Evaluation of the molecular variability and characteristics of Paramecium polycaryum and Paramecium nephridiatum, within subgenus Cypriostomum (Ciliophora, Protista)

Accepted Manuscript Evaluation of the molecular variability and characteristics of Paramecium polycaryum and Paramecium nephridiatum, within subgenus Cypriostomum (Ciliophora, Protista) Ewa Przyboś, Maria Rautian, Alexandra Beliavskaia, Sebastian Tarcz PII: DOI: Reference:

S1055-7903(18)30098-8 https://doi.org/10.1016/j.ympev.2018.12.003 YMPEV 6359

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

16 February 2018 5 November 2018 4 December 2018

Please cite this article as: Przyboś, E., Rautian, M., Beliavskaia, A., Tarcz, S., Evaluation of the molecular variability and characteristics of Paramecium polycaryum and Paramecium nephridiatum, within subgenus Cypriostomum (Ciliophora, Protista), Molecular Phylogenetics and Evolution (2018), doi: https://doi.org/10.1016/j.ympev. 2018.12.003

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Evaluation of the molecular variability and characteristics of Paramecium polycaryum and Paramecium nephridiatum, within subgenus Cypriostomum (Ciliophora, Protista)

Ewa Przybośa, Maria Rautianb, Alexandra Beliavskaiab, Sebastian Tarcza a -

Institute of Systematics and Evolution of Animals, Polish Academy of Sciences,

Sławkowska 17, 31-016 Kraków, Poland b –

Laboratory of Protistology and Experimental Biology, St. Petersburg State University,

Botanicheskaya, 17, 198504 St. Petersburg, Russia

Corresponding author: S. Tarcz; Institute of Systematics and Evolution of Animals, Polish Academy of Sciences; Sławkowska 17, 31-016 Kraków, Poland; FAX: +48 12 422 42 94; e-mail: [email protected]

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Abstract Although some Paramecium species are suitable research objects in many areas of life sciences, the biodiversity structure of other species is almost unknown. In the current survey, we present a molecular analysis of 60 Cypriostomum strains, which for the first time allows for the study of intra- and interspecific relationships within that subgenus, as well as the assessment of the biogeography patterns of its morphospecies. Analysis of COI mtDNA variation revealed three main clades (separated from each other by approximately 130 nucleotide substitutions), each one with internal sub-clusters (differing by 30 to 70 substitutions – a similar range found between P. aurelia cryptic species and P. bursaria syngens). The first clade is represented exclusively by P. polycaryum; the second one includes only four strains identified as P. calkinsi. The third cluster seems to be paraphyletic, as it includes P. nephridiatum, P. woodruffi, and Eucandidatus P. hungarianum. Some strains, previously identified as P. calkinsi, had COI sequences identical or very similar to P. nephridiatum ones. Morphological reinvestigation of several such strains revealed common morphological features with P. nephridiatum. The paper contains new information concerning speciation within particular species, i.e. existence of cryptic species within P. polycaryum (three) and in P. nephridiatum (six).

Keywords: microbial eukaryotes, Paramecium, morpho-molecular analysis, cryptic species, COI haplotypes, biogeography

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1. Introduction Application of molecular techniques, including the availability of nucleotide sequences (for example, from the GenBank database) in the last dozen or so years, permits better evaluation of the complex structure of several protist species (Bass et al., 2016). It is recommended, however, that the obtained DNA sequence information be applied to the background of the genetic, morphological, physiological, and ecological data (Caron, 2013; Dunthorn et al., 2014; Przyboś and Tarcz, 2016; Stoeck et al., 2014). Species from the genus Paramecium (Protista, Ciliophora, Oligohymenophorea) are suitable objects of research in many fields of life sciences including systematics and evolutionary studies. The genus is composed of several

morphological

species

arranged

into

five

subgenera

(Viridoparamecium,

Chloroparamecium, Cypriostomum, Helianter, and Paramecium - Fokin et al., 2004; Kreutz et al., 2012). Morphospecies, in turn, consist of cryptic species; in some of them (e.g. P. bursaria) they are called syngens (generating together) (Sonneborn, 1957). In fact, they may be recognized as biological species (Mayr, 1942) that are reproductively isolated ciliate populations. Inter- and intraspecific relationships are best studied within the Paramecium subgenus, especially within P. aurelia – a complex of 15 cryptic species (Aufderheide et al., 1983; Sonneborn, 1975). It is suggested that they diverged as a result of at least two rounds of whole-genome duplications and about 50% of genes duplicated by the recent whole-genome duplications and subsequent events (Aury, 2006; Johri et al., 2017; McGrath et al., 2014). The majority of species of the P. aurelia complex reveal intra-specific polymorphism; and interspecific relationships within the complex are well understood (Barth et al., 2008; Catania et al., 2009; Przyboś et al., 2007, 2013, 2014, 2015; Tarcz et al., 2013). The other morphospecies of this subgenus are composed of a lower number (3-5) of cryptic species (groups): P. multimicronucleatum (Tarcz et al., 2012), P. caudatum (Krenek et al., 2015), P.

