A g m G M a P b R c d d e N R A A t 1 s m d b f f T t n © K 0 d European Journal of Protistology 47 (2011) 103–123 n Antarctic hypotrichous ciliate, Parasterkiella thompsoni (Foissner) nov. en., nov. comb., recorded in Argentinean peat-bogs: Morphology, orphogenesis, and molecular phylogeny abriela Cristina Küppersa,∗, Thiago da Silva Paivab, Bárbara do Nascimento Borgesc, aria Lúcia Haradad, Gabriela González Garrazae, Gabriela Matalonie Instituto de Limnología Dr. R. A. Ringuelet (CCT LA PLATA CONICET), Av. Calchaquí km 23,5, (1888) Florencio Varela, Buenos Aires rovince, Argentina Laboratório de Protistologia, Departamento de Zoologia, Instituto de Biologia, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, J, Brazil Instituto Socioambiental e dos Recursos Hidricos – Universidade Federal Rural da Amazônia, Belém, PA, Brazil Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Cidade Universitária Prof. José Silveira Netto, Universidade Federal o Pará, Belém, PA, Brazil Grupo de Biodiversidad, Limnología y Biología de la Conservación, 3iA - Instituto de Investigación e Ingeniería Ambiental, Universidad acional de San Martín, Buenos Aires Province, Argentina eceived 19 August 2010; received in revised form 12 January 2011; accepted 19 January 2011 vailable online 3 April 2011 bstract The ciliate Parasterkiella thompsoni (Foissner, 1996) nov. gen., nov. comb. was originally described from Antarctica. In he present study, we report the morphology, morphogenesis during cell division, and molecular phylogeny inferred from the 8S-rDNA sequence of a population isolated from the Rancho Hambre peat bog, Tierra del Fuego Province (Argentina). The tudy is based on live and protargol-impregnated specimens. Molecular phylogeny was inferred from trees constructed by eans of the maximum parsimony, neighbor joining, and Bayesian analyses. The interphase morphology matches the original escription of the species. During the cell division, stomatogenesis begins with the de novo proliferation of two fields of basal odies, each one left of the postoral ventral cirri and of transverse cirri, which later unify. Primordia IV–VI of the proter develop rom disaggregation of cirrus IV/3, while primordium IV of the opisthe develops from cirrus IV/2 and primordia V and VI rom cirrus V/4. Dorsal morphogenesis occurs in the Urosomoida pattern—that is, the fragmentation of kinety 3 is lacking. hree macronuclear nodules are generated before cytokinesis. Phylogenetic analyses consistently placed P. thompsoni within he stylonychines. New data on the morphogenesis of the dorsal ciliature justifies the transference of Sterkiella thompsoni to a ew genus Parasterkiella. 2011 Elsevier GmbH. All rights reserved. eywords: Parasterkiella thompsoni nov. gen., nov. comb.; Stylonychinae; Ontogeny; 18S-rDNA gene; Peat bogs; Argentina ∗Corresponding author. Fax: +54 11 42758564. E-mail address: gkuppers@fcnym.unlp.edu.ar (G.C. Küppers). I 1 932-4739/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. oi:10.1016/j.ejop.2011.01.002 ntroduction The hypotrichous ciliate Sterkiella thompsoni Foissner, 996 was originally described from samples of the moss dx.doi.org/10.1016/j.ejop.2011.01.002 mailto:gkuppers@fcnym.unlp.edu.ar dx.doi.org/10.1016/j.ejop.2011.01.002 104 G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 ho Ham S D I S s t A i m a d ( c L t P b t r b T a p m r l c l b t p a 1 s g M S M 6 S o c d a b ( O i c s a c q p A t Fig. 1. Sampling sites (ponds RH3 and RH5) in Ranc anionia uncinata (Hedwig, 1801) Loeske, 1907 (formerly repanocladus uncinatus) from Signy Island, South Orkney slands, in the maritime Antarctic region (Foissner 1996). ince then, this species has only been recorded from moss amples and in a barren soil fellfield near Casey Sta- ion, Wilkes Land—a very distant location on Continental ntarctica (Petz and Foissner 1997). This ciliate is eas- ly recognizable because of the typical presence of three acronuclear nodules, a distinct feature that is unusual mong oxytrichids (Berger 1999, p. 4). Before the formal escription of S. thompsoni by Foissner (1996), Sudzuki 1964) found an Antarctic ciliate with two to three macronu- lear nodules, recording it under the name Opisthotricha sp. ater, Thompson (1972) described an Oxytricha sp. with hree macronuclear nodules from a rock pool in the Antarctic eninsula. Foissner (1996) regarded these forms as possi- ly conspecific with his new species and consequently S. hompsoni could be widespread throughout the Antarctic egion. Until now, this ciliate has been considered as possi- ly endemic from Antarctica (Berger 1999; Petz et al. 2007). he province of Tierra del Fuego (Argentina) is located bout 1000 km from the Antarctic Peninsula and encom- asses extensive areas of peat bogs dominated by Sphagnum osses. Among aquatic ecosystems, peat bogs are rare envi- onments found in particular climatic conditions; such as ow temperatures, high humidity, and evenly distributed pre- ipitations (Mataloni 1999). In the course of an integrated imnological research, this oxytrichid ciliate was found in two odies of water in the locality of Rancho Hambre. This con- ribution aims at describing the morphology of the Fuegian opulation along with its morphogenesis during cell division nd hypothesizing its molecular phylogeny from analyses of 8S-rDNA genetic sequences. The new data indicate that this w w fi n bre peat bog, Tierra del Fuego Province, Argentina. pecies is a representative of a new genus, Parasterkiella nov. en. aterial and Methods tudy area and samplings The Rancho Hambre peat bog is located in the Tierra ayor Valley, about 50 km from Ushuaia city (54◦47′S, 8◦19′W). This area is dominated by the peat-forming moss phagnum magellanicum Bridel, 1798 and constitutes an mbrotrophic peat bog, since its only source of nutrients omes from precipitations and snow melt. A large number of bodies of water of different sizes, epths, and physicochemical and biological conditions char- cterize the landscape of Rancho Hambre. Two such water odies, named RH3 (54◦44′46.3′′S, 67◦49′32.9′′W) and RH5 54◦44′39.4′′S, 67◦49′27.1′′W), were sampled in April and ctober 2008 and 2009 during the course of a limnological nvestigation (Fig. 1). Water samples were preserved in the old for the establishment of cultures. Temperature, pH, dis- olved oxygen, and conductivity were measured in situ with multiparametric probe (HORIBA, Japan). Photosyntheti- ally active radiation (PAR) was measured with a spherical uantum sensor (Li-COR Biosciences, USA). Water sam- les for dissolved nutrients were filtered through Millipore PFF membrane. Samples for PO4-P and NO3-N concentra- ion measurements were preserved at −20 ◦C until analysis ithin four weeks’ time at the Universidad de Buenos Aires, hile those for determination of NH4-N concentration were xed with sulfuric acid at 4 ◦C. Concentrations of dissolved utrients (NH4-N, NO3-N, PO4-P) were measured with a urnal of s p M b ( P s i m a fi ( fi c m o w t a f d b ( F d L 2 fl t c W W D c w d T a a p m a 1 m