Paramecium jenningsi Species Complex (Ciliophora, Protista) in India: the First Description of New Stands of P. bijenningsi and P. trijenningsi

Paramecium jennngsi (Ciliophora, Protista) is a complex of three cryptic species known from 13 sampling points situated around the world, mainly in the tropics. Two strains recently collected in India were identified as P. bijenningsi and P. trijenningsi from the P. jenningsi complex, based on an analysis of 16 (both nuclear and mitochondrial) loci, strain crosses, and cytological analyses. Current results increase the knowledge about the species range of particular members of the P. jenningsi complex.

Microbial eukaryotes are one the of the main components of tropical food webs in different ecosystems. However, our knowledge of their biodiversity is still significantly underestimated (MED-INGER et al. 2010). Even in Paramecium (one of the best studied genera of microbial eukaryotes), the problem of under-sampling is still a concern in less frequently studied parts of the world, particularly the southern hemisphere (FOKIN 2010(FOKIN /2011 Similarly, data concerning the occurrence of particular species from the tropics are rare. One of the Paramecium morphospecies with preference for a warm climate and therefore poorly studied as concerns its range and biodiversity is P. jenningsi (DILLER & EARL 1958). Although up to now it was collected in only 13 sampling sites distributed worldwide but situated mainly in the tropics (PRZYBOOE et al. 1999), it was suggested (PRZYBOOE et al. 1999(PRZYBOOE et al. , 2003MACIEJEWSKA 2007) and then confirmed by strain crosses, morphological and molecular analyses (PRZYBOOE & TARCZ 2013) to be a complex of three cryptic species (PRZYBOOE & TARCZ 2016). Moreover, two of them are known from one (P. primjenningsi) or two (P. bijenningsi) southern Asian locations in contrast to P. trijenningsi collected from the Japanese islands (6 sampling points), Africa (2 sampling points) and Americas (2 sampling points). With reference to the above, every new sampling point of P. jenningsi complex should bring key information about its biogeography and cryptic diversity. In the present study, based on strain crosses, cytological analyses as well as a comparison of 11 nuclear and 5 mitochondrial loci, we showed the Paramecium species affiliation of two strains collected in India in 2014.

Material
The two investigated strains (designated KT1 and MVP1) of the P. jenningsi complex (Table 1) originated from India, Andhra Pradesh, Visakhapatnam. More precisely, the KT1 strain was collected in the Kottura, a freshwater pond (18°5¢34.76"N, 83°8¢10.56"E) on 27/01/2014. The MVP1 strain was sampled in a wastewater stream in MVP Colony, which is an urban neighbourhood of the city of Visakhapatnam (17°44¢14.63"N, 83°20¢16.79"E) on 24/03/2014. The general morphology and nuclear apparatuses in vegetative and autogamous individuals are characterized by cytological preparations (presented in Fig. 1), and the dimensions of particular strains are given in Table 3.

Strain cultivation
Paramecia were cultured in a medium made of dried lettuce and distilled water inoculated with Enterobacter aerogenes according to the methods of SONNEBORN (1950SONNEBORN ( , 1970 and supplemented with 0.8 µg/ml ß sitosterol (Merck, Darmstadt, Germany). Methods used in cytological studies, strain measurements, and crosses, as well as molecular analyses were as in PRZYBOOE and TARCZ (2016).

Molecular methods and data analysis
Paramecium genomic DNA was isolated from vegetative cells at the end of the exponential phase (approx. 1000 cells were used for DNA extraction). Eleven nuclear and five mitochondrial DNA fragments were amplified, sequenced and analyzed. The above techniques as well as the nucleotide sequences of the primers used for amplification and sequencing were described in detail in PRZYBOOE and TARCZ (2016). The DNA sequences of both newly identified strains from India are available from the NCBI GenBank database (see Table 2). The studied sequences were aligned using ClustalW (THOMPSON et al. 1994) as part of BioEdit software (HALL 1999) and checked manually. All of the obtained sequences were unambiguous and incorporated in analysis. Phylograms were constructed for the studied fragments as in PRZYBOOE and TARCZ (2016).

Results and Discussion
Cytological characteristics of strains from the P. jenningsi complex The strains are characterized by two micronuclei (Figs 1A,C) of chromosomal type with a reticular structure (FOKIN 1997;PRZYBOOE et al. 2015) and two macronuclear anlagen with chromatin centres (DILLER & EARL 1958;MITCHELL 1963;PRZYBOOE 1975PRZYBOOE , 1986PRZYBOOE et al. 2003;PRZYBOOE & TARCZ 2013) seen at a later stage of nuclear reorganization in sexual processes (autogamy, conjugation) (Figs 1B,D). Paramecia have the shape of a cigar, with two contractive vacuoles on the dorsal side, usually with 8 long radial canals.