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jenningsi (Przyboś and Tarcz, 2013, 2016), and P. schewiakoffi known to date only from one site (Fokin et al., 2004). Furthermore, over the past several years, two new species have been distinguished: P. caudatum pakistanicus within P. caudatum (Shakoori et al., 2014) and P. grohmannae from Brazil, a relative of P. multimicronucleatum (Paiva et al., 2016). The other Paramecium subgenera are less studied. Subgenus Viridoparamecium consists only of the recently re-described P. chlorelligerum (Kreutz et al., 2012), which was found only in a few (four) stands (Kahl, 1935; Kreutz et al., 2012; Lanzoni et al., 2016; Vuxanovici, 1960 cit. after Kreutz et al., 2012). However, there is no suggestion of the occurrence of cryptic species within P. chlorelligerum, as only two existing populations of the species (one from Germany and the second from Russia) revealed very low genetic variability (Lanzoni et al., 2016). Similarly, the next subgenus Chloroparamecium is composed of only one morphospecies, P. bursaria, which in turn is divided into five syngens (cryptic species) (Greczek-Stachura et al., 2012; Zagata et al., 2015). In the subgenus Helianter, the intra-specific differentiation into five haplogroups (potential cryptic species) was found in P. putrinum (Tarcz et al., 2014) and in P. duboscqui, where three main groups of strains have been distinguished (Boscaro et al., 2012). Additionally, P. buetschli (known from sites in Norway and Finland), a sister taxon to P. putrinum, has been recently described (Fokin, 2017; Krenek et al., 2015). Thus far, the least examined subgenus is Cypriostomum, which is composed of P. polycaryum, P. nephridiatum, P. calkinsi, and P. woodruffi morphospecies (Fokin et al., 2004; Kreutz et al., 2012). Up to now, intra-specific variability was studied only in P. calkinsi (Przyboś et al., 2012) and P. nephridiatum (Fokin et al., 1999). However, the clear separation of some morphospecies, as well as their identification based on morphological features, has been problematic (Fokin, 2010/2011; Fokin & Chivilev, 1999; Fokin et al., 1999).

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Therefore, in order to shed some light on this still under-researched subgenus, we present the results of intra-specific variability based on molecular analysis as well as cytological investigations of some strains representing P. polycaryum (13 strains studied molecularly, 10 among them cytologically) and P. nephridiatum (40 strains studied molecularly, 16 among them cytologically) in comparison to the other Cypriostomum morphospecies. Moreover, detailed molecular analysis of these 60 Cypriostomum strains reveals for the first time the intra- and interspecific relationships as well as the biogeographical patterns within that subgenus.

2. Material and Methods

2.1 Material Strains of Paramecium spp. studied in the present paper representing morphospecies of the Cypriostomum subgenus as well as other subgenera are listed in Table S1 (supplementary material). Strains of P. polycaryum and P. nephridiatum belong to the Culture Collection of Ciliates and their Symbionts (CCCS), located in St. Petersburg State University, Russia. Collection has been registered in WFCC, #1024. Other strains used in the present paper are from the strain collection of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków, Poland.

2.2 Strains cultivation Cypriostomum species can be isolated from fresh or brackish water; in laboratory conditions they can be adopted to a standard culture medium for Paramecium. They were cultured according Sonneborn (1950, 1970) in a medium made of dried lettuce and distilled water, and

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a temperature of 22°C, inoculated with Enterobacter aerogenes, and supplemented with 0.8 mg/ml β-sitosterol.

2.3 Cytological studies The well known taxonomic criteria for the discrimination of either living or fixed and stained cells of particular Paramecium morphospecies are: shape and size of the body, type and number of micronuclei (mi), features of contractile vacuoles (CV) and number of pores per CV, as well as the type of paramecium cell movement (Fokin, 2010/2011). Type and number of micronuclei were initially analyzed on slides temporarily stained using aceto-carmine (Sonneborn, 1970). Permanent slides of vegetative paramecia were fixed with Schaudinn’s fluid with glacial acetic acid (Chen, 1944) and stained with Giemsa’s stain (Merck, Darmstadt, Germany) 10% solution in 0.01 M phosphate buffer (Przyboś, 1978). Paramecia cells were also investigated in phase contrast as well as DIC. Photographs of Giemsa stained (Figs 1-8) paramecia were taken using a Nikon Eclipse E400 microscope equipped with a Nikon DS-Fi2 digital camera; photographs of unstained paramecia were taken with a Leica DM2500 microscope (optical magnification 1250) using differential interference contrast (DIC).

