European Journal of Protistology 47 (2011) 138–143
Short communication
Polymorphism of Paramecium pentaurelia (Ciliophora, Oligohymenophorea) strains revealed by rDNA and mtDNA sequences Ewa Przybo´sa , Sebastian Tarcza,∗ , Magdalena Greczek-Stachurab , Marta Surmacza a
Department of Experimental Zoology, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016 Kraków, Poland b Institute of Biology, Pedagogical University, Podbrzezie 3, 31-054 Kraków, Poland Received 26 July 2010; received in revised form 3 November 2010; accepted 19 November 2010 Available online 1 February 2011
Abstract Paramecium pentaurelia is one of 15 known sibling species of the Paramecium aurelia complex. It is recognized as a species showing no intra-specific differentiation on the basis of molecular fingerprint analyses, whereas the majority of other species are polymorphic. This study aimed at assessing genetic polymorphism within P. pentaurelia including new strains recently found in Poland (originating from two water bodies, different years, seasons, and clones of one strain) as well as strains collected from distant habitats (USA, Europe, Asia), and strains representing other species of the complex. We compared two DNA fragments: partial sequences (349 bp) of the LSU rDNA and partial sequences (618 bp) of cytochrome B gene. A correlation between the geographical origin of the strains and the genetic characteristics of their genotypes was not observed. Different genotypes were found in Kraków in two types of water bodies (Opatkowice—natural pond; Jordan’s Park—artificial pond). Haplotype diversity within a single water body was not recorded. Likewise, seasonal haplotype differences between the strains within the artificial water body, as well as differences between clones originating from one strain, were not detected. The clustering of some strains belonging to different species was observed in the phylogenies. © 2010 Elsevier GmbH. All rights reserved. Keywords: Paramecium pentaurelia; Paramecium aurelia species complex; LSU rDNA; cytB mtDNA; Intra-specific diversity
Introduction The Paramecium aurelia complex is composed of 15 species (Sonneborn 1975; Aufderheide et al. 1983) known world-wide. Some of them are existing all over the world, such as P. primaurelia, P. biaurelia, P. tetraurelia, P. sexaurelia, others seem limited to geographic regions or temperature zones (P. octaurelia, P decaurelia, P. undecaurelia, P. tredecaurelia, P. quadecaurelia, P. sonneborni) (Sonneborn 1975; Aufderheide et al. 1983; Przybo´s and Fokin 2003; Przybo´s et al. 2003, 2005, 2007b,c, 2009b). However, several species ∗ Corresponding
author. E-mail address:
[email protected] (S. Tarcz).
0932-4739/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2010.11.001
that were earlier supposed to have a restricted distribution have recently been shown to be more widespread, e.g., P. novaurelia, which had been known before only from Europe but was found later in the Asian part of Turkey (Przybo´s 1998) and the USA (Przybo´s et al. 2007c). Thus, sampling larger areas and different habitats may bring new data on the distribution of the P. aurelia species. This was also the case in P. pentaurelia. This species was recorded in North America, Australia and Europe (warm zone) (Przybo´s and Surmacz 2010). For the first time it was found in Poland (Przybo´s et al. 2008a) in material collected in 2005 (Kraków, a pond in Jordan’s Park). Later, in 2008, it was recorded in the same pond, and also in another pond in Opatkowice/Kraków (Przybo´s et al. 2011). The finding
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of new stands of this species in Poland was unexpected as already 218 habitats out of 483 throughout Europe had been studied in this country since 1959 (Przybo´s et al. 2011). The new strains of P. pentaurelia from Poland originated both from different water bodies situated in the same town and were collected in different years and seasons or from different sampling points of the same pond. These samples were assessed for possible molecular differentiation of P. pentaurelia. Previously, PCR-based fingerprint analyses (RAPD, random amplified polymorphic DNA; ARDRA, amplified ribosomal DNA restriction analysis; RFLP, restriction fragment polymorphism) showed no intra-specific differentiation of known strains of P. pentaurelia in comparison with other species of the P. aurelia complex (Przybo´s et al. 2005, 2007/2008, 2007a; Stoeck et al. 1998, 2000). In contrast, exceptional polymorphism was found in P. tetraurelia, P. octaurelia, and P. dodecaurelia, also confirmed later by sequencing rDNA and COI mtDNA gene fragments (Przybo´s et al. 2008b,c, 2009a,b, 2010; Tarcz et al. 2006). The present study was undertaken to assess the polymorphism within P. pentaurelia, including new strains found in Poland. The possible intra-specific variability of P. pentaurelia was investigated by analysis of gene fragments of LSU rDNA (349 bp) and cytBmtDNA (618 bp). The small LSU rDNA fragment was chosen because it is the most variable rDNA fragment in the Paramecium aurelia species complex (Tarcz PhD thesis 2007, unpublished), and also it corresponds to the variable domain regions D1–D5 (Wylezich et al. 2010).
