Molecular Phylogenetics and Evolution 59 (2011) 510–522

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Molecular Phylogenetics and Evolution

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Phylogeny and classification of the (Protista, Ciliophora), with emphasis on free-living taxa and the 18S rRNA gene ⇑ Peter Vd’acˇny´ a,b,c, , William A. Bourland d, William Orsi c, Slava S. Epstein c,e, Wilhelm Foissner b a Department of Zoology, Comenius University, Mlynská dolina B-1, SK 84215 Bratislava, Slovak Republic b Department of Organismal Biology, University of Salzburg, Hellbrunnerstrasse 34, A 5020 Salzburg, Austria c Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA d Department of Biological Sciences, Boise State University, 1910 University Drive, Boise, ID 83725, USA e Marine Science Center, Northeastern University, 430 Nahant Rd., MA 01908 Nahant, USA article info abstract

Article history: The class Litostomatea is a highly diverse taxon comprising hundreds of species ranging from aer- Received 16 November 2010 obic, free-living predators to anaerobic endocommensals. This is traditionally reflected by classifying the Revised 27 January 2011 Litostomatea into the subclasses Haptoria and Trichostomatia. The morphological classifications of the Accepted 3 February 2011 Haptoria conflict with the molecular phylogenies, which indicate polyphyly and numerous homoplasies. Available online 17 February 2011 Thus, we analyzed the genealogy of 53 in-group species with morphological and molecular methods, including 12 new sequences from free-living taxa. The phylogenetic analyses and some strong morpho- Keywords: logical traits show: (i) body polarization and simplification of the oral apparatus as main evolutionary Body polarization trends in the Litostomatea and (ii) three distinct lineages (subclasses): the Rhynchostomatia comprising Diversity Evolution Tracheliida and Dileptida; the Haptoria comprising Lacrymariida, Haptorida, Didiniida, Pleurostomatida Homoplasies and Spathidiida; and the Trichostomatia. The curious Homalozoon cannot be assigned to any of the hap- Oral simplification torian orders, but is basal to a clade containing the Didiniida and Pleurostomatida. The internal relation- Radiation ships of the Spathidiida remain obscure because many of them and some ‘‘traditional’’ haptorids form separate branches within the basal polytomy of the order, indicating one or several radiations and con- vergent evolution. Due to the high divergence in the 18S rRNA gene, the chaeneids and cyclotrichiids are classified incertae sedis. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction other protists or small metazoans such as rotifers and nematodes, while others are endocommensals or parasites in vertebrates, rang- The ciliate class Litostomatea Small and Lynn, 1981 represents a ing from fish to reptiles and mammals. In spite of this morphological very diverse taxon in terms of body size and shape, somatic ciliary and ecological diversification, Litostomatea has been consistently patterns, oral structures, and life histories (Lynn, 2008). The size viewed as a monophylum in both molecular and morphological ranges from about 30 15 lminPseudoholophrya minuta to phylogenies (Foissner and Foissner, 1988; Gao et al., 2008; Leipe 2500 200 lminHomalozoon vermiculare. Likewise, shape varies et al., 1994; Lipscomb and Riordan, 1990, 1992; Small and Lynn, conspicuously (e.g., bursiform, cylindroidal, vermiform, clavate, 1981; Strüder-Kypke et al., 2006, 2007; Vd’acˇny´ et al., 2010, submitted axe-shaped, lanceolate, spatulate), as does the ratio of body length for publication; Wright and Lynn, 1997a,b; Wright et al., 1997). to body width spanning a range of about 1:1–30:1. Many litostom- The monophyletic origin of the Litostomatea is supported by ateans display bizarre morphologies, such as an extensible ‘‘neck’’ five strong apomorphies: (1) the somatic kinetids are single basal (e.g., Lacrymaria), an agile proboscis (e.g., ), toxicyst-bearing bodies with a convergent postciliary ribbon, a short tentacles (e.g., Actinobolina), and a variety of body lobes and spines kinetodesmal fiber, and two transverse microtubule ribbons (Leipe (e.g., Ophryoscolex). The somatic and oral ciliature are no less di- et al., 1992; Small and Lynn, 1981); (2) the cytopharynx is of the verse. For instance, the ciliary rows extend meridionally to spirally rhabdos type, i.e., it is lined by transverse microtubule ribbons and the cilia may form isolated bands and tufts. As concerns life his- (Foissner and Foissner, 1985, 1988; Grain, 1966a,b); (3) the cilia tory, many litostomateans are free-living, voracious predators of of at least one somatic kinety are differentiated to clavate bristles, forming the so-called dorsal brush or ‘‘clavate field’’ (Foissner, 1996; Lynn, 2008); (4) the stomatogenesis is holotelokinetal, ex- ⇑ Corresponding author at: Department of Zoology, Faculty of Natural Sciences, cept for pleurostomatids and trichostomatids where it is monotel- Comenius University, Mlynská dolina B-1, SK 84215 Bratislava, Slovak Republic. Fax: +421 2 60296333. okinetal and cryptotelokinetal, respectively (Cameron and E-mail address: [email protected] (P. Vd’acˇny´ ). O’Donoghue, 2001; Foissner, 1996; Fryd-Versavel et al., 1975);

