Seasonal and spatial variability of species occurrence of the Paramecium aurelia complex in a single natural pond (Protista, Ciliophora)

Hydrobiologia (2011) 663:233–244 DOI 10.1007/s10750-010-0575-2

PRIMARY RESEARCH PAPER

Seasonal and spatial variability of species occurrence of the Paramecium aurelia complex in a single natural pond (Protista, Ciliophora) Ewa Przybos´ • Sebastian Tarcz • Magdalena Greczek-Stachura • Marta Surmacz

Received: 27 July 2010 / Revised: 30 November 2010 / Accepted: 6 December 2010 / Published online: 17 December 2010  Springer Science+Business Media B.V. 2010

Abstract The geographic distribution and temporal occurrence of ciliates are still little known. In the present article, the occurrence of the Paramecium aurelia species complex in a natural pond situated in Krako´w (Opatkowice) was investigated in different seasons in two following years. A sequence of species occurrence of the P. aurelia complex was observed. Always, paramecia were found only in some sampling points among six points sampled each time and not necessarily in the same ones. Paramecia appearing in one habitat (water body) might occupy different niches characterized by various environmental features suitable for paramecia. The following species were found in the pond: P. biaurelia, P. triaurelia, P. tetraurelia, P. pentaurelia, and P. dodecaurelia. The occurrence of some rare species (P. tetraurelia, P. pentaurelia, and P. dodecaurelia) may be connected with migrating birds which can

Handling editor: Christian Sturmbauer E. Przybos´  S. Tarcz (&)  M. Surmacz Department of Experimental Zoology, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016 Krako´w, Poland e-mail: [email protected] M. Greczek-Stachura Institute of Biology, Pedagogical University, Podbrzezie 3, 31-054 Krako´w, Poland

transport paramecia with drops of water from other water bodies. If a species was observed in successive seasons or years, the possible genetic variation was investigated by analysis of sequences of LSU rDNA and mitochondrial cytb gene fragments. Among the studied species (P. biaurelia, P. triaurelia, P. pentaurelia, and P. dodecaurelia) only P. dodecaurelia showed haplotype variation in different seasons and sampling points, probably caused by the colonization of the pond by different populations of paramecia. Keywords Paramecium aurelia  Temporal occurrence  Geographic distribution  LSU rDNA  Cytb mtDNA

Introduction Protists are very numerous and their biodiversity and biocomplexity are much greater than appreciated (Corliss, 2002). Foissner (2008) estimated that there are about 300,000 free-living protist species. There are still … ‘‘basic questions regarding… the diversity, geographic distribution and temporal occurrence of protistan species in natural ecosystems’’ (Caron, 2005). Different methods of evaluation of the above problems have been applied (cf. Mazei, 2008) including molecular methods (Barth et al., 2006, 2008a, b; Duff et al., 2008; Przybos´ et al., 2008a). However, molecular and genetic studies are still

