new paramecium (ciliophora, oligohymenophorea) congeners ... · paramecium buetschlii sp. nov. from...

ORIGINAL ARTICLE

New Paramecium (Ciliophora, Oligohymenophorea) congenersshape our view on its biodiversity

Sascha Krenek & Thomas U. Berendonk & Sergei I. Fokin

Received: 29 September 2014 /Accepted: 23 February 2015 /Published online: 22 March 2015# Gesellschaft für Biologische Systematik 2015

Abstract Paramecium is one of the best known and mostintensely studied ciliate genera. It currently comprises 18mor-phospecies including the P. aurelia complex of 15 siblingspecies. Here, we describe the new morphospeciesParamecium buetschlii sp. nov. from a freshwater pool inNorway, featuring unusual combinations of morphologicalcharacters and a high genetic diversity relative to other con-geners. Three further investigated Paramecium spp. fromGermany, Hungary, and Brazil are treated as cryptic species,because they are difficult to discriminate from other membersof the genus relying on morphological criteria only. However,DNA-based taxonomic markers (18S-rDNA andmitochondrial COI) clearly indicate they are separate species.Due to the lack of an appropriate systematic term within theInternational Code of Zoological Nomenclature fordistinguishing cryptic from valid biological species, we pro-pose the provisional status Eucandidatus as a component ofthe taxonomic name when describing new but cryptic eukary-otes. Based on our data, we postulate that even within Europe

there is a higher biodiversity within this common ciliate groupthat is heavily used in the classroom. By uncovering poten-tially distinct species that have been classified under the samespecies names, our molecular analyses further suggest ahigher current stock diversity in Paramecium than previouslythought.We also would like to emphasize that under-samplingis another major issue in estimating protist diversity. Futurelarge-scale studies based on extensive sampling not only inexotic and remote regions, but also in less frequently sampledareas, will therefore likely improve our understanding of spe-cies richness and diversity.

Keywords Cryptic species . Molecular analysis .

Newmorphospecies . Paramecium biodiversity .

Paramecium buetschlii

Introduction

Knowledge about the distribution and composition of the ge-nus Paramecium has evolved over the last 250 years (see e.g.,Görtz 1988; Fokin 2010/11; Hall and Katz 2011). More than40 descriptions of representatives of this group have beenpublished in the literature for these very well known ciliatessince the work of Müller (1786). However, the number ofvalid morphospecies within the genus Paramecium is muchlower (Table 1) and currently comprises only 18 morphospe-cies including the P. aurelia complex that encompasses 15sibling species.

While some of these species seem to have a cosmopolitandistribution, other Paramecium species are less widely distrib-uted or might even be considered endemic (Fokin et al. 2004;Fokin 2010/11; Przybos et al. 2014). Still, many regions areeither insufficiently sampled or have never been investigated

Electronic supplementary material The online version of this article(doi:10.1007/s13127-015-0207-9) contains supplementary material,which is available to authorized users.

S. Krenek (*) : T. U. BerendonkInstitute of Hydrobiology, Technische Universität Dresden,Zellescher Weg 40, Dresden, Germanye-mail: [email protected]

S. I. FokinDepartment of Biology, Pisa University, Via A. Volta, 4,56126 Pisa, Italy

S. I. Fokin (*)Department of Zoology Invertebrate, St. Petersburg State University,St. Petersburg, Russiae-mail: [email protected]

Org Divers Evol (2015) 15:215–233DOI 10.1007/s13127-015-0207-9

by professional ciliate taxonomists. This suggests that the spe-cies diversity of genus Paramecium may be larger than ourcurrent knowledge suggests. Generally, it is more likely fornew species to be discovered in more remote and thereforerarely investigated areas. This assumption is also supported bythe latest species descriptions for the genus Paramecium.While the last new valid Paramecium spp. from Europe weredescribed in the early twentieth century (Parameciumduboscqui, (Chatton and Brachon 1933); Parameciumnephridiatum, (Gelei 1925, 1938)), the latest new congenershave been discovered in India (Paramecium jenningsi, (Dillerand Earl 1958)), Egypt (Paramecium wichtermani,(Mohammed and Nashed 1968–1969)), Africa (Parameciumafricanum, Paramecium ugandae, Paramecium jankowskii,(Dragesco 1970, 1972)), and China (Parameciumschewiakoffi, (Fokin et al. 2004)). Due to historical reasonspertaining to the development of zoology as a science, thenumber and quality of investigations on ciliate species inEurope are much higher than elsewhere. Therefore, newEuropean Paramecium morphospecies are not expected, as itis very unlikely that taxonomists would have missed suchrecognizable ciliates as paramecia (Fokin et al. 2004).

However, Paramecium chlorelligerum, a largely forgottenspecies described by Kahl (1935) was lately rediscoveredand redescribed from moorland in southern Germany(Kreutz et al. 2012). This indicates the significance of fieldinvestigations in remote and less frequently sampled areas toestimate species diversity and to seek out highly specialized orendemic congeners (Fokin 2010/11; Przybos et al. 2014).

Modern molecular techniques facilitated the redescriptionof P. chlorelligerum and its establishment as a valid morpho-species. Analyses on the small subunit (SSU) rRNA gene(18S-rDNA) as a universal eukaryote marker and/or someother group-specific molecular markers, such as the mitochon-drial cytochrome c oxidase I (COI) or cytochrome b (cob)genes, provide data useful for taxonomic and phylogeneticaims (e.g., Schrallhammer et al. 2006; Greczek-Stachuraet al. 2012; Przybos et al. 2012). These analyses can also beuseful for investigations of genetic, intraspecific, and biogeo-graphic diversity (e.g., Barth et al. 2006; Boscaro et al. 2012;Tarcz et al. 2013). While solely morphological investigationstend to underestimate taxonomic diversity and species rich-ness, molecular markers can unveil cryptic species or molec-ular clades that represent distinct biological entities with onlyminute or no visible phenotypic differences (e.g., Beale andPreer 2008; Funk et al. 2011; Kosakyan et al. 2012). Suchmolecular methods allow taxonomic differentiation withincomplexes of cryptic species that are morphologically andsuperficially seen cosmopolitan species, but actually representmultiple diverse species with smaller geographic ranges and/or different ecological demands (e.g., Skaloud and Rindi2013; Fehlauer-Ale et al. 2014; Baker et al. 1995). To properlyunveil such cryptic diversity with molecular markers, the useof a meaningful range of available sequence data, however, isessential (Boscaro et al. 2012). This suggests that thoroughmolecular data of the whole genus should be investigated, asfar as possible.

Further, there is currently no common convention on howto describe, delimit, and formally name new cryptic species.While often morphology and/or anatomy have to be includedas the primary source of data for species descriptions, someauthors claim, Btaxonomy, as a science, should not be restrict-ed to certain types of data^ (Cook et al. 2010) and that BDNAcan even serve as the type element^ (Kadereit et al. 2012).Since cryptic species share a similar morphology, DNA se-quences represent the only source for alpha taxonomy andspecies delimitation. Tautz et al. (2003) argue that DNA-only descriptions would require a separate naming system,while others refer to clade numbers when studying crypticspeciation (Leliaert et al. 2009), resulting in species com-plexes, such as Tetrahymena pyriformis, that exhibit a highmolecular and ecological diversity (Simon et al. 2008). Onthe other hand, there are also arguments for the formal namingof cryptic species in order to avoid merging highly divergentgenetic lineages (de Leon and Nadler 2010; Kadereit et al.

Table 1 Current valid Paramecium morphospecies with its respectivereferences and subgenus affiliation

Species Subgenus Reference

P. africanum Parameciuma Dragesco 1970

P. aurelia complexb Paramecium Sonneborn 1975,Aufderheide et al. 1983

P. bursaria Chloroparamecium Focke 1836

P. calkinsi Cypriostomum Woodruff 1921

P. caudatum Paramecium Ehrenberg 1838

P. chlorelligerum Viridoparamecium Kahl 1935; Kreutz et al.2012

P. duboscqui Helianter Chatton and Brachon1933

P. jankowskii Parameciuma Dragesco 1972

P. jenningsi Paramecium Diller and Earl 1958

P. multimicronucleatum Paramecium Powers and Mitchell 1910

P. nephridiatum Cypriostomum Gelei 1925

P. polycaryum Cypriostomum Woodruff and Spencer1923

P. pseudotrichium Cypriostomuma Dragesco 1970

P. putrinum Helianter Claparède and Lachmann1858

P. schewiakoffi Paramecium Fokin et al. 2004

P. wichtermani Parameciuma Mohammed and Nashed1968-1969

P. woodruffi Cypriostomum Wenrich 1928

P. ugandae Parameciuma Dragesco 1972

aNo molecular data available for support of affiliationb Currently a complex of 15 morphologically similar sibling species

216 S. Krenek et al.

2012). This, however, would not allow the differentiation ofprovisional cryptic species from true species.

