New Stands of Species of the Paramecium aurelia Complex ( Ciliophora , Protista ) in Europe , Asia , and Oceania ( Guam )

New stands of species of the Paramecium aurelia complex in Europe (Poland – P. novaurelia, Spain – P. primaurelia), Asia (Russia, Altay – P. biaurelia, P. novaurelia and Kamchatka – P. pentaurelia, Iran – P. primaurelia), and Oceania (Guam island – P. octaurelia) are presented in the paper. Especially interesting is a stand on Guam, the largest and southernmost island in the Marianas Archipelago (Australia with Oceania), as well as a stand in Ispahan, Iran. Both regions were sampled for the first time for the presence of the P. aurelia spp. Results of the paper are in agreement with the opinion that some species of the P. aurelia complex are cosmopolitan (as P. primaurelia and P. biaurelia), other species such as P. octaurelia seem limited to tropical or subtropical regions, and P. novaurelia can also be found outside of Europe. Besides the P. aurelia spp., other species of Paramecium genus were identified in the collected water samples, i.e. P. caudatum (Ispahan, Iran and Bia3owie¿a, Poland); P. polycaryum (Guam), and P. multimicronucleatum-like (Guam).

Paramecia belonging to the Paramecium aurelia spp.complex are model organisms applied in several studies concerning general biology, among other also evolution, and possibility of adaptation to different environmental conditions and colonization of some fresh water bodies.Knowledge of biogeography seems fundamental for understanding evolution (LOWE 2015).Studies on the occurrence and distribution of particular species of the P. aurelia complex have been carried out for many years (SONNEBORN & DIPPELL 1946;BEALE & PREER 2008) and revealed that among 15 known (SONNEBORN 1975;AUFDERHEIDE et al. 1983) species of the complex, some are cosmopolitan (as P. primaurelia, P. biaurelia, P. tetraurelia, and P. sexaurelia), and others limited to particular regions, environments, or habitats (SONNEBORN 1975).However, only some parts of the world have been sampled intensively, such as Europe (PRZYBOOE & SURMACZ 2010;PRZYBOOE et al. 2014a;RAUTIAN et al. 2014;PRZYBOOE & PRAJER 2015), and the USA in North America where the majority of species of the P. aurelia complex were recorded (SONNEBORN 1975;BEALE & PREER 2008).Other continents were studied only occasionally, including Central and South America, Australia, and Africa (PRZYBOOE & FOKIN 2000;PRZYBOOE & SURMACZ 2010;PRZYBOOE et al. 2014b;RAUTIAN et al. 2014;PRZYBOOE & PRAJER 2015).Huge territories of Asia are still unexplored, more intensive sampling was done only in Japan and the Asiatic part of Russia (PRZYBOOE et al. 2013a;RAUTIAN et al. 2015).The southern hemisphere, generally, still awaits investigation (FOKIN 2010(FOKIN /2011)).
Different models of ciliate distribution have been proposed, including the models of ubiquity (FINLAY & FENCHEL 2004;FENCHEL 2005) et al. 2008).Some molecular studies also support the latter model (EPSTEIN & LOPEZ-GARCIA 2008), i.e., the concordance of some protist species distributions with biogeography.The dispersal of paramecia is also a very important aspect concerning their biogeography.Paramecia can only be transferred by animals or human activities with some drops of water, as cysts are unknown (LANDIS 1988;GUTIERREZ et al. 1998;BEALE and PREER 2008).BEALE and PREER (2008) presented a hypothesis that Paramecium might be an old species which already existed before the separation of continents from Pangea and distributed by movement of the continents.The same argument (split of Pangea) was suggested by FOISSNER (2006) andFOISSNER et al. (2008) concerning dispersal of micro-organisms.
The other aspect is the existence of climatic zones limiting the occurrence of some sensitive species, suggested by SONNEBORN (1975), and recently proven (PRZYBOOE & PRAJER 2015).The authors analyzed new and published data (Table 3 in PRZYBOOE & PRAJER 2015) and confirmed that some species were recorded only in warm climate zones, including P. quadecaurelia (PRZYBOOE et al.   2013b), P. sonneborni (PRZYBOOE et al. 2014a; PRZY-BOOE et al. 2015), as well as, e.g., P. sexaurelia, P. octaurelia, and P. tredecaurelia (PRZYBOOE & PRAJER 2015).Knowledge on paramecium species occurrence around the world and filling all existing "white spots" may increase our understanding of the geographical distribution of protists.
Identification of new strains of species of the P. aurelia complex collected in Europe, Asia, and Oceania (Guam) was the aim of this paper.Especially interesting is the strain isolated from Guam, the largest and southernmost island in the Marianas Archipelago (Australia with Oceania, G£ODEK 1967), as well as the strain from Ispahan, Iran.Both regions were sampled for the first time.

