Phylogenetic relationships of the ciliate class Heterotrichea (Protista, Ciliophora, Postciliodesmatophora) inferred from multiple molecular markers and multifaceted analysis strategy

Molecular Phylogenetics and Evolution 78 (2014) 118–135

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Phylogenetic relationships of the ciliate class Heterotrichea (Protista, Ciliophora, Postciliodesmatophora) inferred from multiple molecular markers and multifaceted analysis strategy Shahed Uddin Ahmed Shazib a, Peter Vd’acˇny´ b,⇑, Ji Hye Kim a, Seok Won Jang a, Mann Kyoon Shin a,⇑ a b

Department of Biological Science, University of Ulsan, Ulsan 680-749, South Korea Department of Zoology, Comenius University, 84215 Bratislava, Slovakia

a r t i c l e

i n f o

Article history: Received 28 February 2014 Revised 7 May 2014 Accepted 9 May 2014 Available online 21 May 2014 Keywords: 18S rRNA gene Fabreidae fam. n. Gruberiidae fam. n. ITS1-5.8S rRNA-ITS2 region

a b s t r a c t The ciliate class Heterotrichea is defined by somatic dikinetids bearing postciliodesmata, by an oral apparatus consisting of a paroral membrane and an adoral zone of membranelles, as well as by features of nuclear division involving extramacronuclear microtubules. Although phylogenetic interrelationships among heterotrichs have been analyzed several times, deeper nodes of the heterotrichean tree of life remain poorly resolved. To cast more light on the evolutionary history of heterotricheans, we performed phylogenetic analyses of multiple loci (18S rRNA gene, ITS1-5.8S rRNA-ITS2 region, and 28S rRNA gene) using traditional tree-building phylogenetic methods and statistical tree topology tests as well as phylogenetic networks, split spectrum analysis and quartet likelihood mapping. This multifaceted approach has shown that (1) Peritromus is very likely an adelphotaxon of all other heterotrichs; (2) Spirostomum and Anigsteinia are sister taxa and their common monophyletic origin is strongly supported by a uniquely posteriorly-thickened paroral membrane; (3) the monotypic family Chattonidiidae should be suppressed because its type genus clusters within the family Condylostomatidae; and (4) new families are needed for Gruberia and Fabrea because their affiliation with Spirostomidae and Climacostomidae, respectively, is not supported by molecular phylogenies nor the fine structure of the paroral membrane. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The ciliate class Heterotrichea Stein, 1859 is characterized by somatic dikinetids associated with postciliodesmata, i.e., stacked postciliary microtubule ribbons that form distinct fibres close to the right of the kineties, and by an oral apparatus consisting of a paroral membrane and a prominent adoral zone of membranelles (Lynn, 2008). Postciliodesmata are also a property of the class Karyorelictea, which is the main reason why these two classes were united into the subphylum Postciliodesmatophora. However, these two groups can be distinguished based on the division properties of their macronuclei. In the Heterotrichea, macronuclei divide with external microtubules, while in the Karyorelictea they do not

⇑ Corresponding authors. Address: Department of Zoology, Faculty of Natural Sciences, Comenius University, Mlynská dolina B-1, 842 15 Bratislava, Slovakia (P. Vd’acˇny´). Address: Department of Biological Science, College of Natural Sciences, University of Ulsan, Ulsan 680-749, South Korea (M.K. Shin). E-mail addresses: [email protected] (P. Vd’acˇny´), [email protected] (M.K. Shin). http://dx.doi.org/10.1016/j.ympev.2014.05.012 1055-7903/Ó 2014 Elsevier Inc. All rights reserved.

divide. In contrast, macronuclei of all other ciliates divide with intramacronuclear microtubules and, therefore, they were united into the subphylum Intramacronucleata (Hirt et al., 1995; Lukashenko, 2009; Lynn, 1996; Lynn and Small, 2002). The first well-founded phylogeny of heterotrichs was provided by Jankowski (1964) who considered the genus Blepharisma to be the most primitive member of heterotrichs. Based on comparative morphological data, Jankowski (1964) speculated that ‘‘vorticellization’’ of buccal cavity of Blepharisma could have given rise to Fabrea, Climacostomum, and Stentor via a Condylostoma-like stage whose buccal cavity became widened. Further, he speculated that Gruberia and Spirostomum could have evolved from some other Blepharisma species by polymerization of adoral membranelles. With advent of molecular phylogenetic techniques, relationships among heterotrichs have been analyzed several times, but many Jankowski’s hypotheses remained unanswered because deeper nodes of the heterotrichean tree of life were poorly resolved and received only weak statistical support (Gong et al., 2007; Miao et al., 2005, 2009; Rosati et al., 2004; Schmidt et al., 2007; Thamm et al., 2010). Moreover, the phylogenetic positions of

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several heterotrichean families and genera were conflicting and the families Spirostomidae, Climacostomidae and Blepharismidae, as defined by Lynn (2008), were not recognized as monophyletic. Given the limitations of single-gene phylogenies, we adopted a multiple loci approach to unravel genealogical relationships among heterotrichs. Moreover, we used phylogenetic networks (Bryant and Moulton, 2004; Huson, 1998; Huson and Bryant, 2006), split spectrum analysis (Wägele and Mayer, 2007), and quartet likelihood mapping (Schmidt et al., 2002) to assess the information content of our alignments. We also analyzed diagnostic morphological features of the sequenced genera to reconstruct heterotrichean morphological evolution, using the likelihood method in combination with the Markov k-state 1-parameter evolutionary model (Maddison and Maddison, 2007; Pagel, 1999; Schulter et al., 1997).

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2.3. Sequence processing and alignment Sequence fragments were checked and assembled into contigs using Geneious ver. 6.1.6 created by Biomatters (http://www.geneious.com/). Because sequences obtained from various individuals of the same species did not show any polymorphisms, we did not perform cloning. Alignments were constructed in Mafft ver. 7.0 using the Q-INS-I strategy that takes into consideration the secondary structure of RNA molecules (Katoh and Toh, 2008). Resulting alignments were checked, manually refined, and masked according to the column scores calculated with aid of the computer program G-blocks ver. 0.91b (Castresana, 2000; Talavera and Castresana, 2007). In total, five different alignments were assembled; their characteristics are reported in Table 2. 2.4. Phylogenetic analyses

2. Materials and methods 2.1. Sample collection and processing Fifteen heterotrichean and a single karyorelictean species were collected from a variety of habitats and localities in South Korea (Table 1). Specimens from clonal cultures were used for morphological identification and molecular studies in order to ensure that the identified species is the same which was analyzed molecular biologically. Taxa were identified according to morphological criteria, using a combination of detailed live observation and Wilbert’s (1975) protargol impregnation method. For molecular studies, one or more cells from individual species were washed several times to remove contaminants and then transferred into 1.5 ml microtubes with a minimum volume of sterile water. To test the quality and reliability of obtained sequences, at least two microtubes were prepared as described above for each species studied.

2.2. DNA extraction, PCR and sequencing Genomic DNA was extracted using the RED Extract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s instruction. Reaction volume was reduced to one tenth for single-cell samples, and to one fifth for samples containing more than two cells. Eukaryotic universal forward EukA (50 -AAC CTG GTT GAT CCT GCC AG-30 ) and reverse EukB (50 -CAC TTG GAC GTC TTC CTA GT-30 ) primers (Medlin et al., 1988) served to amplify the 18S rRNA gene using the polymerase chain reaction (PCR). PCR amplifications were performed using a TaKaRa ExTaq DNA polymerase kit (TaKaRa Bio-medicals, Otsu, Japan) under the following conditions (Chen and Song, 2001): 1 cycle (5 min at 94 °C); 5 cycles (1 min at 94 °C, 2 min at 56 °C, 2 min at 72 °C); 35 cycles (1 min at 94 °C, 2 min at 62 °C, 2 min at 72 °C); and 1 cycle (10 min at 72 °C). The ITS1-5.8S-ITS2 region was amplified using ITS-F (50 -GTT CCC CTT GAA CGA GGA ATT C-30 ) and ITS-R (50 -TAC TGA TAT GCT TAA GTT CAG CGG-30 ) primers, with the following cycling parameters (Goggin and Murphy, 2000): 1 cycle (1 min at 94 °C); 30 cycles (15 s at 94 °C, 30 s at 63 °C, 1 min at 72 °C); and 1 cycle (5 min at 72 °C). The D1D2 fragment of the 28S rRNA gene was amplified with D1D2-fwd1 (50 -AGC GGG AGG AAA AGA AAC T-30 ) and D1D2-rev2 (50 -ACG ATC GAT TTG CAC GTC AG-30 ) primers, with the following cycling conditions (Sonnenberg et al., 2007): 1 cycle (4 min at 94 °C); 45 cycles (20 s at 94 °C, 20 s at 52.5 °C and 90 s at 72 °C; 1 cycle (8 min at 72 °C). Successful amplification was confirmed by electrophoresing PCR products on 1.2% agarose gels. PCR products were directly sequenced on an ABI 3730 automatic sequencer (Macrogen Inc., Seoul, Korea), using PCR primers as sequencing ones.