2.4 Molecular techniques Paramecium genomic DNA was isolated from vegetative cells at the end of the exponential phase (approximately 1000 cells were used for DNA extraction) using a NucleoSpin Tissue Kit (Macherey-Nagel, Germany), according to manufacturer’s instructions for DNA isolation from cell cultures. The only modification, in order to concentrate the sample, was centrifugation of the cell culture for 20 min at 13,200 rpm. The supernatant was then removed, and the remaining cells were suspended in lysis buffers and proteinase K. Both the

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quantity and purity of the extracted DNA were evaluated with a NanoDrop-2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Fragments of rDNA and COI genes were amplified, sequenced, and analyzed. The rDNA fragment was amplified with a forward ITS1 universal eukaryotic primer (5’TCCGTAGGTGAACCTGCGG-3’ (White et al., 1990)) and a reverse 3pLSU primer (5’CAAGACGGGTCAGTAGAAGCC-3’ using the protocol previously described by (Tarcz et al., 2012)). The COI fragment of mitochondrial DNA was amplified with the primer pair forward

F388dT

(5’-TGTAAAACGACGGCCAGTGGwkCbAAAGATGTwGC-3’)

reverse

and (5’-

R1184dT

CAGGAAACAGCTATGACTAdACyTCAGGGTGACCrAAAAATCA-3’)

using

the

protocol previously described by Strüder-Kypke and Lynn (2010). For all analyzed DNA fragments, PCR amplification was carried out in a final volume of 40 μL containing 30 ng of DNA, 1.5 U Taq-Polymerase (EURx, Poland), 0.8 μL of 20 μM of each primer, 10 Χ PCR buffer, and 0.8 μL of 10 mM dNTPs. In order to assess the quality of amplification, PCR products were electrophoresed in 1% agarose gel for 30 min at 85 V with a DNA molecular weight marker (Mass Ruler Low Range DNA Ladder, Thermo Fisher Scientific, USA). For purifying PCR products, 5 µl of each were mixed with 2 µl of Exo-BAP Mix (EURx, Poland), and then incubated at 37°C for 15 min and afterwards at 80°C for another 15 min. Cycle sequencing was done in both directions with BigDye Terminator v3.1 chemistry (Applied Biosystems, USA). The primers used in the PCR reactions were again used for sequencing

the

rDNA

region

and

the

primer

pair

forward

M13F

(5’-

TGTAAAACGACGGCCAGT-3’) and reverse M13R (5’-CAGGAAACAGCTATGAC-3’) (Messing, 1983; Strüder-Kypke and Lynn, 2010) was used for sequencing the COI fragment. The details of the sequencing procedure are from Tarcz et al. (2012). The sequences of both studied fragments are available in the NCBI GenBank database (see supplementary Table S1).

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2.5 Data analysis Sequences were examined using Chromas Lite (Technelysium, Australia) to evaluate and correct chromatograms. Alignments of the studied sequences were performed using BioEdit software (Hall, 1999) and checked manually. All the obtained sequences were unambiguous and were used for analyses. Phylograms were constructed for the studied fragments by means of Mega v6.0 (Tamura et al., 2013), using neighbor-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML). All positions containing gaps and missing data were eliminated. NJ analysis was performed using the Mega v6.0 program by bootstrapping with 1000 replicates. 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; trees were sampled every 100 generations. The both (rDNA and COI) trees for BI analysis were visualized with TreeView 1.6.6 (Page, 1996). The number of segregating sites (S), number of haplotypes (h), haplotype diversity (Hd), and nucleotide diversity (Π) were identified with DnaSP v5.10.01 (Librado and Rozas, 2009). Identification of substitution models for ML analysis was done using Mega v6.0 (Tamura et al., 2013). Mega 6.0 identified the TN93+G for the ITS1-5.8S-ITS2-5’ end of LSU rDNA and the HKY+G+I for the COI mtDNA as the best nucleotide substitution models for maximum likelihood tree reconstruction. The haplotype network, which presented the distribution and relationships among haplotypes of Cypriostomum strains, was reconstructed by means of the Median Joining method (Bandelt et al., 1999), as implemented in the PopART software v. 1.7 (Leigh and Bryant, 2015).

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3. Results and Discussion

Although ciliates, like many other groups of microbial eukaryotes, are one of the main components of trophic food webs in different ecosystems, they are still significantly underestimated in the field of biodiversity (Medinger et al., 2010; Weisse, 2014). The above problem results from two factors: the complex structure of the species (Caron, 2013) as well as under-sampling of many ecosystems (Fokin, 2010/2011). This is especially visible in the case of Paramecium morphospecies which are classified as Cypriostomum subgenus, and raises the question of degree of biodiversity corresponding to subgenus-species-cryptic species levels.

3.1 Characteristics of morphospecies from subgenus Cypriostomum Paramecium polycaryum (Woodruff and Spencer, 1923) is the fresh-water species with cell length 70 to 110 μm as well as morphology typical for “bursaria” group (Wenrich, 1928). The nuclear apparatus is composed of oval macronucleus and usually four (occasionally one to eight) “blind” micronuclei; two contractile vacuoles may be observed in P. polycaryum cell (Fokin, 1997; Fokin and Chivilev, 1999; Vivier, 1974; Woodruff and Spencer, 1923). The appearance of autogamy has been noticed (Diller, 1954; Fujishima, 1988) within this species. Our data concerning studied P. polycaryum cells (Table S1) confirm the above characteristics. Cytological analysis of Giemsa-stained 12 strains (in each 20 studied individuals) showed a variable number of micronuclei (from one to four, usually three to four) (Fig. 1), with homogenous chromatin. They are located rather close to the macronucleus, with a diameter of about 3µm. Two contractile vacuoles with short and thin canals, as well as with one pore within the CV, have been observed (Fig. 2). In the current studies, both vegetative and autogamic P. polycaryum cells have been observed (Fig. 3).

