Autoecological and molecular approach to the species problem in the Euplotes vannus-crassus-minuta group (Ciliophora, Hypotrichida)

Europ.]. Protisto!' 26, 142-148 (1990) Octo ber 19, 1990

Eu ropean Journal o f

PROTISTOLOGY

Autoecological and Molecular Approach to the Species Problem in the Euplotes vannus-crassus-minuta Group (Ciliophora, Hypotrichida) Alessandro Gianni and Luciana Piras Dipartimento di Scienze dell'Ambiente

e del

Territorio, Universita di Pisa, Plss, Italy

SUMMARY The long stan ding species problem in the Eup lotes uannus-crassus-minu ta gro up is addressed here by means of restriction enzyme rONA maps and salinity and temperature tolerance tests performed on several strains of these morph ospecies (i.e. morph ologically identified species). The rationale in collecting such data (which add to others existing in literature) is to provide a broad data base to allow a multi-integrated appro ach to the problem at hand. The autoecological parameters considered and the rON A maps proved to be different in the three morphospecies. Both recent literature and o ur data suggest the existence of E. uannus, E. crassus and E. minuta as sepa rate evolutionary ent ities. Conceptual and pr actical implicat ions of our findings are discussed.

Introduction

The situation of the Euplotes vannus-crassus-minuta group is paradigmatic of the problems that can arise in the identification of unicellular organisms, which frequ entl y display slight morphological differences among congener ic species. According to Curds [1] the correct nomenclature of th e species involved is E. vannus (M iiller, 1786 ) M inkjewicz, 1901 ; E. crassus (Duja rdin, 1841 ) Kahl, 1932 and E. m inuta Yocum, 1930. Nowadays, Eupl otes identification is based mainly on silver stai ning. Since this techn ique was firstly used for Eup lotes taxonomy onl y in 196 0 by Tuffrau [21], it is difficult to be completely sure th at these morphospecies correspo nd to th ose described by Miiller, Dujardin and others. Hence, one may say that the modern Euplotes ta xonomy has restarted after Tuffrau [21]. In the section A of the genus Euplotes, comprising E. vannus, E. crassus and E. minuta (and th e less studied E. balticus and E. cristatus), the dor sal subpellicular network (dargyrome ) consists of single rows of poligons between adjacent lines of dorsal bristles (kineties) [1]. Other morphological parameters (cell size, above all) are commonly used to discriminate th ese morphospecies [1, 2 1, 23 ] which have long been con sidered sepa rate ta xa. Important analyses 0932-4739/90/0026-0142$3.50/0

like those on the struc ture of their mating systems were performed separat ely on E. vannus [8], E. crassus [9], and E. minuta [16]. However, after an accidental inversion of E. crassus and E. vannus body lengths (in Tuffrau [21]; see Valbonesi et al. [22] and Schlegel et al. [18]), a considerable confusion in the identification of such morphospecies ha s been generated, especially when the existence of an E. vann us Complex of sibling species was reported: strains attributed to both E. crassus and E. vannus morphotyp es were said to belong to the same biological species (repro ductive unit) and no morphological and electrophoretic differentiation was found among th em (reviewed in Genermont et al. [6]). Recently, Valbon esi et al. [23] reaffirmed th at E. vannus, E. crassus and E. m inuta are three distin ct "evo lution ary ent ities", on the basis of multivariate morphom etrics, mating relation ship s and isoenzyme electr ophor etic pattern an alysis. Addition al evidences of a clear distin ction of E. crassus from E. m inu ta (unfortunately, no E. vannu s strain was studied) was lat er given by Schlegel et al. [18] in a larg e survey of th e ph ylogenetic relationships within the genus Euplotes, on the basis of enzyme electr ophor esis. Here we report autoccological and molecular dat a on © 1990 by Gustav Fischer Verlag, Stuttgart

Species Problem in Marine Euplotes . 143

strains morphologically characterized according to Valbonesi et al. [23]. We investigated salinity and temperature tolerances and determined a partial restriction enzyme map of their macronuclear rDNA fragments to test the real existence of discontinuities among the morphospecies. This appears clear from our data which support the idea that they really constitute three separate entities. Material and Methods