N H N t r a a t s a g D s g e s a P B p a i R M c e fi a c k ( B l b ( a 1 ( ( “ B ( f G.C. Küppers et al. / European Jo pectrophotometer (Hach Company, USA), with the appro- riate reactants for each analysis. orphology In the laboratory, the samples were kept in a culture cham- er, under controlled temperature (4–10 ◦C) and photoperiod 12:12 light/dark cycles). Raw cultures were established in etri dishes by enriching samples with wheat kernels to timulate growth of bacteria as a food source for the cil- ates. The ciliates were observed in vivo under the stereo icroscope and bright-field microscope. In order to visu- lize the infraciliature and the nuclear apparatus, cells were xed in Bouin solution and silver-impregnated with protargol Wilbert 1975). Measurements were taken under the bright- eld microscope with a calibrated ocular micrometer. Living ells were free-hand sketched, based on live observations and icrographs. Impregnated cells were illustrated with the aid f a drawing tube attached to the microscope. Observations ere performed under the stereo microscope at magnifica- ions of 20× and 40×, and under the bright-field microscope t 40×, 100×, 400×, and 1000×. Resting cysts of Parasterkiella thompsoni were obtained rom old cultures, by scratching the bottom of the Petri ish with a micropipette. Cysts were observed in vivo by right-field microscopy, stained with methyl-green pyronin Foissner 1991), and impregnated with protargol. Voucher slides of Parasterkiella thompsoni from Tierra del uego (accession numbers MLP66, MLP67, MLP68) were eposited in the Colección de Invertebrados of the Museo de a Plata, Argentina, with relevant cells marked. The general terminology used is after Berger (1999, 2006, 008) and Lynn (2008). The terminology concerning body exibility is according to Foissner and Stoeck (2008) and for he oral apparatus after Foissner and Al-Rasheid (2006). The irri and developing primordia were numbered according to allengren (1900). Resting cysts were described following alker and Maugel (1980) and Berger (1999). NA extraction, amplification, and sequencing Specimens of Parasterkiella thompsoni were picked from ultures from pool RH3 and transferred to embryo dishes, here the isolates were thoroughly washed with mineral and istilled water, then fixed with 70% (v/v) aqueous ethanol. he individuals were then submitted to DNA extraction ccording to Sambrook et al. (1989). The 18S-rDNA was mplified via PCR, through the use of the Hypotricha-specific rimers designed by Paiva et al. (2009). Amplified frag- ents were purified according to Sambrook et al. (1989) nd sequenced by the dideoxyterminal method (Sanger et al. 977) by means of an ABI 3130 (Applied Biosystems) auto- atic sequencer. The resulting sequence was uploaded to the CBI/GenBank and is available under the accession code M569264. t a 1 a Protistology 47 (2011) 103–123 105 ucleotide matrix assembly and alignment The nucleotide matrix used in the present study con- ained 29 representatives of supposedly related taxa (not estricted to species with 18 frontal–ventral–transverse cirri) s ingroup, plus nine urostylid sequences as outgroup. This pproach allows a proper accessing of the monophyly of he stylonychines to the inclusion of P. thompsoni in uncon- trained phylogenetic analyses (Kitching et al. 1998; Nixon nd Carpenter 1993). The nucleotides were aligned through the so-called pro- ressive approach of multiple-sequence alignment (Feng and oolittle 1987) implemented in the Clustal X 1.81 computer oftware (Thompson et al. 1997), with the adoption of the ap-opening and -extension parameters determined in Paiva t al. (2009). The resulting matrix was further modified in the oftware BioEdit v7.0.5 (Hall 1999) for manual refinement nd trimming. hylogenetic analyses Phylogenetic hypotheses were constructed under ayesian, likelihood, and parsimony frameworks, thus ermitting the assessment of data sensitivity to different nalytic criteria. A Bayesian phylogenetic inference (BI) was performed n the computer software MrBayes 3.1.2 (Huelsenbeck and onquist 2001). The BI was based on two Markov chain onte Carlo (MCMC) simulations that were run with four hains of 1,000,000 generations, of which trees were sampled ach 200 generations (temperature of heat chains = 0.2). The rst 100,000 generations were then discarded as burn-in, and majority-rule consensus of the remaining trees was used to alculate the Bayesian posterior probabilities of the recovered inships (Schneider 2007). Under the likelihood framework, the software PhyML 3.0 Guindon and Gascuel 2003) was used to build an initial ioNJ (Gascuel 1997) pairwise-distance tree, which had its ikelihood improved via “subtree pruning and regrafting” ranch-swapping moves to generate a maximum-likelihood ML) tree. To evaluate the stability of the nodes obtained, nonparametric bootstrap analysis was conduced based on 000 replicates. Both BI and ML analyses made use of the GTR + I =0.6671) + G (=0.5653) nucleotide-substitution model Rodriguez et al. 1990), which was chosen according to the minimum theoretical information criterion” (Akaike 1974; os and Posada 2005), via the software MODELTEST 3.7 Posada and Crandall 1998). Finally, a maximum parsimony (MP) analysis was per- ormed in the software PAUP* 4b10 (Swofford 2003), hrough the use of only parsimony-informative characters nd gaps scored as a “fifth base” (Giribet and Wheeler 999; Schneider 2007) along with the successive-weighting pproach (Farris 1969). Initially, the “parsimony ratchet” 1 urnal of ( s t t p r o o t t p J r a r a p R P O o F a s r v 5 06 G.C. Küppers et al. / European Jo Nixon 1999) strategy implemented in PAUP* via the acces- ory software PaupRat (Sikes and Lewis 2001) was adopted o speed up the search for fundamental most-parsimonious rees, based on 250 iterations with 15% of the characters erturbed. The characters were then reweighted after their escaled consistency index (base weight = 10), and a further rdinary heuristic search was conduced with 300 replicates f random sequence addition. This reweighting and heuris- ic search cycle was successively applied until the length of he optimal trees stabilized (Kitching et al. 1998). Node sup- ort in MP was assessed via 1000 jackknife replicates (with AC emulation enabled in PAUP*). The “tree bisection and econnection” branch swapping algorithm was employed in ll MP-tree searches. The optimal trees obtained under each framework were ooted a posteriori according to the outgroup position (Nixon r k g igs 2–7. Morphostatic specimen (2–4) and resting cysts (5–7) of Parast nd 5) and protargol impregnation (3, 4, 6, and 7). (2) Ventral view of a rep ide and nuclear apparatus of the same specimen. Arrowheads mark mic idges on the surface of the resting cyst. (7) Fused macronucleus and mic acuole; Ma, macronucleus; Mi, micronuclei; 1–4, dorsal kineties 1–4 (k 0 �m (3 and 4). Protistology 47 (2011) 103–123 nd Carpenter 1993), visualized, and edited for aesthetics and ublishing in the software MEGA 4 (Tamura et al. 