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Paramecium nephridiatum was described for the first time by Gelei (1925), and later redescribed by him in 1938; however, it was considered as a non-valid species and, in turn, rediscovered and characterized by Fokin et al. (1999) with the application of microscopic and molecular methods (RAPD). Paramecium nephridiatum occurs mainly in brackish water. Its cell (average length 145μm) is of the “bursaria“ type (Wenrich, 1928), and is characterized by multiple contractile vacuole pores. The nuclear apparatus is composed of ovoid macronucleus and three to four spherical micronuclei (Fokin et al., 1999) of “endosomal” type (Fokin, 1997; Fokin and Chivilev, 1999). All the 16 strains (20 individuals in each) (Table S1) presently studied by us showed characteristic features of P. nephridiatum in shape and size of the cell and number of micronuclei. Cytological analysis of Giemsa-stained strains showed a variable number of micronuclei (from one to three, usually two to three) (Fig. 4A) with diameter about 4-5 µm, characterized by non-homogenous chromatin with more compact zones visible on DIC photographs (Fig. 4B). Micronuclei are usually located at some distance from macronucleus. In the current survey, we noticed two contractile vacuoles with short but distinct collecting canals (Fig. 5A), which when filled, looked like bubbles. The other small vacuoles which surround the CV were also visible. Usually, more than one CV pore has been observed (Fig. 5B). Furthermore, both vegetative and autogamic P. nephridiatum cells were observed during the current study (Fig. 6). Paramecium calkinsi. For comparative cytological analysis we used the strain SH10I from Salt Lake City, Utah (obtained by courtesy of S. Krenek, Institute of Hydrobiology, Technical University, Dresden, Germany). The nuclear apparatus was composed of an ellipsoidal macronucleus and two micronuclei (sometimes only one) of the endosomal type, situated near the macronucleus. These were observed (approximately 20 individuals studied) on Giemsa stained slides (Fig. 7A) and on DIC photographs (Fig. 7B), as in Fokin (1997); Fokin and Chivilev (1999); Fokin (2010/2011). Two (Fig. 8A) contractile vacuoles with short canals and

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usually one, but sometimes two, pores were observed (Fig. 8B). Long cilia at the posterior end of the cells seem characteristic feature for this species (Fig. 8A). Paramecium woodruffi belonging to the subgenus Cypriostomum was represented in the current study only by the COI sequence of one strain: OLI-Parame-wood-01 (Strüder-Kypke and Lynn, 2010). It is the largest (length 120-210 μm) species among the “bursaria” group, and, based on Wenrich (1928), shows a typical scattered arrangement of vesicular micronuclei. However, according Fokin and Chvilev (1999) and Fokin (2010/2011) these are the “endosomal” type. Individuals of this species possess up to eight small micronuclei, usually three or four (Wichterman, 1986); Kościuszko (1986), however, observed up to ten “vesicular” micronuclei. Paramecium woodruffi is characterized by the occurrence of two contractile vacuoles (Vivier, 1974) with only one pore in each one, and with 8-16 collecting canals with ampoules (Fokin and Chivilev, 1999). Paramecium woodruffi is mainly a brackish water species but can also occur in freshwater habitats (Kościuszko, 1986). Eucandidatus Paramecium hungarianum has been described as “typical woodruffi shaped”. Its existence as a separate taxon has been proposed based on molecular data obtained from only one strain (Krenek et al., 2015). Currently, no significant morphological features are known that might discriminate Eucandidatus P. hungarianum from the other Cypriostomum morphospecies (Krenek et al., 2015). Due to the fact that morphospecies belonging to the Cypriostomum subgenus are not as common as some other representatives of Paramecium subgenus, e.g. P. caudatum, there is a high probability that they may be not properly identified. This is of special concern for strains of morphologically similar species which might occupy the same ecological niches. Therefore, distinguishing between P. calkinsi, P. nephridiatum, and P. woodruffi might be problematic (Boscaro et al., 2012; Fokin and Chivilev, 1999; Melekhin et al., 2017a,b; Przyboś et al., 2012), particularly in assessing the degree of possible internal morphological

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variability of the investigated morphospecies. Application of more detailed methods (DIC) in the current studies permitted reinvestigation of some strains (a total of nine; see Table S1) previously identified as P. calkinsi (Przyboś et al., 2012), leading to their classification as P. nephridiatum (Table S1). According to DIC analysis performed in the current survey, these strains have comparatively large (4-5 µm) micronuclei with distinct non-homogenous chromatin, as well as more and less condensed zones (Fig. 4A). Each P. nephridiatum cell had two CV with very short canals, which were well seen at diastole stage. In the systole stage, many vacuoles that surround the CV can be seen (Fig. 5A). In contrast, strain SH10I, designated as P. calkinsi, has micronuclei lacking more condensed lumps and with longer CV canals (Fig. 8A). The number of CV pores seems to be more variable and not as significant for distinguishing these two morphospecies. In summary, different morphological methods may lead to different results, and furthermore, the number of pores can be different in different cells of the same strain or even in different CV of the same cell. We have also noted that the CV pore number varies at different salinities and probably in different media (Rautian, unpublished). Therefore, in such cases application of molecular analyses helps to resolve the systematic/taxonomic problem.