1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.02.016 P. Vd’acˇny´ et al. / Molecular Phylogenetics and Evolution 59 (2011) 510–522 511 and (5) the micronucleus conspicuously increases in size during orders and families remained a difficult enterprise for both mor- the first maturation division, and conjugation is heteropolar, ex- phologists and molecular taxonomists. The Haptoria always ap- cept for the pleurostomatid haptorians in which it is homopolar peared polyphyletic and their relationships remained unclear (Raikov, 1972; Vd’acˇny´ and Foissner, 2008; Xu and Foissner, 2004). (e.g., Gao et al., 2008; Pan et al., 2010; Strüder-Kypke et al., 2006, Recently, significant progress has been made in understanding 2007; Vd’acˇny´ et al., 2010, submitted for publication). Likewise, the deep phylogeny of the litostomateans. The class phylogenies based on morphological characteristics failed to pro- was found to be sister of the Litostomatea both forming the infra- duce a consistent topology (Corliss, 1974; Foissner and Foissner, phylum Lamellicorticata (Vd’acˇny´ et al., 2010). A resolution at the 1988; Grain, 1994; Jankowski, 2007; Lipscomb and Riordan, base of the Litostomatea recognized three main lineages: Rhyn- 1990, 1992; Lynn and Small, 2002; Fig. 1). chostomatia, Haptoria, and Trichostomatia (Vd’acˇny´ et al., submit- For better understanding of the haptorian evolution, we: (i) se- ted for publication). In contrast, the phylogeny of the haptorian quenced the 18S rRNA gene of 12 free-living Litostomatea, (ii) de-

Fig. 1. Five classifications of the Litostomatea. Names of subclasses and classes in bold. Foissner and Foissner (1988) used ultrastructure and details of the oral and somatic infraciliature. The system of Lipscomb and Riordan (1990) was based on a cladistic analysis of 46 ultrastructural and morphological characters. Both Grain (1994) and Lynn (2008) based their systems on ultrastructural and morphological data. Grain (1994) raised the subclasses to classes, calling them Litostomatea (=Haptoria) and Vestibuliferea (=Trichostomatia). In the present study, we propose a refined system using 18S rRNA gene sequences and morphological characteristics. 512 P. Vd’acˇny´ et al. / Molecular Phylogenetics and Evolution 59 (2011) 510–522

Table 1 Characterization of new 18S rRNA gene sequences of 12 litostomatean (arranged alphabetically).

