ZOOLOGICAL SCIENCE 24: 123–129 (2007) © 2007 Zoological Society of Japan Biogeography and Karyotypes of Freshwater Planarians (Platyhelminthes, Tricladida, Paludicola) in Southern Brazil Tanise Knakievicz1*, Adriana Helena Lau2, Daniel Prá1 and Bernardo Erdtmann3 1Programa de Pós-Graduação em Genética e Biologia Molecular. Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre-RS, Brasil 2Universidade Luterana do Brasil (ULBRA), Canoas-RS, Brasil 3Instituto de Biotecnologia da Universidade de Caxias do Sul (UCS), Caxias do Sul-RS, Brasil In the Tricladida (Platyhelminthes), the incidence of different biotypes identified by several ploidy levels is very common. Planarians collected in the State of Rio Grande do Sul were identified using cytogenetics. Different species distributions were observed with respect to Rio Grande do Sul’s geomorphology, which could have been caused by their different microhabitats. Girardia tigrina and G. anderlani consisted of diploid and triploid individuals, whereas G. schubarti showed diploids, triploids, and mixoploids; for all these species, individuals of different ploidies were sympatric. Only for diploid G. anderlani were B chromosomes observed. These B chromosomes seem to have an irregular segregational behavior during mitosis, and possibly also during meiosis. However the processes (e.g., selection, mutation) of maintaining 2n, 3n, and 2n/3n individuals within natural populations of G. schubarti remain to be clarified. Key words: B chromosomes, karyotypes, mixoploidy, polyploidy, taxonomy the subjects of studies on morphology, karyology, and tax- INTRODUCTION onomy (Kawakatsu et al., 1981, 1982, 1985, 1993; Vreys et In Tricladida, the presence of different biotypes identi- al. 2002), reproduction and life-cycle (Knakievicz et al., fied by several ploidy levels is very common. Diploid and 2006), and ecotoxicology (Guecheva et al., 2001, 2003; triploid individuals were reported in Dugesia japonica Preza et al., 2001; Prá et al., 2005). However, little is known (Tamura et al., 1995, 1998), Schmitea lugubris (Gremigni et about their biogeography and ecology. al., 1980; Storhas et al., 2000), Girardia anceps (Benazzi This work aimed to obtain preliminary data on the kary- and Benazzi-Lentati, 1976), Polycelis nigra (Beukeboom et otype variation and geographical distribution of G. schubarti, al., 1998), and Schmidtea polychroa (Storhas et al., 2000). G. tigrina, and G. anderlani in the State of Rio Grande do Mixoploids were found in Schmidtea polychroa (Benazzi Sul for future experimental approaches based on modern and Benazzi-Lentati, 1976), Girardia schubarti (Kawakatsu biomolecular techniques and/or applications to environmen- et al., 1980), G. anderlani (Kawakatsu et al., 1983), and tal monitoring. Dugesia ryukyuensis (Tamura et al., 1998). Tetraploids and MATERIALS AND METHODS pentaploids occur in Schmidtea polychroa (Beukeboom et al., 1996) and G. schubarti (Kawakatsu et al., 1984). In the Sampling order Paludicola, B chromosomes are present in polyploid Specimens were collected in lotic and lentic freshwater environ- ments at various sites in Rio Grande do Sul from 1996 to 2000. populations (Beukeboom et al., 1994; Benazzi and Benazzi- Worms were detached from pebbles with small brushes and trans- Lentati, 1976). These data represent significant material for ported to the laboratory in plastic containers filled with water. In evolutionary studies on ploidy levels. some cases, different sites of the same lake or creek were sampled. Six species of planarians have been identified in Rio Previously published data on the distribution of species of Girardia Grande do Sul (Southern Brazil): Girardia (Cura) schubarti Genus in Brazil are shown in Table 1. (Marcus, 1946), Girardia tigrina (Girard, 1850), Girardia Culturing anderlani (Kawakatsu et al., 1983), Girardia uroriogran- In the laboratory, planarians were cultivated in atoxic plastic deana (Kawakatsu et al., 1992), Girardia arndti (Marcus, containers filled with reconstituted water (0.228 mM NaHCO3, 0.013 1946), and Girardia biapertura (Sluys, et al., 1997). The first mM KCl, 0.060 mM MgSO4, 0.090 mM CaSO4) (Cabridenc, 1979) three, G. schubarti, G. tigrina, and G. anderlani, have been and fed weekly with small chunks of bovine liver. The water was immediately changed after feeding. Populations were kept in a dark * Corresponding author. Phone: +51-3308-6070; environment at the optimal temperature for each species, 18–22°C Fax : +51-3308-7309; for G. tigrina (Gee et al., 1998) and 13–15°C for G. schubarti E-mail: [email protected] (Knakievicz, et al., 2006). Only G. anderlani did not adapt to labo- doi:10.2108/zsj.24.123 ratory conditions before karyotyping. 