3.2 Analysis of ITS1-5.8S-ITS2-5’LSU rDNA and COI mtDNA sequences

During the current studies, we isolated DNA fragments from not yet examined 18 strains of Cypriostomum species: P. calkinsi (1), P. nephridiatum (6), and P. polycaryum (11). The remaining data were downloaded from GenBank database, which gave altogether 32 ribosomal and 60 mitochondrial DNA sequences of Cypriostomum subgenus. For phylogenetic tree reconstructions, additional GenBank records for the other Paramecium subgenera and two Tetrahymena species (used as an outgroup) have been included in both the

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datasets, which ultimately involved 79 ribosomal and 113 mitochondrial DNA sequences (for details, see Table S1). After trimming, for sequence comparison we used the rDNA region containing the 3’ end of SSU rDNA-ITS1-5.8S-ITS2-5’ end of LSU rDNA (1041-1098 bp) and cytochrome c oxidase subunit I (637-638 bp). The values of intraspecific haplotype diversity (Hd) were 0.927 for the rDNA fragment (N=32) and 0.985 for COI (N=60), which indicates great differentiation among the studied Cypriostomum strains. However, variability in each species differed (Table S2). In the case of the ribosomal DNA fragment, the greatest number of segregating sites (S) as well as nucleotide diversity (π) were observed within P. calkinsi (N=3), in comparison to P. nephridiatum (N=17) and P. polycaryum (N=12). Although strains CyL 1-24 and CyL 8-33 (obtained from GenBank) form a monophyletic clade on rDNA tree together with the studied morpho-molecularly Paramecium calkinsi strain SH10I (Fig. 9), they may constitute separate morphospecies within Cypriostomum as suggested in (Melekhin et al., 2017a,b). In turn, COI mtDNA revealed the greatest number of segregating sites (S) in P. nephridiatum (N=40) in comparison to P. calkinsi (N=4) and P. polycaryum (N=14). Nucleotide diversity (π), as in the case of the rDNA fragment, was highest in P. calkinsi. Due to the fact that the ribosomal fragment studied herein consisted of alternating coding and noncoding segments, variable and conserved regions were distributed unevenly (Figure S1). It is not unusual that a significant number of segregating sites (S) were located in ITS1, ITS2 noncoding regions (Fig. S1), as was reported in other Paramecium species (e.g. Coleman, 2005). However, some nucleotide differences can be observed in 3’ SSU rDNA (within P. calkinsi) and in 5.8S gene (between particular Cypriostomum morphospecies). Finally, a part of the LSU rDNA gene also revealed important variability of nucleotide positions, especially in the 3’ end of studied rDNA fragment (Fig. S1), which corresponds with D1-D2 domain

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characterized by a significant volatility, and which is proposed as a DNA barcoding marker for ciliates (Stoeck et al., 2014).

3.3 Inter- and intraspecific relationships of Cypriostomum species based on the comparison of ITS1-5.8S-ITS2-5’LSU rDNA and COI mtDNA sequences

Based on the obtained rDNA and COI data and the constructed trees, we confirmed that subgenus Cypriostomum forms a monophyletic clade well separated from the other Paramecium subgenera (Figs 9, 10). Analysis of the rDNA tree (Fig. 9) revealed the existence of two welldefined monophyletic clades consisting of P. nephridiatum (17 strains), P. polycaryum (12 strains) and a clade of P calkinsi (3 strains). The general topology of the COI tree (Fig. 10) is similar to the rDNA tree. The P. calkinsi clade (4 strains) is rather distant from the other Cypriostomum species, and contains two subclades; then P. polycaryum (14 strains divided into three subclades) and finally the largest cluster which consists of P. nephridiatum (40 strains) and one strain of P. woodruffi and one strain of Eucadidatus P. hungarianum. Paramecium nephridiatum, which forms on the COI tree a paraphyletic taxon is also divided into several subclades and branches. In turn, it contrasts with previous intraspecific Paramecium nephridiatum analyses where the studied strains of different geographical origin were characterized by an identical diagnostic DNA fingerprint band pattern (Fokin et al., 1999). To further examine the interspecific relationships within the Cypriostomum subgenus we constructed a haplotype network based on the 60 COI sequences dataset. Analysis of the