Taxa Collection site Culture No. of cells Sequence No. of Average pairwise GC conditionsa picked length (nt) clones distance between content (%) sequenced clones (%) Apobryophyllum schmidingeri Foissner Germany, terrestrial mosses NFP 30 1640 22 0.21 (0.00–0.50) 43.0 and Al-Rasheid, 2007 Arcuospathidium namibiense tristicha Germany, terrestrial mosses NFP 15 1639 3 0.40 (0.30–0.50) 43.1 Foissner et al., 2002 Arcuospathidium sp.b Australia, leaf litter NFP 70 1639 21 0.30 (0.00–0.60) 43.1 Balantidion pellucidum Eberhard, USA, Idaho, Boise, garden water tank ES 20 1634 – – 43.3 1862c Cultellothrix lionotiformis (Kahl, 1930) Finland, Pyhä-Luosto NP, forest soil NFP 20 1638 – – 43.3 Foissner, 2003c Enchelyodon sp. 2b USA, Idaho, Boise, floodplain soil NFP 50 1641 18 0.33 (0.10–0.70) 43.4 Enchelys gasterosteus Kahl, 1926 Jamaica, bromeliad tank ES 10 1636 21 0.25 (0.00–0.50) 43.3 Fuscheria sp.b,c USA, Idaho, Boise, ephemeral puddle ES 20 1638 – – 41.9 Protospathidium muscicola Dragesco Botswana, floodplain soil NFP 35 1639 13 0.22 (0.00–0.40) 43.3 and Dragesco-Kernéis, 1979 Semispathidium sp.b South Africa, Krüger NP, floodplain soil NFP 18 1641 20 0.24 (0.00–0.50) 43.4 Spathidium sp.b USA, Idaho, Boise, floodplain soil NFP 8 1641 20 0.20 (0.00–0.40) 43.3 Trachelophyllum sp.b USA, Idaho, Boise, floodplain soil NFP 20 1641 23 0.28 (0.00–0.60) 43.3

a ES – environmental sample, NFP – non-flooded Petri dish culture. b These species are new and their descriptions are in preparation. c PCR products sequenced directly.

scribe the haptorian lineages and homoplasies, and (iii) discuss the cycles (denaturing at 95 °C for 45 s, annealing at 55 °C for 1 min, morphological apomorphies of the Litostomatea and Haptoria. This and extension at 72 °C for 2.5 min), and final extension at 72 °C resulted in a refined classification of the Litostomatea. for 10 min. To check the quality of the amplified DNA, PCR prod- ucts were run on a 1% agarose gel. The resulting PCR products were 2. Material and methods cloned into the vector plasmid pCR 2.1 using the TOPO TA Cloning kit (Invitrogen, Carlsbad, CA, USA). Plasmids were sequenced bi- 2.1. Collection and sample processing directionally using M13 forward and reverse primers supplied with the kit and the internal primer Euk528F (Elwood et al., 1985)at Twelve free-living litostomateans from the orders Haptorida Beckman Coulter Genomics (Danvers, MA, USA) to obtain the and Spathidiida were sampled mainly in terrestrial and semi-ter- full-length 18S rRNA gene sequences. The 18S rRNA gene PCR restrial habitats around the world (Table 1). Most species se- products for B. pellucidum, C. lionotiformis, and Fuscheria sp. were quenced were cultivated, using the non-flooded Petri dish directly sequenced at Sequetech Corporation (Mountainview, CA, method described in Foissner et al. (2002). There are three excep- USA), using the amplification primers and two internal primers. tions: Balantidion pellucidum was isolated in a garden water tank in the town of Boise, Idaho, USA; Fuscheria sp. was found in a tempo- 2.3. Sequence processing and alignments rary rainwater puddle from the surroundings of Boise; and Enchelys gasterosteus occurred in a bromeliad tank from Jamaica. Species The sequence fragments were imported into Chromas ver. 2.33 were identified using live observation, protargol impregnation, (Technelysium Pty Ltd.) to check for data quality and trim the 50 and SEM (Foissner, 1991). Half of the species studied represent and 30 ends. Trimmed sequences were assembled into contigs new taxa whose descriptions are in preparation. Eight to seventy using BioEdit (Hall, 1999). The consensus sequences, based on se- cells were picked with a micropipette, washed at least twice in quences from 3 to 23 clones (Table 1), were created in BioEdit with sterile spring water to remove contaminants, and transferred into an inclusion threshold frequency of 90% identity. These consensus 180 ll ATL buffer (Qiagen, Hildesheim, Germany). Samples were sequences were subsequently aligned to litostomatean 18S rRNA stored at +1 to +3 °C pending DNA extraction. sequences available in the ARB-package (Ludwig et al., 2004). The alignment was manually corrected according to the secondary 2.2. DNA extraction, PCR amplification, and molecular cloning structural features of the 18S rRNA molecule. Ambiguously aligned and hyper variable regions were masked, using a sequence align- Prior to DNA extraction, Proteinase K 20 ll (20 mg/ml; Qiagen, ment filter created for the alignment in ARB. Valencia, CA, USA) was added and the samples were incubated at 56 °C for 1 h. Genomic DNA of nine species was extracted using a 2.4. Phylogenetic analyses DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA), while that of B. pellucidum, Cultellothrix lionotiformis, and Fuscheria sp. was To determine the phylogenetic positions of the twelve newly isolated with the modified chelex protocol (Strüder-Kypke and sequenced haptorids and spathidiids, we analyzed an 18S rRNA Lynn, 2003). The 18S rRNA gene was PCR-amplified using the uni- gene sequence alignment containing 1408 unambiguously aligned versal forward and reverse eukaryotic primers EukA and EukB nucleotide characters of 49 representative rhynchostomatian, hap- (Medlin et al., 1988). The amplification reaction contained 10– torian, and trichostomatian taxa (Table 2). The program Modeltest 20 ng of DNA template, 2.5 U HotStar Taq DNA polymerase (Qia- (Posada and Crandall, 1998) was used to determine the best fit gen, Valencia, CA, USA), 200 lM of dNTP, and 0.5 lM of each oligo- model of nucleotide substitution under the Akaike Information Cri- nucleotide primer. The final volume was adjusted to 50 ll with terion (AIC). The best fit model was the Second Transition Model sterile distilled water. PCR conditions were as follows: initial hot with invariable sites and gamma distribution (TIM2 + I + C) with start denaturation at 95 °C for 15 min, 30 identical amplification the following parameter values: gamma distribution shape param- P. Vd’acˇny´ et al. / Molecular Phylogenetics and Evolution 59 (2011) 510–522 513