Dimensions of strains
The dimensions (length and width of cells and macronuclei) of the strains (fixed, Giemsa-stained) and diameter of micronuclei are given in Table 3. The average length of the cells in the strain MVPI equals 248.03 ìm, and 228.15 ìm in the strain KTI. The diameter of micronuclei did not show much variation between the strains (3.12 -3.85 ìm in both strains).

Biology -the appearance of sexual processes
Autogamy (leading to homozygosity of all genes) was observed in strains that were starved for 16-20 fissions (as an inter-autogamous period between successive autogamies) in daily isolated lines (d.i.l.) cultivated at 27°C in a medium enabling three fissions daily. In turn, conjugation was obtained on the sixth or seventh day of clone culture at 27°C at a rate of three fissions daily. The cytoplasmic type of mating type inheritance is characteristic for the species.

Identification of species and results of strain crosses
Strain MVPI was identified as P. bijenningsi and strain KTI as P. trijenningsi, based on conjugation between the studied strains and the reference strains of the particular species, i.e. the strain CS from China, Shanghai, reference for P. bijenningsi, and the strain JH from Japan, Honshu Island, Hagi, reference for P. trijenningsi. The percentage of surviving hybrid clones in F1 and F2 was high (Table 4). The results of the strain crosses are in concordance with the results of multi-locus analyses of the studied strains of the P. jenningsi complex (Figs 2,3). Table 2 GenBank accession numbers to studied DNA fragments nucDNA 1 nucDNA 2 nucDNA 3 nucDNA 4 nucDNA 5 nucDNA 6 nucDNA 7 nucDNA 8 nucDNA 9 nucDNA 10nucDNA 11 mtDNA 1 mtDNA 2 mtDNA 3 mtDNA 4 mtDNA 5 rDNA

Phylogenetic analysis
All applied methods (NJ -neighbour-joining, MP -maximum parsimony, ML -maximum likelihood, BI -Bayesian inference) of tree reconstruction gave an almost identical topology, so in the current study only neighbour-joining phylograms (Figs 2, 3) providing bootstrap/posterior probability values for the other methods are presented. Due to the lack of nuclear sequences for P. caudatum (outgroup) it was impossible to obtain a rooted tree for the nucDNA dataset.
Based on the two phylogenetic trees (Figs 2, 3) constructed on the basis of two datasets (nucDNA and mtDNA) of 16 sequenced genome fragments, the division of P. jenningsi into two clades (P. bijenningsi and P. trijenningsi ) and one branch (P. primjenningsi) can be seen. The strains from India identified by cytological characteristics as P. jenningsi appear with strong bootstrap support in both trees in two different clades: strain MVPI in the P. bijenningsi cluster and strain KTI in the P. trijen-ningsi cluster. The positions of MVPI and KTI strains (Figs 2,3) are in accordance with the results of strain crosses (Table 4). It is worth noting that strain MVPI based on a comparison of the nucDNA dataset (Fig. 2) is most closely related to the P. bijenningsi strain from Saudi Arabia (SA) but in the case of the mtDNA dataset (Fig. 3) it appears in one subclade with the P. bijenningsi strain from China (CS). A similar situation was observed in the case of the second Indian strain (KTI) which appears in one subclade with the Uganda strain (U) (Fig. 2 -nucDNA tree) and with the Panama strain (PA) (Fig. 3 -mtDNA tree). However, the observed positions did not influence species identification of either of the strains.

Species identification of new Paramecium strains
Due to the absence of a uniform species definition, the abundance of cryptic diversity, and the occurrence of convergent morphology (BOENIGK   BICKFORD et al. 2007) because they can be differentiated based on strain crosses and molecular characteristics but they cannot be differentiated based on morphological features alone. The strains identified in this work as P. bijenningsi and P. trijenningsi were collected and described as P. jenningsi (SERRA et al. 2014) but without detailed identification of cryptic species. In 2014 another Indian strain (designated BJ1) was collected in Chilka Lake, Odisha, presenting a macronuclear endosymbiont "Candidatus Gortzia infectiva" (SERRA et al. 2016), a recently described bacterial species originally retrieved in P. jenningsi strain TS-j1-8 from Thailand (BOSCARO et al. 2013). Based on the results presented here, the future sampling of tropical regions such as India, which are in general poorly investigated in terms of Paramecium diversity, may reveal new data on biodiversity and biogeography of known or even undiscovered morpho-species.