In the present study, we combined classical morphologicalapproaches with thorough molecular analyses to describethree cryptic Paramecium spp. and one apparently new mor-phospecies isolated from freshwater samples of natural habi-tats in Brazil, Hungary, Germany, and Norway. Based on ourfindings, we discuss the current perspective for the biodiver-sity of the genus Paramecium and propose the systematic termEucandidatus to distinguish true biological species or taxo-nomic units from a provisional cryptic species status. Thisterm should be used when describing cryptic fungi, algae,plants, and animals or when using DNA sequences as themain reference for species descriptions.

Materials and methods

Sampling sites, culture conditions, morphological,and morphometric analysis

Paramecia of new (cryptic) species were found in freshwatersamples collected in a pond in the Porto Alegre botanicalgarden (Brazil, 30° 03′ 03″ S, 51° 10′ 45″ W; 2000), from areservoir near by Cranzahl (Germany, 50° 30′ 15″ N, 13° 00′01″ E; 2005) and a pond close to Lake Balaton (Hungary, 46°54′ 31″ N, 17° 53′ 13″ E; 2000). A new morphospecies ofParameciumwas found in a slowly running freshwater streamnear Hakadal (Norway, 2005; 60° 07′ 10″ N, 10° 49′ 56″ E).Established monoclonal strains or populations of the new(cryptic) species, subsequently named as BPA1-1–BPA1-3(Brazil, Porto Alegre); GO1-1–GO1-3 (Germany, Cranzahl);HB2-7–HB2-9 (Hungary, Balaton), and NO6 (Norway,Hakadal) were cultivated at 20 °C on a lettuce or Cerophylmedium inoculated with Enterobacter aerogenes and/or on arice grain medium. Cells from these cultures were used forobservations, measurements and genetic analyses.

Several clones of different Paramecium spp. of variousorigins were also used for comparison with the cortical patternof the new species as well as for molecular analyses (seeTable S1, S2). Single cells of P. chlorelligerum used forDNA analyses were collected from samples of a Sphagnumbog near Constance (Germany; 2012) kindly provided by M.Kreutz. Morphometric data obtained previously for the mainParamecium spp. (Fokin and Chivilev 1999, 2000) were usedas well.

Living cells were immobilized for observation with thehelp of a special immobilization device (Skovorodkin 1990).For studies on conjugating cells, mating reactive cells wereobtained according to Miyake (1968) and mixed together indepression slides (2 ml) at 20 °C or by repeatedly washingthese cultures with SBM buffer or Dryl’s solution. Some spec-imens were fixed in Bouin’s fluid (Bouin 1897) and stained by

the Feulgen procedure, while others were impregnated by thesilver nitrate procedure after Champy’s fixation (Corliss 1953;Foissner 1991). Living and fixed cells were examined andphotographed by bright field and Nomarski interference mi-croscopy using an Orthoplan Leitz microscope (Leitz,Germany) at ×300–1.250 magnifications with a digital cameraCanon S45.

For the morphometric comparison of differentParamecium species, 10–25 properly oriented and well-impregnated specimens were selected from each stock of theherein investigated Paramecium spp. and individually mea-sured. The following measurements were made using thesame microscopes equipped with a micrometer (Carl Zeiss,Germany): cell length, cell width, buccal cavity (BC) length,buccal overture (BO) position, cytoproct (C) position, positionof pores of contractile vacuoles (PCV). The average ratio data(parameter/cell length) for each character were used for com-parative analyses. Additional morphometric data of 12 previ-ously investigatedParamecium species were used to constructa tree based on Euclidean distances (Fokin and Chivilev 1999,2000).

Graphical representation of the similarity matrix was per-formed with multidimensional scaling analysis (MDS;Kruskal and Wish 1978). Cluster analyses were performedusing the unweighted pair-group methods with arithmetic av-erages (UPGMA; Sneath and Sokal 1973) using the programpackage CLASS developed by the Research Institute forNature Conservation of the Arctic and the North (RINCAN),Russia, and the program STATISTICA for Windows(StatSoft, Inc.).

The morphobiological features (i.e., type of micronucleus[MI], type of contractile vacuole [CV] and swimming behav-ior) were represented as a binary presence or absence corre-sponding to 1 or 0, respectively. In such a way, the qualitativefeatures can be represented as quantitative features. This ma-trix was used for measuring morphobiological similarity be-tween the species (as done for morphometric data).

DNA isolation, PCR amplification, and sequencing

For molecular analyses, DNA was extracted from either sev-eral cells of clonal cultures (new species) or from single cells(P. chlorelligerum) using the Chelex®100 (Bio-RadLaboratories, Inc., Germany) method, as described previously(Barth et al. 2006), or a modified DTAB procedure(Gustincich et al. 1991). The resulting DNA solutions weresubsequently used for PCR reactions amplifying the nuclearencoded small subunit (SSU) ribosomal RNA gene (18S-rDNA) and the mitochondrial cytochrome c oxidase I (COI)gene. The 18S-rDNA amplification was performed with theuniversal eukaryotic primers 18S-EukA and 18S-EukB(Medlin et al. 1988). Each PCR reaction contained either2.5–5 μl of Chelex®100 or 1 μl of DTAB extracted DNA as

New Paramecium congeners shape our view on its biodiversity 217

well as 5 pmol of each primer, 1 U Taq-polymerase (GoTaq®,Promega, Germany), 1× PCR buffer (Colorless GoTaq®Reaction Buffer, Promega, Germany) with 3 mM MgCl2 and200 μM dNTPs in a total volume of 25 μl using molecularbiology grade water (Nuclease-Free Water, Promega,Germany) to adjust the volume. PCR conditions were as fol-lows: 5 min initial denaturation (95 °C); 35 cycles of 1 min at95 °C, 1 min at 48 °C and 2min at 72 °C; and a final extensionstep of 5 min (72 °C).

The COI gene sequences were amplified with the primersF199dT-B and R1143dT (Strüder-Kypke and Lynn 2010) underthe following conditions: 4 min initial denaturation (94 °C);5 cycles of 45 s at 94 °C, 75 s at 45 °C and 90 s at 72 °C;followed by 30 cycles of 45 s at 94 °C, 75 s at 55 °C and 90 s at72 °C and a final extension step of 8 min (72 °C). Here, eachPCR reaction contained also either 2.5–5 μl of Chelex®100 or1μl of DTAB extractedDNA aswell as 20 pmol of each primer,1.25 U Taq-polymerase (JumpStart™, Sigma-Aldrich®,Germany), 1× PCR buffer (JumpStart™ reaction buffer,Sigma-Aldrich®, Germany) with 3 mM MgCl2, 200 μMdNTPs and 3 μl BSA (10 mg/μl), for a total volume of 25 μladjusted with nuclease-free water (Promega, Germany).

Purified PCR products (NucleoSpin® Extract II Kit,Macherey-Nagel, Germany) were sent for direct sequencingto EurofinsMWGOperon (Ebersberg, Germany). Sequencingof the 18S-rDNA fragments was performed with the Eukarya-specific PCR primers and the internal primers 18SF590,18SR600, and 18SF1290 (Wylezich et al. 2002). PrimersM13uni(−21) and M13reverse (Messing 1983) were used forsequencing the COI amplification products in both directions.

Comparative sequence analyses and phylogeneticreconstruction

Sequence data were visualized and assembled using the pro-gram Geneious Pro v.6.1.7 (Biomatters Ltd., New Zealand)and compared with the non-redundant sequence databaseusing NCBI-BLAST (Altschul et al. 1990). Calculation ofsequence statistics and estimation of pairwise genetic dis-tances or sequence identities (see supplementary Table S1and S2) were carried out with MEGA6 (Tamura et al. 2013)based on the same alignments used for phylogenetic recon-structions (see below). All complete sequences were depositedin the NCBI GenBank database under the GenBank IDsKM091234–KM091241. The 18S-rRNA gene sequence ofBEucandidatus P. brazilianum^ was already published underAccession No. AJ548822.

The phylogenetic placement of the investigatedParamecium species was assessed using a two-locusbarcoding procedure with 18S-rDNA as the universal eukary-otic barcode and COI as a group-specific barcode (Pawlowskiet al. 2012). The 18S-rDNA sequences were aligned to allother Paramecium SSU rRNA gene sequences available in

NCBI GenBank (effective May 2014) that were nearly fulllength and not identical. Sequences of other Peniculida (namelyApofrontonia dohrni, Frontonia spp., Lembadion bullinum,Stokesia vernalis, and Urocentrum turbo) were included asoutgroup. Multiple sequence alignment was carried out usingthe SILVA INcremental Aligner (SINA) v1.2.11 (Pruesse et al.2012) based on the human-curated SILVA 115 SEED align-ment. The resulting alignment was automatically trimmed usingthe automated1 algorithm of TrimAl v.1.3 (Capella-Gutiérrezet al. 2009) via the Phylemon 2.0 online suite (Sánchez et al.2011). Manual refining and trimming at either end of the se-quences was performed to produce comparable sequencelengths. The final alignment comprised 49 taxa with an overalllength of 1640 characters including 0.5 % internal gaps.

