Sebastian Tarcz

Genetic analysis of the Paramecium aurelia species complex (Protozoa: Ciliophora) by classical and molecular methods

Abstract The Paramecium aurelia complex includes 15 species (sibling species) and is characterised by inbreeding (to varying degrees in different species), causing an increase in intra‐specific differentiation. Investigations into inter‐ and intraspecific differentiation of strains originating from remote habitats within species of the complex were carried out by classical inter‐strain crosses and molecular analyses (RAPD‐PCR fingerprints, ARDRA riboprints, RFLP‐PCR analysis). RAPD analysis showed that all species in the complex possessed characteristic band patterns and the majority were also polymorphic intra‐specifically. A correlation exists between the degree of inbreeding characteristic for a species with differentiation of DNAgenotypes revealed by RAPD analysis within species, where inbreeders showed substantial variability of band patterns and moderate inbreeders were highly similar. RFLP analysis (a 480bp fragment of the gene coding the Hsp 70 protein) with the application of restriction enzyme TruII distinguished among species, while digestion with restriction enzyme AluI distinguished groups of species (clusters) and both enzymes revealed intra‐species polymorphism within P. dodecaurelia. ARDRA riboprinting (using a fragment of SSU‐LSU rDNA, about 2400 bp) with restriction enzymes HhaI, AluI, HinfI, TaqI distinguished groups of species with different band patterns. The majority of enzymes also demonstrated intra‐specific differentiation within P. dodecaurelia. TaqI also revealed intraspecific differences in P. biaurelia and P. tetraurelia. All species in the P. aurelia complex showed a high percentage of surviving hybrid clones in F 1 obtained by conjugation and F2 obtained by autogamy in inter‐strain crosses. A low percentage was observed only in F2 inter‐strain hybrids of P. tredecaurelia, however no cytological changes in the nuclear apparatuses were detected and similar band patterns existed in the studied strains. Future studies, including sequencing of rDNA fragments, may disclose deeper relationships of the species.

Seasonal and spatial variability of species occurrence of the Paramecium aurelia complex in a single natural pond (Protista, Ciliophora)

The geographic distribution and temporal occurrence of ciliates are still little known. In the present article, the occurrence of the Paramecium aurelia species complex in a natural pond situated in Kraków (Opatkowice) was investigated in different seasons in two following years. A sequence of species occurrence of the P. aurelia complex was observed. Always, paramecia were found only in some sampling points among six points sampled each time and not necessarily in the same ones. Paramecia appearing in one habitat (water body) might occupy different niches characterized by various environmental features suitable for paramecia. The following species were found in the pond: P. biaurelia, P. triaurelia, P. tetraurelia, P. pentaurelia, and P. dodecaurelia. The occurrence of some rare species (P. tetraurelia, P. pentaurelia, and P. dodecaurelia) may be connected with migrating birds which can transport paramecia with drops of water from other water bodies. If a species was observed in successive seasons or years, the possible genetic variation was investigated by analysis of sequences of LSU rDNA and mitochondrial cytb gene fragments. Among the studied species (P. biaurelia, P. triaurelia, P. pentaurelia, and P.dodecaurelia) only P.dodecaurelia showed haplotype variation in different seasons and sampling points, probably caused by the colonization of the pond by different populations of paramecia.

Polymorphism of Paramecium pentaurelia (Ciliophora, Oligohymenophorea) strains revealed by rDNA and mtDNA sequences.

Paramecium pentaurelia is one of 15 known sibling species of the Paramecium aurelia complex. It is recognized as a species showing no intra-specific differentiation on the basis of molecular fingerprint analyses, whereas the majority of other species are polymorphic. This study aimed at assessing genetic polymorphism within P. pentaurelia including new strains recently found in Poland (originating from two water bodies, different years, seasons, and clones of one strain) as well as strains collected from distant habitats (USA, Europe, Asia), and strains representing other species of the complex. We compared two DNA fragments: partial sequences (349 bp) of the LSU rDNA and partial sequences (618 bp) of cytochrome B gene. A correlation between the geographical origin of the strains and the genetic characteristics of their genotypes was not observed. Different genotypes were found in Kraków in two types of water bodies (Opatkowice-natural pond; Jordans Park-artificial pond). Haplotype diversity within a single water body was not recorded. Likewise, seasonal haplotype differences between the strains within the artificial water body, as well as differences between clones originating from one strain, were not detected. The clustering of some strains belonging to different species was observed in the phylogenies.