2007). esults arasterkiella nov. gen. Diagnosis. Stylonychinae with undulating membranes in xytricha pattern. With 18 frontal–ventral–transverse cirri, ne left and one right marginal rows of cirri separated poste- iorly, and four dorsal kineties. Caudal cirri present. Dorsal ineties develop without fragmentation. Etymology. Composite of para (Greek, beside) and the eneric name Sterkiella. Feminine gender. erkiella thompsoni from Tierra del Fuego after live observation (2 resentative specimen. (3 and 4) Infraciliature of ventral and dorsal ronuclei. (5) Cross section of a mature resting cyst. (6) Polygonal ronuclei of the kinetosome-resorbing resting cyst. CV, contractile inety 4 is a dorsomarginal row). Scale bars = 30 �m (2 and 5–7), urnal of n a P c ( T o m fi c o p a V M d T 1 a T C B B B B A A P P A A A M M P P M M M M M A F B F P P T R L C D D D D D D R M % G.C. Küppers et al. / European Jo Type species. Parasterkiella thompsoni (Foissner, 1996) ov. comb. (basionym: Sterkiella thompsoni Foissner, 1996). Species assignable. Presently only the type species is ssigned to Parasterkiella. arasterkiella thompsoni (Foissner, 1996) nov. omb. Improved diagnosis. Size in vivo 90–130 �m × 40–60 �m Foissner 1996) or 95–142 �m × 52–70 �m. Body semirigid. hree macronuclear nodules. On average 34 (Foissner 1996) r 37 adoral membranelles, 22 (Foissner 1996) or 29 right arginal cirri, 17 (Foissner 1996) or 22 left marginal cirri, ve transverse cirri, and four dorsal kineties with one caudal irrus each associated with kineties 1 and 3. Oral primordium 2 r p S able 1. Morphometric characterization of Parasterkiella thompsoni from haracter ody, length in vivo ody, width in vivo ody, length ody, width ZM, length nterior body end to anteriormost transverse cirrus, distance ostoral ventral cirrus V/4 to postoral ventral cirrus V/3, distance in betwe osterior body end to posteriormost transverse cirrus, distance nterior body end to first macronuclear nodule, distance nterior macronuclear nodule, length nterior macronuclear nodule, width iddle macronuclear nodule, length iddle macronuclear nodule, width osterior macronuclear nodule, length osterior macronuclear nodule, width acronuclear figure, length acronuclear nodules, number icronuclei, length icronuclei, width icronuclei, number doral membranelles, number rontal cirri, number uccal cirri, number rontoventral cirri, number ostoral ventral cirri, number retransverse ventral cirri, number ransverse cirri, number ight marginal cirri, number eft marginal cirri, number audal cirri, number orsal kineties, number orsal bristles, length ikinetids in dorsal kinety 1, number ikinetids in dorsal kinety 2, number ikinetids in dorsal kinety 3, number ikinetids in dorsal kinety 4, number esting cyst, diameter easurements are in �m and, unless indicated, based on protargol-impregnated spe ; M, median; Max, maximum value; Min, minimum value; n, number of observat Protistology 47 (2011) 103–123 107 riginates de novo from two anarchic fields of basal bodies; rimordia IV and V of the proter develop from cirrus IV/3, nd primordia V and VI of the opisthe develop from cirrus /4. orphology of Parasterkiella thompsoni from Tierra el Fuego Province, Argentina (Figs 2–7, 24–29 and able 1) Size in vivo about 95–142 �m × 52–70 �m, usually 22 �m × 58 �m; length to width ratio 2:1 in vivo and fter protargol impregnation; dorsoventrally flattened about :1. Body with parallel margins, anteriorly and posteriorly ounded, sometimes broadly rounded posteriorly or slightly ointed (Figs 2–4). Well fed organisms rather bursiform. emirigid—i.e., the species cannot be classified unambigu- Tierra de Fuego. X̄ M SD CV Min Max n 122.1 121.2 15.8 12.9 95.0 142.5 10 58.4 56.5 5.4 9.2 52.2 70.0 10 134.4 140.0 22.7 16.8 91.0 168.0 20 63.0 63.0 12.6 20.0 42.0 84.0 20 59.8 63.0 7.7 12.8 42.0 70.0 20 117.1 121.8 17.7 15.1 82.6 145.6 20 en 14.9 15.4 3.2 21.4 8.4 19.6 20 4.3 4.2 0.9 20.9 2.8 5.6 20 37.5 38.5 5.6 14.9 28.0 46.2 20 22.8 23.8 3.6 15.7 15.4 28.0 20 17.9 18.2 2.7 15.0 12.6 22.4 20 18.9 18.9 4.2 22.2 12.6 29.4 20 18.1 18.2 3.5 19.3 12.6 25.2 20 24.2 23.8 4.8 19.8 15.4 33.6 20 17.6 17.1 3.0 17.0 12.6 22.4 20 64.3 64.4 12.0 18.6 44.1 85.4 20 3.0 3.0 0.2 6.6 2 4 50 4.6 4.9 0.5 10.8 3.5 5.6 20 3.9 4.2 0.5 12.8 2.8 4.9 20 2.5 2.0 0.9 36.0 2 6 50 36.8 37.0 2.0 5.4 32 40 20 3.0 3.0 0.0 0.0 3 3 20 1.0 1.0 0.0 0.0 1 1 20 4.0 4.0 0.0 0.0 4 4 20 3.0 3.0 0.0 0.0 3 3 20 2.0 2.0 0.0 0.0 2 2 20 5.0 5.0 0.2 4.0 5 6 20 28.6 28.0 2.4 8.3 25 33 20 21.9 22.0 2.1 9.5 19 27 20 2.0 2.0 0.0 10.0 2 3 20 4.0 4.0 0.0 0.0 4 4 20 4.5 4.5 0.3 6.6 4.2 4.9 10 19.5 20.0 2.1 10.7 15 22 10 20.8 20.0 2.1 10.0 19 24 7 21.1 21.0 1.6 7.5 19 23 6 20.8 21.5 2.4 11.5 17 25 8 45.8 45.2 2.7 5.8 41.0 51.0 10 cimens. AZM, adoral zone of membranelles; CV, coefficient of variation in ions; SD, standard deviation; X̄, arithmetic mean. 108 G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 Figs 8–12. Morphogenesis of ventral side of Parasterkiella thompsoni after protargol impregnation. (8) Oral primordium development from two fields of basal bodies. (9) Enlargement of both basal body fields. Replication bands in macronuclear nodules (arrowhead). (10) Oral primordium as a unique field of basal bodies. (11) Membranelles differentiation and formation of the primordia I–III of the opisthe and the primordia IV, V of the proter. (12) Development of the primordia IV, V of the opisthe and the primordia III, VI of the proter. OP, oral primordium; IV/3, IV/2, V/4, ontogenetically active parental cirri; V/3, ontogenetically inactive parental postoral ventral cirrus; I–VI, frontal–ventral–transverse cirri primordia. Scale bars = 50 �m. Table 2. Origin of the frontal–ventral–transverse cirri primordia during morphogenesis of Parasterkiella thompsoni. Primordium I II III IV V VI Proter UM(I/1) II/2(II/3, II/2, II/1) III/2(III/3, III/2, III/1) IV/3(IV/3, IV/2, IV/1) IV/3(V/4, V/3, V/2, V/1) IV/3(VI/4, VI/3, VI/2, VI/1) Opisthe OP (I/1) OP(II/3, II/2, II/1) OP (III/3, III/2, III/1) IV/2(IV/3, IV/2, IV/1) V/4 (V/4, V/3, V/2, V/1) V/4 (VI/4, VI/3, VI/2, VI/1) Cirri formed per primordium are in parenthesis. OP, oral primordium; UM, undulating membrane. urnal of o O w w A m T m ( b ( e 2 m b a m d t a w c M a t D n 1 l s d s ( t r a c l ( e c a c p 4 4 w w l ( l M b a M a t c I k l r b f b b k r o t o r p I t T c p i o b z f m c s i f b m b m d g o t d o d b t f G.C. Küppers et al. / European Jo usly in the rigid (e.g., Stylonychia) or the flexible (e.g., xytricha) group (see Foissner and Stoeck 2008). Usually ith three macronuclear nodules (one individual out of 50 ith two, and one with four nodules) in central body portion. nterior and posterior macronuclear nodules ellipsoidal; iddle macronuclear nodule almost spherical or ellipsoidal. wo globular micronuclei (one individual out of 50 with six icronuclei), sometimes faintly impregnated with protargol Figs 4, 28). Contractile vacuole in midbody near to the left ody margin, with an anterior and a posterior collecting canal Figs 2, 24). Cortical granules