Parameciom hongarianom^

Krenek, Sascha, Berendonk, Thomas U. & Fokin, Sergei I., 2015, New Paramecium (Ciliophora, Oligohymenophorea) congeners shape our view on its biodiversity, Organisms Diversity & Evolution (New York, N. Y.) 15 (2), pp. 215-233 : 223-225

B Eocandidatos Parameciom hongarianom^

Size in vivo was up to 110× 50 μm, usually 95× 45 μm (Fig. 30). The average size of fixed and impregnated cells was 84.9 ± 6.6 ×37.1 ± 3.4 μm. The number of cilia rows amounted to 44–65 (56.9±4.8 on average). The BC size was 18.5±1.4 μm ( Table 3) with a BC size to cell length ratio of 0.22. The location of the BO was shifted from the cell’ s equator forward to the anterior end of the ciliate (occurring at about 45 % of the cell’ s length). Two contractile vacuoles always had one PCV each (Figs. 32, 33). The number of collecting canals in CV was six to eight (most often seven). As characteristic for Parameciom , the cortex contained numerous typical trichocysts with a homogeneous distribution. The single ovoid or ellipsoidal MA (17.2±1.1×23.4±3.5 μm on average) was usually situated close to the cell’ s equator. Numerous roundish MI (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 vesicolar. The general cell form was typical woodroffi shaped with a width-to-length ratio of 0.45 (Figs. 20, 32). During the swimming 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 some morphobiological features are represented as a dendrogram and topogram according to UPGMA and MDS methods ( Fig. 35a, b ). For these analyses, the same set of characters obtained for other species previously investigated (12 morphospecies of the Parameciom genus) were used together with the data derived from the herein described new Parameciom species. The addition of the four new representatives does not change the previous tree topology much and fits with major morphological characters of the herein described paramecia ( Fig. 35a ). P. boetschlii sp. nov. clusters within the woodroffi subgroup (subgenus Cypriostomom ) but is separated by a quite large Euclidian distance from the rest of the species. Here, P. woodroffi was the nearest congener to P. boetschlii sp. nov. The same topology was indicated by the MDS analysis ( Fig. 35b ).

According to their morphometric and morphobiological features, the other three investigated Parameciom species, namely B Eocandidatos Paramecium brazilianum^, B Eocandidatos Paramecium germanicum^ and B Eocandidatos Paramecium hungarianum^, can be associated to Parameciom moltimicronocleatom , Parameciom caodatom , and Parameciom polycaryom , respectively ( Fig. 35a, b ). While these three Parameciom species show also rather high Euclidian distances to their nearest relatives, they cannot be clearly separated from congeners based on general morphology. For example, while B Eocandidatos P. germanicum^ is closer related to P. caodatom and P. B aorelia^ according to MDS and UPGMA analyses, it rather resembles P. moltimicronocleatom based on its general morphology. However, it could only be distinguished from P. moltimicronocleatom by the morphological type of MI (compact vs vesicolar, respectively), which is not an easy character to perceive because of its small size and the requirement of high quality staining techniques.

Sequence comparison and molecular phylogeny of new Parameciom spp.

Comparative sequence analysis and phylogenetic reconstructions based on 18S-rRNA and mitochondrial COI gene sequences of the herein described new Parameciom spp. gave us trees with different topology ( Figs. 36 , 37).

