Available online at www.sciencedirect.com
ScienceDirect European Journal of Protistology 50 (2014) 236–247
The first European stand of Paramecium sonneborni (P. aurelia complex), a species known only from North America (Texas, USA) Ewa Przybo´sa , Sebastian Tarcza,∗ , Maria Rautianb , Natalia Lebedevab a
Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków 31-016, Sławkowska 17, Poland Laboratory of Protistology and Experimental Zoology, Faculty of Biology and Soil Science, St. Petersburg State University, Saint Petersburg 199034, Universitetskaya nab. 7/9, Russia b
Received 25 November 2013; received in revised form 15 February 2014; accepted 3 March 2014 Available online 18 March 2014
Abstract P. aurelia is currently defined as a complex of 15 sibling species including 14 species designated by Sonneborn (1975) and one, P. sonneborni, by Aufderheide et al. (1983). The latter was known from only one stand (Texas, USA). The main reason for the present study was a new stand of Paramecium in Cyprus, with strains recognized as P. sonneborni based on the results of strain crosses, cytological slides, and molecular analyses of three loci (ITS1-5.8S-ITS2-5 LSU rDNA, COI, CytB). The new stand of P. sonneborni in Europe shows that the species, previously considered endemic, may have a wider range. This demonstrates the impact of under-sampling on the knowledge of the biogeography of microbial eukaryotes. Phylogenetic trees based on all the studied fragments revealed that P. sonneborni forms a separate cluster that is closer to P. jenningsi and P. schewiakoffi than to the other members of the P. aurelia complex. © 2014 Elsevier GmbH. All rights reserved.
Keywords: Ciliophora; Paramecium aurelia species complex; Paramecium sonneborni; Strain crosses; Three-locus analysis
Introduction The species structure of ciliates is complex as several genera contain morphological species consisting of sibling species (Caron 2013; Nanney and McCoy 1976; Przybo´s 1986; Strüder-Kypke and Lynn 2010). Paramecium (Oligohymenophorea, Ciliophora) is one of the best studied ciliate genera, with the P. aurelia species complex being thoroughly examined (Barth et al. 2008; Przybo´s et al. 2007; Schlegel and Meisterfeld 2003). ∗ Corresponding
author at: Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016 Kraków, Poland. Tel.: +48 12 4227006Ext.43; fax: +48 12 422 42 94. E-mail address: [email protected] (S. Tarcz). http://dx.doi.org/10.1016/j.ejop.2014.03.001 0932-4739/© 2014 Elsevier GmbH. All rights reserved.
The P. aurelia complex is currently thought to be composed of 14 species designated by Sonneborn (1975) and of P. sonneborni (Aufderheide et al. 1983), which was added to the complex at a later time (Wichterman 1986). However, according to Corliss and Daggett (1983) “it was never treated first as another numbered syngen”. The latter species was named in honor of Dr. Tracy M. Sonneborn of Indiana University because his scientific studies led to establishing Paramecium as a model organism in genetics, cytology, pharmacology, etc. The species of the complex are reproductively isolated from one another, each has two complementary mating types, and conjugation is possible only between them (Sonneborn 1937). According to Sonneborn (1975), the main criteria for P. aurelia species identification are a mating reaction (conjugation) between the standard strain of a given species and a
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new strain, and viable F1 and recombinant F2 clones. Moreover, isozyme patterns (Allen et al. 1973; Tait 1970) and several molecular methods have revealed the possibility of distinguishing between some species of the P. aurelia complex (Barth et al. 2008; Catania et al. 2009; Przybo´s et al. 2007; Tarcz et al. 2013). Paramecium sonneborni n. sp., a new member of the Paramecium aurelia species complex, was described by Aufderheide et al. (1983) based on nuclear and whole cell morphology (strain ATCC 30995). The species is reproductively isolated from other members of the complex and characterized by unique esterase zymograms (Aufderheide et al. 1983). It was collected from an intermittent stream in College Station, Texas, USA. Only one stand of this species was known until recently. According to Aufderheide et al. (1983), the body length of P. sonneborni (silver-impregnated specimens) ranges from 130 to 186 m, with a mean length of 154.4 m. It belongs to the largest species of the complex, being as large as P. undecaurelia (length of up to 170 m, Sonneborn 1975). Its two micronuclei (mi) are of vesicular type with a mean diameter of 4.9 m (measured in living cells). Conjugation and autogamy have been observed with typically two macronuclear anlagen after sexual processes, and two mating types of the caryonidal determination type. P. sonneborni is characterized by a rapid growth rate (four fissions daily at 27 ◦ C) and long clonal life-span (250 generations). The present study was motivated by the discovery of a new Paramecium stand in Cyprus (Table S1, supplementary material). At first, the paramecia were thought to be similar to P. jenningsi based on cell dimensions and micronucleus diameter (Przybo´s et al. 2003), but subsequent three-locus comparisons (ITS1-5.8S-ITS2-5 LSU rDNA, COI, CytB) suggested that they were new strains of P. sonneborni. The aim of the present study was to characterize these strains cytologically and genetically and assess their phylogenetic relationship to the P. aurelia spp. complex and other species of the genus Paramecium. Our investigations were carried out on three levels, i.e., cellular with the application of genetic methods (strain crosses), sub-cellular (cytological investigations of nuclear apparatuses), and molecular (the three markers mentioned above).
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migratory birds. The water in the creeks was slightly salty (1.4 ppm), but the clones easily adapted themselves to the standard culture conditions for paramecia. These strains from Cyprus belong to the Culture Collection of Ciliates and their Symbionts (CCCS) of St. Petersburg University and are deposited in the Core Facilities Centre “Culture Collections of Microorganisms” of St. Petersburg State University. The reference strain of P. sonneborni (Table S1, ATCC 30995) was obtained by the senior author from Dr. Karl Aufderheide, Texas A&M University, USA. Other strains used for molecular comparisons, representing particular species of the P. aurelia complex as well as other species of the genus Paramecium, were from the strain collection of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Poland (Table S2, supplementary material). Strains of P. polycarium, P. nephridiatum, P. putrinum used in the studies are from the Core Facilities Centre “Culture Collection of Microorganisms” of St. Petersburg State University (Table S2).
Strain cultivation Paramecia were cultured in a medium made of dried lettuce and distilled water inoculated with Enterobacter aerogenes according to the method of Sonneborn (1970) and supplemented with 0.8 mg/L  sitosterol (Merck, Darmstadt, Germany).
Methods used in cytological studies The nuclear apparatuses of individuals in autogamy as well as individuals in the vegetative stage were examined. Slides were temporarily stained with acetocarmine (Sonneborn 1950), or permanent slides were made, fixed with Schaudinn’s fluid with glacial acetic acid (Chen 1944) and stained with Giemsa stain (Merck, Darmstadt, Germany) (10% solution in 0.01 M phosphate buffer) (cf. Przybo´s 1978). Photographs were taken using a Nikon Eclipse E400 microscope equipped with a Nikon DS-Fi2, digital camera. Some photographs were taken with Leika DM2500 microscope (optical magnification 1250) using differential interference contrast (DIC).
Material and Methods Material
Methods of strain measurements
The Cyprus strains were established by cloning individuals from three water samples (CyL 2, CyL 3, CyL 5) collected in 2010 by Natalia Lebedeva. According to the samples, the clones were designated CyL 2-5, CyL 2-6, CyL 2-10, CyL 31, CyL 3-8, CyL 5-6, and CyL 5-8 (Table S1). The collecting sites were small creeks situated on both sides of a road in a suburb of Larnaca (34.9N/33.6E) and running to the Larnaca Salt Lake (about 100 m away), which is a wintering site for
Young cells of clones representing strains designated CyL 2, CyL 3, CyL 5, from different creeks, at a similar clonal age and at the vegetative stage of life, cultivated in excess of food at 27 ◦ C, were fixed and stained with Giemsa stain. The paramecia were measured (cell and macronuclear length and width, micronuclear diameter) using a Nikon Eclipse E400 microscope and Coolview software (Precoptic Co, Poland).
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Strain crosses Sonneborn’s (1970) methods were used to induce conjugation, autogamy and strain crosses. In intra- and interstrain 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 in preparations stained with acetocarmine. Clone survival in both generations was estimated as percentages. According to Chen (1956), clones can be considered surviving after passing 6–7 fissions during 72 h following the separation of conjugation partners or postautogamous caryonids. Inter-strain cross was done between P. sonneborni reference strain and the Cyprus strain (CyL 2-6) representing other clones from that stand. The percentage of surviving clones in interstrain cross and the duration of particular generations of interstrain hybrids were compared. The methods were described in detail in Przybo´s (1975).