The COI alignment was constructed using Muscle (Edgar2004) as implemented in Geneious Pro v.6.1.7 (BiomattersLtd., New Zealand) based on the predicted amino acid trans-lations. Published COI sequences of Paramecium spp. longerthan 600 bp were integrated into the analyses (downloadedfrom NCBI GenBank by May 2014, excluding sequences ofa recent study by Zhao et al. (2013) that investigated COIheteroplasmy in Paramecium). Sequences of other ciliates(namely Colpidium striatum, Cyclidium glaucoma, Halteriagrandinella, L. bullinum, Tetrahymena thermophila andU. turbo) were included as outgroup taxa. This alignmentcontained 237 taxa and was automatically trimmed usingtrimAl v.1.3 (cons=60, gt=0.4, st=0.01, w=3; Capella-Gutiérrez et al. 2009) through the Phylemon webserver(Sánchez et al. 2011) in order to optimize the signal-to-noiseratio. The final alignment had an overall length of 725 char-acters with 0.8 % internal gaps and allowing terminal gaps.

Maximum Likelihood (ML) analyses and BayesianInference (BI) methods were conducted for phylogenies withboth 18S-rDNA and COI alignments. The programjModelTest v.2.0 (Darriba et al. 2012; Guindon and Gascuel2003) implemented by the Phylemon 2.0 online suite(Sánchez et al. 2011) was used to select the most appropriateDNA substitution model for both the 18S-rRNA and COIgene sets required for ML and BI analyses. For both data sets,the General-Time-Reversible (GTR) model considering in-variant sites (I) and gamma-distributed substitution ratesamong sites (Γ) was reported as the best-fit according toAkaike and Bayesian information criteria (AIC and BIC).

ML analyses were conducted using RAxML v.7.6.3(Stamatakis 2006; Stamatakis et al. 2008) with 5000 rapidbootstrap replicates and a simultaneous likelihood search forthe best-scoring ML tree using the GTRGAMMA + I model.BI analyses were performed with MrBayes v.3.2.1 (Ronquistand Huelsenbeck 2003) using the GTR + I + Γ model. Threeindependent runs were carried out for 5,000,000 generationswith a tree sampled every 100 generations. Each run used fourchains, one cold and three heated. From the sampled trees, thefirst 25 % were discarded as a burn-in to ensure stable


likelihood values. The remaining trees of all sampled runswere used to compute the BI consensus tree and the posteriorprobabilities of all branches. The fast hybrid versions ofRAxML and MrBayes were run on the CIPRES ScienceGateway v.3.3 server (Miller et al. 2011). The resulting phy-logenetic trees were visualized and edited for publicationusing FigTree v.1.4.0 (Rambaut 2012) and Corel Draw X6(Corel Corporation Ltd.).

Results

General morphology of new Paramecium spp.

Paramecium buetschlii sp. nov.

Size in vivo was up to 200×55 μm, usually 190×52 μm(Figs. 1–3). The average size of fixed and impregnated cellswas 174.5±7.0×48.8±4.7 μm (Figs. 4, 5). The number ofcilia rows amounted to 70–85 (77.5±3.6 on average). Thebuccal cavity (BC) size was 31.1±1.6 μm which is 0.18 ofthe body length only (Table 2). The buccal overture (BO),ellipsoidal in shape, was located a bit forward to the anteriorend from the cell’s equator and has a normal composition ofbuccal ciliature–quadrilus, two peniculi and endoral mem-brane (Figs. 4, 6). Two contractile vacuoles (CV) had usuallytwo and sometimes three pores (PCV) each, but no collectingcanals (Figs. 1, 2, 5, 7, 14–16). The cytoproct started relativelyfar from BO, close to the posterior end of the cell (Figs. 3, 4,8). The cortex contained numerous uniformly distributedtrichocysts (Figs. 9, 12, 13) that were classically spindle-shaped and about 5–6 μm long in resting condition. The el-lipsoidal or oval macronucleus (MA) (21.1±2.0×41.4±

2.9 μm on average) was situated close to the cell’s equator(Table 2, Figs. 1, 9, 10). A single ellipsoidal micronucleus(MI) had sizes of 2.5–3.2×5.5–6.0 μm (2.9±0.9×5.8±1.8 μm on average) and was usually located close to the MAor in its depression (Table 2, Figs. 1, 9, 10, 12). This singleMIis of the compactmorphological type, but the chromatin of thenucleus always looked fine and homogenous without anyachromatin cape (Figs. 9, 10). The general cell form was in-termediate, between the classical cigar shape and those of

Fig. 1–8 General shape and cortex construction of P. buetschlii sp. nov.1–2Ventral 1 and dorsal 2 views of the same cell. Ampoules of contractilevacuoles (CV) and its pores (wedge-tailed arrows in Fig. 2) as well as themacronucleus (MA) and the micronucleus (large arrow in Fig. 1) arevisible. 3 Deciliated cell (found by chance) from the ventral view.Position of oral aperture (OA), anterial suture (arrowhead) andcytoproct (C) are shown. 4–8 Impregnated cells: ventral 4 and dorsal 5side of different cells and its details; quadrulus (Q), cytoproct (C) andpores of contractile vacuole (PCV). 6 Buccal overture with buccalciliature: endoral membrane (EM) and quadrulus (Q). 7 Two pores ofthe same contractile vacuole. 8 Cytoproct region. 1–3 Living cells, DICcontrast. 4–8 Silver nitrate impregnation. Bars 50μm (1–3), 25μm (4, 5),10 μm (6, 8), 5 μm (7)

Table 2 Morphometric data of Paramecium buetschlii sp. nov.

Characteristics x SD Min Max CV n

Body, length 174.5 7.0 165 185 4.0 24

Body, width 48.8 4.7 45 53 9.6 24

Buccal cavity, length 31.1 1.6 28 34 5.1 21

Macronucleus, length 41.4 2.9 37 45 7.0 20

Macronucleus, width 21.1 2.0 18 24 9.4 20

Somatic ciliary rows, number 77.5 3.6 70 85 4.6 10

Excretory pores, number 2.2 0.52 1 3 23.6 20

Micronucleus, number 1 0 1 1 0 20

Micronucleus, length 5.8 1.6 2.5 6.0 27.6 20

Micronucleus, width 2.9 0.9 2.5 3.2 31.0 20

Data based on Chatton–Lwoff silver impregnated cells. Macro- andmicronuclei were measured from Feulgen-stained ciliates. Measurementsin micrometers

x arithmeticmean, SD standard deviation,Minminimum,Maxmaximum,CV coefficient of variation in percentage, n number of cells investigated


P. woodruffi, which is posteriorly rounded and to some extentdorsoventrally compressed (Figs. 1–5). The width-to-lengthratio was 0.28. When undisturbed, the cells were slowly mov-ing or gliding along substrate using typical cilia beating.During the normal swimming procedure, the cells rotated bothclockwise and counter-clockwise (Table 2, Figs. 1–8, 9–16,and 17–22).

Using artificial treatment of the culture with KCl, acrifla-vine and Ca-poor conditions (Miyake 1968), conjugationcould be induced (Fig. 17). In the prophase of the first meioticdivision, the MI does not manifest a clear parachute stage;however, this species apparently has no crescent MI stage aswell (Fig. 18). Exchange of pronuclei started before the oldMA break down. The old MA became fragmented only afterthe first synkaryon division. The number of oldMA fragmentswas always quite small (15–25) and of diverse dimensions andforms (10–25 μm; Figs. 20–22). The three consecutivesynkaryon divisions resulted in four MA anlagen and four

further MI (Figs. 19–22), but only one of the generative nucleisurvived in progeny.

Despite many attempts, we did not succeeded in establish-ing stable monoclonal cultures, but the ciliate could be main-tained as a multi-clonal population on rice grain medium.Other food sources such as lettuce medium inoculated withE. aerogenes or medium with the freshwater micro-algaChlorogonium sp. never served as a proper medium forP. buetschlii sp. nov. Unfortunately, the stock culture diedoff after 2 years of maintenance under laboratory conditions.