Strains Strains used are listed in Table 1, where their origins are mentioned and it is specified if they were used for the autoecological or rONA analysis or both. Several strains, expecially within the E. vannus morphospecies, have been already studied by Valbonesi et al. [23]. During the analysis, our strains displayed neither autogamy nor selfing (intraclonal conjugation). Therefore, our results concern nonautogamous populations only [3]. With the exception mentioned in Table 1, the strains der ived directly from individuals isolated from the wild. Euplotes cultures were maintained in autoclaved (120°C, 18 min) artificial seawater at 3.5% salinity (Marine-Tropic New salt, Euraquarium, Bologna) and fed with the green algae Dunaliella tertiolecta. Algae were reared in the previously described artificial seawater enriched with Walne medium [24] in air-bubbled culture flasks exposed to a 12 h light/dark cycle at 19 °C. As a rule, Euplotes strains were kept in large test tubes at room temperature (22-24 °C) and fed weekly. To obtain the necessary amount of cells for the rDNA mapping, mass cultures were grown in one liter glass flasks, fed every two days and incubated at 25°C in a thermostatic room.

Table 1. List and origin of strains

Euplotes crassus PB l a, PB 2 ES 9', ES 10 NRl FS 4A, FS 8A SM 1 SS 22 a , SS 52 EC i -, EC 2, EC 3, EC 4 a LD t-, LD 3, LD 4, LD 5 HA 15 b, HA 25 b

Piombino, Italy (1978) Motril, Spain (1986) Sinop, Turkey (1982) Sciacca-Foggia, Italy (1984) Sciacca-S, Marco, Italy (1984) Sciacca-Stazzone, Italy (1987) Porto vecchio, France (1986) Livorno, Italy (198 7) Handbjerg, Denmark (1988)

Euplotes vannus AL2 a, AL3

SB

r-, SB 2 ·

LC 52 a TM Ib

Euplotes minuta CP-Lb 3<, CP-Lb 7 e TI-Lb Ie Szf 1 a RO 7, RO

s-

Alghero, Italy (1984) Santa Barbara, USA (1985) Livorno, Italy (1986) Torre Mileto, Italy (197 9) Capraia, Italy (1985) Tirrenia, Italy (1985) Sciacca-Stazzone, Italy (1982) Roscoff, France (1986)

strains used for rDNA mapping and autoecological tests. strains used for rDNA mapping only . All the other strains were used for autoecological tests only . e F1 strains obtained in laboratory from cross-breeding wild strains. a

b

Autoecological Tests Salinity and temperature tolerances, and the influenc e of different salinity values on thermal tolerances were analyzed. The sterile artificial seawater for the salinity tests was obtained by dilution of a concentrated solution. Salinity wa s checked by an optical salinometer (American Optical Corporation). After a preliminary identification of the optimal salinity ranges of the strains (100% survival), the high and low salinity tests were run separately. In each trial all the strains used were tested for a range of salinity values from complete survival to complete mortality. For each strain, at each salinity value, 9 single cells (24 h starved) were isolated with a handmade micropipette in 0.2 ml of the test medium dispensed into a well of a three-spot hemispheric depression slide. The slides were placed in glass moist chambers to reduce alteration in salinity because of evaporation. These experiments were run in a thermostatic room at 22± 1 °C in the dark. Survivorships were recorded after23-25 h by means of a stereomicroscope (16 or 40 x magnification). According to Martinez [12], cells unable to swim or creep on the bottom of the well (although sometimes showing an uncoordinated ciliary activity) were regarded as dead. The experimental procedure followed for the temperature tolerance tests was similar to that for salinity tests, but cells were placed in media at 2.0, 3.5 or 5.0% salinity (at 22 °C) and then incubated (for 23-25 h) in thermostatic rooms at the test temperature. For each salinity, all the strains were contemporaneously tested for low or high temperature tolerance. From a general point of view salinity and temperature tests differ because only in the first case there was a real shock. As single cells were always isolated from a medium at 3.5% salinity and 22-24 °C of temperature (our standard culturing conditions), the salinity change following isolation in the above described experimental media was sudden, while in the temperature tests the experimental value was attained more slowly. All experimental trials were repeated three times and the observed scattered variations of strain survivorships (of about 10 %) never changed the overall comparisons between species. Therefore, only data from the same replicate were used for the subsequent calculations of the 95 % confidence intervals. The influence of salinity on low and high temperature tolerance was tested at 2.0, 3.5 and 5.0% salinity; these values fall within the full-survivorship range of all the morphospecies. The median lethal doses (LD so) for each strain were graphically estimated after probit transformation [4]. Afterwards, mean LDso and 95% confidence intervals were obtained for each morphospecies for low and high temperatures, at each tested salinity. Within a morphospecies all the strains behaved quite homogeneously except that two groups of E. crassus strains were evidenced in low salinity tolerance tests. Whatever be the meaning of such dichotomy, as the present report is focused on differences among morphospecies, the two groups (i.e. the E. crassus rnorphospecies) were treated as a single entity when their tolerances proved not to be significatively different by 95% confidence intervals.