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scarce in ciliates (Foissner, 2006; Groben et al., 2007; Foissner et al., 2008) but they may bring new data on their biodiversity. It is still unresolved (cf. Lynn, 2008; Caron, 2009) whether protistan species are ubiquitous or endemic regarding their distribution, i.e., show global dispersal (Finlay et al., 1996) or limited geographical range (Foissner, 1999). Morphological and molecular data (according to Schlegel & Meiserfeld, 2003) have shown that many freeliving protists are globally distributed but also patterns in geographical and local distribution may occur. Recently, Bass et al. (2007) used a fastevolving marker (ITS1 rDNA) for studying global diversity and distribution of three clades of cercomonads (Protozoa) and found that ‘‘some taxa show restricted and/or patchy distribution while others are clearly cosmopolitan’’. Essays of Foissner (2006, 2008; Foissner et al., 2008) concerning the geographic distribution of protists put forward the idea of similarity of ciliate biogeography and that of plants and animals. However, they emphasized higher proportions of cosmopolitan species, supporting ‘‘the moderate endemicity model’’, and the fact of the existence of ‘‘numerous patterns of distribution of ciliate morphospecies’’. The problem of geographic distribution and temporal occurrence was also studied in the genus Paramecium (e.g., Fokin et al., 1999, 2004) and widely in the Paramecium aurelia complex (Przybos´ & Fokin, 2000; Przybos´ et al., 2007b, 2008a; and others). The complex is composed of 15 species (Sonneborn, 1975; Aufderheide et al., 1983) known worldwide but some of which are cosmopolitan such as P. primaurelia, P. biaurelia, P. tetraurelia, and P. sexaurelia (Sonneborn, 1975) while others seem limited to geographic regions or temperature zones (P. octaurelia, P decaurelia, P. undecaurelia, P. tredecaurelia, P. quadecaurelia, and P. sonneborni) (Sonneborn, 1975; Przybos´ & Fokin, 2000; Przybos´ et al., 2003a, 2005, 2007a, b, 2009). A species recognized previously as having a distribution limited to Europe (P. novaurelia) was later found in Asiatic Turkey (Przybos´, 1998) and Boston, USA (Przybos´ et al., 2007b). Therefore, sampling from larger areas and different habitats may bring new data concerning the distribution of the P. aurelia species. Probably also many free-living microbial eukaryote morphospecies may consist of multiple ecotypes with different genotypic and phenotypic divergences

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in local niches (Finlay, 2004; Finlay et al., 2006). So we also should take into consideration the existence of local niches in a pond. In this article, the occurrence of species of the P. aurelia complex in a natural pond situated in Krako´w (Opatkowice) was investigated in different seasons in two consecutive years. If a species was observed in successive seasons or years, possible genetic variation was investigated by analysis of sequences of LSU rDNA and mitochondrial cytb gene fragments.

Materials and methods Material Procedures of paramecia collection The sampled pond (designated as Opatkowice pond) is situated in the southern part of Krako´w, former village Opatkowice, since 1973 administratively included into Krako´w as X district called Swoszowice, part of the larger district of Podgo´rze. The area of the pond is ca. 1 ha, it is a fish pond with Cyprinus carpio, settled with ducks, visited also by swans and grebes (Podiceps). It is about 50-years old, before it was swampy terrain, neighboring with a tributary of the small river Wilga, which in turn is a tributary of the Vistula River. A routine procedure of collecting and establishing clones from nature (cf Komala & Przybos´, 1984) was applied. Samples of water with plankton and plant remnants were taken from the surface of littoral waters. From each sampling point 10 samples (tubes) were taken with about 25 ml of water. The pH (6.4–6.8) and the temperature of the water were measured simultaneously, as well as the ambient temperature. Each time the samples were collected from six sampling points (designated I–VI s.p.) situated at a distance of about 5–10 m from each other, along one edge of the pond only because the other sides were inaccessible (Fig. 1) being thickly overgrown by Typha (cattail). All paramecia found in the particular tubes (samples) were isolated and clones established. To the samples in which no paramecia were found, a small amount of medium was added and after a week the samples were again examined. The collecting was done in June and September 2008, and again in May, July, September,

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complementary mating types of the standard strains of species of the P. aurelia complex. The following standard strains were used: strain strain strain strain strain strain

90, Pennsylvania, USA, P. primaurelia, Rieff, Scotland, G.B. P. biaurelia, 324, Florida, USA, P. triaurelia, Sydney, Australia, P. tetraurelia, 87, Pennsylvania, USA, P. pentaurelia, 246, Mississipi, USA, P. dodecaurelia.