124 T. Knakievicz et al. Karyology ysis under a ZEISS Axiophot optical microscope. At least 10 mitotic Regenerating tissue obtained as described by Baguñà et al. cells were analyzed to determine the ploidy level of each individual. (1989) was used for karyotyping. The regenerating tissue (postblast- However, the average was 26 mitotic cells analysed per individual. ema) was dissected and treated with colchicine (0.2% for 3 h). The colchicine-treated blastemas were minced and further disaggregated RESULTS by pipetting several times. They were then incubated for 20 min in a hypotonic KCl solution (0.075 M). Free cells and tissue fragments Biogeography of Girardia in the State of Rio Grande do Sul were sedimented by centrifugation and resuspended in 5 ml of cold For biogeographical and ecological studies, a total of Carnoy’s solution. Slides were prepared by the air-drying method and 140 freshwater planarians were karyotyped from 16 areas in stained with 10% Giemsa (Lamatsch et al., 1998) for standard anal- Rio Grande do Sul (Table 2). The samples comprised G. Table 1. Data published about the distribution of the species of Girardia Genus in Brazil. City/Statea Species Reference Arroio do Meio/RS G. ururiograndeana Kawakatsu et al., 1992 Arroio Grande/RS G. ururiograndeana Kawakatsu et al., 1992 Caxias do Sul/RS G. schubarti Kawakatsu et al., 1980 Constantina/RS G.sp Kawakatsu et al., 1986 Dois Irmãos/RS G. anderlani, G. schubarti and G. sp Hensel, 1980; Kawakatsu et al., 1976, 1980, 1986 Encruzilhada do Sul/RS G. anderlani Kawakatsu et al., 1986 Montenegro/RS G. schubarti Hensel, 1980; Kawakatsu et al., 1980 Nova Petrópolis/RS G. sp Kawakatsu et al., 1980 Novo Harmburgo/RS G. schubarti Kawakatsu et al., 1980 Pelotas/RS G. anderlani, G. schubarti and G. tigrina Kawakatsu et al., 1986 São Francisco de Paula/RS G. schubarti and G. tigrina Kawakatsu et al., 1986 São Leopoldo/RS G. schubarti and G. tigrina Kawakatsu et al., 1980 Sapiranga/RS G. schubarti Hensel, 1980 Botucatu/SP G. schubarti Kawakatsu et al., 1983 Campos do Jordão/SP G. schubarti Kawakatsu et al., 1976 Cantareira/SP G. schubarti Kawakatsu et al., 1983 Cidade Universitaria/SP G. tigrina Kawakatsu et al., 1981 Monte Alegre/SP G. schubarti Kawakatsu et al., 1976 Salesópolis/SP G. schubarti Kawakatsu et al., 1976 São Carlos/SP G. schubarti and G. tigrina Kawakatsu et al., 1982 Salvador/BA G. tigrina Preza and Smith, 2001 Serra da Mantiqueira/RJ G. schubarti Kawakatsu et al., 1976 aRio Grande do Sul state (RS), São Paulo state (SP), Rio de Janeiro state (RJ), and Bahia state (BA). Table 2. Sampling sites and karyotypic classification of the collected planarians in Rio Grande do Sul state. Species/ karyotypea Site G. schubarti G. tigrina G. anderlani 2n=8 3n=12 2n/3n 2n=16 3n=24 2n=18 3n=27 Arambaré – – – 3 – – – Caçapava do Sul – – – – – 5 – Dois Irmãos 2 – – – – – – Erechim 1 1 1 – – 1 2 Flores da Cunha 6 – – – – 7 – Glorinha – – – 1 – – – Jacutinga – 12 ––––– Porto Alegre ––– 13 ––– Salvador do Sul – – – 1 2 5 – Santa Maria 6 – 2 – – 2 – Santo Ângelo – 9 6 – – – – São Francisco de Paula 16 – 3 – – 1 – São José dos Ausentes 6 – – – – – – Severiano de Almeida 10 – 1 – – 3 – Uruguaiana 5 – 1 – – 2 – Viamão – – – – – 1 – Total 54 22 15 18 2 27 2 anumber of planarians (identified from of the analyses of at least ten methaphasic cells per individual). Karyotopes of Brazilian Planarians 125 schubarti, G. tigrina, and G. anderlani, taken from distinct creeks of the Basaltic Rock Plateau, a geomorphological geomorphological regions of Rio Grande do Sul. region extending from São Paulo State to the northern half Girardia schubarti occurred under stones in well-oxy- of Rio Grande do Sul (Tables 1 and 2; Fig. 1b). genated lotic water bodies. It was found predominantly in Girardia tigrina was detected among vegetation in lentic Fig. 1. Geographic distribution and karyotypes of freshwater planarians in the State of Rio Grande do Sul, Brazil. The black dots indicate where diploid planarians were found. The black arrows indicate where triploids or mixoploids were also found. (a) Morphogeological regions of Rio Grande do Sul State (map adapted from Ramgrab et al., 2000). (b) Distribution of collected Girardia schubarti specimens. (c) Distribution of collected Girardia tigrina specimens. (d) Distribution of collected Girardia anderlani specimens. (e, f) 2n and 3n karyotypes of G. schubarti. (g, h) 2n and 3n karyotypes of G. tigrina. (i, j) 2n and 3n karyotypes of G. anderlani. 126 T. Knakievicz et al. water bodies. It was found in lakes of the Serra Geral’s mixoploids (18%) (Table 2). scarp, the Central Depression, the Coastal Plain (Fig. 1c), For G. tigrina, diploid (2n=16; Fig. 1g) and triploid and northern Uruguay (Table 1). (3n=24; Fig. 1h) specimens were collected (Fig. 1c). The Girardia anderlani was detected in sympatry with G. sample population consisted of 18 diploids (65%) and two schubarti in some places located at the borders of lakes and triploids (35%) (Table 2). streams. Girardia anderlani was also found in the Central Diploid (2n=18; Fig. 1i) and triploid (3n=27; Fig. 1j) spec- Depression, Coastal Plain, and Crystalline Shield regions imens of G. anderlani were collected (Fig. 1d). The sample (Figs. 1d). population consisted of 27 diploids (93%) and two triploids These geographical distributions indicated by our data (7%) (Table 2). B chromosomes were found in a diploid G.