For all five alignments, the GTR + I + U was the best-fit evolutionary model selected by jModeltest ver. 2.0.1 under the Akaike Information Criterion (Guindon and Gascuel, 2003; Posada, 2008). This model was implemented in Bayesian inference performed in MrBayes ver. 3.2.1 (Ronquist and Huelsenbeck, 2003). Analyses were run twice with four MCMC chains (one cold and three heated) for 1 million generations with a sample frequency of 100 generations. The first 25% sampled trees were discarded as burn-in prior to constructing a 50% majority rule consensus tree. Maximum likelihood (ML) analyses were carried out using PhyML ver. 3.0 (Guindon et al., 2010) under the best selected evolutionary model (GTR + I + U). Maximum parsimony (MP) trees were constructed in PAUP ver. 4.0 (Swofford, 2003). The support for branching patterns in both the ML and MP trees was assessed using non-parametric bootstrapping with 1000 replicates. To assess whether differences in log likelihoods between the best and alternative tree topologies (Table 3) were statistically significant, we performed the approximately unbiased, weighted Shimodaira-Hasegawa, and weighted Kishino-Hasegawa tests for all five datasets separately using CONSEL ver. 0.1j (Shimodaira, 2002, 2008; Shimodaira and Hasegawa, 2001). In total, 24 unrooted, constrained ML trees were built in PAUP using the ML criterion and a heuristic search with TBR branch swapping and 10 random sequence addition replicates. Site-wise likelihoods for the best unconstrained ML trees and all constrained ones were calculated in PAUP under the best evolutionary model (GTR + I + U) with the parameters suggested by jModeltest for each alignment. A P-value < 0.05 was chosen to reject the null hypothesis that the log likelihoods of the constrained and best unconstrained trees were not significantly different. To visualize all possible phylogenetic trajectories that could be inferred from the 18S-63 and CON-17 alignments, phylogenetic networks were generated using the computer program SplitsTree ver. 4 (Huson, 1998; Huson and Bryant, 2006). All networks were constructed using the neighbornet algorithm with uncorrected distances, and their reliability was assessed with 1000 bootstrap replicates (Bryant and Moulton, 2004). To estimate the amount of untransformed phylogenetic signal present in the largest 18S-63 alignment, we employed the computer program SAMS. This software identifies split-supporting nucleotides without reference to a tree or evolutionary model and, therefore, is independent of any a priori assumptions (Wägele and Mayer, 2007). Results of the split spectrum analysis were visualized as described in Wägele et al. (2009), whereby the numbers of split-supporting positions for the ingroup partition are shown above and those for the outgroup partition below the horizontal axis. In addition, various supporting positions, as defined by Wägele and Rödding (1998), were distinguished by

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Table 1 List of studied species, their origin and characterization of their 18S rRNA gene, ITS1-5.8S-ITS2 region, and 28S rRNA D1D2 region. Taxon

Gruberia sp. Loxodes vorax Stokes, 1885 Spirostomum ambiguum (Müller, 1786) Ehrenberg (1835) Spirostomum caudatum (Müller, 1786) Delphy, 1939 Spirostomum minus Roux, 1901 Spirostomum teres Claparéde and Lachmann, 1858 Stentor amethystinus Leidy, 1880 Stentor coeruleus Pallas, 1766 Ehrenberg, 1831 [pop. 1] Stentor coeruleus Pallas, 1766 Ehrenberg, 1831 [pop. 2] Stentor muelleri Ehrenberg, 1831 Stentor roeselii Ehrenberg, 1835 Peritromus sp.

ITS1-5.8S-ITS2 region

D1D2 of 28S rRNA gene

Length (nt)

18S rRNA gene GC (%)

GB accession number

Length (nt)

GC (%)

GB accession number

Length (nt)

GC (%)

GB accession number

References for species identification

Meonmulkkak (freshwater pond) in Seonheul-ri, Jocheon-eup, Jeju-si, Jeju-do (E126°43’09’’ N30°31’35’’) Geowugae (intertidal sandflat) in Pyoseon-myeon, Seogwipo-si, Jeju-do (E126°50’51’’ N33°19’13’’)

1606

47.2

KJ651813

509

47.9

KJ651847

686

52.0

KJ651831

Giese (1973), Kahl (1932)

1591

48.5

KJ651814

500

53.4

KJ651848

685

56.1

KJ651832

Foissner et al. (1992), Kahl (1932), Repak (1972)

Jukbyeon harbor (coastal seawater) in Jukbyeon-ri, Jukbyeonmyeon, Uljin-gun, Gyeongsangbuk-do (E129°25’44’’ N37°03’32’’) Geowugae (intertidal sandflat) in Pyoseon-ri, Pyoseon-myeon, Seogwipo-si, Jeju-do (E126°50’45’’ N33°19’22’’) Taehwagang River (brackish water) in Samsan-dong, Nam-gu, Ulsan (E129°21’17’’ N35°32’41’’) Gomso Saltern (hypersaline water) in Gomso-ri, Jinseo-myeon, Buan-gun, Jeollabuk-do (E126°37’02’’ N35°42’30’’) Saltmarsh in Ojo-ri, Seongsan-eup, Seogwipo-si, Jeju-do (E126°55’06’’ N33°27’33’’) Mugeocheon Stream (freshwater) in Mugeo-dong, Nam-gu, Ulsan (E129°15’42’’ N35°32’34’’) Meonmulkkak (freshwater pond) in Seonheul-ri, Jocheon-eup, Jeju-si, Jeju-do (E126°43’09’’ N30°31’35’’)

1565

47.7

KJ651827

–

–

–

–

–

–

Song et al. (2003)

1542

46.8

KJ651815

–

–

–

631

52.5

KJ651845

Chen et al. (2007)

1537

46.8

KJ651816

500

47.6

KJ651849

632

53.3

KJ651833

Shao et al. (2006)

1598

47.5

KJ651817

489

50.1

KJ651850

674

54.5

KJ651834

1600

46.0

KJ651818

505

48.9

KJ651851

659

54.8

KJ651835

1546

47.4

KJ651829

546

42.3

KJ651860

591

48.2

KJ651844

1598

48.6

KJ651819

500

53.6

KJ651852

686

57.0

KJ651836

Kahl (1932), Song and Wilbert (1997) Kahl (1932), Lynn and Small (2002) Foissner et al. (1995), Kahl (1931) Foissner et al. (1992), Kahl (1932)

Freshwater pond in Nansan-ri, Seongsan-eup, Seogwipo-si, Jejudo (E126°49’33’’ N33°24’43’’)

1597

48.1

KJ651820

497

51.9

KJ651853

685

55.8

KJ651837

Foissner et al. (1992)

Puddle of rainwater in Gukdang-1-ri, Gangdong-myeon, Gyeongju-si, Gyeongsangbuk-do (E129°18’04’’ N35°59’25’’) Freshwater puddle within stream in Gusu-ri, Eonyang-eup, Uljugun, Ulsan (E129°11’04’’ N35°32’57’’) Jeonggol Reservoir (freshwater, littoral) in Mugeo-dong, Namgu, Ulsan (E129°14’50’’ N35°32’34’’) Seohongcheon Stream (brackish water) near Cheonjiyeon Waterfall in Seogwipo-si, Jeju-do (E126°33’34’’N33°14’41’’) Seoul Forest Park (freshwater pond) in Seongsu-dong, Seongdong-gu, Seoul (E127°02’18’’ N37°32’42’’) Freshwaer pond in Sinjeon-ri, Sanyang-eup, Tongyeong-si, Gyeongsangnam-do (E128°24’38’’ N34°46’48’’) Seoul Forest Park (freshwater pond) in Seongsu-dong, Seongdong-gu, Seoul (E 127°02’18’’ N 37°32’42’’) Saltmarsh in Ojo-ri, Seongsan-eup, Seogwipo-si, Jeju-do (E126°55’06’’ N33°27’33’’)

1597

48.5

KJ651821

497

52.3

KJ651854

686

57.6

KJ651838

1598

48.7

KJ651822

494

53.0

KJ651855

683

57.8

KJ651839

1560

46.5

KJ651828

–

–

–

–

–

–

Foissner et al. (1992), Kahl (1932) Foissner et al. (1992), Kahl (1932) Foissner et al. (1992)

1606

45.3

KJ651823

509

46.0

KJ651856

686

48.5

KJ651840

1606

45.1

KJ651825

428

47.0

KJ651858

686

48.3

KJ651842

1605

45.2

KJ651824

442

47.5

KJ651857

669

47.7

KJ651841

1605

45.5

KJ651826

507

48.1

KJ651859

669

49.2

KJ651843

1604

44.5

KJ651830

–

–

–

692

51.2

KJ651846

Foissner et al. (1992), Kahl (1932) Foissner et al. (1992), Kahl (1932) Foissner et al. (1992), Kahl (1932) Foissner et al. (1992), Kahl (1932) Lynn and Small (2002), Kahl (1932), Shao et al. (2009)

S.U.A. Shazib et al. / Molecular Phylogenetics and Evolution 78 (2014) 118–135

Blepharisma musculus Penard, 1922 Climacostomum virens (Ehrenberg, 1838) Stein, 1859 Condylostoma curva Burkovsky, 1970 Condylostoma minutum Bullington, 1940 Condylostoma spatiosum Ozaki and Yagiu in Yagiu, 1944 Fabrea salina Henneguy, 1980

Collection site (in South Korea)

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S.U.A. Shazib et al. / Molecular Phylogenetics and Evolution 78 (2014) 118–135 Table 2 List of datasets analyzed in the present study.

a b

Dataset

No. of taxa

No of. characters

No of. PICa

Descriptionb

18S-63 18S-17 ITS-14 D1D2-17 CON-17

63 17 14 17 17

1477 1523 377 436 1959

405 235 84 180 415

18S rRNA gene sequences of 60 heterotrichs and three karyorelicteans 18S rRNA gene sequences of 16 heterotrichs and a single karyorelictean ITS1-5.8S-ITS2 region sequences of 13 heterotrichs and a single karyorelictean First two domains (D1D2) of the 28S rRNA gene of 16 heterotrichs and a single karyorelictean Concatenation of the 18S-17 and D1D2-17 datasets

PIC, parsimony-informative characters. Karyorelictean sequences served as outgroup.