absent. Cytoplasm transpar- nt, with several refractive cytoplasmic inclusions, such as .0–3.5 �m long crystals and 5–7 �m-sized lipid globules, ainly along the margins of the cell. Food vacuoles with acteria, Chlorella-like algae, flagellates, small ciliates, and lso wheat starch in the individuals from cultures. Moves oderately to very fast, crawling on the bottom of the Petri ish, sometimes remaining still for a while. Somatic ven- ral ciliature composed of 18 frontal–ventral–transverse cirri, rranged in the typical oxytrichid pattern (Figs 3, 28). Rarely ith six transverse cirri. Thick fibres associated with ventral irri, mainly with the enlarged frontal and transverse cirri. arginal cirri about 12 �m in length in vivo; transverse cirri bout 17 �m in length in vivo and extending beyond pos- erior end of cell; caudal cirri about 14 �m long in vivo. orsal bristles about 4–5 �m long after protargol impreg- ation, invariably arranged in four kineties. Dorsal kinety slightly shortened anteriorly, kineties 2 and 3 almost as ong as body, and kinety 4 posteriorly shortened. Dikinetids urrounded by oblique fibres, distinctly impregnated around ikinetids of rows 2 and 3. Usually two delicate and incon- picuous caudal cirri at the rear ends of dorsal kineties 1 and 3 Figs 4, 29). One specimen out of 20 with three caudal cirri at he ends of dorsal kineties 1–3. Adoral zone of membranelles epresenting 44.5% of total body length (calculated on the verage values measured on silver-impregnated cells). Buc- al cavity rather large and deep, with a conspicuous hyaline ip covering proximal part of adoral zone of membranelles Figs 24, 25). On average 37 adoral membranelles, with lat- ral membranellar cilia extending to the right of the buccal avity; distal membranelles about 18 �m long in vivo. Paroral nd endoral intersect each other optically behind the buc- al cirrus; both membranes composed of dikinetids. Short haryngeal fibres extend obliquely backwards. Resting cysts of kinetosome-resorbing type, on average 5.8 �m in diameter, golden to light brown colored at 0–100× (Figs 5–7, 26, 27 and Table 1). Surface slightly rinkled, formed by 0.5–1.0 �m high polygonal ridges. Cyst all 1.5–2.0 �m thick, with a 0.5–1.0 �m thin outer hya- ine (mucous?) layer; stains red with methyl-green pyronin with its ridges intensely stained). Cytoplasm with 3–5 �m ipid droplets and 1.0–1.5 �m refractive globular inclusions. acronucleus organized in a single reniform or rather dumb ell-shaped mass. Resting cysts were viable even 14 months fter encystment. i m a Protistology 47 (2011) 103–123 109 orphogenesis during cell division (Figs 8–23, 30–48 nd Table 2) Stomatogenesis of the opisthe begins with the prolifera- ion of basal bodies near the anteriormost postoral ventral irrus IV/2, and near the leftmost transverse cirri II/1 and II/1, resulting in the formation of two anarchic fields of inetosomes (Figs 8, 9, 30–33). As new basal bodies pro- iferate, these two fields enlarge posteriad and anteriad, espectively, and become a unified anarchic field of basal odies (Figs 10, 34). Primordium I of the opisthe develops rom the oral primordium, and detaches as a streak of basal odies which proliferates anteriad. Some presumptive mem- ranelles develop anteriorly, through the alignment of paired inetosomes in two rows. The macronuclear nodules show eplication bands at this stage. Primordia II and III of the opisthe are also formed from the ral primordium (Figs 11, 35, 37). The posteriormost fron- oventral cirrus IV/3 disaggregates to form primordia IV-VI f the proter (Figs 11, 12, 36, 38, 40). The frontoventral cir- us III/2 dedifferentiates and generates primordium III of the roter (Figs 12, 39). The buccal cirrus produces primordium I of the proter, while the paroral dedifferentiates distally o become primordium I of the proter (Figs 13, 14, 41). he anterior part of the oral primordium of the opisthe ontinues with the differentiation of membranelles in a osteriad direction. A third row of kinetosomes is added n the anterior two-rowed membranelles. Primordia I–III f the opisthe lengthen and presumptive undulating mem- ranes align longitudinally and parallel to the forming adoral one of membranelles. Primordium IV of the opisthe arises rom the disaggregated postoral ventral cirrus IV/2 and pri- ordia V and VI from the disaggregated postoral ventral irrus V/4 (Figs 12–14, 42, 43). Consequently, a total of ix frontal–ventral–transverse cirri primordia are produced n each divider. Cirri II/1, III/1, IV/1, V/1–3, VI/1–4, and the rontal cirri are not involved in primordia formation and will e resorbed in later stages of division or in the postdivider. Two marginal rows primordia develop within each parental arginal row; the anterior right primordium is generated y the third or fourth cirrus, while the posterior right pri- ordium is generated from the cirrus behind the presumptive ivisional furrow. The two left marginal rows primordia are enerated somewhat later than the right ones, and the anterior ne develops from the first left cirrus (Figs 15, 44). The dorsal kineties primordia develop intrakinetally within he dorsal rows 1–3 at the level of the marginal row primor- ia (Fig. 16). In the following stage of division, the endoral f the proter dedifferentiates distally. Whether the endoral edifferentiates completely could not be observed. New cirri egin to segregate within the streaks of both proter and opis- he. The membranelles of the opisthe are almost completely ormed. The undulating membrane primordium of the opisthe s parallel to the adoral zone of membranelles and the left- ost frontal cirrus is segregated (Figs 15, 44). The parental doral zone of membranelles is fully retained in the proter 110 G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 Figs 13–16. Morphogenesis of ventral (13–15) and dorsal (16) side of Parasterkiella thompsoni after protargol impregnation. (13) Devel- opment of primordium VI of the opisthe and primordium II of the proter. Arrowhead marks parental cirrus V/3. (14) Development of the primordia I, II of the proter and right marginal cirri primordium of the opisthe (arrowhead). Parental cirrus V/3 is resorbed. (15) Segregation of new cirri. Arrowheads mark the primordia of the right and left marginal cirri; asterisk marks dedifferentiation of the undulating membranes of the proter. (16) Proliferation of the dorsal row primordia 1–3 in the same specimen as in (15). The macronucleus remains tripartite and micronuclei become spindle-shaped. I, II, V, VI, frontal–ventral–transverse cirri primordia; 1–3, dorsal kinety primordia. Parental cirri, white; new cirri, black. Scale bars = 50 �m. G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 111 Figs 17, 18. Morphogenesis of ventral (17) and dorsal (18) side of Parasterkiella thompsoni after protargol impregnation. (17) Late divider s irri co d specim ( white; t V t d T o t o a T b b c ( t d b p a e t p l o t c c m o a I a o n m ( howing the opisthe membranelles and frontal–ventral–transverse c