Molecular methods Paramecium genomic DNA was isolated from vegetative cells at the end of the exponential phase (approx. 1000 cells were used for DNA extraction) using a NucleoSpin Tissue Kit (Macherey-Nagel, Germany), according to the manufacturer’s instructions for DNA isolation from cell cultures. The only modification was centrifugation of the cell culture for 20 min at 13,200 rpm. Then the supernatant was removed and the remaining cells were suspended in lysis buffers and proteinase K. Fragments of rDNA, COI, and CytB genes were sequenced and analyzed. First, rDNA fragments were amplified with ITS1 and ITS4 universal eukaryotic primers (White et al. 1990) and ITS3zg and 3pLSU primers developed with OligoAnalyzer 3.1 (http://www.eu.idtdna. com/analyzer/applications/oligoanalyzer) (Table S3, supplementary material). F388dT and R1184dT primers (Table S3) and the protocol previously described by StrüderKypke and Lynn (2010) were used for the amplification of the COI fragment of mitochondrial DNA. In some cases, when the above COI pair of primers did not yield a welldefined product, the internal primer CoxH10176 (Barth et al. 2006) was used instead of R1184dT. To amplify the CytB gene fragment, the primer pair CytBF/PaCytR and the protocol previously described by Barth et al. (2008) were used. PCR amplification for all analyzed DNA fragments was carried out in a final volume of 40 L containing 30 ng of DNA, 1.5 U Taq-Polymerase (EURx, Poland), 16 pmol of each primer, 10 × PCR buffer, and 8 pmol of dNTPs. In order to assess the quality of the amplification, PCR products were electrophoresed in 1% agarose gel for 45 min at 85 V with a DNA molecular weight marker (Mass Ruler Low
Range DNA Ladder, Fermentas, Lithuania). NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Germany) was used for purifying PCR products. In some PCR products, additional sub-bands were obtained apart from the main band. In these cases, 30 L of each PCR product was separated on 1.8% agarose gel (100 V/60 min) with a DNA molecular weight marker (Mass Ruler Low Range DNA Ladder, Fermentas, Lithuania). Then the band representing the examined fragment was cut out and purified. Sequencing was done in both directions with the application of BigDye Terminator v3.1 chemistry (Applied Biosystems, USA). The primers used in PCR reactions were applied for sequencing the rDNA region or a part of CytB gene, and the primer pair M13F/M13R was used for sequencing the COI fragment (Table S3). The sequencing reaction was carried out in a final volume of 10 L containing 3 L of template, 1 L of BigDye (1/4 of the standard reaction), 1 L of sequencing buffer, and 1 L of 5 M primer. Sequencing products were precipitated using Ex Terminator (A&A Biotechnology, Poland) and separated on an ABI PRISM 377 DNA Sequencer (Applied Biosystems, USA). The sequences are available in the NCBI GenBank database (see Tables S1 and S2, supplementary material).
Data analyses Sequences were examined using Chromas Lite (Technelysium, Australia) to evaluate and correct chromatograms. Alignments of the studied sequences were performed using ClustalW (Thompson et al. 1994) as part of BioEdit software (Hall 1999) and checked manually. All of the obtained sequences were unambiguous and were used for analyses. Phylograms were constructed for the studied fragments by means of Mega v5.2 (Tamura et al. 2011), using neighborjoining (NJ) (Saitou and Nei 1987), maximum parsimony (MP) (Nei and Kumar 2000), and maximum likelihood (ML) (Felsenstein 1981). All positions containing gaps and missing data were eliminated. NJ analysis was performed using Mega v5.2 program by bootstrapping with 1000 replicates (Felsenstein 1985). MP analysis was evaluated with the min-min heuristic parameter (at level 2) and bootstrapping with 1000 replicates. Bayesian inference (BI) was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003); analysis was run for 5,000,000 generations and trees were sampled every 100 generations. All trees for BI analysis were visualized with TreeView 1.6.6 (Page 1996). Analysis of haplotype diversity (Hd) and its sampling variance (SD), nucleotide diversity () and polymorphic sites (Nei 1987) was done with DnaSP v5.10.01 (Librado and Rozas 2009). Analysis of nucleotide frequencies, p-distance estimation and identification of substitution models for ML analysis were done using Mega v5.2 (Tamura et al. 2004, 2011).
0.62 0.67 0.49 0.42 2.5 3.32 2.98 2.95 3.08 2.77 5.41 6.17 3.03 17.51 25.24 30.95 15.54 23.91 5.26 11.45 6.25 9.16 51.51 55.28 61.50 49.67 55.48 7.54 10.83 10.63 7.04 52.27 73.40 70.39 52.12 65.3 17.86 23.29 14.07 16.91 ATTC 30995 CyL 2-6 CyL 3-1 CyL 5-6 Mean for Cyprus strains 1. 2. 3. 4. 5.
185.91 220.00 198.89 163.18 194.02
SD Average diameter of MIC [m] SD Average width of MAC [m] SD Average length of MAC [m] SD Average cell width [m] SD Average length of cells [m]
Vegetative individuals belonging to the Cyprus strains (CyL 2-6, CyL 3-1, and CyL 5-6 were studied) are characterized by two atypical vesicular micronuclei (Fig 1b). The micronuclei of P. sonneborni look atypical in comparison with the other vesicular morphotypes of the P. aurelia spp. as they are larger and have an internal structure (Fig. 1b, d). They also appear away from each other and aside from the macronucleus in the cell (Fig 2a, c). According to Fokin (1997), vesicular micronuclei of the P. aurelia spp. “stained by the Feulgen reaction have a chromatin mass as a tiny ring with a clear center . . . surrounded by karyolymph” (cf. Figs 14 and 24 in his paper). In turn, P. jenningsi reveals a chromosomal type of micronuclei with a reticular structure – both mi are compared in Fig 27, Fokin (1997). It is broadly accepted that in all species of the P. aurelia complex, including P. sonneborni, micronuclei are of the vesicular type (Vivier, 1974; Aufderheide et al., 1983; Fokin, 1997; Fokin, 2010/2011). Two new macronuclear anlagen in the later stages of reorganization of nuclear apparatuses during autogamy and conjugation have darker chromatin centers (Fig 2b, d), indicating chromatin accumulation. The Cyprus strains are characterized by a pointed posterior end, similarly as the P. sonneborni reference strain (Aufderheide et al. 1983) (Fig 2a, b, c, d). The dimensions (cells, macronuclei, micronuclei) of the studied P. sonneborni strains (cells fixed and stained with Giemsa stain) are presented in Table 1. The cell size of P. sonneborni strains is comparable to the dimensions of the largest strains of P. jenningsi (cf. Przybo´s et al. 2003), i.e., the strain from China, designated as CS (cell length 191 m), and the strain from Saudi Arabia, designated as SA (cell length 186 m). However, the diameter of P. sonneborni micronuclei is smaller (2.5–3.32 m) than that observed in P. jenningsi strains (e.g., 4.9 m in the strain from India, designated as IB). Autogamy, resulting in homozygosity for all genes, appeared in all cultures of Cyprus and Texas P. sonneborni strains starved for 14 to 20 fissions in daily isolated lines cultivated at 27 ◦ C in a medium enabling 3 fissions per day (clonal age). Conjugation in mature clones in all the studied Cyprus strains was obtained on the 7th day of culture at 27 ◦ C, at a rate of three fissions daily, similarly as in the reference strain of P. sonneborni. Intrastrain crosses were possible between all Cyprus strains, and the percentage of surviving hybrid clones was high in both generations. Caryonidal mating type inheritance was found to be characteristic of all of these strains.
Strain
Cytological characteristics of the Cyprus strains and their biology
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No.
Results
Table 1. Paramecium sonneborni strain dimensions (Giemsa stain). For better orientation, dimensions of P. sonneborni reference strain (ATTC 30995) and mean dimension for the Cyprus strains studied are highlighted in gray.
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Fig. 1. General morphology of vegetative individuals representing P. sonneborni. DIC microscope. Paramecium sonneborni, strain from Cyprus (clone CyL 2-6) – (a) two vesicular micronuclei (mi), two contractile vacuoles (cv) with 8 channels, (b) structure of vesicular micronuclei (mi). Paramecium sonneborni, reference strain (ATTC 30995) – (c) contractile vacuole (cv) with channels, (d) structure of vesicular micronuclei (mi).