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network (Fig. 11) revealed the existence of three main clusters, which correspond to P. polycaryum, P. calkinsi and P. nephridiatum + P. woodruffi + Eucandidatus P. hungarianum. Analysis of intraspecific variation within the P. polycaryum morphospecies revealed the existence 10 COI haplotypes (PpCOI_01 – PpCOI_10) which might be divided into three haplogroups named Pp A, Pp B and Pp C. Haplogroup Pp B is situated in the central part of the P. polycaryum network and it is independently connected with haplogroup Pp A and Pp C (Fig. 11). For easier analysis and comparison of the results published in various papers as well as for future Paramecium studies we maintain the haplotypes numbering method as in Barth and colleagues (2006). The distances (numbers of nucleotide substitutions) between particular P. polycaryum haplogroups are almost the same as observed between some of the cryptic species of the well-studied Paramecium aurelia complex (see Fig. 5 in Przyboś et al., 2016). In the case of P. calkinsi (differing from P. polycaryum by 110-119 substitutions) three haplotypes (PcsCOI_01, PcsCOI_02 and PcsCOI_03) are visible (Fig. 11). The distance between them (61-70 substitutions) is comparable to the distance between P. aurelia cryptic species (Fig. 5 in Przyboś et al., 2016). This allows one to hypothesize that aforementioned haplotypes also might represent reproductively isolated groups within P. calkinsi. Our results are in line with previous surveys, where the existence of two syngens (cryptic species) within P. calkinsi has been reported (Wichterman, 1986), and with the new molecular data (Sabaneyeva et al. 2018) showing intra-specific variability of the species. The most complicated, and at the same time interesting, relationship has been observed in the P. nephridiatum-P. woodruffi-Eucandidatus P. hungarianum clade, which is separated from P. polycaryum by 135 substitutions. Within that cluster six subclusters (highlighted grey rectangles) can be observed containing from 1 to 21 haplotypes and named Pn A, Pn B, Pn C, Pn D, Pn E, and Pn F (Fig. 11). These entities (we refrain from calling them haplogroups, since 4 of the 6 clusters contain only a single haplotype) may correspond to P. nephridiatum

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cryptic species, because of the number (about 60-70) of nucleotide differences between them similar or even greater than between some members of the P. aurelia complex. However, the same or even smaller distance (as between P. aurelia species) has been observed between some entities of P. nephridiatum and haplotypes representing P. woodruffi or Eucadidatus P. hungarianum. Currently, it is not possible to study the morphology of these strains; in the case of the latter, there are no clear morphological features to discriminate them from the other Paramecium morphospecies (Krenek et al., 2015), so we suppose that this should be a topic for further, more detailed studies. Without comparing at least two to three strains of each species it would be impossible to evaluate the degree of moropho-molecular variability within P. woodruffi or Eucadidatus P. hungarianum and between them and P. nephridiatum cryptic species. Analysis of the COI network (Fig. 11) revealed three levels molecular of variability. First, morphospecies (P. calkinsi, P. nephridiatum, P. polycaryum, but without P. woodruffi) are separated by a "distance" about 130 nucleotide substitutions. Secondly, within the morphospecies one can also see diversity that is not-random, but has internal structure. The diversity is represented by different haplogroups (or single haplotypes) differing by 30-70 substitutions. Similar distances occur between cryptic species of P. aurelia complex (Przyboś et al., 2016). And third, the “distance” within haplogroups is approximately a few dozen substitutions. Some exception has been noticed in the case of morphospecies P. woodruffi, in which the distance to P. nephridiatum sublasters Pn B, Pn C, and Pn D amounts from 61 to 75 substitutions – similar to the distance between P. nephridiatum subclasters (Fig. 11). However, the currently observed situation is not new in Paramecium genus, and has been reported from the reciprocal relationships between the P. aurelia and P. jenningsi complexes (Przyboś and Tarcz, 2016).

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3.4 The phylogeographic patterns within Cypriostomum species

The two issues mentioned above – the complex species structure (Caron, 2013) as well as under-sampling problem (Fokin, 2010/2011) – may not only interfere with the correct assessment of biodiversity but also with phylogeographic patterns within Cypriostomum subgenus. Although the presently studied strains were collected from distant sampling points (Table S1), we are aware that the current analyses are limited only to 32 known locations: most of them were from the Western Palearctic ecozone (15), then the Eastern Palearctic ecozone (8), the Central Palearctic ecozone (3), and finally the Afrotropical, Nearctic and Oceanian ecozones (2). The distribution of microbial eukaryotes, including ciliates, is known to a much lesser extent than for plants and animals (Azovsky et al., 2016). It is currently an intensely debated issue with two proposed hypotheses, i.e., the “ubiquity model” (UM) (Fenchel and Finlay, 2004; Finlay et al., 2006) and the “moderate endemicity model” (MEM) (Foissner, 2006; Foissner et al., 2008). According to the current state of knowledge, it seems that within the Paramecium genus, there are species with a wide range of distribution (Tarcz et al., 2018) as well as species known only from one (Fokin et al., 2004) or two (Przyboś et al., 2014) localities. However, due to a lack of molecular data, so far it has not been clear which of the above hypotheses better describes the Cypriostomum subgenus. Although Paramecium polycaryum is not as common in freshwater habitats, it has a worldwide distribution: 14 strains are studied here, divided in to three haplogroups. It has been isolated from the Afrotropical, Nearctic, Palearctic, and Oceanian ecozones (Fig. 11, Table S1). It is in line with the previous data on biogeography of that species, which has been additionally observed in the Neotropical ecozone (Dominicana) (Fokin et al., 2004; Woodruff

