-044 Cracow, Poland. Tel: þ 48 1266

31764. Fax: þ 48 124228107. Email: a.slomka@iphils.uj.edu.pl; aneta.slomka@uj.edu.pl

Plant Biosystems, 2014Vol. 148, No. 4, 602–608, http://dx.doi.org/10.1080/11263504.2013.788576

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microevolutionary processes leading to speciation

(Schubert 2007; Raskina et al. 2008; Kirkpatrick

2010;Heslop-Harrison& Schwarzacher 2011). Plants

with large genomes are at a selective disadvantage at

heavy metal polluted sites (Vidic et al. 2009; Temsch

et al. 2010). On the other hand, in particular cells of

plants, the ploidy level may increase by endomitosis

and/or reduplication up to 16C, as in Cardaminopsis

halleri growing in Zn/Pb soils (Rostanski et al. 2005).

FISH for specific rDNA sequences is a good method

for genome research, visualizing chromosome

rearrangements during mitotic stages and in inter-

phase nuclei (Garrido et al. 1994; Maluszynska &

Juchimiuk 2005; Jiang & Gil 2006), as chromosomal

alterations are usually assisted by rDNA locus

dynamics (Raskina et al. 2004, 2008). Some

karyotypic changes, especially those enhanced under

stress, allow species with plastic genomes to survive as

new forms or even new species in times of rapid change

(Belyayev et al. 2010).

The present research, part of long-term studies

on the pseudometallophyteV. tricolor colonizing areas

in Europe polluted by heavy metals (Słomka et al.

2008, 2010, 2011a, 2011b, 2012), is aimed at

determining whether heavy metal tolerance is

accompanied by the structural alteration of chromo-

somes and karyotype repatterning.

Materials and methods

Plant material

Seeds for chromosome analyses were collected from

plants growing at a non-polluted site (Zakopane

meadow, ZM) and an area contaminated with heavy

metals (Bukowno mine waste heap in Olkusz area,

BH), both in southern Poland. The soil of ZM

contains relatively low amounts of heavy metals

(means: Zn 83 ppm, Pb 30 ppm, Cd 1ppm), whereas

the soil of BH is metal-rich (Zn 6725 ppm, Pb

1491 ppm, Cd 85ppm) (Słomka et al. 2008).

Seed germination and seedling pretreatment

We applied a specific treatment to improve the very

low frequency of seed germination of the plant. After

8 weeks of cooling at 48C, V. tricolor seeds from BH

and ZM were sterilized in commercial bleach diluted

with sterilized water (1:3 v/v) for 10min and rinsed

several times in sterilized water, then sown on 1%

agar and on moist filter paper and kept in an

experimental chamber under a 16-h photoperiod at

248C/188C. Seedlings for DAPI staining, chromo-

mycin staining, FISH, and Ag-NOR staining were

treated with 0.02M water solution of 8-hydroxyqui-

noline for 4 h at room temperature and fixed in a

mixture of ethanol and glacial acetic acid (3:1 v/v).

Metaphase plates were analyzed from 21 seedlings

from BH and 18 from ZM.

Slide preparation

Fixed seedlings were rinsed in 10mM citric acid–

sodium citrate buffer (pH 4.8) and enzymatically

digested [20% v/v pectinase (Sigma), 1% w/v

Calbiochem cellulase, 1% w/v Onozuka (Serva)

cellulase] for 2.5 h at 378C and finally rinsed in

10mM citric acid–sodium citrate buffer for 15min.

Preparations were made from root tips squashed in a

drop of 45% acetic acid, dry-iced and air-dried.

Slide staining

Chromomycin A3/DAPI staining

Double fluorescent staining with CMA3 (chromo-

mycin A3) and DAPI (40,6-diamidino-2-phenylin-

dole) was done according to the procedure of

Schweizer (1976). Slides were stained with 0.5mg/

ml CMA3 solution (Sigma) for 1 h in the dark, briefly

rinsed in distilled water, air-dried, and mounted in

Vectashield (Vector Laboratories) containing 2.5mg/

ml DAPI (Sigma). The chromosome spreads were

observed with an Olympus Provis AX epifluores-

cence microscope using the proper filter set.