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1975), all showing intra-specific polymorphism. P. multimicronucleatum was used as an outgroup.
Molecular methods Fragments of 5 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 4 (supplementary materials). PCR amplification was carried out in a final volume of 40 l containing: 4 l of DNA, 1.5 U Taq-Polymerase (Qiagen, Germany), 0.6 l 10 mM of each primer, 10× PCR buffer, 0.6 l of 10 mM dNTPs in a T-personal thermocyclerTM (Biometra GmbH, Germany). The protocol for amplification of the 5 LSU rDNA was previously described in Tarcz et al. (2006). The cytB fragment was amplified as previously described in Barth et al. (2008b). For purification, NucleoSpin Extract II (Macherey-Nagel, Germany) was used. Cycle sequencing was done in both directions by application of the BigDye Terminator v3.1 chemistry (Applied Biosystems, USA). The primers used in PCR reactions were applied for sequencing. 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 mM primer. Sequencing products were precipitated using Ex Terminator (A&A Biotechnology, Poland) and separated on a ABI PRISM 377 DNA Sequencer (Applied Biosystems, USA). Sequences are available from the NCBI GenBank database (Tables 1 and 3).
Data analysis
Materials and Methods Procedures of collecting paramecia are described in details in supplementary materials.
Culturing and identification of strains Paramecia were cultured on a lettuce medium inoculated with Enterobacter aerogenes and identified according to the methods of Sonneborn (1970) by mating tests with the standard strains of the particular species. The strains used in the present study are listed in Tables 1–3 (supplementary materials). The strains include P. pentaurelia originating from Poland, Kraków from different ponds—an artificial pond (different years and seasons of collection or different clones from one sampling point) and a natural pond (different sampling points), other European countries (Italy, Spain, Russia), Asia (Russia, Siberia), and the USA (Tables 1–3). Other strains represent different species of the P. aurelia complex belonging to different groups of mating type inheritance, karyonidal (P. primaurelia, P. novaurelia), clonal (P. tetraurelia, P. dodecaurelia), and synclonal (P. tredecaurelia) (Sonneborn
Sequences were visually examined 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. Phylograms were constructed for the studied fragments in Mega version 4.1 (Tamura et al. 2007), using the Neighbor-Joining method (NJ) (Saitou and Nei 1987) and Maximum Parsimony (MP) (Nei and Kumar 2000). The NJ analysis was performed using a Kimura 2-parameter correction model (Kimura 1980) by bootstrapping with 1000 replicates (Felsenstein 1985). The MP analysis was evaluated with the Min-mini heuristic parameter (level = 2) and by bootstrapping with 1000 replicates. For analyses, appropriate nucleotide substitution models were determined using ModelTest Server 1.0 (Posada 2006) in conjunction with PAUP*4.0b (Swofford 2002). A Bayesian (BI) analysis was performed in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). The analysis was run for 5,000,000 generations, and trees were sampled every 100 generations. All trees were reconstructed with Tree View 1.6.6 (Page 1996). The haplotype diversity, nucleotide diversity and analysis of variable nucleotide positions were calculated in DnaSP v. 5.10.01
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(Librado and Rozas 2009). The nucleotide frequencies and transition/transversion rate ratios were computed in Mega version 4.1 (Tamura et al. 2004, 2007).