Results of strain crosses and survival after conjugation Both interstrain and intrastrain crosses were carried out for P. sonneborni. The strain CyL 2-6 was used as representative for all other Cyprus strains in crosses with the P. sonneborni reference strain, as all Cyprus strains were shown to be identical by molecular and genetic studies. The strain CyL 2-6 was conjugated with the reference strain of P. sonneborni (ATTC 30995), which led to 52% of hybrid clones surviving in F1, and 80–90% in F2 (the latter were obtained by autogamy from F1 hybrids) (Table 2). Crosses were repeated five times. The Cyprus strains were all recognized as belonging to P. sonneborni based on the results of crosses (supported by molecular analyses). Neither the P. sonneborni reference strain nor the Cyprus strains conjugated with any P. jenningsi strains representing three syngens (Przybo´s and Tarcz 2013) (Table S4, supplementary material). The crosses were carried out because P. jenningsi and P. sonneborni look similar in terms of morphological features, and therefore in the beginning the P. sonneborni strain from Cyprus was recognized as P. jenningsi.
Phylogenetic analysis of P. sonneborni strains based on comparison of ribosomal and mitochondrial DNA fragments The phylogenetic trees (NJ, MP, ML, BI) constructed based on analysis of the sequenced markers (ITS1-5.8SITS2-5 LSU rDNA, COI, CytB) show the mutual relationship of the two studied P. sonneborni strains from both the USA (ATTC 30995) and Cyprus as well as their position in respect of the P. aurelia spp. complex and the other species of the subgenus Paramecium (with Tetrahymena used as an outgroup) (Figs 3–5). As was mentioned above, due to identical DNA sequences, the strain CyL 2–6 was used as representative for all other Cyprus strains. On the ribosomal tree (Fig 3), most of the studied species are grouped into a compact, monophyletic cluster exhibiting little variability and consisting of representatives of the P. aurelia species complex, P. sonneborni, P. jenningsi, and P. schewiakoffi divided into two subgroups. The first contains the well-separated P. sonneborni clade (the Cyprus strain and the reference strain are molecularly identical). The next two branches are P. schewiakoffi (Sh1-38) and P. jenningsi (CS). Finally, a clade containing two strains of P. jenningsi (IB,
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Fig. 2. Paramecium sonneborni, vegetative and autogamous individuals. Giemsa stain. Paramecium sonneborni, strain from Cyprus (clone CyL 2-6) – (a) vegetative individual, macronucleus (Ma), two micronuclei (mi), (b) autogamous individual, fragmented old macronucleus (OMa), two macronuclear anlagen with chromatin accumulation (A). Paramecium sonneborni, reference strain (ATTC 30995) – (c) young vegetative individual, macronucleus (Ma), two micronuclei (mi), (d) autogamous individual, fragmented old macronucleus (OMa), two macronuclear anlagen with accumulated chromatin (A).
M) and the P. sexaurelia strain (159) from the P. aurelia spp. complex can be seen. In turn, the second subgroup consists of the other representatives of the P. aurelia species complex. Close to the group described above are two branches formed
by P. caudatum (CA) and P. multimicronucleatum (BR). They all form a monophyletic clade of the subgenus Paramecium. The phylogenetic tree constructed on the basis of COI mtDNA comparison (Fig 4) shows the best resolution among
Table 2. Results of Paramecium sonneborni strain crosses, percentage of surviving clones of inter-strain hybrids. No.
Strain designation
Strain origin
1.
P. sonneborni CyL 2-6 × P. sonneborni ATCC 30995 P. sonneborni CyL 2-6 × P. sonneborni CyL 2-6 P. sonneborni ATCC 30995 × P. sonneborni ATCC 30995
Cyprus × USA, Texas
2. 3.
F1 obtained by conjugation
F2 obtained by autogamy
52
90
Cyprus × Cyprus
100
100
USA, Texas × USA, Texas USA
100
98
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Fig. 3. Phylogram constructed for P. sonneborni strains and for other species of the genus Paramecium (two strains of Tetrahymena were used as an outgroup), based on a comparison of sequences from ITS1-5.8S-ITS2-5 LSU rDNA fragment using the Neighbor Joining method. Bootstrap values for Neighbor Joining, Maximum Parsimony analysis, Maximum Likelihood and posterior probabilities for Bayesian Inference are presented. Bootstrap values less than 50% (posterior probabilities less than 0.50) were not shown. All positions containing gaps and missing data were eliminated. There were 1003 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 5.2 (NJ/MP/ML) and Mr Bayes 3.1.2 (BI).
all studied DNA fragments. As in the case of the ribosomal tree, the most distinct is the group comprising the P. aurelia spp. complex, P. sonneborni, P. jenningsi, and P. schewiakoffi. It is worth noting that P. sonneborni forms a clade that is distant from all P. aurelia species, while close to P. schewiakoffi (Sh1-38) and the sister group formed by three strains of P. jenningsi (CS, IB, M), representing three different syngens. However, in the case of the CytB mtDNA tree (Fig 5), P. sonneborni strains appear in one clade with the P. jenningsi
strain from China (CS) and next to the P. schewiakoffi (Sh1-38) branch. The two other P. jenningsi strains (from Madagascar – M and India – IB), representing different syngens from the Chinese one, are separated from P. sonneborni by two species of the P. aurelia complex (P. sexaurelia and P. dodecaurelia). Similarly, as in the rDNA tree, P. sonneborni strains are close to P. jenningsi, P. schewiakoffi, and P. sexaurelia strains. On the mitochondrial trees, the P. sonneborni strains from Europe and North America differ slightly (Figs 4, 5).
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Fig. 4. Phylogram constructed for P. sonneborni strains and for other species of the genus Paramecium (two strains of Tetrahymena were used as an outgroup), based on a comparison of sequences from COI mtDNA fragment using the Neighbor Joining method. Bootstrap values for Neighbor Joining, Maximum Parsimony analysis, Maximum Likelihood and posterior probabilities for Bayesian Inference are presented. Bootstrap values less than 50% (posterior probabilities less than 0.50) were not shown. All positions containing gaps and missing data were eliminated. There were 608 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 5.2 and Mr Bayes 3.1.2 (BI).
Analyses of the studied Paramecium sonneborni sequences Gene sequences encoding ITS1-5.8S-ITS2-5 LSU rDNA (1064 bp), cytochrome c oxidase subunit I (638 bp), and cytochrome b gene (618 bp) were obtained from eight strains of Paramecium sonneborni. For comparison of molecular distances and interspecific relationships, 28 other Paramecium strains (20 for CytB gene) and two Tetrahymena strains were used. All positions containing gaps and missing data were eliminated. A total of 991 positions in the final dataset of the rDNA fragment, 608 positions for COI, and 618 for CytB were used for tree reconstruction. There was no intraspecific haplotype diversity between the two studied strains of P. sonneborni in the case of ribosomal fragment comparison. In turn, haplotype diversity in mitochondrial fragments was Hd = 0.25 (SD = 0.18) for COI and for CytB. The present
analysis of P. sonneborni strains showed the existence of one haplotype for the rDNA fragment and two haplotypes for both mitochondrial sequences. The levels of nucleotide diversity were π = 0.00157 for COI and π = 0.00081 for CytB. The nucleotide frequencies were A = 0.305, T = 0.288, C = 0.174, and G = 0.233 for rDNA; A = 0.238, T = 0.343, C = 0.234, and G = 0.185 for COI; and A = 0.217, T = 0.321, C = 0.255, and G = 0.207 for CytB. In the studied fragments, we found 0/4/2 (rDNA/COI/CytB) variable positions among P. sonneborni strains (none of them was parsimony informative). Mega 5.2 identified the TN93 + G model for ITS1-5.8S-ITS2-5 LSU rDNA (G = 0.27), T92 + G for COI mtDNA (G = 0.43), and TN93 + G + I for CytB mtDNA (I = 0.45, G = 0.56) as the best nucleotide substitution models for maximum likelihood tree reconstruction. It is noteworthy that distance matrixes for all the studied DNA fragments reveal closer relationships between P. sonneborni, P. jenningsi, and P. schewiakoffi than
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Fig. 5. Phylogram constructed for P. sonneborni strains and for other species of the genus Paramecium (two strains of Tetrahymena were used as an outgroup) based on a comparison of sequences from CytB mtDNA fragment using the Neighbor Joining method. Bootstrap values for Neighbor Joining, Maximum Parsimony analysis, Maximum Likelihood and posterior probabilities for Bayesian Inference are presented. Bootstrap values less than 50% (posterior probabilities less than 0.50) were not shown. All positions containing gaps and missing data were eliminated. There were 618 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 5.2 and Mr Bayes 3.1.2 (BI).
between P. sonneborni and any other member of the P. aurelia species complex. Details of variation among all the studied Paramecium sonneborni strains and other Paramecium strains used in the present study are showed in Tables S5–S7 (supplementary material).