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and Spencer, 1923; Yang and Shi, 2007). Seven of ten COI haplotypes (PpCOI_01, PpCOI_03, PpCOI_05, PpCOI_07, PpCOI_08, PpCOI_09, PpCOI_10) were found in one locality as unique haplotypes, and the remaining 3 haplotypes have a wider range with the Palearctic ecozone (PpCOI_04, PpCOI_06) or were shared between two distant ecozones (PpCOI_02). On the other hand, two P. polycaryum haplotypes (PpCOI_08, PpCOI_09), representing two haplogroups (Pp B and PpC) have been observed in a relatively small area (Guam island). Current analyses revealed that Paramecium nephridiatum is also a widespread species, but restricted to the Palearctic ecozone. Additionally, that species has been reported on the east coast of the USA (Nearctic ecozone) (Fokin et al., 1999). However, there are no molecular data for that strain. The “P. nephridiatum part” of the network revealed the existence of 22 unique haplotypes (Fig. 11). The remaining five haplotypes contain from two to eleven COI sequences from one locality; there is only one haplotype (Pn CO_04) that originates from distant populations. Despite the small number of analyzed sequences, P. nephridiatum discloses a potential variability of local populations: haplotypes Pn CO_07 and Pn CO_08 have been isolated from Lake Baikal, Olchon Island (this study, see Table S1), and 18 haplotypes from Qingdao (Zhao et al., 2013, see Table S1). The molecular variability of local populations has been recently obtained from the other microbial eukaryotes, such as the ciliate Tetrahymena thermophila (Zufall et al., 2012) or the alga Klebsormidium (Ryšánek et al., 2014). The intraspecific relationships of P. calkinsi (Fig. 11) showed the existence of three haplotypes: PcsCOI_01 and PcsCOI_03 from Western Palearctic, and PcsCOI_02 from Nearctic ecozones. The results obtained are in concordance with previous data concerning the place of origin of P. calkinsi (Fokin et al., 1999).

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Finally, two strains of P. woodruffi and Eucandidatus P. hungarianum have been identified in the Western Palearctic ecozone. In the case of the latter, there is the only one strain of that taxon (Krenek et al., 2015). However, P. woodruffi, in addition to the Palearctic, has also been reported in the Nearctic ecozone (Fokin et al., 1999). Based on the current molecular analyses, as well as existing literature data, it can be assumed that probably all Cypriostomum morphospecies have a wide range of occurrence. However, without a detailed sampling of the thus far poorly studied brackish or even marine environments, it will be difficult to decide whether some of the Cypriostomum morphospecies may be more frequently observed in waters with a higher salinity. The currently analyzed haplotype dataset, in addition to a large variability, indicates the existence of cryptic species and essentially no population structure within particular members of Cypriostomum subgenus (Fig. 11).

3.4 Future studies on Cypriostomum biodiversity

Although the present study increases our knowledge of biodiversity structure of the least examined Paramecium subgenus, there are still some issues that should be resolved in future studies. First, an analysis based on more than one strain of Eucandidatus P. hungarianum and P. woodruffi is necessary to verify their relations to P. nephridiatum morphospecies, as well as to assess their internal variability and to test for the occurrence of cryptic species. Secondly, cryptic species within P. nephridiatum and P. polycaryum delineated by current molecular analyses should be verified by mating tests. It is suggested that the number of species within genus Paramecium including Cypriostomum subgenus may be higher. Their affiliation, however, cannot be confirmed due to a lack of living strains, [P. africanum, P. jankowski, P. wichtermani, P. ugandae (all from subgenus

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Paramecium)] or P. pseudotrichium from subgenus Cypriostomum (cf. Krenek et al., 2015). On the other hand, several new cryptic species in different Paramecium subgenera have been distinguished solely by DNA fragment comparison, as studied in the current survey of Eucandidatus P. hungarianum (Krenek et al., 2015). Moreover, recent studies based on morpho-molecular features combination have revealed that species biodiversity within the subgenus Cypriostomum might be more complicated than anticipated (Beliavskaia et al., 2016; Melekhin et al., 2017a,b).

Acknowledgements The authors would like to thank “Molecular and cellular technologies” and “Cultivation of Microorganisms” resource centers of St. Petersburg State University for providing technical support. The authors would also like to thank the Research Coordination Network for Ciliate Biodiversity (IRCN-BC) and Guam University for permission to collect Ciliates on Guam, as well as the other colleagues who helped in collecting Paramecia in the areas studied. The authors would like to thank Sascha Krenek (Institute of Hydrobiology, Technical University, Dresden, Germany) for providing the strain SH10I of Paramecium calkinsi from Salt Lake City, Utah. Molecular analyses performed for the current study were supported by the statutory funds of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków, Poland.