Strain crosses

Fig. 1 Distribution of sampling points designated by Roman numerals and occurrence of species of the Paramecium aurelia complex in the Opatkowice pond, Krako´w in several sampling seasons in 2008 and 2009. Numbers refer to the particular species; 2—P. biaurelia, 3—P. triaurelia, 4—P. tetraurelia, 5—P. pentaurelia, 12—P. dodecaurelia. Explanation in Table 1

and November 2009 (Table 1). The data concerning temperature and pH of water as well as ambient temperature are presented in details in Table 2. Methods Identification of Paramecium morphospecies Paramecia were analyzed on slides stained using aceto-carmine (Sonneborn, 1970) and identified to the particular species of Paramecium by the type and number of micronuclei (Vivier, 1974). All paramecia from the P. aurelia complex have a nuclear apparatus with two vesicular micronuclei, many micronuclei in P. multimicronucleatum, while species belonging to P. caudatum contain one compact micronucleus. Culture and identification of species of the P. aurelia complex Paramecia were cultured (in a medium made of dried lettuce and distilled water, inoculated with Enterobacter aerogenes), and identified according to the methods of Sonneborn (1970). Investigated clones matured for conjugation were mated with the reactive

In the strain crosses, the F1 generation was obtained by conjugation and F2 by autogamy (using the method of daily isolation lines). The occurrence of the desired stage of autogamy (specimens at the stage of two macronuclear anlagen) was examined on preparations stained with aceto-carmine. Survival of clones in both generations was estimated as percentages. According to Chen (1956), clones can be considered as surviving after passing 6–7 fissions during 72 h after separation of partners of conjugation or postautogamous caryonids. The methods were described in detail in Przybos´ (1975). Methods used in molecular studies Paramecium genomic DNA was isolated (500 ll of cell culture was used for DNA extraction) from vegetative cells at the end of the exponential phase using the NucleoSpin Tissue Kit (Macherey–Nagel, Germany) as described by Przybos´ et al. (2003b). The strains used in molecular studies are listed in Table 1. Fragments of the 50 LSU rDNA (349 bp) and mitochondrial cytb (618 bp) were sequenced and analysed. The primers used for PCR amplification of both fragments are listed in Table 3. For amplification of 50 LSU rDNA, a forward primer was constructed using Oligoanalyzer 3.0.1 The reverse primer—LR6—is the universal eukaryotic primer.2 PCR amplification was carried out in a final volume of 40 ll containing: 4 ll of DNA, 1.5 U TaqPolymerase (Qiagen, Germany), 0.6 ll 10 mM of each primer, 109 PCR buffer, and 0.6 ll of 10 mM

1 2

http://scitools.idtdna.com/analyzer/. http://www.biology.duke.edu/fungi/mycolab/primers.htm.

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Table 1 Species of the Paramecium aurelia complex in natural pond, Opatkowice, Krako´w

Date of collection

18 June 2008

9 September 2008 21 May 2009 2 July 2009

Sampling points

Species

Accesion numbers

III

P. pentaurelia

HM001314

HM001337

HM001315

HM001338

V V

P. triaurelia

HM001316 HM229428

HM001339 HM229419

I

P. triaurelia

HM229427

HM229418

III

P. biaurelia

HM229424

HM229415

III

P. biaurelia

HM229425

HM229416

HM229426

HM229417

HM229430

HM229421

P. dodecaurelia

II V

Organisms sequenced in this study are highlighted in bold

Table 2 The data concerning temperature, pH of water and ambient temperature during collection

13 November 2009

CytB mtDNA

IV

V 21 September 2009

LSU rDNA

HM229432

HM229423

VI

P. tetraurelia

–

–

I

P. dodecaurelia

HM229429

HM229420

HM229431

HM229422

IV

Date of collection

pH of water

Water temperature (C)

Ambient temperature (C)

18 June 2008

6.5

19.8

23.5

9 September 2008

6.7

18

22

21 May 2009

6.4

18

20

2 July 2009

6.8

22.5

25

21 September 2009

6.7

18.3

24.2

13 November 2009

6.4

6.6

11

Table 3 Primers used in this study Fragment

Primer

Sequence 50 –30

References

LSU rDNA

LSUF

50 -CCCGTATTTGGTTAGGACT-30

Tarcz et al. (2006)