DNA Extraction Cells harvested from mass cultures according to Valbonesi ct al. [23] were resuspended in a small volume of sterile synthetic seawater. Two volumes of lysis buffer (0.01 M Tris-HCI, 0.5 M EDTA, 1% SDS; pH 9.5) prewarmed at 60°C were added, and the sample kept for 15 min at 37°C, followed by the addition of 0.1 mg/ml of proteinase K (Boehringer, Mannheim) and incubation at37 °C for 5-8 h. After 1: 1 dilution with 0.1 x NET (0.5 M NaCl, 0.05 M EDTA, 0.05 M Tris -HCI; pH 8.5) the DNA was phenol and chloroform extracted and precipitated with ethanol.

144 . Gianni and Piras DNA was dissolved in 0.5 ml of 0.1 X NET, incubated with 0.1 mg/ml of ribonuclease A (Sigma Chemical Co., St. Louis, MO) at 3rc for 1 h and finally re-extracted and precipitated as above. The whole procedure is basically that described by Nielsen et at. [13] with minor modifications.

Restriction enzymes and gel electrophoresis Restriction enzymes were obtained from Boehringer and used according to the supplier's recommendations. Agarose was purchased from Biorad (Richmond, CAl. Gels were run in TAE buffer (0.04 M Tris-acetare, 0.001 M EDTA; pH 8.3) for 4 h, at 100V.

region of Tetrahymena thermopbila rONA , while pRP 11 contains only a portion located at the 3 ' end, within the 26 S rONA and was used to assess the 5 '-3' polarity of the rONA fragments (see Fig. 4 for details). After hybridization the membranes were serially washed for 15 min each in 2 X SSC, 1 X SSC and 0.1 X SSC at room temperature, and placed at - 70 "C for 24 h expo sition time with AGFA Curix RP 1, using intensifying screens. In orderto perform a second hybr idization, the membranes were dehybridized by incubation in 0.4 M NaOH at 45 "C for 30 min and then placed in 0.2 MTris-HCI, 0.1 % SDS, 0.1 X SSC at 45 °C for 30 min. Hybridization followed as previously described .

Hybridization

Results

Native macronuclear DNA fragments were transferred from gels to Hybond-N nylon membrane (Amersham, Little Chalfont, UK) following the Southern procedure [17]. The membrane was prehybridized in 50 % formam ide, 6 X SSC, 5 X Denhardt solution (100 X Denhardt solution = 2% bovine serum albumine, 2% FICOLL and 2% polyvinylpyrrolidone) [2], 1% SDS and 0.02 mg/ml of sheared calf thymus DNA, at 37 "C, Hybridization was carried out in the same conditions, adding as a probe the gel-purified insert of pGY 17 or pRP 11 plasmids (kindly provided by Prof. Jan Engberg, Panum Institute, Copenhagen) 32P-labelled by nick-translation to initial specific activity of 10 8 cpm/ug, The insert of pGY 17 comprises most of the coding

Salinity and Temperature Tolerance Salinity tolerances of the three morphospeciesare shown in Figs. lA (low-salinity) and IB (high-salinity). Both graphs display a similar pattern with E. crassus more tolerant than E. uannus, in turn slightly superior to E. minuta. Two data points for E. crassus are reported only at 0.4% salinity, were significative differences between two clearly distinct groups of strains exist. Survivorships after temperature tolerance tests are shown in Figs. 2A (low-temperature) and 2B (high100

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Fig. 2. Low (A) and high (B) tem perature tolerances of E. uannus (.A.), E. crassus (e) and E. minuta (.). Bars represent 95 % confidence intervals.