orsal row primordia and condensed macronucleus of the same arrowheads). Ma, macronucleus; Mi, micronucleus. Parental cirri, hough a cryptic reorganization could not be excluded (see oss and Foissner 1996). Some extra cirri were observed next o the right anterior and/or posterior marginal row primor- ia, although these cirri could also be parental (Figs 19–21). hese extra cirri seem to be resorbed later since they were not bserved in interphase specimens. In late divisional stages, he streaks differentiate into cirri (Table 2). The adoral zone f the opisthe is completely formed and curves to the right nteriorly, typically adopting the shape of a question mark. he undulating membranes have already split longitudinally, ut are still parallel to each other (Figs 17, 45). The new cirri egin to migrate to form the characteristic 18-cirri pattern. Dorsal kineties primordia are lengthened, and one caudal irrus is formed at the end of dorsal kineties 1 and 3 Figs 18, 46). One dorsomarginal row differentiates from he right marginal row primordium (Figs 17, 44). Parental ikinetids remain among the new developing dorsal kineties, ut these parental kineties are resorbed later or in the ostdivider. The three macronuclear nodules condense in a single mass nd the micronuclei become elliptic. The macronuclear mass O l mpletely formed. Arrowheads mark dorsomarginal rows. (18) The en as in (17). Caudal cirri are formed only on kineties 1 and 3 new cirri, black. Scale bar = 50 �m. longates and divides into two pieces, one for the proter and he other for the opisthe. Later, the macronuclear mass of the roter divides again into two nodules, with the anterior being arger than the posterior; while the macronuclear mass of the pisthe divides into an anterior rounded nodule and a pos- erior enlarged nodule. Both enlarged macronuclear nodules onstrict again, resulting in the typical tripartite macronu- leus for each divider. Meanwhile, the micronuclei divide itotically (Figs 18–21, 45–48). The undulating membranes f the proter reorganize and the membranes of both the proter nd the opisthe intersect each other optically (Figs 21, 48). n the postdivider, the cirri have already achieved their ppropriate positions but some parental marginal (proter) r transverse cirri (opisthe) are still not resorbed. The uclear apparatus already consists of the typical three acronuclear nodules and the two or three micronuclei Figs 22, 23). ccurrence and ecology Parasterkiella thompsoni was found in two small, shal- ow water pools (about 500 m2, 0.33 m deep) from a Fuegian 112 G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 Figs 19–23. Morphogenesis of ventral (19–22) and dorsal (23) side of Parasterkiella thompsoni after protargol impregnation. (19) Late divider with macronucleus in two masses and dividing micronuclei. Arrowhead marks extra cirri. (20) Late divider with two macronuclear m opisth m ivider. m cirri, w p d o d g asses in each divider and crossed undulating membranes of the acronuclear nodules and crossed undulating membranes in each d arks unresorbed parental marginal cirri. CC, caudal cirri. Parental eat bog dominated by Sphagnum magellanicum. Low con- uctivity and pH values (Table 3) are typical of this type f environment (Mataloni 1999), as well as low contents of issolved nutrients. Waters were well illuminated and oxy- d w e. Arrowheads mark extra cirri. (21) Very late divider with three Arrowheads mark extra cirri. (22 and 23) Postdivider. Arrowhead hite; new cirri, black. Scale bars = 50 �m. enated, and both ponds presented a 10 cm-thick ice cover uring October 2009. Remarkably, during April 2009, Chlorella-like green algae ere densely packed in the cytoplasm of P. thompsoni G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 113 Figs 24–27. Interphase morphology (24 and 25) and resting cyst (26 and 27) of Parasterkiella thompsoni in vivo (24–26) and after protargol impregnation (27). (24) Ventral view of a specimen with food. (25) Ventral view of a slender specimen. Arrowhead marks the conspicuous buccal lip. (26) Optical cross section of a mature resting cyst. Arrowhead marks a ridge on the cyst wall. (27) View of polygonal ridges on the surface of the resting cyst. CV, contractile vacuole; Ma, macronucleus. Scale bars = 30 �m. Table 3. Physicochemical variables in sampling sites RH3 and RH5, where Parasterkiella thompsoni was found. Environmental variable RH3 RH5 Temperature (◦C) 7.1 (3.2–11.4) 5.4 (1.7–7.5) pH 4.5 (4.4–4.7) 4.5 (4.4–4.8) Conductivity (�S cm−1) 14 (10–17) 11 (5.5–18) Dissolved oxygen (mg L−1) 8.8 (8.7–8.9) 8.7 (8.7–8.8) PAR (� photons m−2 s−1) 707.4 (410.3–1,184) 635.5 (244.8–1026) DIN (�g L−1) 30.4 (10–51) 42.9 (31.3–54.4) PO4-P (�g L−1) 80 (30–130) 30 (20–40) Total N (�g L−1) 6800 (5500–9400) 6033 (4900–8300) Total P (�g L−1) 143 (90–170) 307 (80–420) T n paren a sphoru o c a o D k b T ( he arithmetic mean is followed by minimum and maximum observations i ctive radiation; PO4-P, phosphate; Total N, total nitrogen; Total P, total pho btained from the pool RH5. Protargol impregnation of these ells showed no difference from the specimens lacking the lgae (i.e., cells from RH5 obtained during other sampling ccasions and from RH3). Unfortunately, we could not obtain NA from the specimens with algae, thus we assume both inds of specimens to be conspecific until additional data ecome available. t A a theses (n = 3). DIN, dissolved inorganic nitrogen; PAR, photosynthetically s. he 18S-rDNA data and phylogenetic analyses Figs 49–51 and Table 4) The obtained 18S-rDNA fragment of Parasterkiella hompsoni had 1621 nucleotides and a CG content of 44.81%. fter aligning and trimming, the nucleotide matrix yielded total of 1671 characters, of which 1332 were constant and 114 G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 Figs 28–35. Interphase morphology (28 and 29) and morphogenesis (30–35) of Parasterkiella thompsoni after protargol impregnation. (28) Ventral infraciliature. Arrowhead marks micronucleus. (29) Dorsal kineties. Arrowhead marks an extra dikinetid, possibly a still unresorbed parental dikinetid. (30 and 31) Very early divider. Arrowhead marks the beginning of stomatogenesis, with proliferation of basal bodies near the postoral ventral cirri (30); detail of the proliferation of basal bodies near postoral ventral cirri (31, arrowhead). (32) Very early divider showing proliferation of a second field of basal bodies near the transverse cirri. Arrowheads mark both fields of basal bodies. (33) Detail of enlarged fields of basal bodies. Arrowhead marks replication band in macronucleus. (34) Detail of unique field of basal bodies of an early divider. (35) Early to middle divider showing the oral primordium. Arrowhead marks disaggregated cirrus IV/2. PO, postoral ventral cirri. Scale bars = 20 �m (31 and 34), 30 �m (28–30, 32, 33, and 35). G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 115 Figs 36–42. Middle dividers of Parasterkiella thompsoni after protargol impregnation. (36) Disaggregating cirrus IV/3 and primordium IV of the proter (arrowhead). (37) Oral primordium and primordia I–III of the opisthe. (38) Primordia IV, V of the proter (arrowhead). (39) P (41) Pr I I, fron 3 2 o s c n w t t o m A n R P t E p a rimordium III of the proter. (40) Primordia IV–VI of the proter. –VI of the opisthe; postoral ventral cirrus V/3 still unresorbed. I–V 9–42), 30 �m (37 and 38). 