BEucandidatus Paramecium germanicum^

Size in vivo was up to 250×90 μm, usually 210×70 μm(Fig. 23). The average size of fixed and impregnated cellswas 190.4±13.4×60.6±4.0 μm. The number of cilia rows

Fig. 9–16 Morphological features of P. buetschlii sp. nov. 9–12 Nuclearapparatus and buccal region of the ciliate. 9 Buccal cavity and part ofmacronucleus (MA) with micronucleus (MI). Trichocysts along of cortexare also visible. 10 Nuclear apparatus. 11 Buccal overture. 12 Buccalciliature and nuclear apparatus. 13 Posterior end of the ciliate with nu-merous trichocysts. 14 Pores of CV. 15–16) CV dynamic. Systole (15)and diastole (16). 9, 11–16) Living cells. DIC contrast. 10 Feulgen-stained nuclear apparatus. Bars 10 μm (9–13), 5 μm (14–16)

Fig. 17–22 Nuclear reorganization in P. buetschlii sp. nov. duringconjugation. 17 Early micronuclear migration. 18 Prophase of the firstmeiotic division of the micronucleus. 19 The third synkaryon division inexconjugant. Four spindles of synkaryon derivates are marked by wedge-tailed arrows; three of the four products indicated by large arrows(micronuclear anlagen) and by arrowheads (macronuclear anlagen).20–21 Exconjugant cell with eight products of synkaryon division andfragments of the old macronucleus (bold arrow) under differentmagnification. 22 Exconjugant cell with more developed macronuclearanlagen. Bars 15 μm (17), 10 μm (18, 19), 30 μm (20), 15 μm (21, 22)


Table 3 Morphometric data of the three new described cryptic species of Paramecium

Characteristics Statistics BEucandidatus P. germanicum^ BEucandidatus P. brazilianum^ BEucandidatus P. hungarianum^

Body, length x 190.45 202.1 84.9

SD 13.4 12.6 6.6

Min 185.0 182.5 72

Max 210.0 225.0 98

CV 7.0 6.2 7.8

n 20 25 20

Body, width x 60.65 57.6 37.1

SD 4.0 5.2 3.4

Min 49.0 45.1 30

Max 69 64.8 43

CV 6.6 9.0 9.2

n 20 25 20

Buccal cavity, length x 37.8 39.6 18.5

SD 1.5 3.5 1.4

Min 34.0 35.0 16

Max 40.0 42.0 21

CV 4.0 9.3 7.3

n 20 20 15

Macronucleus, length x 55.45 48.1 23.0a 23.4

SD 5.4 2.0 2.5a 3.5

Min 45 45 12a 15

Max 68 52 25a 28

CV 9.8 3.6 12.6a 15.0

n 20 20 50a 15

Macronucleus, width x 42.6 30.2 17.2

SD 4.9 2.7 1.1

Min 35 24 14

Max 50 35 18

CV 11.4 9.1 6.1

n 20 20 15

Somatic ciliary rows, number x 86.8 93.0 56.9

SD 4.6 5.5 4.8

Min 80 80 44

Max 95 100 65

CV 5.3 5.9 8.4

n 16 15 20

Excretory pores, number x 1 1.1 1

SD 0 0.3 0

Min 1 1 1

Max 1 2 1

CV 0 27.3 0

n 16 20 20

Micronucleus, number x 2.7 1.48 3

SD 0.4 0.5 0.7

Min 2 1 2

Max 4 2 4

CV 21.9 33.8 23.3

n 15 20 20

Micronucleus, size x 1.8 3.4 1.7


was 80–95 (87.0±4.6 on average). The BC size was 37.8±1.5 μm (ratio of BC size to the cell length, 0.20). Location ofthe BO was close to the cell’s equator (Fig. 24). Two contrac-tile vacuoles had always 1 PCV each and a number ofcollecting canals, 7–10 (most often 9). The cytoproct wassituated in the middle between BO and the posterior end ofthe ciliate. The cortex contained numerous typical trichocysts.The ellipsoidal MA (42.6±4.9×55.45±5.4 μm on average)was situated close to the cell’s equator. A number of roundedMI (2–4) with sizes of 1.6–2.0 μm (1.8±0.2 μm on average)were usually located around and close to the MA (Table 3,Fig. 25) and belong to the compact morphological type(Figs. 25, 26). The general cell form had the classical cigarshape; however, cultured cells usually were rather fat, with awidth-to-length ratio of 0.39 (Fig. 23). During the normalswimming procedure, the ciliate always rotated counter-clockwise (left spiral swimmer) (Table 3, Figs. 23–26).

BEucandidatus Paramecium brazilianum^

Size in vivo was up to 270×80 μm, usually 220×65 μm. Theaverage size of the cells at fixed and impregnated conditionwas 202.1±12.6×57.6±5.2 μm (Fig. 27). The number of ciliarows was 80–100 (93.0±5.5 on average). The BC size was39.6±3.5 μm, with a BC size to cell length ratio of 0.195(Table 3). The BO was situated very near to the cell’s equator,but beneath it. Two contractile vacuoles usually had one PCVeach, but sometimes one of the CV had two pores (4 % ofindividuals). The number of collecting canals in CV was fiveto nine (most often seven). The cytoproct was situated in themiddle between the BO and the posterior end of the ciliate(Fig. 27). The cortex contained quite numerous, typical tricho-cysts. Sometimes, an ellipsoidal MA (30.2±2.7×48.1±2.0 μm on average) was situated close to the cell’s equator,but very often (up to 40 % of cells) it was present by tworounded pieces of equal or slightly different size (12–25 μm;Table 3, Figs. 28, 29). One or two rounded MI with sizes of3.0–4.0 μm (3.4±0.4 μm on average) were usually locatednear by the MA. The morphological type of the MI resembled

the vesicular type, but was not very typical (Fig. 29). Thegeneral cell form had the classical cigar shape, with a width-to-length ratio of 0.27 (Fig. 27). Brief observations of thenuclear reorganization process for this species (data are not

Fig. 23–26 Morphology of BEucandidatus P. germanicum^. 23 Livingcell with visible food vacuoles (FV) and macronucleus (MA). DICcontrast. 24Ventral view of silver nitrate-impregnated cell with cytoproct(C). 25Nuclear apparatus of the Feulgen-stained cell with indications formicronuclei (small arrows). 26 Nuclear apparatus. Feulgen-stained cell,large magnification. Bars 40 μm (23), 35 μm (24), 4.5 μm (25), 6 μm(26)

Table 3 (continued)

Characteristics Statistics BEucandidatus P. germanicum^ BEucandidatus P. brazilianum^ BEucandidatus P. hungarianum^

SD 0.2 0.4 0.1

Min 1.6 3.0 1.5

Max 2.0 4.0 1.9

CV 11.1 11.7 5.9

n 15 20 20

Data based on Chatton–Lwoff silver impregnated cells. Macro- and micronuclei were measured from Feulgen-stained ciliates. Measurements inmicrometers

x arithmetic mean, SD standard deviation, Min minimum, Max maximum, CV coefficient of variation in percentage, n number of cells investigatedaMorphometry of the macronuclear fragments


shown) indicate some extraMA anlagen in exconjugants. Thiscould be a reason for the occurrence of bi-macronuclear cells.During the typical swimming procedure, the ciliate alwaysrotated counter-clockwise (left spiral swimmer) (Table 3,Figs. 27–29).

BEucandidatus Paramecium hungarianum^

Size in vivo was up to 110×50 μm, usually 95×45 μm(Fig. 30). The average size of fixed and impregnated cellswas 84.9±6.6×37.1±3.4 μm. The number of cilia rowsamounted to 44–65 (56.9±4.8 on average). The BC size was18.5±1.4 μm (Table 3) with a BC size to cell length ratio of0.22. The location of the BOwas shifted from the cell’s equatorforward to the anterior end of the ciliate (occurring at about45 % of the cell’s length). Two contractile vacuoles alwayshad one PCV each (Figs. 32, 33). The number of collectingcanals in CV was six to eight (most often seven). As character-istic for Paramecium, the cortex contained numerous typical

trichocysts with a homogeneous distribution. The single ovoidor ellipsoidal MA (17.2±1.1×23.4±3.5 μm on average) wasusually situated close to the cell’s equator. Numerous roundishMI (2–4) with sizes of 1.5–1.9 μm (1.7±0.1 μm on average)were usually located close to and around the MA (Table 3,Figs. 30, 31). The morphological type of the MI was vesicular.The general cell form was typical woodruffi shaped with awidth-to-length ratio of 0.45 (Figs. 20, 32). During the swim-ming procedure, the ciliate always rotated counter-clockwise(left spiral swimmer) (Table 3, Figs. 30–34).