Ag-NOR staining

The transcriptional activity of 45S (18-5.8-25S)

rRNA genes was determined using silver staining

(Hizume et al. 1980). Slides were incubated in borate

buffer (pH 9.22, Merck) for 10min, dried, and then

moisten with 50% silver nitrate (Merck) in redistilled

water. Slides were covered with nylon mesh and

incubated in a humid chamber at 428C for 20min,

washed in distilled water, air-dried, and mounted in

Entellan.

Fluorescence in situ hybridization

Two probes were used in this study: (1) 5S rDNA

isolated from Triticum aestivum – pTa 794 (Gerlach

& Dyer 1980) labeled with rhodamine-4-dUTP, and

(2) 25S rDNA isolated from Arabidopsis thaliana

(Unfried & Gruendler 1990) labeled with digox-

ygenin-11-dUTP by nick translation (Roche). 25S

rDNA was used to determine the localization of 45S

rRNA genes (18S-5.8S-25S rDNA). The labeling

procedure is described in detail in Hasterok et al.

(2001).

FISH was applied according to the method

described by Schwarzacher and Heslop–Harrison

(2000) with some modifications. The slides were

pretreated with RNase (100mg/ml), post-fixed in 1%

aqueous formaldehyde in PBS buffer, washed in 2

£ SSC, dehydrated in ethanol, and air-dried. The

Karyological instability of Viola tricolor 603

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hybridization mixture consisted of 2.0–3.0 ng/ml ofeach probe, 50% deionized formamide, 10% dextran

sulfate, 0.5% SDS (sodium dodecyl sulfate), 2

£ SSC, and salmon sperm blocking DNA in a 50–

100 £ excess of DNA probes. Slides with chromo-

somes and predenatured probe (758C for 10min)

were denatured together at 728C for 4.5min in an

in situ thermal cycler (Hybaid). Hybridization was

carried out overnight at 378C in a humid chamber.

After post-hybridization washes (10% formamide in

0.1 £ SSC for 2 £ 4min at 428C), immunodetection

of digoxigenated probes was carried out with FITC-

conjugated anti-digoxigenin antibodies (Roche)

according to the standard protocol. Dehydrated

preparations were mounted and counterstained

in Vectashield (Vector Laboratories) containing

2.5mg/ml 40,6-diamidino-2-phenylindole (DAPI,

Sigma). The chromosome spreads were observed

with an Olympus Provis AX epifluorescence micro-

scope using the proper filter set. Images were

captured with a Hamamatsu C5810 CCD camera

and were processed with Adobe Photoshop 7.0 and

Micrografx (Corel) Picture Publisher.

The x 2 test in Statistica ver. 7.0 was used for

comparison of chromosome rearrangement

frequencies.

Chromosome measurement and karyotype formula

For chromosome measurements, four to six well-

spread metaphase plates with similarly condensed

chromosomes and standard chromosome number

(2n ¼ 26) were selected. Chromosomes were classi-

fied according to Levan et al. (1964). Chromosome

length measurements were carried out using Image

Tool ver. 3.0, and the chromosome grouping in

homologous pairs and calculations in Mr Karyo ver.

3.10 (by Tokarski & Joachimiak). In the ideograms,

chromosomes are arranged by length, not by

centromere position.

Results

Interpopulation karyotype variability

Interpopulation variability in the karyotype structure

of V. tricolor was found. The karyotype of seedlings

from ZM consisted of 22 metacentric, 2 submeta-

Table 1. Chromosome length and morphology in karyotypes of Viola tricolor from non-metallicolous (ZM) and metallicolous (BH)

populations.