22 were parsimony-informative. The 18S-rDNA sequence f P. thompsoni differs by up to 3.5% from the analogous equences of Sterkiella species (Table 4). The phylogenetic patterns obtained from BI, MP, and ML onsistently hypothesized a monophyly of the Stylonychi- ae (Figs 49–51). The internal kinships of the ingroup ere slightly variable as a result of different analytic cri- eria. The BI and ML results yielded almost identical opologies, differing because of a consensus compromise f the relationships within the group formed by Cyrtohy- ena citrina (Berger and Foissner, 1987) Foissner, 1989, b l s imordia II–VI of the proter. (42) Oral primordium and primordia tal–ventral–transverse cirri primordia. Scale bars = 20 �m (36 and frokeronopsis aurea (Foissner and Stoeck, 2008) Foiss- er et al., 2010, Onychodromopsis flexilis Stokes, 1887, ubrioxytricha ferruginea (Stein, 1859) Berger, 1999, and araurostyla weissei (Stein, 1859) Borror, 1972 in the BI ree. The genera Sterkiella Foissner et al., 1991, Stylonychia hrenberg, 1830, and Tetmemena Eigner, 1999 are all poly- hyletic. Parasterkiella thompsoni was consistently placed mong the stylonychines. In BI and ML trees, P. thompsoni ranched after the Laurentiella + Stylonychia ammermanni- emnae-mytilus group; while in the MP trees both groups witched their positions, so that P. thompsoni split at the 116 G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 Figs 43–48. Morphogenesis of ventral (43–45, 47, and 48) and dorsal (46) side and behavior of nuclear apparatus of Parasterkiella thompsoni after protargol impregnation. (43) Middle divider with six frontal–ventral–transverse cirri primordia; arrowheads mark primordium VI of the proter (white) and of the opisthe (black). (44) Cirri segregation in a middle divider; arrowheads mark the formation of dorsomarginal kineties. (45) Late divider with macronucleus fused in a single mass (white arrowhead); black arrowhead marks the dorsomarginal kinety of the opisthe. (46) Late divider with caudal cirri at the ends of dorsal kineties 1 and 3 (black arrowheads); white arrowheads mark elongated macronucleus and dividing micronuclei. (47) Late divider with two macronuclear masses each in the proter (arrowheads) and the opisthe. (48) Late divider with three macronuclear nodules in the proter (arrowheads) and opisthe. Scale bars = 50 �m. G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 117 Stylonychia notophora FM209297 Tetmemena pustulata 1 AF396973 Tetmemena pustulata 2 X03947 Onychodromus grandis AJ310486 Sterkiella nova 2 AF508771 Sterkiella nova 1 X03948 Tetmemena bifaria FM209296 Steinia sphagnicola AJ310494 Gastrostyla steinii AF164133 Pattersoniella vitiphila AJ310495 Histriculus histrio FM209294 Styxophrya quadricornuta X53485 Pleurotricha lanceolota AF164128 Sterkiella histriomuscorum 1 AF164121 Sterkiella histriomuscorum 2 AF508770 Parasterkiella thompsoni Laurentiella strenua AJ310487 Stylonychia ammermanni FM209295 Stylonychia mytilus AM086661 Stylonychia lemnae 1 AM086652 Stylonychia lemnae 2 AM260994 Rubrioxytricha ferruginea AF370027 Paraurostyla weissei 1 AF164127 Paraurostyla weissei 2 AJ310485 Onychodromopsis flexilis AM412764 Cyrtohymena citrina 2 AY498653 Cyrtohymena citrina 1 AF164135 Afrokeronopsis aurea EU124669 Oxytricha longa AF164125 Pseudokeronopsis flava DQ227798 Pseudokeronopsis rubra EF535729 Urostyla grandis 1 AF164129 Urostyla grandis 2 EF535731 Diaxonella trimarginata DQ19095 Metaurostylopsis salina EU220229 Metaurostylopsis sinica EU220227 Apokeronopsis bergeri DQ777742 Apokeronopsis crassa DQ359728 0.05 85 98 76 * * *87 83 85 *71 * * * * * 98 76 * * * * * * * * * 9872 96 Stylonychinae Urostylida Oxytrichinae Neokeronopsidae Fig. 49. Majority rule (50%) consensus of the Bayesian trees remaining after burn-in. The numbers associated with nodes indicate the posterior p es smal t 2; sca b f p ( T b 1 2 3 4 5 D robabilities (shown in %). Asterisks indicate full support, and valu he species names. Mean ln L = −6734.644; standard deviation = 0.3 ase of the Stylonychinae. The divergence of P. thompsoni rom its sister-taxa had low bootstrap and jackknife sup- ort values (Figs 50, 51), but high Bayesian probability Fig. 49). able 4. Model-corrected pairwise genetic distances (in %) etween species of Sterkiella and Parasterkiella thompsoni. Species 1 2 3 4 Sterkiella histriomuscorum 1 AF164121 S. histriomuscorum 2 AF508770 <0.1 S. nova 1 X03948 2.2 2.1 S. nova 2 AF508771 2.2 2.1 0.8 Parasterkiella thompsoni HM569264 1.9 1.9 3.3 3.5 I d t s b p c e F f F ler than 50% are omitted. NCBI accession codes are provided after le bar: five substitutions per 100 nucleotide positions. iscussion nterphase morphology and resting cyst The morphology of Parasterkiella thompsoni from Tierra el Fuego matches the original description of the Antarc- ic population by Foissner (1996). At first, Foissner (1996) upposed that the Antarctic specimens, with an unusual num- er of macronuclear nodules, could represent a teratological opulation of species having normally two or four macronu- lear nodules, such as Sterkiella histriomuscorum (Foissner t al., 1991) Foissner et al., 1991 or S. cavicola (Kahl, 1935) oissner et al., 1991, respectively. Specimens with two or our macronuclear nodules were also observed in Tierra del uego, although with a very low frequency. 118 G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 Stylonychia notophora Tetmemena pustulata 2 Tetmemena pustulata 1 Onychodromus grandis Sterkiella nova 2 Sterkiella nova 1 Tetmemena bifaria Steinia sphagnicola Gastrostyla steinii Pattersoniella vitiphila Histriculus histrio Styxophrya quadricornuta Pleurotricha lanceolata Sterkiella histriomuscorum 1 Sterkiella histriomuscorum 2 Parasterkiella thompsoni Laurentiella strenua Stylonychia ammermanni Stylonychia mytilus Stylonychia lemnae 1 Stylonychia lemnae 2 Onychodromopsis flexilis Paraurostyla weissei 1 Paraurostyla weissei 2 Cyrtohymena citrina 2 Rubrioxytricha ferruginea Cyrtohymena citrina 1 Afrokeronopsis aurea Oxytricha longa Pseudkeronopsis flava Pseudokeronopsis rubra Urostyla grandis 1 Urostyla grandis 2 Diaxonella trimarginata Metaurostylopsis salina Metaurostylopsis sinica Apokeronopsis bergeri Apokeronopsis crassa 85 63 62 99 83 77 99 99 85 99 70 58 53 98 72 87 57 97 66 56 62 96 0.05 * * * Stylonychinae Oxytrichinae Urostylida Neokeronopsidae F odes i v ar: five m P r m O r u ( 2 a p A a w A O w t ( 1 h i a s o s c p 1 ig. 50. Maximum likelihood tree. The numbers associated with n alues smaller than 50% are omitted. ln L = −6648.801467. Scale b Other authors described Antarctic oxytrichids with three acronuclear nodules that were considered conspecific with