Table 2 List of ciliate species with GenBank accession numbers of corresponding 18S rRNA gene sequences included in the phylogenetic analyses. Sequences obtained during this study are in bold.

Species name GB number Species name GB number Species name GB number Amphileptus aeschtae EU242510 Enchelyodon sp. 1 U80313 Ophryoscolex purkynjei U57768 Amphileptus procerus AY102175 Enchelyodon sp. 2 JF263446 Pelagodileptus trachelioides AB558117 Amylovorax dehorityi AF298817 Enchelys gasterosteus JF263447 Phialina salinarum EU242508 Apobryophyllum schmidingeri JF263441 Enchelys polynucleata DQ411861 Protospathidium muscicola JF263449 Arcuospathidium cultriforme DQ411860 Entodinium caudatum U57765 Pseudoamphileptus macrostoma AY102173 Arcuospathidium muscorum DQ411859 Epispathidium papilliferum DQ411857 Pseudomonilicaryon fraterculum HM581677 Arcuospathidium namibiense tristicha JF263442 Eudiplodinium maggii U57766 Rimaleptus mucronatus HM581675 Arcuospathidium sp. JF263443 Fuscheria sp. JF263448 Semispathidium sp. JF263450 Balantidion pellucidum JF263444 Homalozoon vermiculare L26447 Siroloxophyllum utriculariae L26448 coli AF029763 intestinalis U57770 Spathidium sp. 1 Z22931 Bandia cribbi AF298824 Lacrymaria marina DQ777746 Spathidium sp. 2 JF263451 Bitricha tasmaniensis AF298821 Litonotus paracygnus EU242509 Spathidium stammeri DQ411862 Cultellothrix lionotiformis JF263445 Loxophyllum jinni EF123708 Teuthophrys trisulca africana DQ411863 Dasytricha ruminantium U57769 Loxophyllum rostratum DQ190465 Trachelius ovum HM581673 nasutum U57771 Macropodinium yalabense AF042486 Trachelophyllum sp. JF263452 Dileptus sp. DQ487195 Monodinium sp. DQ487196 Diplodinium dentatum U57764 Monomacrocaryon terrenus HM581674

Table 3 Log likelihoods and P-values of AU (approximately unbiased), SH (Shimodaira–Hasegawa), and WKH (weighted Kishino–Hasegawa) tests for tree comparisons considering different topological scenarios. Significant differences (P-value < 0.05) between the best unconstrained and constrained topologies are in bold.