Chromosome numberChromosome length (mm) Longer arm length (mm) Arm ratio

Chromo-

some type

ZM BH ZM BH ZM BH ZM BH

1 9.70 ^ 0.38 7.68 ^ 0.32 5.04 ^ 0.23 4.16 ^ 0.32 1.08 ^ 0.06 1.18 ^ 0.02 M m

2 8.07 ^ 0.70 6.09 ^ 0.48 4.29 ^ 0.31 3.28 ^ 0.08 1.13 ^ 0.09 1.17 ^ 0.08 m m

3 7.12 ^ 0.27 6.08 ^ 0.13 4.02 ^ 0.18 4.00 ^ 0.16 1.30 ^ 0.14 1.92 ^ 0.05 m sm

4 6.54 ^ 0.21 6.00 ^ 0.14 3.80 ^ 0.28 4.40 ^ 0.12 1.39 ^ 0.23 2.75 ^ 1.21 m sm

5 6.30 ^ 0.37 5.12 ^ 0.32 3.97 ^ 0.48 2.88 ^ 0.32 1.71 ^ 0.35 1.28 ^ 0.34 m m

6 6.23 ^ 0.48 5.10 ^ 0.2 3.73 ^ 0.53 4.30 ^ 0.2 1.50 ^ 0.24 2.98 ^ 0.12 m sm

7 6.21 ^ 1.23 4.96 ^ 0.16 4.99 ^ 1.38 3.2 ^ 0.32 4.09 ^ 1.14 1.53 ^ 0.32 st m

8 5.84 ^ 0.42 4.46 ^ 0.63 3.47 ^ 0.41 2.78 ^ 0.43 1.46 ^ 0.21 1.65 ^ 0.24 m m

9 5.57 ^ 0.44 4.00 ^ 0.16 3.03 ^ 0.38 2.4 ^ 0.08 1.20 ^ 0.18 1.5 ^ 0.24 m m

10 5.45 ^ 0.38 3.84 ^ 0.01 3.11 ^ 0.17 2.08 ^ 0.16 1.32 ^ 0.33 1.18 ^ 0.06 m m

11 5.25 ^ 0.31 3.83 ^ 0.02 3.52 ^ 0.36 2.16 ^ 0.08 2.02 ^ 0.35 1.28 ^ 0.23 sm m

12 5.07 ^ 0.46 3.76 ^ 0.08 2.71 ^ 0.33 2.00 ^ 0.08 1.14 ^ 0.11 1.13 ^ 0.04 m m

13 5.00 ^ 0.41 3.60 ^ 0.08 2.84 ^ 0.23 2.1 ^ 0.02 1.31 ^ 0.14 1.82 ^ 0.33 m sm

Note: Classification of chromosome types taken from Levan et al. (1964). For population abbreviations see materials and methods.

1 2 3 4 5 6 7 8 9 10 11 12 13

1 2 3 4 5 6 7 8 9 10 11 12 13

A

B

Figure 1. Ideograms of Viola tricolor from non-metallicolous ZM

(A) and metallicolous BH (B) populations. 1 scale unit < 1.2mm.

604 A. Słomka et al.

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centric, and 2 subtelocentric chromosomes

(2M þ 20m þ 2sm þ 2st) (Table I, Figures 1A and

2A). The karyotype of the material from polluted

area BH was more uniform and contained only

metacentric and submetacentric chromosomes

(18m þ 8sm) (Table I, Figure 1B).

Chromosome rearrangements

Altered karyotypes in 28% of the 76 metaphase

plates from ZM and 29% of the 59 from BH, both

within and between plants, without interpopulation

differences (l 2 ¼ 0.03, 0.5 , P , 0.9) were

observed. The most common chromosome

mutations were chromosome fissions leading to the

formation of separate acentric fragments and

translocation of them (Figure 2B–D), which resulted

in increased or decreased chromosome numbers in

some cells (Figure 2D).

In plant material from the non-metallicolous as

well as the metallicolous populations, intra- and

interindividual variability in the number, size, and

distribution of 25S rDNA and 5S rDNA loci was

found. For both populations, the most frequent were

three to five hybridization signals of 25S rDNA and

six to eight signals of 5S rDNA (Figure 2E–I) on

chromosomes. Usually four, rarely three or five 25S

rDNA loci, were terminally located (Figure 2E–I).

Only two of the 25S rDNA loci were transcriptionally

active as confirmed by silver staining (Figure 2J) and

adjacent to GC-rich but not AT-rich heterochroma-

tin, as shown by double DAPI/CMA3 staining

(Figure 2K,L). The odd numbers of 25S rDNA

loci were likely a result of chromosome rearrange-

Figure 2. Standard metaphase plate (A), chromosome structural mutations (B–D), distribution of rDNA loci (E–I), and marker

chromosomes (J–L) in karyotype ofViola tricolor plants from non-metallicolous ZM (A–E, J) and metallicolous BH (F–I, K, L) populations.