Table 3 Log likelihoods and P-values of the AU (approximately unbiased), WSH (weighted Shimodaira-Hasegawa), and WKH (weighted Kishino-Hasegawa) tests to determine the plausibility of different topological scenarios. Significant differences (P-value < 0.05) between the best unconstrained and constrained topologies are indicated in bold.

a b

Topology

Alignmenta

Log likelihood (ln L)

D (ln L)b

AU

WSH

WKH

Conclusion

Best maximum likelihood tree (unconstrained)

18S-17 18S-63 ITS-14 D1D2-17 CON-17

6292.8223 9338.6450 1917.1893 3305.6761 9770.0004

– – – – –

0.900 0.692 0.838 0.901 0.998

0.988 0.960 0.951 0.977 0.995

0.815 0.618 0.595 0.806 1.000

– – – – –

Sister relationship of Peritromus and all other heterotrichs

18S-17 18S-63 D1D2-17 CON-17

6295.1711 9340.7616 – –

2.35 2.12 – –

0.164 0.438 – –

0.556 0.803 – –

0.185 0.381 – –

Not rejected Not rejected Shown in the best tree Shown in the best tree

Monophyly of the family Spirostomidae sensu Lynn (2008) (i.e., Spirostomum + Gruberia)

18S-17 18S-63 ITS-14 D1D2-17 CON-17

6367.7076 9420.7439 – 3309.0459 9842.8080

74.89 82.10 – 3.37 72.81

7e-07 4e-04 – 0.286 2e-04

1e-04 0.001 – 0.460 8e-06

0.000 2e-04 – 0.194 0.000

Rejected Rejected Shown in the best tree Not rejected Rejected

Monophyly of the family Climacostomidae (i.e., Climacostomum + Fabrea)

18S-17 18S-63 ITS-14 D1D2-17 CON-17

6320.3916 9375.2898 1921.3073 3316.2798 9805.6003

27.57 36.65 4.12 10.60 35.60

0.004 0.011 0.088 0.108 0.003

0.030 0.048 0.147 0.225 0.012

0.012 0.016 0.128 0.100 0.004

Rejected Rejected Not rejected Not rejected Rejected

Monophyly of the family Blepharismidae sensu Lynn (2008) (i.e., Blepharisma + Anigsteinia)

18S-17 18S-63 D1D2-17 CON-17

6406.6524 9454.8667 3326.1914 9905.9422

113.83 116.22 20.52 135.94

9e-06 1e-73 0.016 2e-43

0.000 0.000 0.040 0.000

0.000 0.000 0.017 0.000

Rejected Rejected Rejected Rejected

Monophyly of genera with peristomial kineties (i.e., Stentor + Maristentor + Condylostentor + Climacostomum + Fabrea)

18S-63

9509.0934

170.45

3e-05

2e-05

2e-05

Rejected

For abbreviations and further alignment details, see Table 2. Difference between log likelihoods of constrained and best (unconstrained) tree.

color: binary positions by red, asymmetrical by orange, and noisy by yellow. We also investigated phylogenetic relationships among heterotrichean lineages and families using the quartet-based maximum likelihood technique. This method allows taxa to be partitioned into four clusters whose phylogenetic relationships are inferred by plotting the relative frequencies of the likelihoods for each possible topology among them in an equilateral triangle. The three tips of the triangle represent three well-resolved quartets, the three rectangles on the sides of the triangle are quartets with network evolution (i.e., with conflicting signal), and the central region of the triangle represents star-like evolution (i.e., noisy signal; Nieselt-Struwe and von Haeseler, 2001). We analyzed four alignments (18S-63, 18S-17, D1D2-17, and CON-17) by quartet likelihood mapping, assuming the GTR model and parameters estimated with Tree-Puzzle ver. 5.2 (Schmidt et al., 2002). Analyses consisted of sampling neighbor-joining trees for all possible quartets. 2.5. Reconstruction of ancestral morphologies Last common ancestors of individual heterotrichean families and lineages were reconstructed using the likelihood method and

the Markov k-state 1-parameter evolutionary model (Pagel, 1999; Schulter et al., 1997). Reconstruction analyses included 19 morphological characters that are considered to be phylogenetically and taxonomically important for members of the class Heterotrichea (Lynn, 2008). Characters and character states in the taxa are summarized in Table 4, and their distributions in the taxa are provided in Table 5. By convention, a code 0 was assigned to the supposed plesiomorphic states, while a code 1 was applied to the supposed apomorphic states. Plesiomorphic and apomorphic character states were allocated according to Guo et al. (2008) who, however, did not provide Hennigian argumentation for this classification. Nevertheless, labeling of character states can be arbitrary for the present type of reconstruction analyses because their single parameter is the rate of change and any particular change from one state to another is equally probable (Maddison and Maddison, 2007). Plesiomorphic/ apomorphic nature of character states can be deduced from their proportional likelihoods calculated at ancestral nodes by looking on changes of character states over the phylogenetic tree. For this reason, Hennigian discussion of plesiomorphic/apomorphic nature of character states is not necessary. Proportional likelihoods of character states at family ancestral nodes of the phylogenetic tree shown in Fig. 2 given the distribution

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Table 4 Phylogenetically important characters and their states in heterotrichean ciliates. No.

Character

Plesiomorphic

Apomorphic

1. 2. 3. 4. 5. 6. 7. 8. 9.

Body, contractility Ciliary rows during body contraction Dorsal ciliature Dorsal cilia Frontal membranelles Adoral zone of membranelles (AZM) Orientation of AZM Paroral membrane, number Paroral membrane, localization

Yes (coded 0) Meridional (coded 0) U-shaped kinety (coded 0) Bristle-like (coded 0) No (coded 0) Not closed (coded 0) Along main body axis (coded 0) 1 (coded 0) Right margin of the buccal cavity (coded 0)

10.

Paroral membrane, length

As long as AZM (coded 0)

11. 12.

No (coded 0) Spaced evenly throughout (coded 0)

14. 15. 16.

Paroral membrane, fragmentation Paroral membrane, spacing of basal bodies Paroral membrane, orientation of basal bodies Peristomial ciliature, developed Peristomial ciliature, structure Peristomial ciliature, localization

No (coded 1) Helical (coded 1) Holotrichous (coded 1) Ordinary (coded 1) Yes (coded 1) Almost closed (coded 1) Along anterior body end (coded 1) 2 (coded 1) Deep inside the buccal cavity (coded 1) Deep inside the feeding tube (coded 2) Shortened anteriorly (coded 1) Strongly shortened (coded 2) Yes (coded 1) More narrowly spaced in posterior region (coded 1)

17. 18. 19.

Peristomial lobes Buccal cavity Lorica

13.

Character states

Same throughout the paroral membrane (coded 0) No (coded 0) Arranged in kineties (coded 0) Central area or bottom of the buccal cavity (coded 0) No (coded 0) Inconspicuous (coded 0) No (coded 0)

of character states in the terminal taxa (Table 5) were calculated using the computer program Mesquite ver. 2.73 (Maddison and Maddison, 2007). The most likely character states were used to reconstruct ancestral morphologies, taking into consideration the actual morphologies of the terminal taxa. 3. Results 3.1. Sequence information Eighteen new 18S rRNA gene sequences, 14 new ITS1-5.8S-ITS2 region sequences, and 16 new sequences from the D1D2 region of the 28S rRNA gene were obtained during the course of this study.

Different in anterior and posterior region of the paroral membrane (coded 1) Yes (coded 1) Scattered basal bodies (coded 1) Upper wall of the buccal cavity (coded 1) Yes (coded 1) Conspicuously deep (coded 1) Yes (coded 1)

Sequence length, GC content, and GenBank accession numbers of these sequences are provided in Table 1. 3.2. Tree-building phylogenetic analyses 3.2.1. Phylogenies inferred from the 18S rRNA gene We analyzed two 18S rRNA gene alignments using three different phylogenetic algorithms (maximum likelihood, Bayesian inference, and maximum parsimony). The first alignment (designated 18S-17) contained 17 species that we also had D1D2 sequence information for. The second alignment (designated 18S-63) included 63 species, and we used this alignment to investigate the effect of low taxon sampling on phylogenetic inferences.