Topology Log likelihood Difference to best AU SH WKH Conclusion (–ln L) tree (–ln L) Best maximum likelihood tree (unconstrained) 5906.2455 – 0.724 0.948 0.578 – Monophyly of Homalozoon, didiniids and pleurostomatids 5923.7895 17.54 0.196 0.593 0.145 Not rejected Sister relationship of Homalozoon and pleurostomatids 5914.1958 7.95 0.227 0.825 0.106 Not rejected Sister relationship of didiniids and pleurostomatids 5918.3176 12.07 0.338 0.738 0.221 Not rejected Didiniids belong to the order Haptorida 5934.4408 28.19 0.010 0.312 0.015 Rejected Didiniids belong to the order Spathidiida 5969.6132 63.37 1e–004 0.021 0.008 Rejected Homalozoon belongs to the order Spathidiida 5972.5596 66.31 0.003 0.024 0.015 Rejected Monophyly of lacrymariids and haptorids sensu stricto 5929.6651 23.42 0.028 0.428 0.039 Rejected Monophyly of lacrymariids and pleurostomatids 5914.7489 8.50 0.160 0.805 0.106 Not rejected Monophyly of haptorids sensu stricto and ‘‘traditional’’ haptorids 5975.5102 69.26 3e–009 0.007 0.002 Rejected Monophyly of spathidiids, ‘‘traditional’’ haptorids and trichostomatians 5907.0875 0.84 0.618 0.932 0.422 Not rejected Monophyly of spathidiids and ‘‘traditional’’ haptorids 5915.0885 8.84 0.385 0.901 0.201 Not rejected Monophyly of spathidiids and haptorids sensu stricto 5971.5885 65.34 0.003 0.012 0.007 Rejected Sister relationship of spathidiids and trichostomatids 5959.2886 53.04 0.002 0.029 0.012 Rejected Monophyly of trichostomatids and free-living haptorians with 6049.5997 143.35 0.001 0.000 0.000 Rejected oralized somatic monokinetids Monophyly of the genus Arcuospathidium 5925.9314 19.69 0.149 0.559 0.115 Not rejected Monophyly of the genus Spathidium 5931.3853 25.14 0.021 0.384 0.040 Rejected Monophyly of the genus Enchelys 5958.6282 52.38 0.009 0.019 0.005 Rejected Monophyly of the genus Enchelyodon 6024.2720 118.03 6e–041 0.000 0.000 Rejected

eter C = 0.6200; proportion of invariable sites I = 0.6380; base fre- tested by the bootstrap approach, using 1000 pseudoreplicates quencies A = 0.3008, C = 0.1660, G = 0.2492, T = 0.2840; and rate and a heuristic search algorithm. Support values from all tree matrix for the substitution model [AC] = 2.1350, [AG] = 4.9465, building methods were annotated onto the tree with the best [AT] = 2.1350, [CG] = 1.000, [CT] = 6.1471, and [GT] = 1.0000. A log-likelihood score which was chosen for presentation. Bayesian inference (BI) tree was computed in MrBayes (Ronquist and Huelsenbeck, 2003), using the model suggested by Modeltest and the Markov Chain Monte Carlo (MCMC) algorithm. The chain 2.5. Test of hypotheses length was 5000,000 generations with trees sampled every 1000 generations. The first 25% of trees were considered burn-in trees In addition to the best ML tree, 16 unrooted trees with enforced and were discarded prior tree reconstruction. A 50% majority rule topological constraints (Table 3) were built in PAUP⁄, using ML cri- consensus of the remaining trees was used to calculate posterior terion and heuristic search with TBR and 10 random sequence probabilities (PP) of the branching pattern. The maximum likeli- addition replicates. The site-wise likelihoods for the best uncon- hood (ML) analysis was computed on the CIPRES Portal V 1.15 strained ML tree and all constrained trees were calculated in PAUP⁄ (http://www.phylo.org), using RAxML with settings as described under the TIM2 + I + C model with parameters as suggested by in Stamatakis et al. (2008). The neighbor-joining (NJ) tree was con- Modeltest (see above). The reliability of the constrained trees structed using PAUP⁄ ver. 4.0b8 with randomly added species and was analyzed in likelihood frameworks through the approximately tree bisection-reconnection (