Discussion New data on the biogeography of Paramecium sonneborni To date, P. sonneborni was thought to be an extremely rare species of the P. aurelia species complex with only one strain found in Texas, USA (Aufderheide et al. 1983). It is worth emphasizing that the accuracy of identification of the P. sonneborni strain from Cyprus was checked by three approaches: genetic crosses, cytological slides, and three-locus analysis. Therefore, the finding of a new stand of P. sonneborni in
Europe (Cyprus) shows that the species, previously considered endemic, may have a wider range. This demonstrates the impact of under-sampling on the knowledge of the biogeography of microbial eukaryotes. This also gives yet another impulse for the discussion concerning their distribution (cosmopolitan – Finlay et al. 2006 versus endemic, or rather representing “the moderate endemicity model” – Foissner et al. 2008). Evidence in support of both hypotheses can be found in the Paramecium genus (Greczek-Stachura et al. 2012; Przybo´s and Surmacz 2010; Sonneborn 1975). Moreover, this stimulates the debate on how ciliate species, including species in the genus Paramecium, spread. They need some water for dispersal because they do not produce cysts (Beale and Preer 2008). As was mentioned above, under-sampling is one of the main problems in studies of free-living microorganisms, such as Paramecium. Some territories are still not sampled at all or sampled insufficiently (Fokin 2010/2011; Przybo´s and Fokin 2000; Przybo´s and Surmacz 2010) and future
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studies in those territories will probably lead to new data. It is worth noting that, despite the large distance, the two populations of P. sonneborni (Cyprus and USA) do not differ from each other (rDNA), or differ slightly (COI and CytB). The obtained results may be an argument for the global dispersal of P. sonneborni, as it was suggested by, for example, ITS1 rDNA analysis in cercomonad Protozoa (Bass et al. 2007), where identical sequences were obtained from widely separated sites. Similar results were obtained in ITS sequence comparison of the spirotrich ciliate Meseres corlissi originating from distant populations in Austria and Australia (Weisse et al. 2008). On the other hand, the fact that both P. sonneborni sampling sites are characterized by warm climate suggests that this species may be restricted to the warm zone, similarly to two other members of the P. aurelia species complex, i.e., P. quadecaurelia (Przybo´s et al. 2013a) and P. tredecaurelia (Przybo´s et al. 2013b). In connection to the fact that studies on the occurrence of the Paramecium species have been limited to the Palearctic and Nearctic (mainly USA) ecozones, more intense sampling of the tropics might reveal that P. sonneborni is a cosmopolitan species, found in the warm zone on different continents, similarly to, for example, P. quadecaurelia (Przybo´s et al. 2013a). Similarly as in the case of P. tredecaurelia (Przybo´s et al. 2013b), the obtained results may suggest that in the past the P. sonneborni population (predecessors of the present population) probably went through a bottleneck, and its current distribution is the result of a recent dispersal by natural or anthropogenic factors. Another explanation for the low level of genetic diversity despite the huge distance between the two collecting sites may be a slow rate of mutation of the studied DNA fragments revealed in some species of the P. aurelia complex (Catania et al. 2009). A comparatively low mutation rate, as recently shown for P. tetraurelia (Sung et al. 2012), and high selective pressures for a certain temperature zone may lead to reduced intraspecific genetic variation as well.
Low viability of inter-strain crosses of the two P. sonneborni strains In spite of a lack of (rDNA) or very low (mtDNA) genetic variation shown by the three DNA fragments (Tables S5–S7), and the absence of any disorder in nuclear apparatuses observed on cytological slides, the present results revealed a notably low viability (52%) of F1 inter-strain hybrids of the two P. sonneborni strains (Table 2). Similarly, low viability of the F2 generation was obtained in inter-strain crosses of P. tredecaurelia (Przybo´s et al. 2007, 2013b; Rafalko and Sonneborn 1959). Currently, it is difficult to explain the discordance between the low molecular variability of the strains (or its absence) and the low viability of inter-strain hybrids. Perhaps this might be due to the gradual emergence of a reproductive barrier between the studied
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strains. The influence of epigenetic factors (in cytoplasm) cannot be excluded. Future sampling may lead to collecting other strains of P. sonneborni, which would allow to test whether the low viability of the F1 generation observed in the present study is a characteristic feature only for crosses of the reference and Cyprus strains.
Should Paramecium sonneborni be a member of the P. aurelia species complex? Insights from molecular, genetic, and cytological studies The new taxonomy of the genus Paramecium proposed by Kreutz et al. (2012), based on the 18S rRNA gene sequence, divided the genus into five subgenera, including Fokin’s subgenera, i.e., Chloroparamecium, Helianter, Cypriostomum, and Paramecium, plus a new subgenus, Viridoparamecium, containing the redescribed species P. chlorelligerum, P. sonneborni appeared in this tree as a distinct branch in one clade with the P. aurelia spp. complex, P. jenningsi, and P. schewiakoffi. In turn, our phylograms (Figs 3–5) of the Paramecium subgenus showed a more pronounced divide between P. sonneborni and the remaining species of the P. aurelia complex. The discovery of a new P. sonneborni strain in Cyprus (Table S1) created the possibility to conduct for the first time intra-specific, molecular analysis of this species and compare its phylogenetic position with those of the other members of the P. aurelia species complex and other closely related morphospecies, i.e., P. jenningsi and P. schewiakoffi. Previous studies (based on the mitochondrial CytB gene), which used only the one known reference strain (ATTC 30995), revealed a closer relationship of P. sonneborni to P. jenningsi and P. schewiakoffi than to the P. aurelia species complex (Barth et al. 2008). An unclear relationship between P. jenningsi and the P. aurelia complex was also indicated by comparison of a fragment of the hsp70 gene (Hori et al. 2006). The most recent data analysis (Tarcz et al. 2013) based on two independent loci (ITS1-5.8S-ITS2-5 LSU rDNA and COI mtDNA) for over a hundred strains of the P. aurelia species complex showed P. sonneborni to be a distinct branch on both trees. The investigated strains of P. sonneborni are characterized by no (ribosomal) or slight (mitochondrial) variability of the studied DNA sequences. As can be seen from all the obtained trees (Figs 3–5), P. sonneborni forms a separate cluster (together with the closely related strains of P. jenningsi and P. schewiakoffi), which is distinct from the majority of P. aurelia species. Previous analyses revealed a paraphyletic relationship between the P. aurelia species complex and the closely related P. schewiakoffi and P. jenningsi (Tarcz et al. 2013). Although all of these species belong to the subgenus Paramecium (Fokin et al. 2004) and are true biological species (strain crosses did not reveal any possibility of mating between P. sonneborni and P. jenningsi - see Table S4), the phylogenetic relationships between them still remain unclear. All dendrograms (both current and previous) concerning the
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genus Paramecium show a monophyletic clade containing the P. aurelia species complex together with P. schewiakoffi, P. jenningsi, and P. sonneborni. They are related to each other more closely than to any other Paramecium morphospecies. Aury and colleagues (Aury et al. 2006) suggested that the recent whole genome duplication (WGD) event occurred after the divergence of P. tetraurelia from P. caudatum and P. multimicronucleatum and before the explosion of speciation events that gave rise to the P. aurelia complex together with P. schewiakoffi, P. jenningsi, and P. sonneborni. This may support the hypothesis that P. sonneborni, as well as P. jenningsi and P. schewiakoffi, might belong to the “aurelia” complex potentially sharing all whole genome duplication events. Currently, it is difficult to definitively exclude P. sonneborni from the P. aurelia species complex based on cytological studies. P. sonneborni strains have vesicular micronuclei (Fig 1a, b, d) similar to those of the species belonging to the P. aurelia complex. However, the structure of the macronuclear anlagen (Fig 2b, d) visible at the later stages of nuclear apparatus reorganization following conjugation or autogamy is different from that appearing in the P. aurelia complex and similar to P. jenningsi and P. schewiakoffi strains (Fokin et al. 2004). In our opinion, that problem requires more detailed molecular studies with the application of a greater number of independently inherited DNA sequences. Then, the results may allow to exclude P. sonneborni from the P. aurelia complex and simultaneously constitute a sister group (P. sonneborni–P. jenningsi–P. schewiakoffi) to the remaining fourteen species of the P. aurelia complex.
Acknowledgements This study was partly supported by a grant from the National Centre of Science, Cracow, Poland, No. 2012/05/B/NZ8/00387. It was also carried out in the context of the CNRS-supported European Research Group “Paramecium Genome Dynamics and Evolution” and the European COST Action BM 1102, as well as supported by grant RFBR 13-04-01714 to M.R. We thank Dr Alexey Potekhin (Department of Microbiology, Faculty of Biology and Soil Science, St. Petersburg State University, Russia) for strains of P. polycarium, P. nephridiatum, P. putrinum, from the Core Facilities Centre “Culture Collection of Microorganisms” of St. Petersburg State University used in the studies. Finally, we would like to thank two anonymous reviewers for their constructive comments.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ejop.2014.03.001.