Table 5 Distribution of character states in heterotrichean genera. For characters and character states, see Table 4. A dash (–) indicates an inapplicable character. Taxon

Character states

References

1–5

6–10

11–15

16–19

Lineage I Peritromus

10000

00100

0000-

-000

Rosati et al. (2004), Song and Wilbert (1997)

Lineage II Anigsteinia Spirostomum

0–110 11110

00000 00000

01000100-

-000 -000

Own observations Foissner et al. (1992)

Lineage III Climacostomum

0-111

00022

00010

0010

Augustin and Foissner (1992)

Lineage IV Chattonidium Condylostoma Condylostomides Condylostentor

10111 10111 0-111 10110

11010 00010 00000 11010

00000000000000010

-010 -010 -010 1010

Modeo et al. (2006) Chen et al. (2007), Kim et al. (2012) Foissner et al. (2002), da Silva Neto (1994) Chen et al. (2007)

Lineage V Blepharisma Eufolliculina Fabrea Folliculina Gruberia Maristentor Stentor

0-110 10110 0-110 10110 10110 10110 10110

00000 11000 00001 11000 00000 11002 11000

0010000010010 0000100000011 00010

-000 -101 0000 -101 -000 0100 0000

Giese (1973) Mulisch (1987) Song and Packroff (1996/97) Song et al. (2003) Own observations Lobban et al. (2002) Foissner and Wölfl (1994), Foissner et al. (1992)

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At first glance, the branching pattern of phylogenetic trees inferred from the 18S-17 dataset was inconsistent with that inferred from the 18S-63 alignment (cp. Figs. 1 and 2). This mismatch was very likely caused by taxon undersampling and/or the lack of sufficient phylogenetic signal in the 18S rRNA gene to resolve deep heterotrichean relationships (see Section 3.5. Split spectrum analysis). Moreover, the majority of deeper nodes had poor nodal support, thus the phylogenetic relationships between the major heterotrichean lineages were unresolved. Only a very few relationships received strong support from all phylogenetic analyses. These included the monophylies of the genera Spirostomum and Stentor, as well as the sister relationships of Spirostomum and Anigsteinia and of Stentor and Blepharisma. Analysis of the small dataset indicated that the genus Climacostomum was a sister taxon of all other heterotricheans, whereas analysis of the large dataset indicated that Peritromus was the adelphotaxon of all other heterotricheans. The former topology was, however, very poorly sustained (44% ML bootstrap support and posterior probability of only 0.60). The latter topology received moderate support in the MP tree (70%), poor support in the ML tree (52%), and was not recognized in the Bayesian tree. The families Spirostomidae, Blepharismidae, and Climacostomidae, as defined by Lynn (2008), were not monophyletic in any of the 18S rRNA gene datasets. Specifically, (i) Anigsteinia did not cluster together with Blepharisma but with Spirostomum; (ii) Gruberia did not form a monophyletic group with Spirostomum; and (iii) Climacostomum and Fabrea were not sister taxa. To summarize, the phylogenetic trees computed from the 18S63 alignment showed that there are five main heterotrichean lineages: lineage I comprising the family Peritromidae; lineage II uniting the genera Spirostomum and Anigsteinia; lineage III containing the family Climacostomidae with a single genus Climacostomum; lineage IV representing the family Condylostomatidae with four genera (Condylostoma, Condylostomides, Condylostentor, and Chattonidium); and lineage V that contained six families: Blepharismidae, Gruberiidae fam. n., Fabreidae fam. n., Folliculinidae, Maristentoridae, and Stentoridae (Fig. 2). 3.2.2. Phylogenies inferred from the ITS1-5.8S-ITS2 region and the D1D2 region of the 28S rRNA gene The ITS-14 alignment included a single karyorelictean and 13 heterotrichean ITS1-5.8S-ITS2 region sequences. Nodal support values in the resulting phylogenetic trees were very poor, except for the monophylies of the genera Spirostomum (100% ML, 1.00 BI, 99% MP) and Stentor (100% ML, 1.00 BI, 85% MP). In accordance with 18S rRNA gene phylogenies, Blepharisma was classified as the sister taxon of Stentor although with very poor support (59% ML, 0.55 BI). All other phylogenetic relationships within heterotricheans remained basically unresolved (Fig. 1). Phylogenetic trees inferred from the D1D2-17 alignment, which contained a single karyorelictean and 16 heterotrichean sequences of the first two domains of the 28S rRNA gene, were much better correlated with the 18S rRNA gene trees than with the ITS1-5.8SITS2 region trees. Specifically, (i) Peritromus branched off first (60% ML, 0.99 BI, 79% MP); (ii) the genus Spirostomum was monophyletic (98% ML, 1.00 BI, 100% MP) and clustered together with Anigsteinia (86% ML, 1.00 BI, 65% MP); and (iii) Stentor was monophyletic (98% ML, 1.00 BI, 100% MP). Monophylies of the families Blepharismidae, Spirostomidae, and Climacostomidae, as defined by Lynn (2008), were not recovered in the D1D2 trees. The single substantial difference between the 18S rRNA and D1D2 trees was the phylogenetic position of Blepharisma. This genus was the sister taxon to Stentor in the 18S rRNA gene trees, while it was the sister taxon of {Stentor + Fabrea} in the D1D2 trees. However, statistical support for the latter topology was very poor (50% ML, 0.75 BI, 54% MP) and therefore should be interpreted with caution (Fig. 1).

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3.2.3. Phylogenies inferred from the concatenated alignment Altogether, we had sequence information from the 18S rRNA gene and the first two domains of the 28S rRNA gene available for one karyorelictean and 16 heterotrichean species. As both genes are parts of the major rRNA transcript, we concatenated them generating a single alignment that we designated CON-17. Topologies of trees inferred from this concatenated alignment matched the trees based on the D1D2-17 and 18S-63 alignments very well (cp. Figs. 1 and 2). Only the positions of Climacostomum, Gruberia and Condylostoma conflicted between trees inferred from the CON-17 and 18S-63 datasets, which is very likely a result of taxon undersampling. 3.3. Topology testing Because statistical support for most deep branching nodes was comparatively poor, we tested several alternative topologies using the AU, WKH, and WSH tests (Table 3). A sister relationship of Peritromus and all other heterotrichs could not be excluded for the trees inferred from the 18S rRNA gene alignments (P-values > 0.05), and was already shown in the best D1D2-17 and CON-17 trees. Monophyly of the family Spirostomidae, as defined by Lynn (2008), was firmly rejected (P-values < 0.001) for trees inferred from the 18S rRNA gene and concatenated alignments. However, it could not be excluded in trees inferred from the D1D2-17 alignment, which is very likely a result of taxon and/or character undersampling. The same applied to the monophyly of the family Climacostomidae, i.e., a sister relationship between Climacostomum and Fabrea was rejected for trees inferred from the 18S rRNA gene and concatenated alignments, but not for those based on the D1D2-17 alignment. Monophyly of the family Blepharismidae, as defined by Lynn (2008), was consistently rejected by all three statistical topology tests for trees inferred from the 18S rRNA gene, the first two domains of the 28S rRNA gene, and the concatenated alignment. Monophyly of genera with peristomial kineties was firmly rejected for trees inferred from the largest 18S rRNA gene alignment. 3.4. Phylogenetic networks Because reticulate evolutionary relationships cannot be captured by phylogenetic trees, we constructed phylogenetic networks for all alignments. The results were qualitatively similar to those obtained for the phylogenetic trees, therefore we only present the neighbornet split graphs inferred from the CON-17 and 18S-63 alignments (Figs. 3 and 4). These analyses showed several distinct relationships that were well supported by a set of long parallel edges and bootstrap values ranging from 89% to 100%: (i) sister relationship of Peritromus and Loxodes; (ii) monophyly of the genus Spirostomum; (iii) sister relationship of Spirostomum and Anigsteinia; (iv) monophyly of the genus Stentor; and (v) sister relationship of Stentor and Blepharisma. Moreover, the phylogenetic network computed from the 18S-63 alignment showed the same five main heterotrichean lineages as the phylogenetic trees generated from the same alignment (cp. Figs. 2 and 4). However, deep heterotrichean nodes were not unambiguously resolved in the split graphs, as reflected by the star-like central part that completely lacked treeness and was full of short parallelograms (Fig. 4). 3.5. Split spectrum analysis This method has been used to investigate the untransformed information content of alignments, because it is independent of evolutionary substitution models and tree-building algorithms. This approach can be used to determine the exact number of

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Fig. 1. Maximum likelihood (ML) phylogenetic trees inferred from 18S-17, ITS-14, D1D2-17 and CON-17 alignments. Results from Bayesian inference (BI) and maximum parsimony (MP) bootstrap analysis were mapped onto the ML tree. A dash indicates either mismatch in branching pattern or posterior probability value below 0.50/ maximum parsimony bootstrap value below 50%. All sequences, except for those of Anigsteinia clarissima, were obtained during this study. Scale bars indicate number of nucleotide substitutions.

nucleotides that support individual splits or nodes in phylogenetic networks or phylogenetic trees. We computed the split spectrum for the 18S-63 alignment; the most relevant splits with respect to the 18S rRNA gene tree shown in Fig. 2 are plotted in Fig. 5. The best split supports the monophylies of the genus Loxodes (62 binary and 163 asymmetric positions) and of the class Heterotrichea (62 binary, two asymmetric, and 108 noisy positions). Monophyly of lineage I, i.e., the family Peritromidae, was the second-best supported split (10 binary and 95 asymmetric positions). The third-best split supported monophyly of lineage III, that is, the family Climacostomidae, with seven binary and 73 asymmetric positions. Monophyletic origin of lineage II, i.e., Anigsteinia + Spirostomum, was documented by split No. 5 with four binary, 29 asymmetric, and 26 noisy positions. The next column represented the split between lineages II + III + IV + V (four binary, three asymmetric, and 35 noisy positions) and Loxodes + lineage I (four binary, nine asymmetric, and two noisy positions), supporting the sister relationship between the family Peritromidae and all other heterotrichean families. Monophyly of lineage IV was represented by split No. 41, which was supported by two binary, three asymmetric and seven noisy positions. Monophyletic origin of lineage V (split No. 139) was corroborated by two asymmetric and four noisy positions, while common ancestry of lineages IV and V (split No. 84) was supported by one binary, two asymmetric, and six noisy positions. A node uniting lineages III, IV, and V (split No. 57) was supported by two asymmetric and eight noisy positions.