Multivariate morphometric analysis

Comparisons of mean values of morphometric data and somemorphobiological features are represented as a dendrogramand topogram according to UPGMA and MDS methods(Fig. 35a, b). For these analyses, the same set of charactersobtained for other species previously investigated (12

Fig. 27–29 Morphology of BEucandidatus P. brazilianum^. 27 Ventralview of silver nitrate-impregnated cell. 28 General view of the Feulgen-stained cell with macronucleus (MA) fragmentation and two surroundingmicronuclei (indicated by arrows). 29 Nuclear apparatus of the cell withfragmented MA and surrounding micronuclei (arrows) in higher magni-fication. Bars 30 μm (27, 28), 15 μm (29)

Fig. 30–34 Morphology of BEucandidatus P. hungarianum^. 30 Livingciliate with visible contractile vacuoles (CV) and macronucleus (MA).DIC contrast. 31–32 Ventral (31) and dorsal (32) views with indicationsfor cytoproct (C) and pores of the contractile vacuoles (PCV). Silvernitrate impregnation. 33 Nuclear apparatus of the ciliate with MA andsurrounding micronuclei (indicated by arrows). Feulgen-stained cell. 34Nuclear apparatus with MA and MI (arrows). Large magnification. Bars15 μm (30), 13 μm (31, 32), 20 μm (33), 10 μm (34)


morphospecies of the Paramecium genus) were used togetherwith the data derived from the herein described newParamecium species. The addition of the four new represen-tatives does not change the previous tree topology much andfits with major morphological characters of the herein de-scribed paramecia (Fig. 35a). P. buetschlii sp. nov. clusterswithin the woodruffi subgroup (subgenus Cypriostomum)but is separated by a quite large Euclidian distance from therest of the species. Here,P. woodruffiwas the nearest congener

to P. buetschlii sp. nov. The same topology was indicated bythe MDS analysis (Fig. 35b).

According to their morphometric and morphobiological fea-tures, the other three investigated Paramecium species, namelyBEucandidatus Paramecium brazilianum^, BEucandidatusParamecium germanicum^ and BEucandidatus Parameciumhungarianum^, can be associated to Parameciummultimicronucleatum , Paramecium caudatum , andParamecium polycaryum, respectively (Fig. 35a, b). While the-se three Paramecium species show also rather high Euclidiandistances to their nearest relatives, they cannot be clearly sepa-rated from congeners based on general morphology. For exam-ple, while BEucandidatus P. germanicum^ is closer related toP. caudatum and P. Baurelia^ according to MDS and UPGMAanalyses, it rather resembles P. multimicronucleatum based onits general morphology. However, it could only be distin-guished from P. multimicronucleatum by the morphologicaltype of MI (compact vs vesicular, respectively), which is notan easy character to perceive because of its small size and therequirement of high quality staining techniques.

Sequence comparison and molecular phylogeny of newParamecium spp.

Comparative sequence analysis and phylogenetic reconstruc-tions based on 18S-rRNA and mitochondrial COI gene se-quences of the herein described new Paramecium spp. gaveus trees with different topology (Figs. 36, 37).

According to the 18S-rDNA analyses, P. buetschlii sp. nov.represents a sister taxon to Paramecium putrinum with a se-quence similarity of 95.6 % (Fig. 36, Table S1). The averagegenetic distance to P. duboscqui, another member of theHelianter subgenus like P. putrinum, is comparably low(0.048 vs 0.051, cf. Table S1) indicating that P. buetschlii sp.nov. belongs to theHelianter subgenus. However, as shown inFig. 36 the Helianter subgenus does not form a monophyleticclade within our analyses. While the relationship ofP. buetschlii sp. nov. to P. putrinum is fairly supported bythe tree reconstruction methods used, the two more basalbranch nodes are not supporter by either method. The othernew cryptic species, namely BEucandidatus P. brazilianum^and BEucandidatus P. germanicum^ belong to theParamecium subgenus , whi le BEucandidatus P.hungarianum^ appears to be a member of the Cypriostomumsubgenus (Fig. 36). Here, BEucandidatus P. germanicum^ isinferred to be a sister species to P. caudatum with a sequencesimilarity of 97.8 %. BEucandidatus P. brazilianum^, on theo t h e r h a n d , c l u s t e r s w i t h i n o n e o f t h e t w oP. multimicronucleatum clades. However, it should be stressedthat both P. multimicronucleatum clusters exhibit a compara-tively large averaged genetic distance of 0.021 with an aver-aged sequence similarity of only 98.0 %. BEucandidatus P.hungarianum^, however, seems to be a sister species to

Fig. 35 Dendrogram (a) for hierarchical clustering (UPGMA) andtopogram (b) for non-metric multidimensional scaling (MDS) ofmorpho-metric and morphobiological characteristics of 16 Paramecium species.AU P. aurelia, BU P. bursaria, CA P. caudatum, CL P. calkinsi, DU P.duboscqui, JE P. jenningsi, MU P. multimicronucleatum, NR P.nephridiatum, PB BEucandidatus P. brazilianum^, PG BEucandidatus P.germanicum^, PH BEucandidatus P. hungarianum^, PN P. buetschlii sp.nov., PO P. polycaryum, PU P. putrinum, SH P. schewiakoffi, WO P.woodruffi


P. woodruffi and P. nephridiatum with a comparatively highsequence similarity of 99.5 % each.

Using the mitochondrial COI gene as a molecular marker,the position of P. buetschlii sp. nov. is the most basal in theParamecium genus with representatives of P. putrinum andP. duboscqui as closest relatives (Fig. 37). Interestingly, theinclusion of P. buetschlii sp. nov. and the other new crypticParamecium species into the COI dataset dramaticallychanged the relationships between the different Parameciumsubgenera, resulting in Chloroparamecium appearing moreclosely related to Paramecium than to Helianter (data notshown, but cf. Fig. 4 in Boscaro et al. 2012). According tothe COI tree topology, BEucandidatus P. germanicum^ isplaced at a very basal position in the Paramecium subgenus(subgroup Bcaudatum^) with three P. multimicronucleatumstrains as apparently closest relatives (averaged genetic

distance 0.2), which cluster outside of all other so farhaplotyped P. multimicronucleatum strains (see Fig. 37). Theposition of BEucandidatus P. hungarianum^, based on its COIgene sequence, is similar to its 18S-rDNA phylogenetic posi-tion within the Cypriostomum subgenus with a close relation-ship to P. woodruffi (strain BB-5, sequence similarity 89.2 %).However, as shown in Fig. 37, P. calkinsi does not form amonophyle t i c c lade and the c loses t r e l a t ive toBEucandidatus P. hungarianum^ is actually P. calkinsi strainBOB130-7 from Olkhon Island in Lake Baikal (Przybos et al.2013) with a sequence similarity of 94.8 %.

Differential diagnosis of P. buetschlii sp. nov.

1. Size in vivo is up to 200×55 μm, usually 190×52 μm(fixed cell, 175×49 μm). Number of kineties is 70 to 85.

Paramecium

Cypriostomum

Helianter

Chloroparamecium

Viridoparamecium

0.05

“Eucandidatus Paramecium hungarianum”

Paramecium primaurelia JZ1-4


Paramecium multimicronucleatum FT2

Paramecium caudatum FT6

Frontonia lynni

Paramecium multimicronucleatum TH105

Paramecium putrinum PG-5

Paramecium tetraurelia 51

Paramecium sp. para200

Apofrontonia dohrni

Paramecium duboscqui IG2-1

Paramecium polycaryum TR-5

Paramecium multimicronucleatum YM25

“Eucandidatus Paramecium brazilianum”

Paramecium bursaria PB-SW1

Paramecium sp. paraGC

Paramecium bursaria OK1

Stokesia vernalis

Paramecium duboscqui BB8

Paramecium schewiakoffi Ch1

Paramecium bursaria CCAP 1660/10

Frontonia leucas

Paramecium caudatum Isn4

Paramecium nephridiatum BB3-9

Paramecium multimicronucleatum LSA

Frontonia mengi

“Eucandidatus Paramecium germanicum”

Paramecium bursaria CCAP 1660/12

Paramecium jenningsi FT3

Paramecium duboscqui TuAI-2

Paramecium chlorelligerum Constance

Paramecium multimicronucleatum Pm

Paramecium multimicronucleatum FT1

Paramecium multimicronucleatum FT5Paramecium multimicronucleatum FT4

Frontonia vernalis

Paramecium chlorelligerum SC1

Urocentrum turbo

Paramecium woodruffi BB-5

Lembadion bullinum

Paramecium jenningsi Jap1-1

Paramecium duboscqui strain Ku4-8

Frontonia magna

Frontonia tchibisovae

Paramecium calkinsi GN5-1

Paramecium caudatum FT8Paramecium caudatum FT7

Frontonia didieri

0.90/67

0.65/58

0.98/89

0.68/58

1.00/88

0.99/97

0.48/--

0.97/71

1.00/64

0.88/82

1.00/96

0.92/71

1.00/89

1.00/94

1.00/93

1.00/98

0.35--0.59/66

0.59/--

0.86/43

0.25/--

*

** *

*

*

*

*

*

*

*

*

*

*

Fig. 36 SSU rDNA tree topology. Phylogenetic reconstruction of thegenus Paramecium based on 18S-rDNA sequences inferred by BayesianInference analysis. The alignment contained 49 taxa and 1640 sites in-cluding gaps. Sequences of new or cryptic species characterized withinthis study are shown in boldface. Sequences of other Peniculida served asoutgroup. Numbers at nodes (occasionally indicated by an arrow) repre-sent support values for the Bayesian Inference andMaximum Likelihood

analyses, respectively, while asterisks denote full support (1.00/100) fromboth tree reconstruction methods. The scale bar corresponds to 0.05nucleotide substitutions per site. Squared brackets to the right of the treedesignate the five different subgenera Chloroparamecium ,Cypriostomum, Helianter, Paramecium, and Viridoparamecium as pro-posed previously (Fokin et al. 2004; Kreutz et al. 2012)


2. Body has slightly sharp posterior end; the anterior part ofbody is evenly narrowed and rounded. The form of thecell is intermediate between the classical cigar shape andthe woodruffi type. The oval buccal overture is situated abit forward from the cell equator.