Species Problem in Marine Eup lotes . 145

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146 . Gianni and Piras

temperature). The last shows the same tolerance pattern as for salinity tolerances (E. crassus, E. vannus, E. minuta), while at low temperature the two less tolerant morphospecies are not significativelydifferent. These data refer to tests at 3.5% salinity, but similar results (at both high and low temperatures) were observed at 2.0% and 5.0% salinity (data not shown). Influence of salinity on temperature tolerances

In Fig. 3 the influence of salinity on thermal tolerances of the three morphospecies are reported as 95% confidence intervals around mean LDso with the exception of E. crassus low temperature tolerances. All the strains of this morphospecies displayed high survivorship at 0 °C but no cell survived at -2°C. Hence, we did not estimate the LDso and percent survivorships at O°C are reported in Fig. 3: no effect of salinity appears at this temperature. In both E. van nus and E. minuta a decrease in low temperature tolerances at 2.0% and 5.0% if compared to 3.5% salinity data was clear. At high temperature, for all morphospecies, thermal tolerances recorded at 3.5 and 5.0% are similar and both significatively superior than those recorded at 2.0% salinity. The inhibition by low salinity (1.5%) of E. crassus growth at high temperature (30°C) has already been reported [12]. rDNA Maps

In hypotrichous ciliates the macro nuclear DNA is organized in small fragments bearing one or few genes [11]. In our experiments the insert of pGY 17 (containing sequences from 17 S, 5.8 Sand 26 S Tetrahymena rDNA, see Fig. 4) always hybridized in a single band with undigested Euplotes DNA. Due to this fact, and comparing the sizes of the Euplotes fragments (about 6.8 or 7.5 kilobases, kb) with the expected sum of the length of such rDNAs (about 6 kb), we assume that in these Euplotes morphospecies each rDNA molecule comprises a single copy of, respectively, 19 S, 5.8 Sand 25 S rDNA genes, according to other data known for hypotrichous ciliates [19]. In order to map the macronuclear rDNA, we used several restriction enzymes. Six of them (Bam HI, Kpn I, I I I

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Eco RV, Xba I, Dra I and Dde I) did not digest these Euplotes rDNAs, while Rsa I and Alu I were unsuitable producing many small restriction fragments. Hence, the maps are based on restriction fragments generated by Eco RI, Hin dIll and Bgl II digestions, either partial or complete, and in double digestions with the same enzymes. First of all, the size of the rDNA fragment of the three morphospecies proved to be different. Figs. 5A and Bshow the typical results obtained after Eco RI digestion. The 5' end is about 0.7 kb shorter in E. minuta than in E. crassus and E. vannus (this difference was appreciable even in undigested rDNA). The 3' end in E. crassus is longer than in E. vannus and E. minuta as evidenced by hybridization with pRP 11 probe which extends further on the 3' side than pGY 17 (Fig. 4). The size difference (about 0.1 kb) is not easily appreciable in undigested rDNA and one might suppose it to be the consequence of an Eco RI site producing a 0.1 kb restriction fragment too short and probably external to our probe, so that it may not be detected in our autoradiograms. Nevertheless, Bgl II generates 3' rDNA fragments of about 1.6 kb in E. crassus and 1.5 kb in both E. vannus and E. minuta and we deduce that 3' -end size differences do really exist. The maps in Fig. 6 show that the restriction sites of the analyzed enzymes are identical in E. crassus and E. vannus, but E. minuta has an additional Hin dIll site. This site seems to be located within the region coding for the 26 S rDNA. Finally, our mapping experiments have not revealed differences among strains of the same morphospecies in spite of the great distances among certain locations of origin. Discussion The idea that the three morphospecies of the so-called Euplotes uannus-crassus-minuta group must be considered as separate evolutionary entities is strongly supported by auto ecological and molecular data. Although one may be surprised by the unusual lumping of such different disciplines we maintain that the convergence of data from largely different sources can help to solve particularly entangled systematic problems. Because many specialists currently working on these Euplotes morphospecies may not be familiar with multivariate morphometry and protein electrophoresis and ceo-physiology and molecular biology but only with some of these fields, we think it is important to give different diagnostic traits of every morphospecies. In our minds autoecological parameters (above all salinity tolerance) can be the simplest tools for the rapid identification of the members of this group, a difficult task when strains of the morphologically similar E. vannus and E. crassus morphospecies are considered. Within the E. vannus Complex (comprising the E. vannus and E. crassus morphospecies) tolerances to high and low temperature and increase or decrease of salinity have been reported to be very similar among different sibling species [6]. Because of the clear autoecological differences among our E. vannus and E. crassus strains, we feel that Valbo-