3.6. Four-cluster likelihood analyses To unravel deep heterotrichean phylogenetic relationships, we split the taxa into four groups: (i) Loxodes which was considered the outgroup [designated as L]; (ii) all members of the family Peritromidae, i.e., lineage I [designated as P]; (iii) all members of lineage II, i.e., Spirostomum + Anigsteinia [designated as S]; and (iv) the rest of the heterotrichean taxa, i.e., lineages III, IV, and V [designated as H s.s.]. For all analyzed alignments, four-cluster likelihood analyses supported a sister relationship between Loxodes and Peritromus and a sister relationship between lineage II and the bulk of lineages III–V. This arrangement was corroborated by 96.0% of data points for the 18S-17 alignment, 99.1% of data points for the 18S-63 alignment, 94.0% of data points for the D1D2 alignment, and 100% of data points for the CON-17 alignment (Fig. 6). Thus, these quartet-mapping results strongly support the sister relationship of Peritromus and all other heterotrichs, when trees are rooted with Loxodes. To get a better picture of the phylogenetic relationships among lineages III, IV, and V, we grouped taxa in the 18S-63 alignment into the following quartets: (i) Loxodes + all members of lineages I and II, i.e., Loxodes + Peritromus + Spirostomum + Anigsteinia [designated as A]; (ii) all members of lineage IV, i.e., Condylostoma, Condylostomides, Condylostentor, and Chattonidium [designated as B]; (iii) all members of lineage III, i.e., Climacostomum [designated as C]; and (iv) all members of lineage V, i.e., Blepharismidae,

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Fig. 2. Maximum likelihood (ML) phylogenetic trees inferred from the 18S-63 alignment. Results from the Bayesian inference (BI) and maximum parsimony (MP) bootstrap analysis were mapped onto the ML tree. A dash indicates either mismatch in branching pattern or a posterior probability value below 0.50/maximum parsimony bootstrap value below 50%. Roman numerals in parentheses denote affiliation of the heterotrichean families with the five main heterotrichean evolutionary lineages. Sequences in bold face were obtained during this study. Scale bars indicate one substitution per 10 nucleotide positions.

Gruberiidae fam. n., Fabreidae fam. n., Folliculinidae, Maristentoridae, and Stentoridae [designated as D]. Quartet mapping analyses supported the branching pattern of lineages as shown in Fig. 2, i.e., that lineage III branched off after the group uniting Loxodes and lineages I and II and that lineage IV was sister to lineage V by 50.4% of data points. Sister relationship of lineage IV and the group uniting Loxodes and lineages I and II on one hand and sister relationship of lineages III and V on the other one was supported by 37.4% of data points, while the sister relationship of lineages III and

IV and the sister relationship of lineage V and the group uniting Loxodes and lineages I and II was supported by only 7.0% of data points (Fig. 7). 3.7. Reconstruction of heterotrichean morphological evolution The most likely morphological states of the ancestors of all heterotrichean families and lineages were found using the likelihood method in combination with the Markov evolutionary model. This

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Fig. 3. Phylogenetic network inferred from the CON-17 alignment using the neighbornet algorithm and uncorrected distances. Numbers along edges are bootstrap support values coming from 1000 replicates. Values < 50% are not shown. The scale bars indicate one substitution per one hundred nucleotide positions.

procedure was based on the ML tree shown in Fig. 2 and the distribution of morphological characters in the terminal taxa as given in Table 5. Results of the reconstruction analyses are summarized schematically in Fig. 8. All morphological apomorphies recognized by this approach were mapped onto the branches of this genealogical tree. Reconstruction analyses indicated that the stem species of the class Heterotrichea had (1) a contractile body (proportional likelihood pL = 0.8170); (2) a holotrichous ciliature bearing ordinary cilia (pL = 0.6663); (3) an inconspicuous, flat buccal cavity (pL = 0.9995); (4) a single continuous paroral membrane that was composed of evenly spaced dikinetids throughout (pL = 0.6806– 0.9958); and (5) an adoral zone of membranelles that was as long as the paroral membrane and extended along the main body axis

(pL = 0.9444–0.9893). This ancestor did not display any frontal membranelles (pL = 0.9614), peristomial ciliature (pL = 0.8298), or peristomial lobes (pL = 0.9996). This basic ground pattern remained virtually unchanged along the whole stem of the heterotrichean tree of life, from which emerged all main five lineages recognized in this study. The last common ancestor of lineage I, i.e., of the family Peritromidae, had a short additional paroral membrane (pL = 0.9996) and a special dorsal ciliary pattern, including only a single peripheral kinety (pL = 0.9999) that bore bristle-like cilia (pL = 0.9997). The two latter features are also found in the genus Loxodes, which is the outgroup taxon. However, likelihood reconstruction analyses indicated that these features evolved convergently in Peritromus and Loxodes, while the maximum parsimony reconstruction

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Fig. 4. Phylogenetic network inferred from the 18S-63 alignment using the neighbornet algorithm and the uncorrected distances. Numbers along edges are bootstrap support values from 1000 replicates. Values < 50% are not shown. Scale bars indicate one substitution per 100 nucleotide positions.

analyses regarded these characters as plesiomorphies already present in the stem heterotrichean species. Parsimony results thus strongly suggested that Peritromus possesses several important ancient plesiomorphies. The progenitor of lineage II, which unites the genera Anigsteinia and Spirostomum, was morphologically very similar to the stem species of the Heterotrichea but its paroral membrane became distinctly thickened at the posterior end due to the narrow spacing of basal bodies (pL = 0.9869). Moreover, the body of Anigsteinia became acontractile (pL = 1.0000), while the somatic ciliature started to form a helical pattern during body contraction in Spirostomum (pL = 0.9989). The stem species of lineage III, containing only the family Climacostomidae, very likely evolved (1) a frontal membranelle near the right wall of the buccal cavity (pL = 0.9981) and (2) a peristomial ciliature composed of several regularly arranged kineties, extending at the bottom of the deep buccal cavity (pL = 0.9972– 0.9999). The deep buccal cavity and frontal membranelles are also characteristics of members of the family Condylostomatidae, while

peristomial ciliature is a feature of the genus Condylostentor and the families Stentoridae and Maristentoridae. The reconstruction analyses strongly indicated that these characters evolved convergently. Lineage IV, which unites members of the family Condylostomatidae, possibly evolved from the stem heterotrichean species by formation of several frontal membranelles (cirri) arranged in a series along the right border of the deep buccal cavity (pL = 0.4072– 0.8920). The last common ancestor of lineage V very likely maintained the stem morphology. As indicated by the short internodes and edges in the phylogenetic trees and networks, this lineage split rather rapidly into six families that are, however, well defined by morphological apomorphies. Specifically, the paroral membrane of the family Gruberiidae became fragmented throughout (pL = 0.9999), while the paroral membrane of the Fabreidae shortened and became fragmented only in its anterior portion (pL = 1.0000). In contrast, the paroral membrane of the Blepharismidae differentiated into at least two distinct files each

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Fig. 5. Support of selected relevant splits calculated from the 18S-63 alignment with the aid of the computer program SAMS (Wägele and Mayer, 2007). Column height represents the number of clade-supporting positions, i.e., putative primary homologies. Column parts above the y-axis represent the ingroup partition, while those below the axis correspond to the outgroup partition.