3. Buccal cavity is relatively short, only 0.18 of the celllength.

4. Two CV without collecting canals are around the centralampulla. Each CVopens in dorsal pellicle by 2 or 3 CVPeach.

5. One MI, ellipsoidal, quite large with 6×3 μm, is situatedalways near the MA or in its cavity. It is of the compacttype, but chromatin of the nucleus is very homogenouswithout apochromatin cape, as occurs in P. caudatum.

6. In prophase of the first meiotic division, the MI does notmanifest a clear parachute or crescent stage.

7. The ciliate can rotate clockwise and counter-clockwise.

According to two molecular markers investigated (18S-rDNA and mtCOI), this species is a member of the subgenusHelianter, as a sister species of P. putrinum (18S-rDNA, seeFig. 36) or at the very basal position of the genus (COI, seeFig. 37). Morphometry, however, indicates clustering of thisspecies within the Cypriostomum subgenus (the woodruffisubgroup, see Fig. 35a).

Etymology It is named in honour of one of the founders of theEuropean protistology, Professor at the University ofHeidelberg, Johann Adam Otto Bütschli (1848–1920).

Type location Oslo city suburb, Norway, a slowly runningfreshwater stream near Hakadal (60° 7′ 10″ N, 10° 49′ 56″ E).

Type slides Four slides (one holotype, three paratypes) withsilver nitrate-impregnated specimens have been deposited inthe collection of microscopical slides of the Protistology andExperimental Zoology Laboratory, Department of ZoologyInvertebrate, St. Petersburg State University, Russia.

Type culture Unfortunately, living stock cultures are not pres-ent any longer, due to maintaining difficulties and extinctionof the stock culture.

Sequence availability The nucleotide sequences of the smallsubunit rRNA and the mitochondrial cytochrome c oxidase Igene were deposited in the NCBI GenBank database underaccession numbers KM091234 and KM091238, respectively.

Discussion

Given that Paramecium species have been investigated forover 250 years and are present in many school curricula in

Europe, it is surprising that the current biodiversity of thegenus is not fully known or assessed. This is illustrated byour description of a new morphospecies, P. buetschlii sp.nov., from a freshwater pool in Norway as well as by ourinvestigation of three Paramecium sp. stocks from Germany,Hungary, and Brazil, which could at least be treated as crypticspecies according to the results of combined morphologicaland molecular methods. This already illustrates that under-sampling and/or insufficient species identification seems tobe a major issue in estimating species diversity, probably formany micro-eukaryotes. It also re-emphasizes that combiningtraditional morphological approaches with comprehensive ge-netic analyses is necessary to uncover cryptic, but also truebiological species.

New morphospecies P. buetschlii sp. nov.

Among the four herein described and discussed Parameciumspp., P. buetschlii sp. nov. can be clearly accepted as a newvalid morphospecies. The set of morphological features of thisciliate unmistakably separates it from the rest of all knownparamecia (Figs. 1–8, 9–16, 17–22, and 38). It has unusualcombinations of morphological characters. While the cell sizeof P. buetschlii sp. nov. is comparable to members of theParamecium subgenus (almost the same as P. jenningsi,P. schewiakoffi or some P. caudatum stocks (Fig. 38; Fokinet al. 2004; Fokin and Chivilev 2000; 1999)), the general cellshape did not exactly fit the cigar-shaped form typical forParamecium subgenus representatives. Further, the micronu-cleus (MI) structure is different from all of the above-mentioned species. It is quite large and belongs to the compactmorphological type, but has no achromatin cape, as hasP. caudatum, and neither looks like the chromosomal type of

�Fig. 37 Mitochondrial COI tree topology. Phylogenetic tree of the genusParamecium based on mtCOI gene sequences inferred by BayesianInference analysis. The alignment contained 237 taxa and 725 sitesincluding gaps. Sequences of new or cryptic species characterizedwithin this study are shown in boldface. The root was placed betweenParamecium and other ciliate species. Numbers at nodes (occasionallyindicated by an arrow) represent support values for the BayesianInference and Maximum-Likelihood analyses, respectively. Asterisks atnodes denote full support (1.00/100) from both tree reconstructionmethods. The scale bar represents an estimated genetic distance of 0.5.Sequences of identical species clustering together were collapsed andnode support values were omitted for better tree visualization. Anuncollapsed electronic version can be found under TreeBASE accessionno. TB2:S16104. The collapsed clade named “Paramecium ‘aurelia’complex” includes sequences of P. jenningsi and P. schewiakoffi thatare intrinsically not included within the P. aurelia species complex.Squared brackets to the right of the tree designate the five differentsubgenera Chloroparamecium, Cypriostomum, Helianter, Paramecium,and Viridoparamecium as proposed previously (Fokin et al. 2004; Kreutzet al. 2012). Symbols after species names mark discordant haplotypeswith § conceivably misidentified and # potentially further crypticspecies, or ‡ strains that were erroneously named under their respectiveAccession Nos


0.5

Tetrahymena thermophila

Paramecium nephridiatum D-Kusel

Paramecium polycaryum TR-5

Paramecium calkinsi Ch3

Cyclidium glaucoma

Paramecium duboscqui IG2-1 ‡

“Eucandidatus Paramecium germanicum”

Paramecium chlorelligerum SC1

Paramecium caudatum US-LOU

Paramecium nephridiatum Pn3

Paramecium calkinsi GN5-1 ‡

Colpidium striatum

§Paramecium chlorelligerum GC

Paramecium nephridiatum WS97-1

Paramecium polycaryum US-Was1

Paramecium calkinsi PRO165-1

Halteria grandinella

Paramecium polycaryum Khb7-20

Paramecium calkinsi BOB130-7

Paramecium calkinsi IP-4

Paramecium duboscqui 702

Paramecium duboscqui TuAI-2

§Paramecium woodruffi PrS

Paramecium caudatum INL-1

“Eucandidatus Paramecium hungarianum”

Paramecium calkinsi KUZ62-1

Paramecium duboscqui BB8

#Paramecium multimicronucleatum TRB 101-4

Paramecium woodruffi BB-5

Paramecium caudatum INP-3

Paramecium caudatum SWF-1A

Paramecium caudatum INK-1

Urocentrum turbo

#Paramecium multimicronucleatum V105-2

Lembadion bullinum

Paramecium duboscqui AWH9-4Paramecium duboscqui In05

#Paramecium multimicronucleatum TRB 101-1

§Paramecium calkinsi BM1-11


Paramecium multimicronucleatum

Paramecium caudatum haplogroup PcCOI_a

Paramecium caudatum CTJ-P1

Paramecium bursaria

Paramecium putrinum

Paramecium 'aurelia'complex

Paramecium

Cypriostomum

Helianter

Chloroparamecium

Viridoparamecium

1.00/93

1.00/44

0.53/--

0.65/--

0.96/--

0.95/43

1.00/97

0.91/38

1.00/67

0.81/65

1.00/59

0.48/43

1.00/72

1.00/990.99/80

0.62/70

0.83/990.97/74

0.96/60

0.75/55

0.95/88

0.99/51

0.94/60

0.92/40

0.77/21

1.00/99

1.00/99

0.9999

0.95/--§Paramecium multimicronucleatum Kr113-3

Paramecium caudatum ASN-1Paramecium caudatum 1998Paramecium caudatum BRA-RIO

1.00/98

0.64/--

0.96/92

0.92/99

0.57/--0.65/--

0.94/910.71/630.29/59

**

*

**

*

***

*

* *

*

*

*


P. jenningsi and P. schewiakoffi. Additionally, the MI chroma-tin is very fine and homogenous (Figs. 9, 10, 12), resemblingthe structure of the germinal nucleus of P. putrinum.

According to its cortex peculiarities, P. buetschlii sp. nov.fits better within the Cypriostomum or Helianter subgenera.The cytoproct starts halfway between the buccal overture(BO) and the posterior end of the cell and terminates veryclose to the cell’s end (Figs. 3, 4, 8). Each contractile vacuole(CV) has several pores like in P. nephridiatum (Figs. 2, 5, 7,14). The position of the BO is shifted a bit forward to theanterior end of the cell, but not as much as in the case ofP. putrinum, rather similar to P. polycaryum (Figs. 3, 4). Thestructure of the collecting canals-free contractile vacuoles(CV) (Figs. 15, 16) fits very well with both of the Helianterrepresentatives, P. duboscqui and P. putrinum. Further pecu-liarities could be found within the nuclear reorganization pro-cess of P. buetschlii sp. nov.; neither a crescentMI stage nor aclear parachute stage (for MI progamic phase) could be de-tected, while only a small number of old macronucleus (MA)fragments are unique as well (Fokin et al. 2001).