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ScienceDirect European Journal of Protistology 50 (2014) 236–247
The first European stand of Paramecium sonneborni (P. aurelia complex), a species known only from North America (Texas, USA) Ewa Przybo´sa , Sebastian Tarcza,∗ , Maria Rautianb , Natalia Lebedevab a
Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków 31-016, Sławkowska 17, Poland Laboratory of Protistology and Experimental Zoology, Faculty of Biology and Soil Science, St. Petersburg State University, Saint Petersburg 199034, Universitetskaya nab. 7/9, Russia b
Received 25 November 2013; received in revised form 15 February 2014; accepted 3 March 2014 Available online 18 March 2014
Abstract P. aurelia is currently defined as a complex of 15 sibling species including 14 species designated by Sonneborn (1975) and one, P. sonneborni, by Aufderheide et al. (1983). The latter was known from only one stand (Texas, USA). The main reason for the present study was a new stand of Paramecium in Cyprus, with strains recognized as P. sonneborni based on the results of strain crosses, cytological slides, and molecular analyses of three loci (ITS1-5.8S-ITS2-5 LSU rDNA, COI, CytB). The new stand of P. sonneborni in Europe shows that the species, previously considered endemic, may have a wider range. This demonstrates the impact of under-sampling on the knowledge of the biogeography of microbial eukaryotes. Phylogenetic trees based on all the studied fragments revealed that P. sonneborni forms a separate cluster that is closer to P. jenningsi and P. schewiakoffi than to the other members of the P. aurelia complex. © 2014 Elsevier GmbH. All rights reserved.
Keywords: Ciliophora; Paramecium aurelia species complex; Paramecium sonneborni; Strain crosses; Three-locus analysis
Introduction The species structure of ciliates is complex as several genera contain morphological species consisting of sibling species (Caron 2013; Nanney and McCoy 1976; Przybo´s 1986; Strüder-Kypke and Lynn 2010). Paramecium (Oligohymenophorea, Ciliophora) is one of the best studied ciliate genera, with the P. aurelia species complex being thoroughly examined (Barth et al. 2008; Przybo´s et al. 2007; Schlegel and Meisterfeld 2003). ∗ Corresponding
author at: Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016 Kraków, Poland. Tel.: +48 12 4227006Ext.43; fax: +48 12 422 42 94. E-mail address: [email protected] (S. Tarcz). http://dx.doi.org/10.1016/j.ejop.2014.03.001 0932-4739/© 2014 Elsevier GmbH. All rights reserved.
The P. aurelia complex is currently thought to be composed of 14 species designated by Sonneborn (1975) and of P. sonneborni (Aufderheide et al. 1983), which was added to the complex at a later time (Wichterman 1986). However, according to Corliss and Daggett (1983) “it was never treated first as another numbered syngen”. The latter species was named in honor of Dr. Tracy M. Sonneborn of Indiana University because his scientific studies led to establishing Paramecium as a model organism in genetics, cytology, pharmacology, etc. The species of the complex are reproductively isolated from one another, each has two complementary mating types, and conjugation is possible only between them (Sonneborn 1937). According to Sonneborn (1975), the main criteria for P. aurelia species identification are a mating reaction (conjugation) between the standard strain of a given species and a
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new strain, and viable F1 and recombinant F2 clones. Moreover, isozyme patterns (Allen et al. 1973; Tait 1970) and several molecular methods have revealed the possibility of distinguishing between some species of the P. aurelia complex (Barth et al. 2008; Catania et al. 2009; Przybo´s et al. 2007; Tarcz et al. 2013). Paramecium sonneborni n. sp., a new member of the Paramecium aurelia species complex, was described by Aufderheide et al. (1983) based on nuclear and whole cell morphology (strain ATCC 30995). The species is reproductively isolated from other members of the complex and characterized by unique esterase zymograms (Aufderheide et al. 1983). It was collected from an intermittent stream in College Station, Texas, USA. Only one stand of this species was known until recently. According to Aufderheide et al. (1983), the body length of P. sonneborni (silver-impregnated specimens) ranges from 130 to 186 m, with a mean length of 154.4 m. It belongs to the largest species of the complex, being as large as P. undecaurelia (length of up to 170 m, Sonneborn 1975). Its two micronuclei (mi) are of vesicular type with a mean diameter of 4.9 m (measured in living cells). Conjugation and autogamy have been observed with typically two macronuclear anlagen after sexual processes, and two mating types of the caryonidal determination type. P. sonneborni is characterized by a rapid growth rate (four fissions daily at 27 ◦ C) and long clonal life-span (250 generations). The present study was motivated by the discovery of a new Paramecium stand in Cyprus (Table S1, supplementary material). At first, the paramecia were thought to be similar to P. jenningsi based on cell dimensions and micronucleus diameter (Przybo´s et al. 2003), but subsequent three-locus comparisons (ITS1-5.8S-ITS2-5 LSU rDNA, COI, CytB) suggested that they were new strains of P. sonneborni. The aim of the present study was to characterize these strains cytologically and genetically and assess their phylogenetic relationship to the P. aurelia spp. complex and other species of the genus Paramecium. Our investigations were carried out on three levels, i.e., cellular with the application of genetic methods (strain crosses), sub-cellular (cytological investigations of nuclear apparatuses), and molecular (the three markers mentioned above).
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migratory birds. The water in the creeks was slightly salty (1.4 ppm), but the clones easily adapted themselves to the standard culture conditions for paramecia. These strains from Cyprus belong to the Culture Collection of Ciliates and their Symbionts (CCCS) of St. Petersburg University and are deposited in the Core Facilities Centre “Culture Collections of Microorganisms” of St. Petersburg State University. The reference strain of P. sonneborni (Table S1, ATCC 30995) was obtained by the senior author from Dr. Karl Aufderheide, Texas A&M University, USA. Other strains used for molecular comparisons, representing particular species of the P. aurelia complex as well as other species of the genus Paramecium, were from the strain collection of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Poland (Table S2, supplementary material). Strains of P. polycarium, P. nephridiatum, P. putrinum used in the studies are from the Core Facilities Centre “Culture Collection of Microorganisms” of St. Petersburg State University (Table S2).
Strain cultivation Paramecia were cultured in a medium made of dried lettuce and distilled water inoculated with Enterobacter aerogenes according to the method of Sonneborn (1970) and supplemented with 0.8 mg/L  sitosterol (Merck, Darmstadt, Germany).
Methods used in cytological studies The nuclear apparatuses of individuals in autogamy as well as individuals in the vegetative stage were examined. Slides were temporarily stained with acetocarmine (Sonneborn 1950), or permanent slides were made, fixed with Schaudinn’s fluid with glacial acetic acid (Chen 1944) and stained with Giemsa stain (Merck, Darmstadt, Germany) (10% solution in 0.01 M phosphate buffer) (cf. Przybo´s 1978). Photographs were taken using a Nikon Eclipse E400 microscope equipped with a Nikon DS-Fi2, digital camera. Some photographs were taken with Leika DM2500 microscope (optical magnification 1250) using differential interference contrast (DIC).
Material and Methods Material
Methods of strain measurements
The Cyprus strains were established by cloning individuals from three water samples (CyL 2, CyL 3, CyL 5) collected in 2010 by Natalia Lebedeva. According to the samples, the clones were designated CyL 2-5, CyL 2-6, CyL 2-10, CyL 31, CyL 3-8, CyL 5-6, and CyL 5-8 (Table S1). The collecting sites were small creeks situated on both sides of a road in a suburb of Larnaca (34.9N/33.6E) and running to the Larnaca Salt Lake (about 100 m away), which is a wintering site for
Young cells of clones representing strains designated CyL 2, CyL 3, CyL 5, from different creeks, at a similar clonal age and at the vegetative stage of life, cultivated in excess of food at 27 ◦ C, were fixed and stained with Giemsa stain. The paramecia were measured (cell and macronuclear length and width, micronuclear diameter) using a Nikon Eclipse E400 microscope and Coolview software (Precoptic Co, Poland).
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Strain crosses Sonneborn’s (1970) methods were used to induce conjugation, autogamy and strain crosses. In intra- and interstrain 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 in preparations stained with acetocarmine. Clone survival in both generations was estimated as percentages. According to Chen (1956), clones can be considered surviving after passing 6–7 fissions during 72 h following the separation of conjugation partners or postautogamous caryonids. Inter-strain cross was done between P. sonneborni reference strain and the Cyprus strain (CyL 2-6) representing other clones from that stand. The percentage of surviving clones in interstrain cross and the duration of particular generations of interstrain hybrids were compared. The methods were described in detail in Przybo´s (1975).