LR6

50 -CGCCAGTTCTGCTTACC-30

CytB mtDNA a

0

Universal eukaryotic primera 0

CytF

5 -GGWACMATGCTRGCTTTYAG-3

PaCytR

50 -TACTCCGTGAAGGTTAGGCTG-30

Barth et al. (2008a) Barth et al. (2008b)

http://www.biology.duke.edu/fungi/mycolab/primers.htm

dNTPs in a T-personal thermocycler TM (Biometra GmbH, Germany). A small fragment of LSU rDNA was chosen because it is the most variable rDNA fragment in the Paramecium aurelia species complex (Tarcz PhD thesis, unpublished) and also covers a fragment of variable domain regions D1–D5 (Wylezich et al., 2010). The protocol for amplification of the 50 LSU rDNA fragment consisted of initial denaturation at 94C,

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followed by 34 cycles of denaturation at 94C for 45 s, annealing at 50C for 60 s, and extension at 72C for 60 s, with final extension at 72 for 5 min. To amplify the cytB region (618 bp) of mitochondrial DNA, CytF, and PaCytR primers (Table 3) and a protocol previously described by Barth et al. (2008a, b) were used. After amplification, the PCR products were electrophoresed in 1% agarose gels for 45 min at 85 V with a DNA molecular weight marker (XIV Roche,

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France). For purification, 30 ll of each PCR product was separated on a 1.8% agarose gel (100 V/60 min). Then, the band representing the examined fragment was cut out and purified using the NucleoSpin Extract II (Macherey-Nagel, Germany). Cycle sequencing was done in both directions with application of the BigDye Terminator v3.1 chemistry (Applied Biosystems, USA). The primers used in PCR reaction were applied for sequencing. Sequencing reactions were carried out in a final volume of 10 ll containing: 3 ll of template, 1 ll of BigDye (1/4 of standard reaction), 1 ll of sequencing buffer, and 1 ll of 5 mM primer. Sequencing products were precipitated using Ex Terminator (A&A Biotechnology, Poland) and separated on ABI PRISM 377 DNA Sequencer (Applied Biosystems, USA). Sequences are available at the NCBI GenBank database (for the accession numbers see Table 1). P. biaurelia, P. triaurelia, P. pentaurelia, and P. dodecaurelia strains found in the Opatkowice pond and originating from different seasons, years or sampling points were examined for genetic variation. All studied strains in this study are highlighted in bold in Table 1. Data analysis Sequences were checked by eye using Chromas Lite (Technelysium, Australia) to evaluate and correct chromatograms. Alignment and consensus of the studied sequences were performed using Clustal W (Thompson et al., 1994) in the BioEdit program (Hall, 1999) and checked manually. All obtained sequences were unambiguous and were used for analysis. Trees were constructed for the studied fragments in Mega version 4.1 (Tamura et al., 2007), using the Neighbor Joining method (NJ) (Saitou & Nei, 1987), Maximum Parsimony (MP) (Nei & Kumar, 2000). The NJ analysis was performed using the Kimura 2-parameter correction model (Kimura, 1980) by bootstrapping with 1,000 replicates (Felsenstein, 1985). The MP analysis was evaluated with the Min-mini heuristic parameter (level = 2) and bootstrapping with 1,000 replicates. The Bayesian inference (BI) was performed in MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The analysis was run with 5,000,000 generations and trees were sampled every 100 generations. All trees were reconstructed with Tree View 1.6.6 (Page, 1996).

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Haplotype diversity values, nucleotide diversity, and analysis of variable nucleotide positions were carried out using DnaSP v. 5.10.01 (Librado & Rozas, 2009). The nucleotide frequencies and transition/transversion rate ratios were calculated with Mega version 4.1 (Tamura et al., 2004, 2007).