Does high intraspecific variability of two genome fragments indicate a recent speciation process of Paramecium dodecaurelia (P. aurelia species complex, Ciliophora, Protozoa)?

Species of the Paramecium aurelia complex show different levels of intraspecific polymorphism, with P. dodecaurelia revealing a high level of intraspecific variation. Paramecium dodecaurelia strains originating from distant localities in the Palaearctic, North America (USA), and Oceania (Hawaii) were studied in terms of intraspecific differentiation and the degree of speciation. Sequences of genes encoding the ITS1–5.8S-ITS2–5’ end of LSU rDNA (1063–1097 bp) and cytochrome c oxidase subunit I mtDNA (638–644 bp) were obtained from 33 strains of P. dodecaurelia, other P. aurelia species, and another species of the genus Paramecium, with Tetrahymena sp. used as an outgroup. In phylograms, the majority of P. dodecaurelia strains from the Palaearctic appear in one cluster, while strains from Japan, Hawaii and the USA are grouped in another cluster, together with some strains from Italy and representatives of the P. aurelia species complex. Our results tend to support the hypothesis that P. dodecaurelia seems to be a polyphyletic species with several haplotypes similar to or even shared with other members of the P. aurelia species complex. However, it is still an open question whether the revealed intraspecific differences within P. dodecaurelia are the result of ongoing speciation, or perhaps they just indicate genetic differentiation within a species that has a wide geographic distribution.

Paramecium jenningsi complex: existence of three cryptic species confirmed by multi-locus analysis and strain crosses

Ciliates (Protista) have a complex species structure, which means that in several genera the morphological species are differentiated into cryptic species with isolated gene pools (called syngens). Problems of speciation are well known in the genus Paramecium, and especially in the P. aurelia sibling (cryptic) species complex within it. However, the problem of the existence of such species within P. jenningsi was, until recently, still unsolved. Here we present the results of studies based on an analysis of 16 loci (both nuclear and mitochondrial), strain crosses and cytological preparations. The obtained data allowed us not only to study relationships of the P. jenningsi complex and other morphospecies within the Paramecium subgenus, but also to confirm the existence of three isolated reproductive groups within the former P. jenningsi and to propose binominal names for each of them: P. primjenningsi, P. bijenningsi, and P. trijenningsi. In our view, the studied species meet the criteria of a species complex because they can be differentiated based on strain crosses and molecular characteristics, but they cannot be differentiated based on morphological features alone. http://zoobank.org/urn:lsid:zoobank.org:pub:9209CD4C-A255-4A33-BFC1-CE336244C200

Three-locus analysis in conjunction with strain crosses confirms the existence of reproductively isolated populations in Paramecium jenningsi

Paramecium jenningsi (Diller & Earl, 1958) was formerly considered to be a species with only one syngen (genetic species) based on an inter-strain cross of two strains, cytological analysis, and an investigation of esterases and acid phosphatases. However, the existence of syngens within the species was later suggested by genetic studies, i.e. classical strain crosses of new strains and molecular PCR-based analyses (RAPD, RFLP), as well as by sequencing the H4 gene fragment. This issue still needs to be clarified by the application of molecular markers, genetic tests and cytological preparations. In the present study, we tested 12 strains of P. jenningsi originating from Asia, North America and Africa. Trees reconstructed on the basis of three genome fragments (ITS1-5.8S-ITS2-5’LSU, COI and CytB) show that P. jenningsi is divided into two distinct clusters (PJ1, PJ3) and one branch (PJ2) which correspond to reproductively isolated groups revealed by strain crosses. A study based both on strain crosses and a three-locus comparison gives the opportunity for a more complete identification of the reproductively isolated populations of P. jenningsi and other ciliate species.