Material
Paramecia studied in the present paper representing species of the P. aurelia complex are listed in Table 1.They were collected in Russia, Altay  's methods (1950, 1970) of cultivation and identification of strains were used.Paramecia were cultured at 27°C in a medium made of dried lettuce in distilled water, inoculated with Enterobacter aerogenes and supplemented with 0.8 mg/ml â-sitosterol.New strains were identified as particular species of the P. aurelia complex on the basis of strong conjugation between the studied strain and the reference strain of the species.
The standard strains belong to the collection of the P. aurelia spp.strains in the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków, Poland.
In the inter-strain crosses, the F 1 generation was obtained by conjugation and F 2 by autogamy (using the method of daily isolation lines).Inter-strain crosses were carried out between recently identified strains from Spain, Iran, Russia, Guam and Poland representing particular species of the P. aurelia complex and standard strains of the species (Table 2).
The occurrence of the desired stage of autogamy (specimens at the stage of two macronuclear anlagen) was examined on preparations stained with aceto-carmine.Survival of clones in both generations was estimated as percentages.According to CHEN (1956), clones can be considered as surviving after passing 6-7 fissions during 72 hours after separation of partners of conjugation or postautogamous caryonids.
Other species of Paramecium were also identified in the studied samples based on a comparison of cell size and morphology, number and type of micronuclei (VIVIER 1974), and also morphology of contractile vacuoles and number of CV pores (FOKIN 2010(FOKIN /2011)).

Results and Discussion
New stands of P. primaurelia, P. biaurelia, P. pentaurelia, P. octaurelia and P. novaurelia were recorded in the studied localities (Table 1).Results of pioneer sampling in Iran (Ispahan) and Guam seemed especially intriguing where only single strains of the P. aurelia spp.were collected.P. primaurelia (Iran) and P. octaurelia (Guam), respectively, were recorded there, however, the occurrence of the other species of the P. aurelia complex in these regions cannot be excluded.P. octaurelia seems rather rare, limited to the tropical and subtropical regions, being found in Panama, Uganda, Israel and Georgia (SONNEBORN 1975;PRZYBOOE & PRAJER 2015).A new stand of P. primaurelia was also recorded in Spain (Malaga), which was not sampled before.Previously, several species of the complex were already found in Spain, such as P. primaurelia, P. biaurelia, P. triaurelia, P. tetraurelia, P. pentaurelia, P. sexaurelia , and P. novaurelia, as the result of sampling carried out in different regions of the country The viability of the offspring of the hybrid exconjugant clones in the inter-strain crosses was high in F1 and F2 generations (Table 2) in the studied species, a lower (66%) percentage of surviving clones was observed in F2 hybrids of the Iran strain x standard strain of P. primaurelia from the USA.This may be caused by high genetic divergence between strains originating from these remote habitats.
"Only a small percentage of protist diversity can be discovered when single samples are taken from surroundings.While undersampling may be the cause (FOISSNER 2006), our limited understanding is the problem".

Conclusions
The results of the paper are in agreement with the opinion that some species of the P. aurelia complex are cosmopolitan (such as P. primaurelia and P. biaurelia), other species such as P. octaurelia seem limited to tropical or subtropical regions, and P. novaurelia can also be found outside Europe.
(PRZYBOOE et al. 2013aeviously in the Altay region revealed P. biaurelia and P. pentaurelia in the Altay Foreland and in the Altay Mts(POTEKHIN et al. 2006).One stand of P. pentaurelia was found in Kamchatka, Ust Bolsheretsk, at present.Previously, other species of the P. aurelia spp.were recorded on this territory, namely P. primaurelia, and P. dodecaurelia(PRZYBOOE et al. 2013a).