Results and Discussion Analysis of studied DNA fragments Sequences (349 bp) of the gene encoding the large ribosomal subunit rDNA and cytochrome B (618 bp) from 20 strains of P. pentaurelia were obtained. Four haplotypes in rDNA fragment and three haplotypes in mtDNA were observed. The intra-specific haplotype diversity value was Hd = 0.616 for LSU rDNA and Hd = 0.574 for cytB. Nucleotide diversities amounted to π = 0.01075 (LSU) and π = 0.08426 (cytB), respectively. The nucleotide frequencies were A = 0.298, T = 0.254, C = 0.176, G = 0.271 (LSU) and A = 0.210, T = 0.334, C = 0.249 G = 0.206 (cytB). For the LSU rDNA fragment the transition/transversion rate ratios equaled k1 = 9.843 (purines), k2 = 16.594 (pyrimidines) and for cytB k1 = 5.170 and k2 = 9.261. The overall transition/transversion bias was R = 5.362 (LSU) and R = 3.478 (cytB). The mean divergence over all studied P. pentaurelia sequence pairs was 0.011/0.099 (5 LSU rDNA/cytB). The mean divergence of the 26 studied rDNA fragments obtained from all studied strains (including other Paramecium species) was 0.019/0.137 (LSU rDNA/cytB) and varied from 0.000 to 0.039/0.239 for P. aurelia species complex and 0.098/0.401 for P. multimicronucleatum (Table 5, supplementary material). In the studied fragments we found 8/118 (LSU rDNA/cytB) variable positions (7/95 parsimony informative) between P. pentaurelia strains and 35/230 (LSU rDNA/cytB) variable positions (12/151 parsimony informative) between all studied strains. There were no substitutions between strains from one location (Poland, Kraków, Jordan’s Park; Poland, Kraków, Opatkowice; Italy, Valmarana). ModelTest identified the TnN model for 5 LSU rDNA (I = 0, G = equal rates for all sites) and GTR + G for cytB (I = 0, G = 0.2004), as the best nucleotide substitution model for the MrBayes analyses. Intra-specific polymorphism in P. pentaurelia for both sequenced DNA fragments (mitochondrial cytB and nuclear LSU rDNA) (Fig. 1A and B) was expressed as three main groups of strains within the species. One clade includes strains of P. pentaurelia from Poland, Kraków, Jordan’s Park and a strain from Novosibirsk in the Asiaian part of Russia, as well as the strain of P. dodecaurelia from Kraków, Jordan’s Park. The second clade includes strains from different parts of the world (Europe, Asia, N. America), comprising both strains from Poland, Kraków, Opatkowice, and also very similar strains: 90 of P. primaurelia and 510 of P. novaurelia. The P. pentaurelia strain from Spain is separate on both trees (Fig. 1A and B). One main difference exists between the phylograms: P. tredecaurelia in the rDNA tree is the most distant
from other species of the P. aurelia complex (Fig. 1A), but in the case of the cytB tree, it appears close to the P. pentaurelia strains from Jordan’s Park (Fig. 1B). Analysis of results allowed for the following conclusions. No correlation of the geographical origin of the strains and the genetic characteristics of their genotypes was observed. Different sequence patterns were revealed in Kraków in two different types of water bodies (Opatkowice—natural pond,and Jordan’s Park—artificial pond). No haplotype diversity within one water reservoir, whether natural or artificial, was recorded (Poland, Kraków, Jordan’s Park; Poland, Kraków, Opatkowice; Italy, Valmarana). No seasonal diversity of haplotypes of strains originating from the artificial pond in Jordan’s Park, Kraków was found. The variable rDNA fragment seems to be a more appropriate DNA marker because of its better resolution for studying genetic variability. Analysis of both studied fragments failed to discriminate between particular species of the P. aurelia complex. A comparison of the present results concerning intraspecific differentiation within P. pentaurelia with the previously obtained ones (Przybo´s et al. 2007a), showed that the evaluation of the level of intra-specific differentiation depends on the sensitivity of the molecular methods (sequencing of some gene fragments or methods based on PCR). The studies (Stoeck et al. 2000) carried out with the application of the RAPD method revealed identical band patterns (calculated by the similarity index) of the studied strains from Spain, Hungary and the USA (Pennsylvania, designated strain 87). Thus, only one “genotype” was observed in this species. It was concluded by the authors that this situation is characteristic for weak inbreeders such as P. pentaurelia and that such “high genetic similarity among strains – within species – was caused by rather recent spreading of its strains”. Likewise, no polymorphism was observed within this species when strains from Italy (Sicily), Hungary, Russia (Altay) and the USA (Pennsylvania, strain 87) were used for RAPD analysis (Przybo´s et al. 2005). A newly recorded strain from Novorossiysk in the Black Sea region was also similar (Przybo´s et al. 2007/2008). P. pentaurelia strains from Pennsylvania (strain 87) and Russia (Astrahan Nature Reserve) were also used for ARDRA and RFLP analyses (Przybo´s et al. 2007a) and correspondingly, both strains revealed the same “genotype”. Several strains of P. pentaurelia from Russia (from the Astrahan Nature Reserve, Volgograd region, Altai Mountains and Foreland, and Novorossiysk) were used in studies (Catania et al. 2009) assessing genetic diversity in the P. aurelia species complex. All strains were characterized reproductively (mating reaction with the standard strain of the species) and genetically (ten nuclear and five mitochondrial genes were surveyed), and they all cluster together in a NJ tree. This study (Fig. 1A and B), based on the sequenced gene fragments of 5 LSU rDNA and cytB mtDNA, revealed the intra-specific polymorphism of P. pentaurelia as different
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Fig. 1. Phylogram constructed for P. pentaurelia strains, P. primaurelia, P. tetraurelia, P. novaurelia, P. tredecaurelia and P. multimicronucleatum as an outgroup, based on a comparison of sequences from the 5 LSU rDNA (A) and mitochondrial cytB fragment (B) using the NJ (neighbor joining) method (with application of the Kimura 2-parameter), MP (Maximum Parsimony) analysis and Bayesian Inference (BI). Bootstrap values (posterior probabilities for BI) are presented (NJ/MP/BI) for 1000 replicates. Phylogenetic analyses were conducted in MEGA 4.1 (NJ/MP) and Mr Bayes 3.1.2 (BI).