Molecular methods Paramecium genomic DNA was isolated from vegetative cells at the end of the exponential phase (approx. 1000 cells were used for DNA extraction) using a NucleoSpin Tissue Kit (Macherey-Nagel, Germany), according to the manufacturer’s instructions for DNA isolation from cell cultures. The only modification was centrifugation of the cell culture for 20 min at 13,200 rpm. Then the supernatant was removed and the remaining cells were suspended in lysis buffers and proteinase K. Fragments of rDNA, COI, and CytB genes were sequenced and analyzed. First, rDNA fragments were amplified with ITS1 and ITS4 universal eukaryotic primers (White et al. 1990) and ITS3zg and 3pLSU primers developed with OligoAnalyzer 3.1 (http://www.eu.idtdna. com/analyzer/applications/oligoanalyzer) (Table S3, supplementary material). F388dT and R1184dT primers (Table S3) and the protocol previously described by StrüderKypke and Lynn (2010) were used for the amplification of the COI fragment of mitochondrial DNA. In some cases, when the above COI pair of primers did not yield a welldefined product, the internal primer CoxH10176 (Barth et al. 2006) was used instead of R1184dT. To amplify the CytB gene fragment, the primer pair CytBF/PaCytR and the protocol previously described by Barth et al. (2008) were used. PCR amplification for all analyzed DNA fragments was carried out in a final volume of 40 L containing 30 ng of DNA, 1.5 U Taq-Polymerase (EURx, Poland), 16 pmol of each primer, 10 × PCR buffer, and 8 pmol of dNTPs. In order to assess the quality of the amplification, PCR products were electrophoresed in 1% agarose gel for 45 min at 85 V with a DNA molecular weight marker (Mass Ruler Low
Range DNA Ladder, Fermentas, Lithuania). NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Germany) was used for purifying PCR products. In some PCR products, additional sub-bands were obtained apart from the main band. In these cases, 30 L of each PCR product was separated on 1.8% agarose gel (100 V/60 min) with a DNA molecular weight marker (Mass Ruler Low Range DNA Ladder, Fermentas, Lithuania). Then the band representing the examined fragment was cut out and purified. Sequencing was done in both directions with the application of BigDye Terminator v3.1 chemistry (Applied Biosystems, USA). The primers used in PCR reactions were applied for sequencing the rDNA region or a part of CytB gene, and the primer pair M13F/M13R was used for sequencing the COI fragment (Table S3). The sequencing reaction was carried out in a final volume of 10 L containing 3 L of template, 1 L of BigDye (1/4 of the standard reaction), 1 L of sequencing buffer, and 1 L of 5 M primer. Sequencing products were precipitated using Ex Terminator (A&A Biotechnology, Poland) and separated on an ABI PRISM 377 DNA Sequencer (Applied Biosystems, USA). The sequences are available in the NCBI GenBank database (see Tables S1 and S2, supplementary material).
Data analyses Sequences were examined using Chromas Lite (Technelysium, Australia) to evaluate and correct chromatograms. Alignments of the studied sequences were performed using ClustalW (Thompson et al. 1994) as part of BioEdit software (Hall 1999) and checked manually. All of the obtained sequences were unambiguous and were used for analyses. Phylograms were constructed for the studied fragments by means of Mega v5.2 (Tamura et al. 2011), using neighborjoining (NJ) (Saitou and Nei 1987), maximum parsimony (MP) (Nei and Kumar 2000), and maximum likelihood (ML) (Felsenstein 1981). All positions containing gaps and missing data were eliminated. NJ analysis was performed using Mega v5.2 program by bootstrapping with 1000 replicates (Felsenstein 1985). MP analysis was evaluated with the min-min heuristic parameter (at level 2) and bootstrapping with 1000 replicates. Bayesian inference (BI) was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003); analysis was run for 5,000,000 generations and trees were sampled every 100 generations. All trees for BI analysis were visualized with TreeView 1.6.6 (Page 1996). Analysis of haplotype diversity (Hd) and its sampling variance (SD), nucleotide diversity () and polymorphic sites (Nei 1987) was done with DnaSP v5.10.01 (Librado and Rozas 2009). Analysis of nucleotide frequencies, p-distance estimation and identification of substitution models for ML analysis were done using Mega v5.2 (Tamura et al. 2004, 2011).
0.62 0.67 0.49 0.42 2.5 3.32 2.98 2.95 3.08 2.77 5.41 6.17 3.03 17.51 25.24 30.95 15.54 23.91 5.26 11.45 6.25 9.16 51.51 55.28 61.50 49.67 55.48 7.54 10.83 10.63 7.04 52.27 73.40 70.39 52.12 65.3 17.86 23.29 14.07 16.91 ATTC 30995 CyL 2-6 CyL 3-1 CyL 5-6 Mean for Cyprus strains 1. 2. 3. 4. 5.
185.91 220.00 198.89 163.18 194.02
SD Average diameter of MIC [m] SD Average width of MAC [m] SD Average length of MAC [m] SD Average cell width [m] SD Average length of cells [m]
Vegetative individuals belonging to the Cyprus strains (CyL 2-6, CyL 3-1, and CyL 5-6 were studied) are characterized by two atypical vesicular micronuclei (Fig 1b). The micronuclei of P. sonneborni look atypical in comparison with the other vesicular morphotypes of the P. aurelia spp. as they are larger and have an internal structure (Fig. 1b, d). They also appear away from each other and aside from the macronucleus in the cell (Fig 2a, c). According to Fokin (1997), vesicular micronuclei of the P. aurelia spp. “stained by the Feulgen reaction have a chromatin mass as a tiny ring with a clear center . . . surrounded by karyolymph” (cf. Figs 14 and 24 in his paper). In turn, P. jenningsi reveals a chromosomal type of micronuclei with a reticular structure – both mi are compared in Fig 27, Fokin (1997). It is broadly accepted that in all species of the P. aurelia complex, including P. sonneborni, micronuclei are of the vesicular type (Vivier, 1974; Aufderheide et al., 1983; Fokin, 1997; Fokin, 2010/2011). Two new macronuclear anlagen in the later stages of reorganization of nuclear apparatuses during autogamy and conjugation have darker chromatin centers (Fig 2b, d), indicating chromatin accumulation. The Cyprus strains are characterized by a pointed posterior end, similarly as the P. sonneborni reference strain (Aufderheide et al. 1983) (Fig 2a, b, c, d). The dimensions (cells, macronuclei, micronuclei) of the studied P. sonneborni strains (cells fixed and stained with Giemsa stain) are presented in Table 1. The cell size of P. sonneborni strains is comparable to the dimensions of the largest strains of P. jenningsi (cf. Przybo´s et al. 2003), i.e., the strain from China, designated as CS (cell length 191 m), and the strain from Saudi Arabia, designated as SA (cell length 186 m). However, the diameter of P. sonneborni micronuclei is smaller (2.5–3.32 m) than that observed in P. jenningsi strains (e.g., 4.9 m in the strain from India, designated as IB). Autogamy, resulting in homozygosity for all genes, appeared in all cultures of Cyprus and Texas P. sonneborni strains starved for 14 to 20 fissions in daily isolated lines cultivated at 27 ◦ C in a medium enabling 3 fissions per day (clonal age). Conjugation in mature clones in all the studied Cyprus strains was obtained on the 7th day of culture at 27 ◦ C, at a rate of three fissions daily, similarly as in the reference strain of P. sonneborni. Intrastrain crosses were possible between all Cyprus strains, and the percentage of surviving hybrid clones was high in both generations. Caryonidal mating type inheritance was found to be characteristic of all of these strains.
Strain
Cytological characteristics of the Cyprus strains and their biology
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No.
Results
Table 1. Paramecium sonneborni strain dimensions (Giemsa stain). For better orientation, dimensions of P. sonneborni reference strain (ATTC 30995) and mean dimension for the Cyprus strains studied are highlighted in gray.
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Fig. 1. General morphology of vegetative individuals representing P. sonneborni. DIC microscope. Paramecium sonneborni, strain from Cyprus (clone CyL 2-6) – (a) two vesicular micronuclei (mi), two contractile vacuoles (cv) with 8 channels, (b) structure of vesicular micronuclei (mi). Paramecium sonneborni, reference strain (ATTC 30995) – (c) contractile vacuole (cv) with channels, (d) structure of vesicular micronuclei (mi).