Species Problem in Marine Euplotes . 147

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nesi et al. [23] were right in suggesting that strains of one of the two morphospecies, namely of E. vannus , have never been included in the E. vannus Complex. The same authors stated that this could be due to a relative rarity of E. uannus (in nature) compared to E. crassus. According to our data it seems reasonable to assume that a wide tolerance allows E. crassus to colonize physically unstable environments as very shallow sandy bottoms and tidal pond s, where the majority of sampling is currently performed. Our personal sampling experience supports this view. From November 1986 to February 1987 a tidal pond area and a 5 meters deep sand y bottom (no mor e than 50 m

distant) were sampled near Livorno (Italy). In th e unstable tidal ponds (salinity from 1.4 to 3.6%) E. crassus was one of the dominant species of the benthic microfaun a (our LD strains) but it was never found in the five meter deep area. On the contr ary, one strain of E. vannus (LC 52) was found in the deep site (constant salinity 3.6 %) , none in the tidal ponds [7]. As far as the structure of the rDNA macronuclear fragment of the thre e morphospecies is concerned, the observed size difference was unexpected. In fact, it has been reported that in several hypotrichous species, included Euplot es aediculatus, the size of the rDN A macronuclear fragment is the same (about 7.5 kb) [20]. We do not kno w the origin, and meaning , of the observed size hetero geneity which likely involves flanking, non coding sequences. From our maps the structure of the rDNA in E. vannus seems to be intermediate between the two other morphospecies. Interestingly enough, E. vannus has been reported to be closer than E. minuta to E. crassus regard ing the induction of interspecific mat ing reactions [14, 15] and the general morphometric characteristics [22] but, on the other hand, in spite of a large cell size difference E. vannus and E. minuta strains have 8-9 dor solateral kineties while E. crassus strains generally bear 10-11 kineties [1,18,23]. Hen ce, furth er research work is needed to throw light on the phylogenetic relationships among these Euplotes morphospecies.

148 . Gianni and Piras

Acknowledgements The authors want to thank Prof. R. Nobili (Dip. di Sc. dell'Ambiente, Pisa) and Prof. P, Luporini, Prof. C. Miceli and Dr. A.Valbonesi (Dip. di Biologia Cellulare, Camerino) for valuable discussions and comments. Moreover, we thank Prof. J. Engberg (Panum Institut, Copenhagen) and Prof. T. Fenchel (Marine Biological Laboratory, Helsingor) for their warm (and scientifically formative) hospitality during part of the research. The investigation was supported by Dottorato di Ricerca grants from the Italian Ministero della Pubblica Istruzione.