Results A very interesting sequence of species occurrence of the P. aurelia complex was observed in the studied pond when samples were collected from six sampling points, during several seasons and in two successive years (Table 1). Always, paramecia were found only in some (1, 2, or 3) sampling points (s.p.) among six sampled each time, and not necessarily in the same ones. The following species were found in the studied pond: P. biaurelia, P. triaurelia, P. tetraurelia, P. pentaurelia, and P. dodecaurelia (Fig. 1). The strains established from material collected in June 2008 and from sampling points (s.p.) designated III, IV, and V were all identified as P. pentaurelia. The next sampling in September 2008 revealed only the presence of P. triaurelia and only in s.p. V. Next year, as a result of sampling in May, P. triaurelia was found in the s.p. designated as I, and P. biaurelia in s.p. III. P. biaurelia was also identified in material collected in July 2009 in s.p. III and V, in turn in the samples collected in September 2009 P. tetraurelia was identified in s.p. VI, and P. dodecaurelia in sampling points designated II and V. P. dodecaurelia also was found in the material collected in November 2009 in sampling points I and IV. A high percentage of surviving clones was observed in F1 and F2 generations of inter-strain crosses within particular species. The pH values of the water in all seasons and sampling points were within the range 6.4–6.8 without great differences. Analysis of genetic variation in different seasons, years and sampling points Analysis of the ribosomal DNA fragment Sequences (349 bp) of the gene encoding the Large Ribosomal Subunit rDNA from 12 strains of P. aurelia and one from P. multimicronucleatum were obtained.

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All results are based on the pairwise analysis of 13 sequences of studied Paramecium strains. Analyses were conducted using the Kimura 2-parameter method in MEGA4

P. multimicro_UL 13.

0.073

0.073

0.073

0.070

0.070

0.073

0.073

0.073

0.096

0.096

0.096

0.096

0.345

0.334 0.015

0.000 0.000

0.000 0.000

0.000 0.020

0.020 0.020

0.020 0.020

0.020 0.036

0.036 0.036

0.036 0.039

0.039 0.039

0.039 P. dodec_V_September_2009 12.

0.039

P. dodec_IV_November_2009 11.

0.039

0.334

0.334 0.015

0.015 0.003

0.003

0.000 0.000 0.020

0.020 0.020

0.020 0.020

0.020 0.036

0.036 0.036

0.036 0.039

0.039 0.039

0.039 P. dodec_II_September_2009 10.

0.039

P. dodec_I_November_2009 9.

0.039

0.336

0.336 0.175

0.175 0.179

0.179 0.175

0.175 0.175

0.175

0.000 0.000 0.000

0.000 0.015

0.015 0.015

0.015 0.023

0.023 0.023

0.023 0.023 P. pent_IV_June_2008

P. pent_V_June_2008

7.

P. pent_III_June_2008 6.

8.

P. tri_I_May_2009 P. tri_V_September_2008 4. 5.

0.023

0.336 0.175 0.179 0.175 0.175 0.000 0.000 0.015 0.015 0.023 0.023

P. bi_V_July_2009 3.

0.023

0.000

0.327

0.015 0.015 0.015 0.015

P. bi_III_July_2009 2.

0.015 0.015

0.327

0.322 0.322 0.165 0.165 0.162 0.162 0.162 0.162 0.162 0.162 0.148 0.148 0.148 0.148 0.148 0.148 0.000

0.160

0.160 0.162

0.162 0.162

0.162 0.162

0.162 0.157

0.157 0.157

0.157 0.157

0.157 0.174

0.174 0.174

0.174 0.000

0.000 0.000

P. bi_III_May_2009 1.

0.000

0.327 0.160 0.162 0.162 0.162 0.157 0.157 0.157 0.174 0.174 0.000 0.000

12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2.

