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New Stands of the Paramecium Aurelia Spp. Complex (Protista, Oligohymenophorea) in Ethiopia, Madagascar, Taiwan, and Romania

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New Stands of the Paramecium Aurelia Spp. Complex (Protista, Oligohymenophorea) in Ethiopia, Madagascar, Taiwan, and Romania e-ISSN 1734-9168 Folia Biologica (Kraków), vol. 66 (2018), No 3 http://www.isez.pan.krakow.pl/en/folia-biologica.html https://doi.org/10.3409/fb_66-3.12 New Stands of the Paramecium aurelia spp. Complex (Protista, Oligohymenophorea) in Ethiopia, Madagascar, Taiwan, and Romania Ewa PRZYBOŒ and Sebastian TARCZ Accepted September 10, 2018 Published online November 07, 2018 Issue online November 30, 2018 Original article PRZYBOŒ E., TARCZ S. 2018. New stands of the Paramecium aurelia spp. complex (Protista, Oligohymenophorea) in Ethiopia, Taiwan, Madagascar, and Romania. Folia Biologica (Kraków) 66: 111-119. Knowledge about the occurrence and distribution of species from the Paramecium aurelia complex is important for understanding their evolution. However, sampling of paramecia in the southern hemisphere, and, in general, in the tropics was usually done only occasionally. Therefore, biogeographical information from areas such as Africa, South America and Australia may bring new, interesting data. In the current survey, based on mating test results as well as COI mtDNA analysis we showed the existence of new stands of P. sexaurelia, P. octaurelia, P. novaurelia, P. tredecaurelia, and P. quadecaurelia. In the case of Ethiopia and Taiwan these are the first data in terms of occurrence of the P. aurelia complex. Key words: Ciliates; Paramecium aurelia species complex; biogeography, COI mtDNA, molecular analysis. * Ewa PRZYBOŒ, Sebastian TARCZ , Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, S³awkowska 17, 31-016 Kraków, Poland. E-mail: [email protected] E-mail: [email protected] Ciliates occupy different ecological niches in ciliate communities in ecosystems (CLAMP & which they play an important role as trophic links LYNN 2017). in food webs. Some of them, such as a bac- One of the “flagship” models for biodiversity terivorous organisms, can also clean water by re- studies is Paramecium aurelia – a complex of ducing bacteria populations (LYNN 2008). More 15 sibling species (SONNEBORN 1975; AUFDERHEIDE generally, according to CORLISS (2004), protists et al. 1983). Some of them are cosmopolitan are important members of the biological world. (P. primaurelia, P. biaurelia, P. tetraurelia, and However, their geographical diversity (biogeogra- P. sexaurelia), whereas the occurrence of others is phy) has been under discussion for many years. limited by temperature barriers (SONNEBORN One model supports cosmopolitan (FENCHEL & 1975). Restriction to the warm zone has been re- FINLAY 2004) and the second a moderately en- vealed in some species such as P. quadecaurelia demic distribution (FOISSNER et al. 2008; FOISSNER (PRZYBOŒ et al. 2013b), or P. sonneborni (PRZYBOŒ 2017). et al. 2014, 2015). Among ciliates within the Paramecium genus, Some geographical regions have been sampled there are a few flagship species being the subjects more extensively, such as Europe, the USA in of many studies (ALTERMATT et al. 2014) con- North America, and several localities in Asia, as cerning several general biological problems e.g. Japan, Asiatic Russia, partly India and Israel (cf BEALE &PREER 2008), including biodiversity (PRZYBOŒ &FOKIN 2000; PRZYBOŒ &SURMACZ (e.g. SONNEBORN 1975; GRECZEK-STACHURA 2010). Generally, the northern hemisphere is et al. 2012; PRZYBOŒ &TARCZ 2016). Studies of much better explored than the southern one which the latter issue in connection with taxonomy may still has “white spots” in Africa, South America, bring a more complete understanding of the role of and Australia (FOKIN 2010/2011). Ó Institute of Systematics and Evolution of Animals, PAS, Kraków, 2018 Open Access article distributed under the terms of the Creative Commons Attribution License (CC-BY) OPEN Ð ACCESS http://creativecommons.org/licences/by/4.0 112 E. PRZYBOŒ,S.TARCZ Here we present new data on the geographical Romania (Table 1, Fig. 1). In Taiwan, samples distribution of five members of the Paramecium were collected in Wufeng, district of Taizhong aurelia species obtained from Ethiopia, Taiwan town, from a stream near a Buddhist graveyard. (locations never studied before), Madagascar Taiwan is situated in the monsoon tropical cli- (sampled occasionally), and Romania. matic zone. In Ethiopia, samples of water were collected in Lalibela (altitude about 2630 m a.s.l.) from a cis- Material and Methods tern with papyrus, situated near one of the rock- hewn churches from the XII-XIII century. Ethio- pia has a moderate warm climate as the majority of Material the country is situated at high altitude (FLIS 1967). Paramecia strains studied in the present paper A sample of water was collected in Madagascar, representing the P. aurelia species complex are Isalo National Park from a mountain stream in listed in Table 1. They were collected in: Asia, Tai- a shaded ravine. The park is situated on a plateau, wan; Africa, Ethiopia