According to the 18S-rDNA analyses, P. boetschlii sp. nov. represents a sister taxon to Parameciom potrinom with a sequence similarity of 95.6 % ( Fig. 36 , Table S1). The average genetic distance to P. doboscqoi , another member of the Helianter subgenus like P. potrinom , is comparably low (0.048 vs 0.051, cf. Table S1) indicating that P. boetschlii sp. nov. belongs to the Helianter subgenus. However, as shown in Fig. 36 the Helianter subgenus does not form a monophyletic clade within our analyses. While the relationship of P. boetschlii sp. nov. to P. potrinom is fairly supported by the tree reconstruction methods used, the two more basal branch nodes are not supporter by either method. The other new cryptic species, namely B Eocandidatos P. brazilianum^ and B Eocandidatos P. germanicum^ belong to the Parameciom subgenus, while BEocandidatos P. hungarianum^ appears to be a member of the Cypriostomom subgenus ( Fig. 36 ). Here, B Eocandidatos P. germanicum^ is inferred to be a sister species to P. caodatom with a sequence similarity of 97.8 %. B Eocandidatos P. brazilianum^, on the other hand, clusters within one of the two P. moltimicronocleatom clades. However, it should be stressed that both P. moltimicronocleatom clusters exhibit a comparatively large averaged genetic distance of 0.021 with an averaged sequence similarity of only 98.0 %. B Eocandidatos P. hungarianum^, however, seems to be a sister species to P. woodroffi and P. nephridiatom with a comparatively high sequence similarity of 99.5 % each.

Using the mitochondrial COI gene as a molecular marker, the position of P. boetschlii sp. nov. is the most basal in the Parameciom genus with representatives of P. potrinom and P. doboscqoi as closest relatives (Fig. 37). Interestingly, the inclusion of P. boetschlii sp. nov. and the other new cryptic Parameciom species into the COI dataset dramatically changed the relationships between the different Parameciom subgenera, resulting in Chloroparameciom appearing more closely related to Parameciom than to Helianter (data not shown, but cf. Fig. 4 in Boscaro et al. 2012). According to the COI tree topology, B Eocandidatos P. germanicum^ is placed at a very basal position in the Parameciom subgenus (subgroup B caodatom^) with three P. moltimicronocleatom strains as apparently closest relatives (averaged genetic analyses, respectively, while asterisks denote full support (1.00/100) from both tree reconstruction methods. The scale bar corresponds to 0.05 nucleotide substitutions per site. Sqoared brackets to the right of the tree designate the five different subgenera Chloroparameciom, Cypriostomom , Helianter , Parameciom , and Viridoparameciom as proposed previously ( Fokin et al. 2004; Kreutz et al. 2012)

distance 0.2), which cluster outside of all other so far haplotyped P. moltimicronocleatom strains (see Fig. 37). The position of B Eocandidatos P. hungarianum^, based on its COI gene sequence, is similar to its 18S-rDNA phylogenetic position within the Cypriostomom subgenus with a close relationship to P. woodroffi (strain BB-5, sequence similarity 89.2 %). However, as shown in Fig. 37, P. calkinsi does not form a monophyletic clade and the closest relative to B Eocandidatos P. hungarianum^ is actually P. calkinsi strain BOB130-7 from Olkhon Island in Lake Baikal ( Przybos et al. 2013) with a sequence similarity of 94.8 %.

Figures

Fig. 35 Dendrogram (a) for hierarchical clustering (UPGMA) and topogram (b) for non-metric multidimensional scaling (MDS) of morphometric and morphobiological characteristics of 16 Parameciom species. AU P. aorelia, BU P. borsaria, CA P. caodatom, CL P. calkinsi, DU P. doboscqoi, JE P. jenningsi, MU P. moltimicronocleatom, NR P. nephridiatom, PB BEocandidatos P. brazilianum^, PG BEocandidatos P. germanicum^, PH BEocandidatos P. hungarianum^, PN P. boetschlii sp. nov., PO P. polycaryom, PU P. potrinom, SH P. schewiakoffi, WO P. woodroffi

Fig. 36 SSU rDNA tree topology. Phylogenetic reconstruction of the genus Parameciom based on 18S-rDNA sequences inferred by Bayesian Inference analysis. The alignment contained 49 taxa and 1640 sites including gaps. Sequences of new or cryptic species characterized within this study are shown in boldface. Sequences of other Peniculida served as outgroup. Nombers at nodes (occasionally indicated by an arrow) represent support values for the Bayesian Inference and Maximum Likelihood