Sequences (618 bp) of the gene encoding mitochondrial cytb from 12 strains of P. aurelia and one from P. multimicronucleatum were obtained. In the studied sequence set only P. dodecaurelia showed intraspecific differentiation (3 haplotypes were observed). Apart from this we found one haplotype characteristic for each studied species. The haplotype diversity value was Hd = 0.8974 and nucleotide diversity was p = 0.13837. The nucleotide frequencies were A = 0.223, T = 0.346, C = 0.232 and G = 0.199. The transition/transversion rate ratios were k1 = 7.401 (purines) and k2 = 6.785 (pyrimidines). The overall transition/transversion bias was R = 3.478. In the analyzed rDNA fragment there were 216 variable positions (150 parsimony informative). Divergence between all studied Paramecium strains is presented in Table 4. The phylogram constructed on the basis of cytb dataset reveals four well-separated clades which represent particular species (Fig. 3). Sequences obtained from P. multimicronucleatum (strain ISN-11) were used as an outgroup. The first clade contains P. pentaurelia strains forming the sister clade to P. triaurelia. The

1.

Analysis of mtDNA

Table 4 The number of base substitutions per site from analysis between sequences 50 LSU rDNA (lower-left) and CytB mtDNA (upper-right) is shown

In the studied sequence set we found one haplotype characteristic for each species studied. The interspecies haplotype diversity value was Hd = 0.833 and nucleotide diversity was p = 0.02873. The nucleotide frequencies were A = 0.3, T = 0.26, C = 0.171 and G = 0.27. The transition/transversion rate ratios were k1 = 11.595 (purines) and k2 = 27.309 (pyrimidines). The overall transition/transversion bias was R = 7.493. In the analyzed rDNA fragment there were 34 variable positions (15 parsimony informative). Divergence between all studied Paramecium strains is presented in Table 4. The phylogram constructed on the basis of the 50 LSU rDNA dataset reveals four wellseparated clades which represent particular species of the P. aurelia species complex (Fig. 2). A sequence obtained from P. multimicronucleatum (strain UL) was used as an outgroup. The first clade, containing P. dodecaurelia strains, is the most distant from the others. P. pentaurelia strains form a clade next to P. docecaurelia. The third clade is formed by two strains of P. triaurelia and the last, which is closest to P. multimicronucleatum, contains three strains of P. biaurelia.

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13.

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Fig. 2 Phylogram constructed for P. biaurelia, P. triaurelia, P. pentaurelia, P. dodecaurelia strains, and P. multimicronucleatum as an outgroup, based on a comparison of sequences from 50 LSU rDNA fragment using the Neighbor Joining method. Bootstrap values (1,000 replicates) for Neighbor

Joining, Maximum Parsimony analysis and posterior probabilities (1,000 replicates) for Bayesian Interference are shown. There were a total of 349 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 4.1 (NJ/MP) and Mr Bayes 3.1.2 (BI)

Fig. 3 Phylogram constructed for P. biaurelia, P. triaurelia, P. pentaurelia, P. dodecaurelia strains, and P. multimicronucleatum as an outgroup, based on a comparison of sequences from cytB mtDNA fragment using the Neighbor Joining method. Bootstrap values (1,000 replicates) for Neighbor

Joining, Maximum Parsimony analysis and posterior probabilities (1,000 replicates) for Bayesian Interference are shown. There were a total of 618 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 4.1 (NJ/MP) and Mr Bayes 3.1.2 (BI)

third clade contains P. dodecaurelia whereas the fourth clade is formed by P. biaurelia strains and is the sister group to P. multimicronucleatum. The four strains of P. dodecaurelia are differentiated into three branches (Fig. 3): the first consisted

of strains collected in September (s.p. II) and November 2009 (s.p. I), in the second branch contained a strain from November 2009 (s.p. IV) and the third a strain collected in September 2009 (s.p. V).