Fig. 6. Quartet likelihood mapping showing distribution of phylogenetic signal for three possible relationships among the main heterotrichean lineages, as inferred from the 18S-17, D1D2-17, CON-17, and 18S-63 alignments. The corners of the triangles show the percentage of fully resolved trees. Rectangular areas show the percentage of trees that are in conflict. The central triangle shows the percentage of unresolved star-like trees. L, Loxodes; P, Peritromus (lineage II); S, Spirostomum + Anigsteinia (lineage II); H s.s., Heterotrichea sensu stricto (lineages III + IV + V).

characterized by dikinetids with different orientations (pL = 0.9990). Molecular trees strongly indicated that the families Maristentoridae and Folliculinidae are sister groups. Reconstruction

analyses revealed that the anterior body end of their last common ancestor transformed into lobe-like structures taking along the adoral zone of membranelles (pL = 0.9133). Folliculinidae

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Fig. 7. Quartet likelihood mapping showing the distribution of phylogenetic signal for three possible relationships among the main heterotrichean lineages, as inferred from the 18S-63 alignment. Corners of the triangles show the percentage of fully resolved trees. Rectangular areas show the percentage of trees that are in conflict. Central triangle shows the percentage of unresolved star-like trees. A, Loxodes + lineages I and II; B, lineage III; C, lineage IV; D, lineage V.

evolved a lorica (pL = 1.0000), while Maristentoridae developed a peristomial ciliature in the form of scattered basal bodies at the central area or bottom of the buccal cavity (pL = 1.0000). Moreover, the paroral membrane of Maristentor shortened significantly (pL = 1.0000). Morphological apomorphies of the Stentoridae include an almost closed adoral zone of membranelles extending along the anterior body end (pL = 0.9988) and a peristomial ciliature in the form of distinct kineties localized at the bottom of the buccal cavity (pL = 0.9984–0.9997). 4. Discussion 4.1. Resolution at the base of the class Heterotrichea Jankowski (1964) speculated that Blepharisma could be the most primitive heterotrich. However, the sister relationship of Blepharisma and all other heterotrichs was not recognized in molecular phylogenies that, on the contrary, indicated Blapharisma as a member of the heterotrichean terminal radiation, being a sister taxon of Stentor (Gong et al., 2007; Miao et al., 2005, 2009; Schmidt et al., 2007; Thamm et al., 2010). Our analyses, in accordance with Rosati et al. (2004) and Modeo et al. (2006), indicate that the genus Peritromus is a sister taxon of all other heterotrichs. Specifically, this sister relationship was supported by analyses of the large 18S rRNA, D1D2-17, and CON-17 datasets (Figs. 1 and 2). A similar picture was obtained from phylogenetic network analyses, i.e., Peritromus and Loxodes formed a strongly supported split clearly separated from the rest of the heterotrichs by a distinct set of comparatively long edges (Figs. 3 and 4). Likewise, quartet likelihood mapping analyses consistently supported the sister relationship of Peritromus and Loxodes on one hand and monophyly of the remaining heterotrichs on the other one (supported by 94–100% of data points; Fig. 6). In more detail, the sister relationship of Peritromus and Loxodes was supported by four binary, nine asymmetric, and two noisy nucleotide characters, and is the best-supported deeper node in the 18S rRNA gene phylogenetic trees according to the split spectrum analysis (Fig. 5). Comparative morphological analyses indicate that Peritromus is morphologically nearest to many members of the class Karyorelictea and, especially, to Loxodes. The dorsal body sides of species in these two genera look similar in that only a single peripheral kinety bearing bristle-like cilia is present, while the rest of the dorsal side is unciliated (Foissner 1995/96, 1996; Foissner et al., 1995; Rosati et al., 2004; Song and Wilbert, 1997). In contrast, all other heterotrichs have a holotrichously ciliated dorsal side. According to our likelihood reconstruction analyses, this holotrichous pattern is a property of the last common ancestor of the Heterotrichea, though there was not significant statistical support for this

(pL = 0.6662). Maximum parsimony analyses, however, indicated that the Peritromus/Loxodes-like pattern was ancestral. In our opinion, it is very unlikely that the dorsal ciliary pattern of Peritromus and Loxodes evolved convergently; we believe that the barren dorsal side of Peritromus is homologous to the glabrous stripe of other karyorelicteans (Foissner, 1998). 4.2. Phylogenetic position of Anigsteinia Anigsteinia sequences were not included in previous phylogenetic studies (Gong et al., 2007; Miao et al., 2005, 2009; Rosati et al., 2004; Schmidt et al., 2007; Thamm et al., 2010), therefore the phylogenetic placement of this genus has been inferred based only on morphological data. Isquith (1968) and Isquith and Repak (1974) classified Anigsteinia in the family Spirostomidae. However, in Lynn’s (2008) compendium, Anigsteinia was assigned to the family Blepharismidae. This latter affiliation is not supported by any of our phylogenetic analyses. We found that Anigsteinia always clustered as the sister taxon of the Spirostomum clade with very strong statistical support, which corroborates classification of Isquith (1968) and Isquith and Repak (1974). Blepharisma, the type genus of the family Blepharismidae, clustered within lineage V as an adelphotaxon of the Stentor clade (Figs. 1–4). Furthermore, a sister relationship between Anigsteinia and Blepharisma was firmly rejected by all statistical tree topology tests carried out on four different alignments (Table 3). At first glance, Anigsteinia and Blepharisma have similar body shape, somatic ciliary pattern, and morphology of the adoral zone of membranelles, but they clearly differ in the fine structure of their paroral membranes (Small and Lynn, 1985; Lynn and Small, 2002). Specifically, the posterior end of the paroral membrane is distinctly thickened in Anigsteinia (Fig. 9A, arrowhead), while the paroral membrane of Blepharisma is differentiated into two distinct files each characterized by dikinetids with different orientations (Fig. 9D, arrow). Interestingly, the Anigsteinia-like paroral pattern is a property of the genus Spirostomum (cp. Fig. 9A and B, arrowheads), which is the nearest relative of Anigsteinia in all phylogenetic analyses. Therefore, we exclude Anigsteinia from the Blepharismidae and assign it to the Spirostomidae. Anigsteinia and Spirostomum differ mainly with respect to body contractility (acontractile vs. very contractile). 4.3. Phylogenetic position of Gruberia Gruberia has traditionally been affiliated with the family Spirostomidae due to the characteristics of a highly contractile oblong body and extension of the adoral zone of membranelles to about one third to one half of the body length (Lynn, 2008; Lynn and Small, 2002). Moreover, Jankowski (1964) speculated that

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Fig. 8. Reconstruction of ancestral morphologies of stem species of all heterotrichean families and lineages. Schematic drawings are based on likelihood analysis in combination with the Markov evolutionary model as implemented in the computer program Mesquite (Maddison and Maddison, 2007). Asterisks mark morphological characteristics virtually unchanged from the heterotrichean stem species. Apomorphies are put into boxes. AZM, adoral zone of membranelles; PM, paroral membrane.

Spirostomum evolved from Gruberia by polymerization of adoral membranelles. On the other hand, Fernandez-Leborans (1981) hypothesized that Gruberia evolved from Blepharisma. FernandezLeborans’ speculation is partially supported in that both Gruberia and Blepharisma belong to lineage V (Fig. 2) but Jankowski’s hypothesis and Lynn’s classification are not supported by either the 18S rRNA gene (Gong et al., 2007; Hammerschmidt et al., 1996; Miao et al., 2005, 2009; Modeo et al., 2006; Rosati et al., 2004; Schmidt et al., 2007; Thamm et al., 2010) or by the first two domains of the 28S rRNA gene (Figs. 1 and 2). In contrast, a sister relationship between Gruberia and Spirostomum was revealed by ML analyses of the ITS-14 alignment, albeit with negligible bootstrap support (15%). This grouping was not revealed by maximum parsimony or Bayesian analysis of ITS region sequences. Further, according to statistical tree topology tests, a sister relationship between Gruberia and Spirostomum was rejected for the 18S rRNA gene and the concatenated dataset, but not for the

D1D2-17 alignment, which could be a result of taxon and/or character undersampling (Rosenberg, 2007). Comparative morphological analyses indicated that the paroral membrane structure and the behavior of ciliary rows during body contraction are very different between Gruberia and Spirostomum. Specifically, the paroral membrane of Gruberia is fragmented throughout and lacks the typical posterior thickening which is so characteristic of Spirostomum and its sister taxon, Anigsteinia (Fig. 9A–C). Further, the ciliary rows remain meridional in Gruberia during body contraction, while they assume a helical pattern in Spirostomum. Based on this evidence together with the molecular data, we exclude Gruberia from the family Spirostomidae and erect a new family, Gruberiidae, which is assigned to lineage V. At the present state of knowledge, the family Gruberiidae is monotypic and thus its establishment appears to be basically redundant. However, in the light of the present molecular and morphological comparative analyses, it is not possible to unambig-

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uously assign the genus Gruberia to any established family of lineage V due to the lack of both molecular and morphological synapomorphies (Figs. 2, 4, 5 and 8). Even affiliation of Gruberia with lineage V is corroborated with only two asymmetric and four noisy nucleotide positions (Fig. 5) and, moreover, there is no morphological synapomorphy supporting this classification (Fig. 8). When Gruberia is excluded from lineage V, support for its monophyly increases to two binary, one asymmetric and three noisy nucleotide positions. Monophyly of the gruberiid sequences is very strongly to fully supported in both phylogenetic trees and networks as well as by two binary and 41 asymmetric nucleotide positions in the split spectrum analysis. Moreover, gruberiids are separated from all other heterotrichs by a set of long parallel edges whose length is comparable to that of other recognized heteritrichean families (Fig. 4). In this situation, when affiliation of Gruberia with the Spirostomidae is rejected and reliable classification of Gruberia into known heterotrich families is not possible, the establishment of the family Gruberiidae seems to be justified. 4.4. Phylogenetic position of Fabrea Based on its prominent oral region and inconspicuous paroral membrane, Fabrea was assigned to the family Climacostomidae (Lynn, 2008; Lynn and Small, 2002; Repak, 1972). A close relationship between Climacostomum and Fabrea has never been supported by previous molecular analyses (Gong et al., 2007; Miao et al., 2005, 2009; Schmidt et al., 2007; Thamm et al., 2010). Consistent with these previous studies, members of the family Climacostomidae fell into two different clades, with Fabrea typically nested within lineage V in our analyses. Monophyletic Climacostomidae, as defined by Lynn (2008), was consistently rejected by all statistical tree topology tests for the 18S rRNA gene and the concatenated alignments, but not for the D1D2-17 and ITS-14 alignments. Because the latter two datasets comprised a small number of taxa and characters, caution is warranted when interpreting evolutionary relationships based on analyses of such small datasets (Rosenberg, 2007). The only morphological apomorphy that justifies classification of Fabrea within the Climacostomidae is the presence of peristomial kineties. However, according to the statistical tree topology tests (Table 3), this feature has a highly homoplastic nature. Further, oral ciliary patterns of Fabrea and Climacostomum are quite dissimilar on closer inspection. The paroral membrane of Fabrea is shortened and fragmented anteriorly (Fig. 9G, arrow). In contrast, the paroral membrane of Climacostomum was strongly shortened and dislocated to the end of a long feeding tube. Further, Climacostomum evolved a frontal membranelle near the right wall of the buccal cavity (Fig. 9F; Augustin and Foissner, 1992). Neither frontal membranelle nor long feeding tube are present in Fabrea. Because there is no molecular support for classification of Fabrea within the Climacostomidae, we exclude Fabrea from this family and establish for it a new family, Fabreiidae, which is associated with lineage V. Like the family Gruberiidae, the family Fabreiidae is also monotypic and its name-bearing genus, Fabrea, cannot be assigned to any described lineage V family due to the lack of both molecular and morphological apomorphies. As fabreid sequences form a fully supported cluster that is separated from all other heterotrichs by long parallel edges with length being comparable to that of other recognized heterotrichean families (Fig. 4), we find the establishment of the family Fabreiidae justified at the present state of knowledge. 4.5. Monophyly and classification of lineage IV genera Based on our analyses, we suggest uniting the genera Condylostoma, Condylostomides, Condylostentor, and Chattonidium