Results of strain crosses and survival after conjugation Both interstrain and intrastrain crosses were carried out for P. sonneborni. The strain CyL 2-6 was used as representative for all other Cyprus strains in crosses with the P. sonneborni reference strain, as all Cyprus strains were shown to be identical by molecular and genetic studies. The strain CyL 2-6 was conjugated with the reference strain of P. sonneborni (ATTC 30995), which led to 52% of hybrid clones surviving in F1, and 80–90% in F2 (the latter were obtained by autogamy from F1 hybrids) (Table 2). Crosses were repeated five times. The Cyprus strains were all recognized as belonging to P. sonneborni based on the results of crosses (supported by molecular analyses). Neither the P. sonneborni reference strain nor the Cyprus strains conjugated with any P. jenningsi strains representing three syngens (Przybo´s and Tarcz 2013) (Table S4, supplementary material). The crosses were carried out because P. jenningsi and P. sonneborni look similar in terms of morphological features, and therefore in the beginning the P. sonneborni strain from Cyprus was recognized as P. jenningsi.
Phylogenetic analysis of P. sonneborni strains based on comparison of ribosomal and mitochondrial DNA fragments The phylogenetic trees (NJ, MP, ML, BI) constructed based on analysis of the sequenced markers (ITS1-5.8SITS2-5 LSU rDNA, COI, CytB) show the mutual relationship of the two studied P. sonneborni strains from both the USA (ATTC 30995) and Cyprus as well as their position in respect of the P. aurelia spp. complex and the other species of the subgenus Paramecium (with Tetrahymena used as an outgroup) (Figs 3–5). As was mentioned above, due to identical DNA sequences, the strain CyL 2–6 was used as representative for all other Cyprus strains. On the ribosomal tree (Fig 3), most of the studied species are grouped into a compact, monophyletic cluster exhibiting little variability and consisting of representatives of the P. aurelia species complex, P. sonneborni, P. jenningsi, and P. schewiakoffi divided into two subgroups. The first contains the well-separated P. sonneborni clade (the Cyprus strain and the reference strain are molecularly identical). The next two branches are P. schewiakoffi (Sh1-38) and P. jenningsi (CS). Finally, a clade containing two strains of P. jenningsi (IB,
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Fig. 2. Paramecium sonneborni, vegetative and autogamous individuals. Giemsa stain. Paramecium sonneborni, strain from Cyprus (clone CyL 2-6) – (a) vegetative individual, macronucleus (Ma), two micronuclei (mi), (b) autogamous individual, fragmented old macronucleus (OMa), two macronuclear anlagen with chromatin accumulation (A). Paramecium sonneborni, reference strain (ATTC 30995) – (c) young vegetative individual, macronucleus (Ma), two micronuclei (mi), (d) autogamous individual, fragmented old macronucleus (OMa), two macronuclear anlagen with accumulated chromatin (A).
M) and the P. sexaurelia strain (159) from the P. aurelia spp. complex can be seen. In turn, the second subgroup consists of the other representatives of the P. aurelia species complex. Close to the group described above are two branches formed
by P. caudatum (CA) and P. multimicronucleatum (BR). They all form a monophyletic clade of the subgenus Paramecium. The phylogenetic tree constructed on the basis of COI mtDNA comparison (Fig 4) shows the best resolution among
Table 2. Results of Paramecium sonneborni strain crosses, percentage of surviving clones of inter-strain hybrids. No.
Strain designation
Strain origin
1.
P. sonneborni CyL 2-6 × P. sonneborni ATCC 30995 P. sonneborni CyL 2-6 × P. sonneborni CyL 2-6 P. sonneborni ATCC 30995 × P. sonneborni ATCC 30995
Cyprus × USA, Texas
2. 3.
F1 obtained by conjugation
F2 obtained by autogamy
52
90
Cyprus × Cyprus
100
100
USA, Texas × USA, Texas USA
100
98
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Fig. 3. Phylogram constructed for P. sonneborni strains and for other species of the genus Paramecium (two strains of Tetrahymena were used as an outgroup), based on a comparison of sequences from ITS1-5.8S-ITS2-5 LSU rDNA fragment using the Neighbor Joining method. Bootstrap values for Neighbor Joining, Maximum Parsimony analysis, Maximum Likelihood and posterior probabilities for Bayesian Inference are presented. Bootstrap values less than 50% (posterior probabilities less than 0.50) were not shown. All positions containing gaps and missing data were eliminated. There were 1003 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 5.2 (NJ/MP/ML) and Mr Bayes 3.1.2 (BI).
all studied DNA fragments. As in the case of the ribosomal tree, the most distinct is the group comprising the P. aurelia spp. complex, P. sonneborni, P. jenningsi, and P. schewiakoffi. It is worth noting that P. sonneborni forms a clade that is distant from all P. aurelia species, while close to P. schewiakoffi (Sh1-38) and the sister group formed by three strains of P. jenningsi (CS, IB, M), representing three different syngens. However, in the case of the CytB mtDNA tree (Fig 5), P. sonneborni strains appear in one clade with the P. jenningsi
strain from China (CS) and next to the P. schewiakoffi (Sh1-38) branch. The two other P. jenningsi strains (from Madagascar – M and India – IB), representing different syngens from the Chinese one, are separated from P. sonneborni by two species of the P. aurelia complex (P. sexaurelia and P. dodecaurelia). Similarly, as in the rDNA tree, P. sonneborni strains are close to P. jenningsi, P. schewiakoffi, and P. sexaurelia strains. On the mitochondrial trees, the P. sonneborni strains from Europe and North America differ slightly (Figs 4, 5).
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Fig. 4. Phylogram constructed for P. sonneborni strains and for other species of the genus Paramecium (two strains of Tetrahymena were used as an outgroup), based on a comparison of sequences from COI mtDNA fragment using the Neighbor Joining method. Bootstrap values for Neighbor Joining, Maximum Parsimony analysis, Maximum Likelihood and posterior probabilities for Bayesian Inference are presented. Bootstrap values less than 50% (posterior probabilities less than 0.50) were not shown. All positions containing gaps and missing data were eliminated. There were 608 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 5.2 and Mr Bayes 3.1.2 (BI).
Analyses of the studied Paramecium sonneborni sequences Gene sequences encoding ITS1-5.8S-ITS2-5 LSU rDNA (1064 bp), cytochrome c oxidase subunit I (638 bp), and cytochrome b gene (618 bp) were obtained from eight strains of Paramecium sonneborni. For comparison of molecular distances and interspecific relationships, 28 other Paramecium strains (20 for CytB gene) and two Tetrahymena strains were used. All positions containing gaps and missing data were eliminated. A total of 991 positions in the final dataset of the rDNA fragment, 608 positions for COI, and 618 for CytB were used for tree reconstruction. There was no intraspecific haplotype diversity between the two studied strains of P. sonneborni in the case of ribosomal fragment comparison. In turn, haplotype diversity in mitochondrial fragments was Hd = 0.25 (SD = 0.18) for COI and for CytB. The present
analysis of P. sonneborni strains showed the existence of one haplotype for the rDNA fragment and two haplotypes for both mitochondrial sequences. The levels of nucleotide diversity were π = 0.00157 for COI and π = 0.00081 for CytB. The nucleotide frequencies were A = 0.305, T = 0.288, C = 0.174, and G = 0.233 for rDNA; A = 0.238, T = 0.343, C = 0.234, and G = 0.185 for COI; and A = 0.217, T = 0.321, C = 0.255, and G = 0.207 for CytB. In the studied fragments, we found 0/4/2 (rDNA/COI/CytB) variable positions among P. sonneborni strains (none of them was parsimony informative). Mega 5.2 identified the TN93 + G model for ITS1-5.8S-ITS2-5 LSU rDNA (G = 0.27), T92 + G for COI mtDNA (G = 0.43), and TN93 + G + I for CytB mtDNA (I = 0.45, G = 0.56) as the best nucleotide substitution models for maximum likelihood tree reconstruction. It is noteworthy that distance matrixes for all the studied DNA fragments reveal closer relationships between P. sonneborni, P. jenningsi, and P. schewiakoffi than
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Fig. 5. Phylogram constructed for P. sonneborni strains and for other species of the genus Paramecium (two strains of Tetrahymena were used as an outgroup) based on a comparison of sequences from CytB mtDNA fragment using the Neighbor Joining method. Bootstrap values for Neighbor Joining, Maximum Parsimony analysis, Maximum Likelihood and posterior probabilities for Bayesian Inference are presented. Bootstrap values less than 50% (posterior probabilities less than 0.50) were not shown. All positions containing gaps and missing data were eliminated. There were 618 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 5.2 and Mr Bayes 3.1.2 (BI).
between P. sonneborni and any other member of the P. aurelia species complex. Details of variation among all the studied Paramecium sonneborni strains and other Paramecium strains used in the present study are showed in Tables S5–S7 (supplementary material).