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Discussion The results of this study show that several P. aurelia species may occur in a single water body. The studied pond has suitable conditions (chemical and biological) for the survival of P. biaurelia, P. triaurelia, P. tetrauelia, P. pentaurelia, and P. dodecaurelia. Paramecia occurring in one habitat (water body) might occupy different niches characterized by various environmental features (bacteria, organic matter, and temperature). Similarly, studies on the distribution of the North American Tetrahymena thermophila showed the possibility of ecological specialization, i.e., population structure in local populations (Nanney, 2004; Simon et al., 2007). In turn, seasonal variation in a Protozoan population was observed over a period of 1 year (from August 1999 to July 2000) in Pakistan (Rehman et al., 2007) when it was revealed that Paramecium sp. disappeared in November 1999 and reappeared in March 2000. Earlier, the occurrence of the P. aurelia species was studied in a small pond (which no longer exists) situated in southern Krako´w in different seasons of the year (Przybos´ et al., 1967). P. primaurelia was found only in December, P. biaurelia and P. novaurelia were found in other seasons. Studies carried out in an artificial pond in Jordan’s Park, Krako´w revealed the presence of P. dodecaurelia characterized by only one haplotype in spite of different sampling times (years 2006, 2007) and sampling points situated around the pond (Przybos´ et al., 2008b). In this pond P. pentaurelia was found earlier in the sample collected in 2005 (Przybos´ et al., 2008c) and also later in 2008 (Przybos´ et al., unpubl. data). Findings of a new stand of P. pentaurelia and P. dodecaurelia in the pond were the first records of these species for Poland, which is unexpected because of the relatively dense sampling that has been conducted in this country (217 habitats, see Table 5). Moreover, both species are rather characteristic for warm zones. P. pentaurelia and P. dodecaurelia were found once again in Poland in Opatkowice pond, studied at present. Variation in mitochondrial haplotypes of P. dodecaurelia originating from different seasons and sampling points was revealed in Opatkowice pond. This may have been caused by different founding populations of paramecia transferred into the pond in different seasons. However, as mentioned earlier, in

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another pond in Krako´w this species was represented by only one haplotype in spite of different seasons and years of collection (Przybos´ et al., 2008b). Barth et al. (2006) discovered two mitochondrial haplotypes of P. caudatum in one pond differing in three nucleotide positions. Later, Barth et al. (2008b) also found mitochondrial haplotype diversity of Coleps sp. (Ciliophora: Prostomatida) in a population from one lake. It is worth mentioning that the strains of the compared species of the P. aurelia complex did not show any haplotype diversity in the fragment of 50 LSU rDNA. Genetic differences within P. dodecaurelia strains originating from different seasons were detected in cytb. Similarly, Stru¨der-Kypke & Lynn (2010) found that the ‘‘COI gene (COI) shows considerable genetic divergence between the studied species ‘‘of the P. aurelia complex, and ‘‘only few differences in their SSU rRNA sequences’’. Among species of the P. aurelia complex revealed in the studied pond (Opatkowice pond), P. biaurelia is a common species in Poland (88 habitats among 218 studied) and can be found in different regions. P. tetraurelia was found in 20 habitats (mainly unpolluted), but P. triaurelia was detected before only in a single habitat in the Western Sudetes, and similarly P. pentaurelia as well as P. dodecaurelia were found before also in one habitat (Krako´w, Jordan’s Park). It is interesting that both habitats— the pond in Jordan’s Park and Opatkowice pond—are situated in the city of Krako´w and the distance between them is only about 10 km. Besides the species revealed in the Opatkowice pond, the most frequent species in Poland are P. novaurelia (known from 113 habitats situated in different regions) and P. primaurelia (known from 56 habitats), but these species were not present in the studied pond (Table 5). The occurrence of some rare species such as P. tetraurelia, P. pentaurelia, and P. dodecaurelia in Opatkowice pond may be connected with migrating birds (ducks, grebes) which can transport paramecia with drops of water from other water bodies. Generally, it is accepted that the spread of paramecia may be associated with animals, as they can be transported by migrating birds (Sonneborn, 1975; Coleman, 2005) for long distances, migrating mammals, or by insects such as water beetles (Razowski, 1996) for shorter distances. Human activities also play an important role in the dispersal of microorganisms and cause biogeographic changes, emphasized by

Torun´ region

7 1

Eastern

a

56

Some species appeared together in one habitat

Total number of studied habitats

88

9

1

East Beskids

8 10

15

7

4

4

3

15

10

3

6

1

4

2

1

1

-

P. biaurelia

Middle Beskids

Middle Beskid Plateau

2 2

West Beskids

Tatras

West Beskid Plateau

1 11

Podhale

Carpathians Mts.