References 1 Curds C. R. (1975): A guide to the species of the genus Euplotes (Hypotrichida, Ciliata). Bull. Br. Mus. Nat. Hist. (Zool.), 28, 2-61. 2 Denhardt D. T. (1966): A membrane-filter technique for detection of complementary DNA. Biochem. Biophys. Res. Comm., 23,641-646. 3 Dini F. and Gianni A. (1985): Breeding systems in the Euplotes vannus-crassus-minuta group. Atti Soc. Tosc. Sci. Nat., Mem, (Serie B), 92, 75-83. 4 Finney D. J. (1964): Probit analysis: a statistical treatment of the Sigmoid Response curve. Cambridge University Press, Cambridge, Mass. (USA). 5 Genermont J., Machelon V. et Tuffrau M. (1976): Donnees experimentales relatives au probleme de l'espece dans le genre Euplotes (Cilies Hypotriches). Protistologica, 12, 239-248. 6 Genermont J., Machelon V. and Demar C. (1985): The "uannus" group of the genus Euplotes. Sibling species and related forms: evolutionary significance and taxonomical implications. Atti Soc. Tosc. Sci. Nat., Mem. (Serie B), 92, 53-65. 7 Gianni A. (1989): 11 problema delle specie nel gruppo Euplotes vannus-crassus-minuta (Ciliophora, Hypotrichida). Approccio multidisciplinare. Tesi finale di Dottorato di Ricerca. 8 Heckmann K. (1963): Paarungssystem und genabhangige Paarungstypdifferenzierung bei dem hypotrichen Ciliaten Euplotes crassus O. F. Miiller. Arch. Protistenk., 106, 393-421. 9 Heckmann K. (1964): Experimentelle Untersuchungen an Euplotes crassus. I. Paarungssystem, Konjugation und Determination der Paarungstypen. Z. Vererbungsl., 95, 114-124.

10 Kahl A. (1932): Urtiere oder Protozoa. I. Wimpertiere oder Ciliata (Infusoria), eine Bearbeitung der freilebenden und ectocommensalen Infusorien der Erde, unter Ausschluf der marinen Tintinnidae. Tierwelt Deutschlands, 25, 399-650. 11 Klobutcher L. A. and Prescott D. M. (1986): The special case of the Hypotrichs. In: Gall J. G. (ed.): The molecular biology of ciliated protozoa, pp. 111-154. Academic Press Ine., Orlando. 12 Martinez E. A. (1980): Sensitivity of marine ciliates (Protozoa, Ciliophora) to high thermal stress. Estuar. Coast. Mar. Sci., 10, 369-381. 13 Nielsen H., Simon E. M. and Engberg J. (1985): Updating rDNA restriction enzyme maps of Tetrahymena reveals four new intron-containing species. J. Protozool., 32, 480-485. 14 Nobili R. (1964a): Coniugazione ibrida tra specie di Euplotes (Ciliata, Hypotrichida). Boll. Zool., 31, 1338-1348. 15 Nobili R. (1964b): On conjugation between Euplotesvannus O. F. Miiller and Euplotes minuta Yocom. Caryologia, 17, 393-397. 16 Nobili R. (1966): Mating types and mating type inheritance in Euplotes minuta Yocom (Ciliata, Hypotrichida). J. Protozool., 13, 38-41. 17 Southern E. M. (1975): Detection of specific sequences among DNA separated by gel electrophoresis. J. Mol. Biol., 98, 503-517. 18 Schlegel M., Kramer M. and Hahn K. (1988): Taxonomy and phylogenetic relationship of eight species of the genus Euplotes (Hypotrichida, Ciliophora) as revealed by enzyme electrophoresis. Europ. J. Protistol., 24, 22-29. 19 Swanton M. T., Greslin A. F. and Prescott D. M. (1980): Arrangement of coding and non-coding sequences in the DNA molecules coding for rRNAs in Oxytricha sp. Chromosoma, 77,203-215. 20 Swanton M. T., McCarrol R. M. and Spear B. B. (1982): The organization of macronuclear rDNA molecules in four hypotrichous ciliated protozoans. Chromosoma, 85, 1-9. 21 Tuffrau M. (1960): Revision du genre Euplotes, fondee sur la comparaison des structures superficielles. Hydrobiologia, 15, 1-77. 22 Valbonesi A., Ortenzi C. and Luporini P. (1985): Electrophoretic comparison between Euplotes vannus and E. crassus strains. Atti Soc. Tose. Sci. Nat., Mem. (Serie B), 92, 67-73. 23 Valbonesi A., Ortenzi C. and Luporini P. (1988): An integrated study of the species problem in the Euplotes crassusminuta-vannus group. J. Protozool., 35, 38-45. 24 Walne P. R. (1966): Experiments in the large scale culture of larvae of Ostrea edulis L. Fish. Invest. London, Ser. 2, 25, 1-10.

Key words: Euplotes - Species problem - Autoecology - rDNA maps Alessandro Gianni and Luciana Piras, Dipartimento di Scienze dell' Ambiente e del Territorio, Universita di Pisa, Via A. Volta 4,1-56100 Pisa, Italy