clades of strains in the phylograms. No correlation of the geographical origin of strains and genetic characteristics of their genotypes was observed in one clade composed of strains from Jordan’s Park, Kraków, and the Asian part of Russia (Fig. 1A) and in a second clade with strains from Opatkowice, Kraków, and strains from other continents. It is worth mentioning that strains from Poland, Kraków, artificial pond in Jordan’s Park (PK1—collected July 2005 and PJ1—7 collected May 2008 representing seven clones from one sampling point) showed the same haplotype. Similarly, strains OPAT A, B, C (Poland, Kraków, Opatkowice, natural pond) collected in June 2008 but from different sampling points did not show any molecular differentiation. P. dodecaurelia strains originating from the same pond (Kraków, Jordans’ Park, artificial pond) and studied earlier were also characterized by only one haplotype in spite of different sampling times (years 2006, 2007) and sampling points (A–C) (Przybo´s et al. 2008c). Barth et al. (2008a) however, found mitochondrial haplotype diversity of Coleps sp. (Ciliophora, Prostomatida) in a population from one lake. It is also interesting that the strain of P. dodecaurelia originating from Jordans’ Park, Kraków is in the same clade with the P. pentaurelia strains from the same collecting place. Both studied gene fragments (LSU rDNA and cytB mtDNA) failed to discriminate particular species of the P. aurelia species complex (Fig. 1A and B), although in future studies different markers can be applied. The clustering of other strains belonging to different species was observed in the constructed trees (Fig. 1A and B). As mentioned before, the P. dodecaurelia strain with P. pentaurelia strains in one clade, and P.
primaurelia and P. novaurelia appeared very close with the P. pentaurelia strains in a second clade. P. primaurelia, P. pentaurelia, and P. novaurelia belong to the same caryonidal group of mating type inheritance (Sonneborn 1975). This clustering of strains belonging to different species was also reported previously in several studies, and it seems that it can appear in different species, e.g., between strains of P. octaurelia and P. septaurelia, P. sexaurelia and P. dodecaurelia, and a group composed of P. septaurelia, P. decaurelia, P. tetraurelia, P. octaurelia with similar cytB sequences (Barth et al. 2008a). The authors hypothesized that such clustering may be the result of rapid speciation, and that a separation of species as to their mating similarity is new and incomplete. The clustering of some P. sexaurelia strains (from Japan, China, Puerto Rico) with a P. dodecaurelia strain from Mississippi, USA, was also observed in the other studies (Przybo´s et al. 2010). Catania et al. (2009) noticed an “incongruence between mating behavior and strain genetic profile” as some strains of P. octaurelia and P. septaurelia cluster together. It seems that the majority of species of the P. aurelia complex are polymorphic and that the relationships between species within the P. aurelia complex is more complicated than previously supposed. Relationships of species as presented above may also support the hypothesis formulated by Aury et al. (2006) on the origin of species of the P. aurelia complex and the observation of Barth et al. (2008b) that “species diversity may have originated from the neutral consequences of a whole genome duplication in the common ancestor of P. aurelia”.
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Acknowledgements The authors are greatly indebted to Andrea PereswietSoltan, Ph.D. student in the International Doctoral Studies in Natural Sciences at the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków for collecting water samples in Italy.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejop.2010.11.001.
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