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into lineage IV. A close relationship among these genera was already hypothesized base on morphological findings (e.g., Chen et al., 2007; da Silva Neto, 1994; Foissner et al., 2002; Modeo et al., 2006; Villeneuve-Brachon, 1940), and was later supported by molecular analyses of the 18S rRNA gene (Miao et al., 2009; Modeo et al., 2006; Gong et al., 2007; Schmidt et al., 2007; Thamm et al., 2010). This lineage was monophyletic in all our phylogenetic analyses, although with comparatively poor bootstrap support (71% ML and 68% MP) and posterior probability (0.93; Fig. 2). According to split spectrum analysis, two binary, three asymmetric, and seven noisy nucleotide positions support this node (Fig. 5). However, when the most divergent genus Condylostentor was excluded, support for the monophyly of the core lineage IV genera changed to one binary, six asymmetric, and 12 noisy nucleotide positions (data not shown). Moreover, monophyly of core lineage IV genera was fully supported in the 18S rRNA gene trees (Fig. 2). Thus, based on consideration of both morphological and molecular data, we are confident that Condylostoma, Condylostentor, and Chattonidium are all members of the family Condylostomatidae. Chattonidium has traditionally been classified in the monotypic family Chattonidiidae due to a posteroaxial cavity containing several ciliary organelles (Modeo et al., 2006; Villeneuve-Brachon, 1940). Because our molecular analyses indicated that this structure is diagnostic at the generic but not at the family level, we suppress the family Chattonidiidae and assign the genus Chattonidium to the Condylostomatidae. Classification of Condylostomides remains uncertain, as phylogenetic network analyses indicated a lot of conflicts with regard to its placement within the family Condylostomatidae (Fig. 4). Morphologically, Condylostomides differs significantly from Condylostoma, Condylostentor, and Chattonidium in having an acontractile (vs. contractile) body and a paroral membrane situated at the right margin of the buccal cavity (vs. at the bottom of the cavity; Fig. 9E). Data from additional genes are needed to determine whether these two features justify separation of Condylostomides from other lineage IV genera at the family level. Previous studies (Miao et al., 2009; Modeo et al., 2006; Gong et al., 2007; Schmidt et al., 2007; Thamm et al., 2010) and the present phylogenetic trees and networks have revealed a serious taxonomic problem in that the genera Condylostentor and Chattonidium are placed within the paraphyletic genus Condylostoma (Fig. 2). However, the support for this paraphyletic topology is statistically insignificant, indicating that the monophyly of Condylostoma cannot be excluded. On the other hand Condylostoma can be paraphyletic and thus, in fact, consisting of several morphologically closely similar Condylostoma-like genera and some morphologically distinct genera such as Condylostentor and Chattonidium. Already Jankowski (1964) speculated that the species rich genus Condylostoma represents a polymorphous group that may have given rise to other genera such as Climacostomum and Stentor. Molecular phylogenies do not support this hypothesis, but indicate Condylostentor and Chattonidium to be the result of radiation within Condylostoma. Nevertheless, data from more genes are required to confirm or reject the monophyletic origin of the genus Condylostoma and to clarify its phylogenetic relationships to Condylostentor and Chattonidium. 4.6. Monophyly of lineage V Five main heterotrichean evolutionary lineages were revealed by our analyses. The first four lineages were quite well supported in both phylogenetic trees and networks as well as in split spectrum analyses (Figs. 1–5). However, the monophyletic origin of the fifth lineage, which unites six families (Blepharismidae, Gruberiidae, Fabreidae, Folliculinidae, Maristentoridae, and Stentoridae), was poorly supported (59% ML bootstrap, 63% MP bootstrap,

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Fig. 9. Morphological diversity in heterotrichs after protargol impregnation (A–E, G, H) and in vivo (F). (A and B) Both Anigsteinia (A) and Spirostomum (B) are characterized by a posteriorly-thickened paroral membrane (arrowheads). Note that the somatic ciliary rows become spiral after body contraction in Spirostomum. (C) In Gruberia, the paroral membrane is fragmented throughout (arrow). (D) In Blepharisma, the paroral membrane is differentiated into two distinct files each characterized by dikinetids with different orientations. Arrow marks the place where dikinetids change orientation. (E) In Condylostoma, the paroral membrane extends at the bottom of the deep buccal cavity. (F) Climacostomum strongly reduced the paroral membrane and evolved a frontal membranelle near the right wall of the buccal cavity whose bottom is lined by peristomial kineties. (G) In Fabrea, the adoral zone of membranelles is spiraling and the paroral membrane is rather indistinct because it is fragmented and shortened anteriorly. (H) In Stentor, the adoral zone of membranelles extends along the anterior body end, becoming almost closed and thus delimitating the paroral membrane and peristomial kineties, which are localized at the bottom of the buccal cavity. AZM, adoral zone of mebranelles; FM, frontal membranelle; MA, macronucleus (nodule); PK, peristomial kineties; PM, paroral membrane; SK, somatic kineties. Scale bars indicate 50 lm.

posterior probability of 0.85). According to split spectrum analysis, only two asymmetric and four noisy nucleotide positions support the monophyly of this lineage (Fig. 5). This weak support could have been caused by one or several rapid radiation events that did not allow primary nucleotide homologies to be fixed and/or by incomplete lineage sorting at the rRNA locus. Morphological data do not cast more light on this, because reconstruction analyses indicated that the last common ancestor of lineage V maintained the heterotrichean ground pattern (Fig. 8). In other words, no morphological synapomorphies uniting all members of lineage V have been found. Data from more genes are required to confirm or reject the monophyletic origin of this lineage. 4.7. Morphological homoplasies in heterotrichean evolution Statistical topology tests and reconstruction analyses suggested that frontal membranelles (cirri) and peristomial ciliature evolved independently several times and that body contractility was lost several times in a convergent manner during heterotrichean evolution.

Frontal membranelles are present in lineage III (i.e., Climacostomidae) and in most members of lineage IV (i.e., Condylostomatidae). However, these structures very likely have different ontogenetic origins. In the Climacostomidae, the frontal membranelle could have evolved by migration of the first adoral membranelle (Augustin and Foissner, 1992), while in the Condylostomatidae, the frontal membranelles (cirri) are derived from the anterior end of the paroral membrane anlage (Shao et al., 2006). These findings clearly indicate that these structures are not homologous and should therefore be designated as frontal membranelles in the Climacostomidae, and as frontal cirri in the Condylostomatidae. Peristomial ciliature was found in five of 15 investigated heterotrichean genera, viz., in Climacostomum (Fig. 9F), Condylostentor, Fabrea (Fig. 9G), Stentor (Fig. 9H), and Maristentor. This special ciliature is in the form of distinct kineties in the four former genera (Augustin and Foissner, 1992; Chen et al., 2007; Foissner et al., 1992), and in the form of scattered basal bodies in the latter genus (Lobban et al., 2002). Monophyletic origin of these five genera was consistently rejected by all three statistical tree topology tests

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(Table 3), which likely reflects the homoplastic nature of this morphological structure. According to the reconstruction analyses, the body of the last common ancestor of the class Heterotrichea was contractile. This feature was independently lost in at least one genus across the majority of five main heterotrichean lineages, namely in Anigsteinia from lineage II, in Climacostomum from lineage III, in Condylostomides from lineage IV, and in Blepharisma and Fabrea from lineage V (Augustin and Foissner, 1992; da Silva Neto, 1994; Foissner et al., 2002; Giese, 1973; Song and Packroff, 1996/97). This diffuse pattern of loss is strongly suggestive of the homoplastic nature of this character.