(A) Euploid 2n ¼ 26 metaphase plate stained with DAPI; (B–D) deficiencies (arrows) and translocations (arrowhead) in euploid 2n ¼ 26

(B,C) and aneuploid 2n ¼ 27 (D) metaphase plates; (E–I) euploid 2n ¼ 26 (E–H) and aneuploid 2n ¼ 27 (I) metaphase plates stained with

DAPI with six to eight red 5S rDNA signals on six chromosomes and three to five green 25S rDNA signals on three to five chromosomes; note

subterminally located 5S rDNA loci (E, G–I; arrows) and weak condensed chromatin in nucleolar organization regions (NOR) (H, I;

arrowheads); (J) euploid 2n ¼ 26 metaphase plate with nucleolar organization regions (NOR) on two chromosomes (arrows) after silver

staining; (K,L) euploid 2n ¼ 26 metaphase plates with chromosomes poor in DAPI-stained AT sequences (K; arrows) but rich in

chromomycin-stained GC sequences A3 (L; arrows) – reverse DAPI/chromomycin A3 staining. All bars ¼ 10mm.

Karyological instability of Viola tricolor 605

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ments including rRNA genes, both in euploid

(Figure 2H) and in aneuploid (Figure 2I) cells. 5S

rDNA loci were located the most frequent terminally

but sometimes also subterminally (Figure 2E,G–I).

Their size and distribution were extremely variable

within and between plants in both populations

(Figure 2E–I).

Discussion

These results support the assertion that species from

the evolutionary young taxa including recently

formed hybrid origin species (homoploid speciation)

and allopolyploids are karyologically unstable (Rie-

seberg 2001; Dadejova et al. 2007; Chester et al.

2012; Peruzzi et al. 2012). The differences in

chromosome types with respect to the symmetry in

V. tricolor are not due to heavy metal pollution, as

interpopulation variability has been found in differ-

ent non-polluted populations in Poland

(2M þ 20m þ 2sm þ 2st; present results), Germany

and Italy (4M þ 14m þ 8sm; Lausi & Cusma Verali

1986; Kellner unpubl.), and the Czech Republic

(8M þ 8m þ 4sm þ 4sm-st þ 2st; Krahulcova et al.

1996). However, intraspecific karyotype differen-

tiation is additionally increased by chromosome

variability in plants from metallicolous populations.

The Polish metallicolous population (BH) had a less

differentiated karyotype, consisting exclusively of

meta- and submetacentric chromosomes

(18m þ 8sm), than the metallicolous Italian popu-

lations, which had a slightly asymmetric karyotype

with two pairs of subterminal chromosomes

(4M þ 8m þ 10sm þ 4st) (Lausi & Cusma Verali

1986). An evident impact of polluted environments

on karyotype differences (mean arm ratio and total

length of chromosomes) has been found in other

species such as Myosotis stenophylla growing on

serpentine and non-serpentine soils (Stepankova

1996) but it does not seem to be the rule: Silene

maritima has a stable karyotype in non-polluted and

in metal-polluted populations (Cobon & Murray

1983).

The relatively high karyological variation we

found, 29% in BH and 28% in ZM, revealed as

structural rearrangements of chromosomes within

and between V. tricolor plants from metallicolous and

non-metallicolous populations, adding to the weight

of evidence that plant species with an unstable

karyotype are favored in adverse environmental

conditions, as they can change rapidly (Coulaud

et al. 1999; Nkongolo et al. 2001; Degenhardt et al.