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Figure legends

Figure 1. Paramecium polycaryum. Nuclear apparatus of vegetative individual, two vesicular micronuclei (mi), macronucleus (Ma). Strain Sp 14-1 from Spain, Barcelona, Giemsa stains. Figure 2. Paramecium polycaryum. Contractile vacuole (CV) with short, thin canals. Strain G6-10 from Guam, DIC. Figure 3. Paramecium polycaryum. Autogamous individual. Macronuclear anlagen (A), fragments of old macronucleus (OMa), new micronulei (mi). Strain G6-10 from Guam, Oceania, Giemsa stains. Figure 4A. Paramecium nephridiatum. Nuclear apparatus of vegetative individual, two

endosomal

micronuclei (mi), macronucleus (Ma). A. Strain KUZ 62-1 from Russia, Kamchatka, Giemsa stains. Figure 4B. Paramecium nephridiatum. Nuclear apparatus of vegetative individual, two endosomal micronuclei (mi), macronucleus (Ma). Strain BOR 130-1 from Russia, Baikal, DIC. Figure 5A. Paramecium nephridiatum. Contractile vacuole (CV) with short canals. Strain BOR 130-1 from Russia, Baikal, DIC. Figure 5B. Paramecium nephridiatum. Contractile vacuole (CV) with four pores (arrows). Strain BOR 130-1 from Russia, Baikal, DIC.

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Figure 6. Paramecium nephridiatum. Autogamous individual. Four macronuclear anlagen (A), fragments of old macronucleus (OMa). Strain BOR 120-1 from Russia, Baikal, Giemsa stains. Figure 7A. Paramecium calkinsi. Nuclear apparatus of vegetative individual, one endosomal micronucleus (mi), macronucleus (Ma). Strain SH10I from USA, Salt Lake City, Giemsa strains. Figure 7B. Paramecium calkinsi. Nuclear apparatus of vegetative individual, two endosomal micronuclei (mi), macronucleus (Ma). Strain SH10I from USA, Salt Lake City, DIC. Figure 8A. Paramecium calkinsi. Vegetative individual. Two contractile vacuoles (CV) with canals, long cilia at the posterior end of paramecium. Strain SH10I from USA, Salt Lake City, DIC. Figure 8B. Paramecium calkinsi. Contractile vacuole (CV) with two pores (arrows). Strain SH10I from USA, Salt Lake City, DIC. Figure 9. Phylogenetic tree constructed for 32 Cypriostomum strains, and the other Paramecium subgenera (two Tetrahymena species were used as an outgroup). The tree was constructed on the basis of a comparison of sequences from the ribosomal 3’SSU-ITS1-5.8S-ITS2-5’LSU fragment using the Maximum Likelihood method. Bootstrap values for neighbor joining, maximum parsimony, maximum likelihood, and posterior probabilities for Bayesian inference are presented. Bootstrap values smaller than 50% (posterior probabilities <0.50) are not shown. Dashes represent no bootstrap or posterior value at a given node. All positions

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containing gaps and missing data were eliminated. Phylogenetic analyses were conducted using MEGA 6.0 (NJ/ML) and MrBayes 3.1.2 (BI). The analysis involved 79 nucleotide sequences. There was a total of 975 positions in the final dataset. Figure 10. Phylogenetic tree constructed for 60 Cypriostomum strains, and the other Paramecium subgenera (two Tetrahymena species were used as an outgroup). The tree was constructed on the basis of a comparison of sequences from the mitochondrial COI fragment using the Maximum Likelihood method. Bootstrap values for neighbor joining, maximum parsimony, maximum likelihood, and posterior probabilities for Bayesian inference are presented. Bootstrap values smaller than 50% (posterior probabilities <0.50) are not shown. Dashes represent no bootstrap or posterior value at a given node. All positions containing gaps and missing data were eliminated. Phylogenetic analyses were conducted using MEGA 6.0 (NJ/ML) and MrBayes 3.1.2 (BI). The analysis involved 113 nucleotide sequences. There was a total of 606 positions in the final dataset. Figure 11. Haplotype network of Cypriostomum constructed using the 60 of mitochondrial COI fragments. The network presents reciprocal relationships between, and the origin of Cypriostomum haplotypes identified in current study. The median vectors that represent hypothetical intermediates or unsampled haplotypes, are shown in black circles. Black dots on particular branches represent nucleotide substitutions between particular haplotypes (in the case of over 10 a corresponding number was given). The boundaries between Cypriostomum morphospecies are marked with a dashed line, and potential cryptic species are highlighted by grey rectangles. Analyses were conducted using the Median Joining method in PopART software v. 1.7.

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Highlights  Some Paramecium species are common research objects in various fields of science  At the same time, the biodiversity structure of the others is almost unknown  Here we present a molecular analysis of 60 Cypriostomum strains  Three main clades has been revealed, each one with internal sub-clusters  Their occurence reflects a separation into morphospecies and cryptic species

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Graphical abstract