Ostrawa Basin

Foothills of the Carpathians Mts.

1

Middle

2

2

Western

Sudetes Mts.

Krako´w region

Krako´w-Cze˛stochowa Upland

River Nida Syncline

Kielce region

Kielce Upland

Lublin region

Lublin Upland 4

1

Torun´ Basin

2

Pomeranian

4

P. primaurelia

2

1

1

-

P. triaurelia

20

1

1

1

10

1

4

1

1

-

P. tetraurelia

Number of studied habitats of particular species of the P. aurelia complex

Mazurian

Lake District

Białowie_za

Lowland of the northern Podlasie

Gdan´sk Seabord

Szczecin Seabord

Baltic Seabord

Region

Table 5 Frequency of species occurrence of the Paramecium aurelia complex in Poland

2

2

-

P. pentaurelia

113

1

8

24

9

8

5

1

3

21

6

4

7

2

2

1

1

2

7

P. novaurelia

2

2

-

P. docecaurelia

218

9

14

33

12

12

16

7

1

6

37

14

10

12

2

6

1

6

5

2

3

10a

Total number of habitats in region

Hydrobiologia (2011) 663:233–244 241

123

242

Foissner (2006). Intercontinental ships carrying goods, ballast water and some introductions of tropical water plants and animals should be mentioned. Some examples of the transfer of tropical Paramecium strains into European Botanical Gardens with water borne plants or animals (fish) have been described (Przybos´ & Fokin, 1997). These concern P. sexaurelia found in the Botanical Garden and ZOO of Stuttgart, Germany, as well as P. tetraurelia found in the Botanical Garden of Krako´w, in a fish pond and in pond in the ‘‘Palm House’’ (cf. Komala & Przybos´, 2001). Cysts are also very important vectors for the transfer of microorganisms, however, they are unknown in Paramecium (Gutierrez et al., 1988; Landis, 1988; Beale & Preer, 2008), therefore, some water is necessary for their dispersal. It is probable that different species of the P. aurelia complex which have colonized the studied pond in Opatkowice/ Krako´w were transferred by birds. It seems that the presence or absence of species at different locations and different times is probably not purely random, it might be determined by proper nutrient concentrations (bacteria for paramecia) at certain sampling sites, and there might be a seasonal progression in species appearance as seen in Table 1. The role of competition between species from the ecological niche can not be excluded as supposed by Hairston (1958) and Hairston and Kellermann (1965). Whereas P. primaurelia has a considerably ability to adapt to various environmental conditions, P. biaurelia shows a great tolerance of low temperatures (Hairston, 1958). Also as demonstrated by Hairston and Kellermann (1965) the latter species may also disclose domination or even exert an inhibitory effect on the development of other paramecia (e.g., P. triaurelia). Sometimes, appearance of several species of the P. aurelia complex in the nearest proximity in nature in one ‘‘micro-niche’’ is possible. It was observed (Przybos´, 1993) in the sample of water (30 ml) collected in September 1991 from the River Hue´car with lime-stone bedding, in Cuenca town, region Castile, Spain where three species of the complex, e.g., P. biaurelia, P. triaurelia, and P. pentaurelia were found. Competition, adaptation abilities, and breeding system (inbreeding is characteristic for species of the P. aurelia complex—Sonneborn, 1975; Landis, 1986) play apparently the same role in paramecia in taking

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up ecological niches as in multicellular organisms. These factors might determine the presence of a species at certain sampling site, however, univocal explanation is difficult.

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