Multivariate morphometric analysis affiliated P. buetschliisp. nov. with the Cypriostomum subgenus (Fig. 35a), while inthe MDS analysis its position is close to the Helianter repre-sentative P. duboscqui (Fig. 35b). However, from previousanalyses, we know that cell length is a very weighty charac-teristic (Fokin and Chivilev 2000; 1999). This may supportP. buetschlii sp. nov. being associated with P. woodruffi (al-most the same cell size) from theCypriostomum subgenus, butnot with the Helianter representatives, which are much small-er (cf. Fig. 38).

Molecular analyses using two different markers, 18S-rRNA and mtCOI gene sequences, confirmed thatP. buetschlii sp. nov. is a member of the Helianter subgenus,either as a sister species ofP. putrinum (18S-rDNA, Fig. 36) orat the very basal position of the genus with a close relationship

to P. putrinum and P. duboscqui strains (COI, Fig. 27).However, as shown in our tree analyses, neither the 18S-rDNA nor the mtCOI sequence data can sufficiently resolvethe relationships between the different Parameciumsubgenera. This is indicated by partially low support valuesfor some basal branch nodes, which are prone to changes dueto the inclusion of new sequences. Here, the addition ofP. buetschlii sp. nov. into the COI data set placed theHelianter subgenus at a quite basal position within the genus,while Chloroparamecium, previously been sister to Helianteror at the base (see, e.g., Boscaro et al. 2012 or Tarcz et al.2013), now represents the sister group to subgenusParamecium. Accordingly, multi-gene analyses withconcatenated sequence data can be an effective method toimprove phylogenetic accuracy, and multi-gene trees are morereliable than single-gene trees (Yi et al. 2014). However, dueto often insufficient sampling and/or the use of 18S-rDNA asthe gene marker in ciliate phylogeny, complete datasets ofpotentially more appropriate marker genes are rare and oftendo not allow for a proper tree reconstruction. In the era of next-generation sequencing (NGS) technologies now available(even for ciliatologists) at a reasonable price, future studiesin this field can hopefully more often take advantage ofNGS to unravel phylogenetic ambiguities.

Cryptic diversity in Paramecium

For describing a new morphospecies, it should be sufficient tohave one obvious morphological character that differs fromrelative ciliates (Foissner et al. 1999). In herein investigatedadditional three Paramecium spp. (besides P. buetschlii sp.nov.), we could not identify such distinct morphological fea-tures. Nevertheless, since all of these three newly identifiedspecies could be clearly separated from the rest of all knownparamecia by using morphometric and molecular analysis, we

Fig. 38 Schematical images of major Parameciummorphospecies madeaccording to its morphometric data (reprint of Fig. 1 fromFokin 2010/11).a P. multimicronucleatum, b P. caudatum, c P. jenningsi, dP. schewiakoffi, e P. buetschlii sp. nov., f P. woodruffi, g P. aurelia, h

P. nephridiatum, i P. bursaria, j P. calkinsi, k P. duboscqui, l P. putrinum,m P. polycaryum. Bar 100 μm. © 2010/2011 by Protozoological Societyaffiliated with Russian Academy of Sciences


currently prefer to refer them as cryptic Paramecium species.Future studies on additional eco-physiological parametersand/or phylogeographical patterns, as well as newmorpholog-ical studies or techniques, may be able to reveal presentlyunknown diagnostic structural differences and allow rapidand practical diagnosis of their taxonomic statuses.

This cryptic status should therefore only be provisional,representing a necessary step to establishing formal scientificnames for these newly discovered and described species (deLeon and Nadler 2010; Jörger and Schrödl 2013). While thereis some debate on the general practice on how to describe anddelimit new cryptic species (e.g., reviewed in Bickford et al.2007), there is no official requirement by the InternationalCode of Zoological Nomenclature (ICZN) on how to namethese species in order to indicate their provisional cryptic sta-tus and to distinguish them from valid biological species.Specifically, if DNA sequence data serve as the primary char-acter set for species delineation, the Linnaean system is underquestion (Tautz et al. 2003), because there is no commonpractice on how to include such molecular data in formalspecies descriptions (Goldstein and DeSalle 2011). Whilesome authors argue for a formal scientific naming of crypticspecies to avoid merging morphologically undistinguishable,but genetically divergent lineages (e.g., de Leon and Nadler2010; Kadereit et al. 2012), others prefer to use clade numberswhen studying cryptic species (e.g., Leliaert et al. 2009;Thomas et al. 2014; Skaloud and Rindi 2013). Here, we pro-pose the systematic term BEucandidatus^ as a component ofthe taxonomic name to make a distinction between valid bio-logical species or taxonomic units and the provisional crypticspecies status. We have chosen this fictional term referring tothe provisional status Candidatus (L. candidatus, a candidate)used in prokaryotic nomenclature for formally described bac-teria that are lacking characteristics required for a valid de-scription according to the International Code of Nomenclatureof Bacteria (Murray and Schleifer 1994; Murray andStackebrandt 1995). The term was altered by prefixing Eu toindicate that the newly described candidate species representsa eukaryote. Since the International Code of Nomenclature ofAlgae, Fungi, and Plants (ICN) also do not provide an officialrequirement on how to treat a cryptic species status, we rec-ommend the use of the term Eucandidatus when describingany new cryptic eukaryotic species. In accordance with theprokaryotic term Candidatus, we propose the termEucandidatus preceding a species specific epithet consistingof the genus name followed by a single specific epithet ac-cording to ICN or ICZN rules. Further, we suggest the termEucandidatus to be italicized, while the succeeding speciesname should be in roman type and the entire name in quota-tion, e.g., BEucandidatus Paramecium germanicum^. Thiswould allow an instant differentiation of cryptic species fromvalid species. This indefinite rank Eucandidatus might beestablished as an Appendix to the nomenclatural codes of

ICN and ICZN, according to the ICSP policy for the nomen-clature of bacteria.

However, caution is called for when describing new crypticspecies from DNA-based characters as long as genetic infor-mation of closely related species is not available. Therefore,taxonomists still must carefully check the type material of allknown morphologically related species and may eventuallycompile the missing sequence information to avoid the estab-lishment of duplicate species under separate names. Thoroughgenetic information of all species in one genus, including dataon intraspecific variability, is therefore necessary to unveil thedegree of cryptic diversity and finally declare species valid.On the other hand, there is controversial discussion amongprotistologists as to whether observed DNA variation onlyreflects neutral mutation accumulation and that the phenotypetherefore comprises the only proper characters by which todefine valid protist species (Fenchel and Finlay 2006).Therefore, we would like to encourage discussion not onlyamong protistologists about a change in current taxonomicpractice. Granting DNA-based characters a similar weight asmorphological features, at least in these instances in whichmolecular data are informative enough to justify the absenceof obvious morphological differences, could not only lead to amore practical and straightforward integrative taxonomy, butalso to a better understanding of species diversity and rich-ness. Appropriately, this procedure then certainly needs acommon means to present and indicate the diagnostic molec-ular markers, which will in turn need further in-depth discus-sion among scientist to ensure stability and traceability offuture taxonomy (Jörger and Schrödl 2013).

New cryptic Paramecium species

As stated above, the further three herein investigated parame-cia are rather difficult to discriminate from similar members ofthe genus by morphology. Therefore, they currently cannot beaccepted as clearly separated morphospecies, due to commonformal requirements not fulfilled and are thus treated as cryp-tic species. More precise, BEucandidatus P. hungarianum^actually has no morphological features that deviate signifi-cantly from those of P. polycaryum (Table 3; Fokin andChivilev 1999), a member of the Cypriostomum subgenus.Classification and ordination of the morphometric data ofBEucandidatus P. hungarianum^ also indicated similarity toP. polycaryum. However, they are not identical, as shown byrather large Euclidian distances and non-overlapping coordi-nates in UPGMA and MDS analyses, respectively (Figs. 35a,b). Further, molecular data clearly separated BEucandidatus P.hungarianum^ from P. polycaryum, showing a closer relation-sh ip to o the r Cypr ios tomum member s , name lyP. nephridiatum, P. woodruffi and/or P. calkinsi (Figs. 36, 37).