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body sides, remains meridional during body contraction. Adoral zone of membranelles extends along main body axis and is as long as paroral membrane. Paroral membrane situated at right margin of or at the bottom of buccal cavity. One to several cirri near right wall of buccal cavity. No peristomial ciliature. Buccal cavity broad and deep. Genera assignable. Chattonidium Villeneuve, 1937; Condylostentor Jankowski (1978); Condylostoma Bory de St. Vincent, 1824 [type genus]; Condylostomides da Silva Neto (1994); Copemetopus Villeneuve-Brachon (1940); Dellochus Corliss, 1960 [incertae sedis in Condylostomatidae]; Electostoma Jankowski, 1979; Linostomella Aescht in Foissner, Berger, and Schaumburg, 1999; Predurostyla Jankowski (1978); Procondylostoma Jankowski, 1979.

5. Taxonomic summary 5.4. Family Fabreidae fam. n. Our phylogenetic analyses revealed that the families Blepharismidae, Climacostomidae, and Spirostomidae, as defined by Lynn (2008), are polyphyletic. To establish a more natural classification of the class Heterotrichea, we erect two new monotypic families within the order Heterotrichida: Fabreidae and Gruberiidae. We suppress the monotypic family Chattonidiidae and assign its type genus, Chattonidium, to the family Condylostomatidae. Further, we transfer the genus Anigsteinia to the family Spirostomidae, as already suggested by Isquith (1968) and Isquith and Repak (1974). Family home of molecularly not studied genera follows Lynn (2008). Based on the present reconstruction analyses, we refine the classification of the class Heterotrichea as follows: Class Heterotrichea Stein, 1859 Order Heterotrichida Stein, 1859 5.1. Family Blepharismidae Jankowski in Small and Lynn (1985) Diagnosis. Acontractile Heterotrichida with oblong body. Somatic ciliature holotrichous on both ventral and dorsal body sides. Adoral zone of membranelles extends along main body axis and is as long as paroral membrane. Paroral membrane differentiated into at least two files of differently oriented basal bodies. No peristomial ciliature. Buccal cavity flat and narrow. Genera assignable. Blepharisma Perty, 1849 [type genus]; Parablepharisma Jankowski (2007); Pseudoblepharisma Kahl, 1927. 5.2. Family Climacostomidae Repak, 1972 Diagnosis. Acontractile Heterotrichida with obovate body. Somatic ciliature holotrichous on both ventral and dorsal body sides. Adoral zone of membranelles slightly spiraling. Paroral membrane strongly shortened and dislocated to end of long feeding tube. A frontal membranelle near right wall of buccal cavity. Peristomial ciliature composed of several regularly arranged kineties. Buccal cavity broad and deep, leading to a feeding tube. Genera assignable. Climacostomum Stein, 1859 [type genus]; Pediostomum Kahl (1932) [incertae sedis in Climacostomidae]. Remarks. Jankowski (1978) classified the genus Pediostomum into the monotypic family ‘‘Pediostomatidae’’. Later, Jankowski (2007) transferred Pediostomum into the family Stentoridae, while Lynn (2008) into the family Climacostomidae. Molecular data are needed to solve this problem, but the family ‘‘Pediostomatidae’’ seems to be superfluous at the present state of knowledge. 5.3. Family Condylostomatidae Kahl in Doflein and Reichenow (1929) Diagnosis. Contractile or acontractile Heterotrichida with oblong body. Somatic ciliature holotrichous on both ventral and dorsal

Diagnosis. Acontractile Heterotrichida with anterior body end snout-like and pointed, posterior body end broadly rounded. Somatic ciliature holotrichous on both ventral and dorsal body sides. Adoral zone of membranelles spiraling along main body axis. Paroral membrane shortened and fragmented anteriorly. Peristomial ciliature composed of several regularly arranged kineties. Buccal cavity flat and narrow. Genus assignable. Fabrea Henneguy, 1890 [type genus]. 5.5. Family Folliculinidae Dons, 1914 Diagnosis. Contractile Heterotrichida with trumpet-shaped body residing in a lorica. Anterior body end differentiated into peristomial lobes. Somatic ciliature holotrichous on both ventral and dorsal body sides, remains meridional during body contraction. Adoral zone of membranelles and paroral membrane extend along peristomial lobes and of similar length. No peristomial ciliature. Buccal cavity flat and narrow. Genera assignable. Ampullofolliculina Hadzi, 1951; Ascobius Henneguy, 1884; Atriofolliculina Hadzi, 1951 [nomen nudum]; Aulofolliculina Hadzi, 1951; Botticula Dioni, 1972; Claustrofolliculina Hadzi, 1951; Diafolliculina Hadzi, 1951 [nomen nudum]; Echinofolliculina Dons, 1934; Epifolliculina Hadzi, 1951; Eufolliculina Hadzi, 1951; Folliculina Lamarck, 1816 [type genus]; Folliculinopsis Fauré-Fremiet, 1936 [nomen nudum]; Halofolliculina Hadzi, 1951 [nomen nudum]; Lagotia Wright, 1857; Latifolliculina Hadzi, 1951; Magnifolliculina Uhlig, 1964 [nomen nudum]; Metafolliculina Dons, 1924; Mirofolliculina Dons, 1928; Pachyfolliculina Hadzi, 1951; Parafolliculina Dons, 1914; Pebrilla Giard, 1888; Perifolliculina Hadzi, 1951; Planifolliculina Hadzi, 1951; Platyfolliculina Hadzi, 1938; Priscofolliculina Deflandre and Deunff, 1957 [fossil]; Pseudofolliculina Dons, 1914; Pseudoparafolliculina Andrews and Nelson, 1942; Splitofolliculina Hadzi, 1951 [nomen nudum]; Stentofolliculina Hadzi, 1938; Valletofolliculina Andrews, 1953. Remarks. Some other problematic and poorly known genera associated with the family Folliculinidae are listed in Jankowski (2007). 5.6. Family Gruberiidae fam. n. Diagnosis. Contractile Heterotrichida with oblong body. Somatic ciliature holotrichous on both ventral and dorsal body sides, remains meridional during body contraction. Adoral zone of membranelles extends along main body axis and is as long as paroral membrane. Paroral membrane fragmented throughout. No peristomial ciliature. Buccal cavity flat and narrow. Genus assignable. Gruberia Kahl (1932) [type genus].

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5.7. Family Maristentoridae Miao, Simpson, Fu and Lobban, 2005

References

Diagnosis. Contractile Heterotrichida with trumpet-shaped body. Anterior body end differentiated into peristomial lobes. Somatic ciliature holotrichous on both ventral and dorsal body sides, remains meridional during body contraction. Adoral zone of membranelles extends along peristomial lobes. Paroral membrane distinctly shortened and restricted to proximal portion of adoral zone. Peristomial ciliature composed of scattered basal bodies. Buccal cavity flat and narrow. Genus assignable. Maristentor Lobban, Schefter, Simpson, Pochon, Pawlowski and Foissner, 2002 [type genus].

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5.8. Family Peritromidae Stein, 1867 Diagnosis. Contractile Heterotrichida with ellipsoidal body. Ventral side holotrichous, while dorsal side bearing only a single Ushaped kinety with bristle-like cilia. Adoral zone of membranelles extends along main body axis. Two paroral membranes: first distinctly shortened, second as long as adoral zone of membranelles. No peristomial ciliature. Buccal cavity flat and narrow. Genus assignable. Peritromus Stein, 1863 [type genus]. 5.9. Family Spirostomidae Stein, 1867 Diagnosis. Contractile or acontractile Heterotrichida with oblong body. Somatic ciliature holotrichous on both ventral and dorsal body sides, becomes spiral during body contraction. Adoral zone of membranelles extends along main body axis and is as long as the paroral membrane. Paroral membrane distinctly thickened posteriorly due to narrowly spaced basal bodies. No peristomial ciliature. Buccal cavity flat and narrow. Genera assignable. Anigsteinia Isquith (1968); Diplogmus Mansfeld, 1923 [incertae sedis in Spirostomidae]; Propygocirrus Mansfeld, 1923 [incertae sedis in Spirostomidae]; Spirostomum Ehrenberg, 1834 [type genus]. 5.10. Family Stentoridae Carus, 1863 Diagnosis. Contractile Heterotrichida with trumpet-shaped body. Somatic ciliature holotrichous on both ventral and dorsal body sides, remains meridional during body contraction. Adoral zone of membranelles and paroral membrane extend along anterior body end. Peristomial ciliature composed of several regularly arranged kineties. Buccal cavity flat and narrow. Genera assignable. Heterostentor Song and Wilbert, 2002 [incertae sedis in Stentoridae]; Filistentor Jankowski, 2007; Parastentor Vuxanovici, 1961 [incertae sedis in Stentoridae]; Stentor Oken, 1815 [type genus]; Stentoropsis Dogiel and Bychowsky, 1934 [incertae sedis in Stentoridae]. Acknowledgments We are very grateful to two anonymous reviewers for their valuable comments and to Dr. Choon Bong Kwon for kindly providing us with micrographs and morphological data on Anigsteinia clarissima. The present study was supported financially by the National Institute of Biological Resources (NIBR) of Ministry of Environment, Korea (Project 1834-302) and the National Research Foundation of Korea funded by the Korean government (2012R1A1A2005751) and the Slovak Scientific Grant Agency (VEGA Project 1/0248/13). This work was supported also by the Slovak Research and Development Agency under Contract No. APVV-0436-12.

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