arasterkiella thompsoni (Foissner 1996). Sudzuki (1964) ecorded under the name Opisthotricha sp. a ciliate from oss samples collected in a petrel rookery near Langhovde, ngul Islands. That isolate differs from the Fuegian species ecorded here, as well as from the other described pop- lations, with respect to the high number of micronuclei more than 26 [a possible misinterpretation?] vs. usually –3). Thompson (1972) described and sketched in more detail fter silver impregnation an Oxytricha sp. from water sam- les collected in a rock pool behind Old Palmer station on the ntarctic Peninsula. The specimens from Tierra del Fuego re very similar to those described by Thompson (1972), ho also observed four dorsal rows of bristles. Outside ntarctica, Seshachar and Katsuri Bai (1963) described an xytricha sp. from a laboratory fish tank in Bangalore, India, i n c ndicate bootstrap percentages. Asterisks indicate full support, and substitutions per 100 nucleotide positions. ith three macronuclear nodules, but much larger in size han the populations from Antarctica and Tierra del Fuego 200–450 �m vs. 80–170 �m, Foissner 1996; Thompson 972; this study). Beside the greater size, the Indian isolate as a higher number of micronuclei (7–14 vs. usually 2–3 n P. thompsoni) and, as was pointed out by Foissner (1996) nd Berger (1999), that isolate could represent a different pecies. The resting cysts of stylonychines are known to have vari- us patterns of wall ornamentation (Berger 1999). Our results how that the cysts of P. thompsoni are similar to those of S. avicola and Gastrostyla steinii considering the presence of olygonal ridges on the wall surface (Berger and Foissner 987; Foissner 1987). Generally, the cyst wall of hypotrichs s composed of four layers (Gutiérrez et al. 2003). Unfortu- ately, the occurrence of such layers could not be properly hecked for P. thompsoni in the present study. G.C. Küppers et al. / European Journal of Protistology 47 (2011) 103–123 119 Tetmemena pustulata 1 Tetmemena pustulata 2 Stylonychia notophora Onychodromus grandis Sterkiella nova 1 Tetmemena bifaria Sterkiella nova 2 Steinia sphagnicola Gastrostyla steinii Pattersoniella vitiphila Histriculus histrio Styxophrya quadricornuta Pleurotricha lanceolota Sterkiella histriomuscorum 1 Sterkiella histriomuscorum 2 Laurentiella strenua Stylonychia ammermanni Stylonychia mytilus Stylonychia lemnae 1 Stylonychia lemnae 2 Parasterkiella thompsoni Afrokeronopsis aurea Onychodromopsis flexilis Rubrioxytricha ferruginea Cyrtohymena citrina 1 Cyrtohymena citrina 2 Paraurostyla weissei 1 Paraurostyla weissei 2 Oxytricha longa Pseudokeronopsis flava Pseudokeronopsis rubra Apokeronopsis bergeri Apokeronopsis crassa Metaurostylopsis sinica Metaurostylopsis salina Diaxonella trimarginata Urostyla grandis 1 Urostyla grandis 2 * 64 99 * 85 * * 90 95 53 61 57 67 66 85 * 96 91 94 74 72 84 79 69 99 92 63 73 Stylonychinae Oxytrichinae Urostylida Neokeronopsidae F umber i ed. Tr i D e ( 2 T t ( d i m o 1 g o l o I I d o a h c S 1 b ig. 51. Strict consensus of four maximum parsimony trees. The n ndicate full support, and values smaller than 50% are not display ndex = 0.8631; rescaled consistency index = 0.6077. ivisional morphogenesis The morphogenesis of P. thompsoni shows some differ- nces compared to the morphogenesis of Sterkiella species Berger et al. 1985; Foissner and Berger 1999; Foissner et al. 002; Fryd-Versavel et al. 2010; Petz and Foissner 1997). he most noticeable difference lies in the development of he dorsal ciliature, which occurs in the Urosomoida pattern see Berger 1999). In P. thompsoni the presence of only four orsal rows of bristles results from the lack of fragmentation n dorsal kinety 3 and the development of only one dorso- arginal kinety from the right marginal row primordium, as ccurs in the Oxytrichinae genera Urosoma Kowalewskiego, 882 and Urosomoida Hemberger in Foissner, 1982. Further differences were observed with respect to the ori- in of the oral primordium of the opisthe. The development e 2 f s associated to branches indicate jackknife percentages. Asterisks ee length (in steps) = 2514; consistency index = 0.7041; retention f this primordium in P. thompsoni begins with the pro- iferation of two separated anarchic fields of basal bodies, ne generated near the anteriormost postoral ventral cirrus V/2 and the other near the leftmost transverse cirri II/1 and II/1. In S. cavicola, a small patch of basal bodies, in fact, evelops close to cirrus IV/2 and probably contributes to the ral primordium. This contribution, however, would occur fter the basal bodies originating near the transverse cirri ave proliferated and reached the level of the postoral ventral irri (Foissner et al. 2002). Such pattern differs from that of terkiella histriomuscorum and S. nova Foissner and Berger, 999, where the oral primordium develops solely from basal odies that originate near the leftmost transverse cirri (Berger t al. 1985; Foissner and Berger 1999; Fryd-Versavel et al. 010; Petz and Foissner 1997). Indeed, the pattern of early ormation of the oral primordium observed in P. thompsoni 1 urnal of r v a g N ( H s B p u c F w 1 i o b a o s t F c p s t m t 1 c e l o fi c 1 a a t s b 1 c n p m U s O S S e h t a o F P S i w e c e f t t s c i d r 1 b i t s 2 M t m v 2 a e m t o h b e w r l 20 G.C. Küppers et al. / European Jo arely occurs among the Stylonychinae – e.g., Pattersoniella itiphila Foissner, 1987 – and is apparently more common mong the flexible-body oxytrichids—e.g., Oxytricha longi- ranulosa Berger and Foissner, 1989, Rubrioxytricha indica aqvi et al., 2006, Tachysoma terricola Hemberger, 1985 Berger 1999; Berger and Foissner 1989; Foissner 1987; emberger 1985; Naqvi et al. 2006). With respect to macronucleus development in Oxytricha p. with three macronuclear nodules (Seshachar and Katsuri ai 1963), the typical number of nodules is achieved in the ostdivider about 24 h after fission, whereas in P. thompsoni sually three nodules are generated in each divider before ytokinesis. oundation for the creation of the new genus The genus Sterkiella was created to include those species ith caudal cirri previously assigned to Histriculus Corliss, 960 (Foissner et al. 1991). At the present time, this genus s defined by a combination of characters—e.g., the rigid r semirigid body, curved and intersecting undulating mem- ranes, separated marginal rows of cirri at the posterior end, nd the presence of caudal cirri. All these features, however, ccur in other genera and thus are not unique. The supposed ole autapomorphy of the genus was the de novo formation of he primordia V and VI of the opisthe (Berger 1999). Later, oissner et al. (2002) recognized this origin as a mistake and onfirmed that both primordia V and VI develop from the ostoral ventral cirrus V/4. Foissner (1996) and Berger (1999) considered the clas- ification of the Antarctic ciliate in the genus Sterkiella as entative, since this ciliate possesses characteristics com- on to several oxytrichids. The dorsal ciliature