A similar situation exists for comparisons betweenBEucandidatus P. brazilianum^ and its close relative


P. multimicronucleatum. On the one hand, they are quite sim-ilar in general morphology, but can be separated by morpho-metric analysis (Figs. 35a, b). On the other hand, moleculardata based on 18S-rDNA sequences clearly assignedBEucandidatus P. brazilianum^ to P. multimicronucleatumwithin the Paramecium subgenus (Fig. 36). Within this treereconstruction, P. multimicronucleatum, however, appears astwo well supported, separate clades with an averaged se-quence similarity of only 98.0 %. At the same time, sequencesimilarity between P. primaurelia and P. tetraurelia, for exam-ple, which are valid biological species, is 99.6 % (Strüder-Kypke et al. 2000). This suggests that the second clade mayrepresent a distinct biological entity, which, besides the hereindescribed BEucandidatus P. brazilianum^, consists of severalP. multimicronucleatum strains and one unspecifiedParamecium species (Paramecium sp. para200) that havebeen all isolated from waste water in Pakistan or Italy, respec-tively. Moreover, the size and number of the micronuclei aswell as the presence of fragmented macronuclei inBEucandidatus P. brazilianum^ deviated from the classicalP. multimicronucleatum phenotype (Table 3). Unfortunately,no other sequence data of any strain of these clade represen-tatives are available nor could we produce own data due tolack of sufficient DNAmaterial or a viable cell culture. Hence,we currently have no additional molecular evidence to sepa-rate these two clades as genetic and potentially valid biologi-cally distinct species.

BEucandidatus P. germanicum^, according to its general con-stitution and size, is rather similar to P. multimicronucleatum too,but the former has the compactMImorphological type instead ofthe vesicular generative nuclei of the latter (Figs. 25, 26). TheMImorphological type, hence, might be the one morphologicalcharacter that allows differentiation between species, which isunfortunately not an easy or very practical character to perceive,since it requires excellent staining preparations and practice.Regarding the MI morphological type, BEucandidatus P.germanicum^ could be weakly related to P. caudatum, as shownby the UPGMA analysis (Fig. 35a). Based on its molecular char-acteristics, BEucandidatus P. germanicum^ is positioned incon-sistently within the genus according to the genetic marker used(Figs. 36, 37). In any case, this species does not group withP. multimicronucleatum, but rather with P. caudatum (based on18S-rDNA), or it is located basally relative to the Parameciumsubgenus (based on COI). As stated above, further multi-geneanalyses may be able to help clarify its phylogenetic position too.

Current and future perspective of Paramecium biodiversity

Paramecium species have been investigated for over250 years. Hence, Bit is very unlikely that in Europe orNorth America taxonomists would have missed such obviousciliates as paramecia during checking of water samples^(Fokin et al. 2004). The redescription of P. chlorelligerum

(Kreutz et al. 2012) and the discovery of the new morphospe-cies P. buetschlii sp. nov. herein described prove the opposite.Still, correct identification and not simply finding parameciain water samples is crucial, and protistologists (or hydrobiol-ogists of any kind) must have an adequate taxonomic knowl-edge to accomplish this task. In fact, the classical taxonomybased on traditional morphological examinations is declining,which means that very few people can do this kind of inves-tigation professionally indeed. The redescriptions ofP. duboscqui and P. nephridiatum (Gelei 1938; 1925;Chatton and Brachon 1933; Shi et al. 1997; Fokin et al.1999a, b) are excellent examples of this decline. Both speciesare widely distributed in brackish and (sometimes) freshwaterhabitats almost ubiquitously in the Northern Hemisphere andparticularly in Europe (Fokin et al. 1999a, b). However, fordecades, they were listed as doubtful species and nobody re-corded these ciliates in nature (Wichterman 1953, 1986), asthey were either confused with P. calkinsi and P. woodruffi orjust ignored as strange paramecia (Fokin et al. 1999a, b).

While traditional taxonomists may unfortunately disap-pear, we have faith that protist taxonomy is not dying butevolving in the way it is done. Molecular approaches willeventually make a major contribution to modern taxonomy,but future taxonomists will still need classical methods forverification and validation of the most reliable morphologicalcharacters needed for integrative taxonomy. For example,P. buetschlii sp. nov. at first glance could be treated asP. caudatum, which may be a reason it is not checked precise-ly. Yet, some morphological peculiarities and genetic dataconvinced us to investigate it in more detail. The same holdstrue for the other herein studied Paramecium spp.

On the other hand, molecular tools alone have the potential tounveil morphologically hidden diversity. As shown in our treeanalyses based on 18S-rRNA and COI gene sequences (Figs. 36,37), there seems to be a higher current stock diversity inParamecium than expected. As mentioned above, the two sepa-rated P. multimicronucleatum clades (18S-rDNA, Fig. 36, cf.Buosi et al. 2014) may represent distinct species withBEucandidatus P. brazilianum^ as potential type species of thesister clade to P. multimicronucleatum. Moreover, as shown inFig. 36, there also exists another 18S-rDNA-basedBP. caudatum^ clade sister to BEucandidatus P. germanicum^and P. caudatum (strain Isn4), which consists of strains isolatedfrom waste water in Pakistan. These strains (typed with FT)exhibit a rather large genetic distance from all other thus fardescribed and investigated P. caudatum species (Table S1).Therefore, we consider it to represent a new Paramecium con-gener as well. Further studies on morphology and genetics areneeded to clarify its taxonomic status and phylogeographicalpatterns.

Within our comprehensive analyses based on currentParamecium COI gene sequences available, we may have re-vealed some potential misidentifications (e.g., P. calkinsi BM1-


11 and BOB130-7; P. woodruffi PrS; P. multimicronucleatumKr113-3, V105-2, TRB 101–1 and TRB 101–4; andP. chlorelligerum GC; cf. Fig. 37), which should be carefullychecked again, if the respective strains are still in stock. In par-ticular, the sequence of P. chlorelligerum strain GC clusteredwithin the outgroup, thereby compromising the monophyly ofgenus Paramecium. As already stated by Boscaro et al. (2012),we also urgently call for the use of comprehensive data sets whenanalyzing and publishing new sequence data of single or newspecies. Further, the P. multimicronucleatum strains V105-2,TRB 101–1 and TRB 101–4 investigated by Tarcz et al. (2012)and originating from the Vologda or Irkutsk regions of Russiamay also represent new cryptic or even true biological species.These specimens form a separate cluster with a comparativelylarge genetic distance (cf. Fig. 37, Table S2) to otherParamecium subgenus representatives (see also Boscaro et al.2012). Here, further studies are needed as well to elucidate phy-logenetic and taxonomic statuses. The morphological type ofmicronuclei, for example, may provide clues on the relation toBEucandidatus P. germanicum^ as the most basal congener tothese specimens.

Our molecular analyses further suggest that P. caudatummay represent a complex of cryptic species too, comprisingpotentially distinct genetic entities with different geographicranges and environmental demands. As shown in Fig. 37,there are five different COI haplogroups that do not formone well-supported clade, but rather show uncertain affilia-tions with close relationships. While haplogroup PcCOI_amainly consists of European strains, its sister clade comprisesrepresentatives from different continents. Another cluster in-cludes strains from tropical Indonesia (INL-1, INP-3, INK-1)that show an altered eco-physiology thereby indicating localadaptation and potential geographic constraints (Krenek et al.2012). While former studies using RAPD and ARDRA fin-gerprinting methods have rejected a potential sibling speciesconcept in P. caudatum (Stoeck et al. 2000), further and morecomprehensive analyses on the different varieties and subpop-ulations will shed light on these clades relationships and thispotential cryptic species complex.

Apparently, the species richness of genus Parameciumseems to be higher than our current knowledge suggests andcould be further increased and revised by more careful inves-tigations that use a combination of classical taxonomic andmodern molecular approaches. Moreover, future studiesshould not only investigate remote and exotic territories.Considering less frequently sampled habitats in the OldWorld will conceivably increase our knowledge as well.Such studies can generate more accurate species lists and spe-cies richness estimations, as our present study indicates.

Acknowledgments This work was supported by a grant from the Ital-ian Ministry of University and Research—MIUR (Ministerio Italianodell’ Universita e della Ricerca) to S. I. Fokin and by the German

Research Foundation (DFG, grant BE-2299/3-1,2,3 and BE 2299/5-1)to T. U. Berendonk, as well as by the European Commission FP7-PEOPLE-2009-IRSES project CINAR PATHOBACTER (247658) andby the BMBSCOSTAction BM1102.We are grateful to D. Ammermannfor sampling in Porto Alegri, to Cora Baier and M. Horn for their helpwith 18S-rDNA sequence of BEucandidatus P. brazilianum^, to M.Müll-er, who provided a possibility of our sampling at Balaton lake region andto Dana Barth for providing cells of P. buetschlii sp. nov. and DNAsamples for molecular analyses. Further gratitude is expressed to twoanonymous reviewers whose valuable comments allowed an essentialimprovement of the manuscript.

Conflict of interest The authors have no conflict of interest to declare.

Ethical standards All the experiments in this study comply with thecurrent laws of the country in which they were performed and do notinfringe human and animal rights.

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http://tree.bio.ed.ac.uk/software/figtree/

new paramecium (ciliophora, oligohymenophorea) congeners ... · paramecium buetschlii sp. nov. from...

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