Molecular polymorphism of strains within Paramecium septaurelia (Ciliophora, Oligohymenophorea)

RAPD-PCR fingerprinting and ARDRA riboprinting revealed polymorphism within P. septaurelia strains from Russia (4 strains from Lower Volga Basin), and one strain from USA, Florida. However, the first method showed the existence of four RAPD genotypes while the second revealed only two groups of strains with different band patterns. All studied strains had a high percentage of surviving hybrid clones in the inter-strain crosses, with little differentiation of strains within species. Intra-species differentiation of strains in RAPD band patterns may be connected with the degree of inbreeding for the studied species. Species of the P. aurelia complex can be arranged according to the degree of inbreeding characteristic for each, which is correlated with the degree of DNA polymorphism revealed by the RAPD method from extreme inbreeders (e.g. P. sexaurelia), moderate inbreeders (e.g. P. triaurelia) to weak inbreeders (e.g. P. pentaurelia). P. septaurelia of the P. aurelia complex should be included in the group of extreme inbreeder.

Delimiting Species Boundaries within a Paraphyletic Species Complex: Insights from Morphological, Genetic, and Molecular Data on Paramecium sonneborni (Paramecium aurelia species complex, Ciliophora, Protozoa)

The demarcation of boundaries between protist species is often problematic because of the absence of a uniform species definition, the abundance of cryptic diversity, and the occurrence of convergent morphology. The ciliates belonging to the Paramecium aurelia complex, consisting of 15 species, are a good model for such systematic and evolutionary studies. One member of the complex is P. sonneborni, previously known only from one stand in Texas (USA), but recently found in two new sampling sites in Cyprus (creeks running to Salt Lake and Oroklini Lake near Larnaca). The studied Paramecium sonneborni strains (from the USA and Cyprus) reveal low viability in the F1 and F2 generations of interstrain hybrids and may be an example of ongoing allopatric speciation. Despite its molecular distinctiveness, we postulate that P. sonneborni should remain in the P. aurelia complex, making it a paraphyletic taxon. Morphological studies have revealed that some features of the nuclear apparatus of P. sonneborni correspond to the P. aurelia spp. complex, while others are similar to P. jenningsi and P. schewiakoffi. The observed discordance indicates rapid splitting of the P. aurelia-P. jenningsi-P. schewiakoffi group, in which genetic, morphological, and molecular boundaries between species are not congruent.

Species of the Paramecium aurelia Complex in Russia (Western Region of European Russia) with Molecular Characteristics of Paramecium novaurelia

The presence of P. primaurelia, P. biaurelia, P. triaurelia, and P. novaurelia of the P. aurelia complex was revealed in the studied region of Russia. RAPD-PCR fingerprints (band patterns) of newly identified P. novaurelia strains from Russia were compared to those characteristic for the other chosen European strains of the species. The strains revealed intraspecific polymorphism as several groups of genotypes confirming the existence of polymorphism within P. novaurelia.

Paramecium tredecaurelia: A Unique Non-Polymorphic Species of the P. aurelia spp. Complex (Oligohymenophorea, Ciliophora)

New stands of Paramecium tredecaurelia , a rare species of the P. aurelia spp. complex, were identified in Thailand and Madagascar on the basis of mating reactions and molecular markers (rDNA and mtDNA). Analysis of DNA fragments showed that all P. tredecaurelia strains, the recently recorded ones and the ones known previously from France, Mexico, and Israel, form a monophyletic and well-defined clade in the P. aurelia species trees. All of these strains, collected from different localities around the world, represent identical or nearly identical haplotypes in terms of all the studied DNA fragments. Given the huge distances between particular collection sites, such a low level of variability of the studied sequences may result from a slow rate of evolution in P. tredecaurelia .