resembles he Oxytrichinae genera Urosoma and Urosomoida (Berger 999) and, as confirmed in the present study, the dorsal iliature actually develops in the Urosomoida pattern. Nev- rtheless, with respect to features like the semirigid body, the ack of cortical granules, the adoral zone of membranelles ccupying about 50% of body length, the presence of oblique bres around dorsal cilia, and the morphogenetic inactivity of irrus V/3, this species belongs to the Stylonychinae (Berger 999). This classification is confirmed by our phylogenetic nalyses (see below). In Sterkiella the rightmost dorsal primordium fragments nd forms two dorsal kineties (Foissner et al. 2002). Hence, he lack of fragmentation in the dorsal primordia of P. thomp- oni clearly separates this genus not only from Sterkiella, ut also from all other 18-cirri stylonychine genera (Berger 999). This difference in the morphogenesis of the dorsal iliature is herein regarded as justifiable for the creation of a ew genus, namely Parasterkiella, for S. thompsoni. For the resent, though, P. thompsoni remains monotypic. Among the non-stylonychine 18-cirri genera in which orphogenetic data also confirmed the occurrence of the rosomoida pattern, Parasterkiella is comparable to Uro- a c a t Protistology 47 (2011) 103–123 oma, Urosomoida, and some species currently assigned to xytricha Bory De Saint-Vincent in Lamouroux, Bory De aint-Vincent and Deslongchamps (e.g., O. longa Gelei and zabados, 1950 and O. setigera Stokes, 1891). These gen- ra all differ in a fundamental way from Parasterkiella by aving flexible bodies, by exhibiting participation of the pos- oral ventral cirrus V/3 in primordia formation, and by having n adoral zone of membranelles that occupies less than 50% f the body length (Berger 1999; Berger and Foissner 1988; oissner 1983; Ganner et al. 1987). hylogenetic position The pairwise genetic distances between populations of terkiella histriomuscorum and of S. nova were smaller than nterspecific distances, including P. thompsoni. Nevertheless, e found, in accordance with others (Foissner 2007; Foissner t al. 2003; Schmidt et al. 2007), that such comparisons ould be misleading as an objective criterion for species or ven genus discrimination among stylonychines when per- ormed independently of a morphological study. Although he 18S-rDNA of P. thompsoni differs by up to 3.5% from he analogous sequence from Sterkiella spp., the P. thompsoni equence does so less when compared with the 18S-rDNAs of ertain stylonychines that are morphologically more dissim- lar. For instance, the 18S-rDNA sequence of P. thompsoni iffered by 2.7% from those of Gastrostyla steinii and Lau- entiella strenua (Dingfelder, 1962) Berger and Foissner, 989 – which have conspicuous ventral row[s] of cirri formed y the ventral primordia – and by 2.6% from the correspond- ng nucleotide sequences of Pattersoniella vitiphila; in which he ventral cirri form a midventral complex and multiple dor- al kinety fragmentation occurs (Berger 1999; Foissner et al. 002; Martin et al. 1983). The phylogenetic patterns obtained from BI, MP, and L did not reject the ingroup monophyly with respect to he urostylid outgroup and consistently hypothesized the onophyly of the Stylonychinae, thus corroborating pre- ious analyses along the same lines (e.g., Bernhard et al. 001; Foissner et al., 2004; Hewitt et al. 2003; Paiva et l., 2009; Schmidt et al. 2007, 2008; Shao et al. 2007; Yi t al. 2008, 2009). The MP trees were generally in agree- ent with those obtained by Paiva et al. (2009) concerning he Stylonychinae kinships and the pectinate distribution f its clades. Paraurostyla weissei and Oxytricha longa, owever, occupied slightly different positions, branching efore the cyrtohymenids. The morphologically similar gen- ra Sterkiella, Stylonychia, and Tetmemena (Berger 1999) ere all polyphyletic within the present analyses, thus cor- oborating the results from Schmidt et al. (2008). We found ittle inconsistency regarding the placement of P. thompsoni mong stylonychines. In the BI and ML trees, P. thompsoni onsistently branched after the Laurentiella + Stylonychia mmermanni-lemnae-mytilus group, while in the MP trees P. hompsoni split at the base of the Stylonychinae. Although the urnal of d s w o s t 2 e t a d g s P m T P s P s h B A P D d T a a m d A 8 R A B B B B B B B B B C F F F F F F F F F F F F G.C. Küppers et al. / European Jo ivergence of P. thompsoni from its sister-taxa had low boot- trap and jackknife support values, the Bayesian probability as high. Worthy of note is that the Bayesian probabilities are ften inflated and thus their reliability as a measure of support hould be interpreted with extreme caution, especially when heir conclusions contradict other supports (Cummings et al. 003; Randle and Prickett 2010; Schneider 2007; Simmons t al. 2004). Berger (2006, 2008) established the Dorsomarginalia for he Hypotricha Stein, 1859 with dorsomarginal kineties s supposed morphological synapomorphy. The Oxytrichi- ae (including the Stylonychinae) belong to the former roup, with the fragmentation of dorsal kinety 3 as a robust ynapomorphy (Berger 1999, 2006, 2008). Accordingly, arasterkiella would represent a non-oxytrichid Dorso- arginalia, since fragmentation of dorsal kinety 3 is lacking. he phylogeny of the 18S-rDNA marker, however, places . thompsoni as either a basal (MP) or internal (BI, ML) tylonychine branch. In conclusion, if the classification of . thompsoni in the stylonychines is correct, then the Uro- omoida pattern likely evolved convergently within other ypotrich lineages, as has been previously conjectured by erger (1999). cknowledgements We are grateful to our research colleagues in the project ICT 2006-01697 for their help with the samplings and to rs. Lía Lunaschi and Fabiana Drago for the use of the rawing tube in their laboratory at the Museo de La Plata. he editor and the reviewers are also acknowledged for valu- ble suggestions. Dr. Donald Haggerty, a career investigator nd native English speaker, edited the final version of the anuscript. 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Zool. J. Linn. Soc. 157, 227–236. An Antarctic hypotrichous ciliate, Parasterkiella thompsoni (Foissner) nov. gen., nov. comb., recorded in Argentinean peat-bogs: Morphology, morphogenesis, and molecular phylogeny Introduction Material and Methods Study area and samplings Morphology DNA extraction, amplification, and sequencing Nucleotide matrix assembly and alignment Phylogenetic analyses Results Parasterkiella nov. gen. Parasterkiella thompsoni (Foissner, 1996) nov. comb. Morphology of Parasterkiella thompsoni from Tierra del Fuego Province, Argentina (Figs 2-7, 24-29 and Table 1) Morphogenesis during cell division (Figs 8-23, 30-48 and Table 2) Occurrence and ecology The 18S-rDNA data and phylogenetic analyses (Figs 49-51 and Table 4) Discussion Interphase morphology and resting cyst Divisional morphogenesis Foundation for the creation of the new genus Phylogenetic position Acknowledgements References