2005; Sedel’nikova & Pimenov 2007; Seehausen

et al. 2008). The difference in the frequency of

karyotype alterations in V. tricolor between plants

from the metal-polluted and non-polluted areas was

not significant, unlike in other metal-tolerant plants

with flexible genomes, such as Armeria maritima,

Deschampsia cespitosa, and Larix sibirica (Coulaud

et al. 1999; Nkongolo et al. 2001; Sedel’nikova &

Pimenov 2007). Besides the rearrangement of large

chromosome fragments, also responsible for the

alteration of the V. tricolor karyotype were the

movements of small acentric chromosome fragments

containing rDNA loci, combined with the high intra-

and interindividual variability of the number of those

loci. Rearrangement of rDNA usually is a very rapid

process; it is recognized to be functionally linked with

transposon elements (TE), whose activity in stressful

environmental conditions (e.g. heavy metals) and

genomic processes (e.g. inbreeding and interspecific

hybridization) increases mutation rates by two orders

of magnitude (Fontdevila 1992). The impact of

transposable elements in the environmental adap-

tation of both prokaryotic and eukaryotic organisms

has recently been summarized by Casacuberta and

Gonzalez (2013). The TnAO22 transposon in a

Protobacterium (Achromobacter sp. AO22) is associ-

ated with the mercury resistance system, among the

few such systems reported in a soil bacterium (Ng

et al. 2009). In plants, adaptation to local environ-

ments is associated with TE-induced mutations (e.g.

Kanazawa et al. 2009). Different transposon

elements insert themselves next to or into rDNA

sequences, creating “hot spots” and finally leading to

chromosome splitting (Raskina et al. 2004, 2008).

Simple transfer of rRNA genes to new sites, besides

“traveling” with transposable elements, can also take

place due to ectopic exchange and heterologous

recombination. Irregular clusters, in turn, are targets

for heterologous synapses and recombination with a

possible change of cluster size. Such genetic diversity

emerging with karyotype repatterning enables a

species with a plastic genome to survive as a new

form under intense environmental or genomic

pressure (Belyayev & Raskina 2010; Rebollo et al.

2010). As the differences in chromosome rearrange-

ment frequency between the plant material from the

metalliferous and non-metalliferous sites were neg-

ligible (29% vs. 28%), we may suggest that intense

genomic rather than environmental pressure was

responsible for it. All the observed phenomena in

both the metallicolous and non-metallicolous popu-

lations – chromosome rearrangements, karyotype

diversification (both from this study), variability of

chromosome number, disturbances in mitosis

(Słomka et al. 2011a) – indicate high genetic

instability in this species. The observed chromosomal

rearrangements probably are mildly deleterious, as

they apparently do not reduce plant fitness. Accord-

ing to Rieseberg (2001), such chromosomal

rearrangements seem to protect a part of the genome

from gene flow, fixing a set of alleles simultaneously

and preventing their breakup or loss by crossing back

606 A. Słomka et al.

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to progenitor plants, and surprisingly they do not

favor genetic divergence (Strasburg et al. 2009).

Both present and former (Słomka et al. 2011a)

results on extensive chromosomal alteration together

with great variability in morphological characters and

lack of genetic barriers manifested as ease to hybridize

with other species from Melanium section (Clausen

1921, 1926; Pettet 1964;Marcussen&Karlsson 2010)

prove evolutionary recent origin of V. tricolor.

Conclusions

(1) The karyotype of V. tricolor is variable. Our

present data and literature data indicate that

there is no uniform karyotype formula for plants

from different areas of its distribution.

(2) Divergent karyotypes were found in material

originating from a non-polluted area as well as

from an area polluted with heavy metals,

suggesting no evident impact of heavy metal

pollution on karyotype alteration.

(3) The metalliferous environment did not increase

the number of chromosome rearrangements,

which occurred in both population types at a

high frequency of almost 30%. Genomic rather

than environmental stress seems responsible for

the chromosome alterations.

(4) The genomic instability ofV. tricolor,manifested in

chromosome number variability and chromo-

some repatterning, apparently allows this species

to inhabit and tolerate elevated concentrations of

heavymetals in soils, rather than being an effect of

acquisition of heavy metal tolerance.

Acknowledgements

This work was funded by the Polish Ministry of

Science (Project Nos. 3861/B/P01/2007/33 and

3935/B/P01/2009/36) and by the Jagiellonian Uni-

versity from funds for the statutory activity of the

young scientists (K/DSC/000830), and with the first

author’s financial support from the Polish Science

Foundation.

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