and Madagascar; Europe, 820 to 1240 m a.s.l., built from sandstone forma- Table 1 New stands of species of the Paramecium aurelia complex Clone Collector and date GenBank Species index Collection place Coordinates of collection acc number Ethiopia, Lalibela, ' '' ' '' A. Bielecka, P. sexaurelia Et-4,a cistern 12°01 45.660 N / 39°02 25.512 E November, 2017 MH550415 Madagascar, Isalo ' '' ' '' £. Przyby³owicz, P. octaurelia MaI National Park, stream 22°32 22.8 S / 45°22 52.4 E April, 2018 MH550416 ' '' ' '' E. Przyboœ, June, P. novaurelia RB Romania, Bran, pond 45°30 23.94 N / 25°22 7.04 E 2017 MH550417 Ethiopia, Lalibela, ' '' ' '' A. Bielecka, P. tredecaurelia Et-3,a cistern 12°01 45.660 N / 39°02 25.512 E November, 2017 MH550418 Taiwan, Taizhong, ' '' ' '' P. Wojtal, P. quadecaurelia Tai Wufeng, pond 24°03 60.00 N / 120°41 59.99 E September, 2017 MH550420 Fig. 1. The origin of studied currently Paramecium aurelia species. Paramecium aurelia species complex 113 tions with deep canyons. The island has a tropical Survival of inter-strain hybrids climate with seasonal rainfall (RATAJSKI 1967). It belongs to the afro-tropical region, its fauna is In the inter-strain crosses, the F1 generation was characterized by diverse ecosystems and unique obtained by conjugation and F2 by autogamy (us- wildlife thanks to the origin of the island from the ing the method of daily isolation lines). Inter-strain breakup of the Gondwana supercontinent and fol- crosses were carried out between recently identi- lowing the split from the Indian peninsula. fied strains from Taiwan, Ethiopia, Madagascar, and Romania representing particular species of the In Romania, material was collected from a pond P. aurelia complex and the standard strains of the near Bran castle (“Dracula castle”), Transylvania. species (Table 2). The occurrence of the desired stage of autogamy (specimens at the stage of two macronuclear anlagen, i.e., after finishing the reor- Methods ganization of the nuclear apparatus) was examined on preparations stained with aceto-carmine. The Identification of established strains of P. aurelia spp. type of macronuclear anlagen is also an important feature for taxonomic diagnosis. Survival of SONNEBORN’s methods (1950, 1970) of cultiva- clones in both generations was estimated as per- tion and identification of strains were used. Para- centages. According to CHEN (1956), clones can mecia were cultured at 27°C in a medium made of be considered as surviving after passing 6-7 fis- dried lettuce in distilled water, inoculated with En- sions during 72 hours after separation of partners terobacter aerogenes and supplemented with 0.8 of conjugation or post-autogamous caryonids. mg/ml â-sitosterol (Merck, Darmstadt, Germany). A detailed description of procedures can be found New strains were identified as particular species of in PRZYBOŒ et al. (2013b). the P. aurelia complex on the basis of strong con- jugation between the studied strain and the refer- Molecular techniques ence strain of the species. Paramecium genomic DNA was isolated from The following standard strains were used: vegetative cells at the end of the exponential phase strain 159 of P. sexaurelia from Puerto Rico, (approximately 1000 cells were used for DNA ex- traction) using a Genomic Micro AX Tissue Grav- strain 138 of P. octaurelia from Florida, USA, ity (A&A Biotechnology, Poland), according to strain 205 of P. novaurelia from Edinburgh, the manufacturer’s instructions for DNA isolation Scotland, UK, from cell cultures. Both the quantity and purity of the extracted DNA were evaluated with a strain TaB of P. tredecaurelia from Thailand, NanoDrop-2000 Spectrophotometer (Thermo Sci- Bangkok, entific, Waltham, MA, USA). strain An1-1 of P. quadecaurelia from Africa, The COI fragment (about 800 bp) of mitochondrial Namibia, DNA was amplified with the primer pair forward F388dT strain 328 of P. quadecaurelia from Australia. (5’-TGTAAAACGACGGCCAGTGGwkCbAA AGATGTwGC-3’) and reverse R1184dT (5’- The standard strains belong to the collection of CAGGAAACAGCTATGACTAdACyTCAGGG the P. aurelia spp. of the Institute of Systematics TGACCrAAAAATCA-3’) using the protocol and Evolution of Animals, Polish Academy of previously described by STRÜDER-KYPKE & Sciences, Kraków, Poland. LYNN (2010). For all analyzed DNA fragments, Table 2 Percentage of surviving hybrid clones in inter-strain crosses of the Paramecium aurelia spp. complex Crossed strains: Percentage of surviving hybrid clones in: Species studied x standard of particular species F1 (obtained by conjugation) F2 (obtained by autogamy) P. sexaurelia Et-4,a x 159 76 50 P. octaurelia MaI x 138 98 82 P. novaurelia RB x 205 97 87 P. tredecaurelia Et-3,a x TaB 98 15.7 P. quadecaurelia Tai x AN1-1 82 60 114 E. PRZYBOŒ,S.TARCZ PCR amplification was carried out in a final For the first time the areas of Ethiopia and volume of 40 ìl containing 30 ng of DNA, 1.5 U Taiwan were examined for the occurrence of Taq-Polymerase (EURx, Poland), 0.8 ìlof20ìM Paramecium, in particular the P. aurelia complex. of each primer, 10 × PCR buffer, and 0.8 ìlof Worth noting is the presence of rare species of the 10 mM dNTPs.
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