Discussion New data on the biogeography of Paramecium sonneborni To date, P. sonneborni was thought to be an extremely rare species of the P. aurelia species complex with only one strain found in Texas, USA (Aufderheide et al. 1983). It is worth emphasizing that the accuracy of identification of the P. sonneborni strain from Cyprus was checked by three approaches: genetic crosses, cytological slides, and three-locus analysis. Therefore, the finding of a new stand of P. sonneborni in
Europe (Cyprus) shows that the species, previously considered endemic, may have a wider range. This demonstrates the impact of under-sampling on the knowledge of the biogeography of microbial eukaryotes. This also gives yet another impulse for the discussion concerning their distribution (cosmopolitan – Finlay et al. 2006 versus endemic, or rather representing “the moderate endemicity model” – Foissner et al. 2008). Evidence in support of both hypotheses can be found in the Paramecium genus (Greczek-Stachura et al. 2012; Przybo´s and Surmacz 2010; Sonneborn 1975). Moreover, this stimulates the debate on how ciliate species, including species in the genus Paramecium, spread. They need some water for dispersal because they do not produce cysts (Beale and Preer 2008). As was mentioned above, under-sampling is one of the main problems in studies of free-living microorganisms, such as Paramecium. Some territories are still not sampled at all or sampled insufficiently (Fokin 2010/2011; Przybo´s and Fokin 2000; Przybo´s and Surmacz 2010) and future
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studies in those territories will probably lead to new data. It is worth noting that, despite the large distance, the two populations of P. sonneborni (Cyprus and USA) do not differ from each other (rDNA), or differ slightly (COI and CytB). The obtained results may be an argument for the global dispersal of P. sonneborni, as it was suggested by, for example, ITS1 rDNA analysis in cercomonad Protozoa (Bass et al. 2007), where identical sequences were obtained from widely separated sites. Similar results were obtained in ITS sequence comparison of the spirotrich ciliate Meseres corlissi originating from distant populations in Austria and Australia (Weisse et al. 2008). On the other hand, the fact that both P. sonneborni sampling sites are characterized by warm climate suggests that this species may be restricted to the warm zone, similarly to two other members of the P. aurelia species complex, i.e., P. quadecaurelia (Przybo´s et al. 2013a) and P. tredecaurelia (Przybo´s et al. 2013b). In connection to the fact that studies on the occurrence of the Paramecium species have been limited to the Palearctic and Nearctic (mainly USA) ecozones, more intense sampling of the tropics might reveal that P. sonneborni is a cosmopolitan species, found in the warm zone on different continents, similarly to, for example, P. quadecaurelia (Przybo´s et al. 2013a). Similarly as in the case of P. tredecaurelia (Przybo´s et al. 2013b), the obtained results may suggest that in the past the P. sonneborni population (predecessors of the present population) probably went through a bottleneck, and its current distribution is the result of a recent dispersal by natural or anthropogenic factors. Another explanation for the low level of genetic diversity despite the huge distance between the two collecting sites may be a slow rate of mutation of the studied DNA fragments revealed in some species of the P. aurelia complex (Catania et al. 2009). A comparatively low mutation rate, as recently shown for P. tetraurelia (Sung et al. 2012), and high selective pressures for a certain temperature zone may lead to reduced intraspecific genetic variation as well.
Low viability of inter-strain crosses of the two P. sonneborni strains In spite of a lack of (rDNA) or very low (mtDNA) genetic variation shown by the three DNA fragments (Tables S5–S7), and the absence of any disorder in nuclear apparatuses observed on cytological slides, the present results revealed a notably low viability (52%) of F1 inter-strain hybrids of the two P. sonneborni strains (Table 2). Similarly, low viability of the F2 generation was obtained in inter-strain crosses of P. tredecaurelia (Przybo´s et al. 2007, 2013b; Rafalko and Sonneborn 1959). Currently, it is difficult to explain the discordance between the low molecular variability of the strains (or its absence) and the low viability of inter-strain hybrids. Perhaps this might be due to the gradual emergence of a reproductive barrier between the studied
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strains. The influence of epigenetic factors (in cytoplasm) cannot be excluded. Future sampling may lead to collecting other strains of P. sonneborni, which would allow to test whether the low viability of the F1 generation observed in the present study is a characteristic feature only for crosses of the reference and Cyprus strains.
Should Paramecium sonneborni be a member of the P. aurelia species complex? Insights from molecular, genetic, and cytological studies The new taxonomy of the genus Paramecium proposed by Kreutz et al. (2012), based on the 18S rRNA gene sequence, divided the genus into five subgenera, including Fokin’s subgenera, i.e., Chloroparamecium, Helianter, Cypriostomum, and Paramecium, plus a new subgenus, Viridoparamecium, containing the redescribed species P. chlorelligerum, P. sonneborni appeared in this tree as a distinct branch in one clade with the P. aurelia spp. complex, P. jenningsi, and P. schewiakoffi. In turn, our phylograms (Figs 3–5) of the Paramecium subgenus showed a more pronounced divide between P. sonneborni and the remaining species of the P. aurelia complex. The discovery of a new P. sonneborni strain in Cyprus (Table S1) created the possibility to conduct for the first time intra-specific, molecular analysis of this species and compare its phylogenetic position with those of the other members of the P. aurelia species complex and other closely related morphospecies, i.e., P. jenningsi and P. schewiakoffi. Previous studies (based on the mitochondrial CytB gene), which used only the one known reference strain (ATTC 30995), revealed a closer relationship of P. sonneborni to P. jenningsi and P. schewiakoffi than to the P. aurelia species complex (Barth et al. 2008). An unclear relationship between P. jenningsi and the P. aurelia complex was also indicated by comparison of a fragment of the hsp70 gene (Hori et al. 2006). The most recent data analysis (Tarcz et al. 2013) based on two independent loci (ITS1-5.8S-ITS2-5 LSU rDNA and COI mtDNA) for over a hundred strains of the P. aurelia species complex showed P. sonneborni to be a distinct branch on both trees. The investigated strains of P. sonneborni are characterized by no (ribosomal) or slight (mitochondrial) variability of the studied DNA sequences. As can be seen from all the obtained trees (Figs 3–5), P. sonneborni forms a separate cluster (together with the closely related strains of P. jenningsi and P. schewiakoffi), which is distinct from the majority of P. aurelia species. Previous analyses revealed a paraphyletic relationship between the P. aurelia species complex and the closely related P. schewiakoffi and P. jenningsi (Tarcz et al. 2013). Although all of these species belong to the subgenus Paramecium (Fokin et al. 2004) and are true biological species (strain crosses did not reveal any possibility of mating between P. sonneborni and P. jenningsi - see Table S4), the phylogenetic relationships between them still remain unclear. All dendrograms (both current and previous) concerning the
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genus Paramecium show a monophyletic clade containing the P. aurelia species complex together with P. schewiakoffi, P. jenningsi, and P. sonneborni. They are related to each other more closely than to any other Paramecium morphospecies. Aury and colleagues (Aury et al. 2006) suggested that the recent whole genome duplication (WGD) event occurred after the divergence of P. tetraurelia from P. caudatum and P. multimicronucleatum and before the explosion of speciation events that gave rise to the P. aurelia complex together with P. schewiakoffi, P. jenningsi, and P. sonneborni. This may support the hypothesis that P. sonneborni, as well as P. jenningsi and P. schewiakoffi, might belong to the “aurelia” complex potentially sharing all whole genome duplication events. Currently, it is difficult to definitively exclude P. sonneborni from the P. aurelia species complex based on cytological studies. P. sonneborni strains have vesicular micronuclei (Fig 1a, b, d) similar to those of the species belonging to the P. aurelia complex. However, the structure of the macronuclear anlagen (Fig 2b, d) visible at the later stages of nuclear apparatus reorganization following conjugation or autogamy is different from that appearing in the P. aurelia complex and similar to P. jenningsi and P. schewiakoffi strains (Fokin et al. 2004). In our opinion, that problem requires more detailed molecular studies with the application of a greater number of independently inherited DNA sequences. Then, the results may allow to exclude P. sonneborni from the P. aurelia complex and simultaneously constitute a sister group (P. sonneborni–P. jenningsi–P. schewiakoffi) to the remaining fourteen species of the P. aurelia complex.
Acknowledgements This study was partly supported by a grant from the National Centre of Science, Cracow, Poland, No. 2012/05/B/NZ8/00387. It was also carried out in the context of the CNRS-supported European Research Group “Paramecium Genome Dynamics and Evolution” and the European COST Action BM 1102, as well as supported by grant RFBR 13-04-01714 to M.R. We thank Dr Alexey Potekhin (Department of Microbiology, Faculty of Biology and Soil Science, St. Petersburg State University, Russia) for strains of P. polycarium, P. nephridiatum, P. putrinum, from the Core Facilities Centre “Culture Collection of Microorganisms” of St. Petersburg State University used in the studies. Finally, we would like to thank two anonymous reviewers for their constructive comments.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ejop.2014.03.001.
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