Constraints on Phylogenetic Interrelationships Among Four Free

Acta Protozool. (2017) 56: 255–281 www.ejournals.eu/Acta-Protozoologica ACTA doi:10.4467/16890027AP.17.023.7825 PROTOZOOLOGICA

Constraints on Phylogenetic Interrelationships among Four Free-living Litostomatean Lineages Inferred from 18S rRNA gene-ITS Region sequences and Secondary Structure of the ITS2 molecule

Ľubomír RAJTER, Peter VĎAČNÝ

Comenius University in Bratislava, Department of Zoology, Bratislava, Slovakia

Abstract. We investigated interrelationships between four free-living litostomatean lineages, using 18S rRNA gene and ITS region sequences as well as the secondary structure of the ITS2 molecules. Our phylogenetic analyses confirmed the deep split of free-living litostomateans into Rhynchostomatia and Haptoria represented here by Haptorida, Pleurostomatida, and Spathidiida. This bifurcation is also corroborated by the signature of the rhynchostomatian and haptorian ITS2 molecules. Specifically, the consensus stems of helices II and III are longer by one base pair in Rhynchostomatia, while the terminal loops of both helices are longer by one or two nucleotide/-s in Haptoria. A close relationship of Pleurostomatida and Haptorida is favored by quartet likelihood-mapping and supported by a 5’-AG vs. CU-3’ motif in the variable part of helix II and by two morphological apomorphies, i.e., meridionally extending somatic kineties and a non-three-rowed dorsal brush. Although monophyletic origin of Spathidiida is poorly supported in phylogenetic trees, the unique motif 5’-GA vs. UC-3’ present in the consensus helix II stem could be an important molecular synapomorphy of spathidiids, apart from the ancestrally anteriorly curved somatic kineties and the three-rowed dorsal brush. The peculiar family Pseudoholophryidae has very likely found its phylogenetic home among spathidiids, as an early branching lineage.

Key words: dorsal brush, Haptoria, molecular synapomorphies, morphological evolution, Pseudoholophryidae, Rhynchostomatia

INTRODUCTION that are used to seize other protists and microscopic metazoans. Over 400 free-living litostomatean species have been described from a variety of habitats all Free-living ciliates of the class Litostomatea Small around the globe (e.g., Kahl 1930a, 1930b, 1931; Foiss- and Lynn, 1981 represent apex predators in micro- ner et al. 1995, 1999, 2002; Kreutz and Foissner 2006; bial food webs both in aquatic and terrestrial habitats Foissner and Xu 2007; Lin et al. 2009; Vďačný and (Foissner et al. 1995, 1999, 2002; Lynn 2008). They Foissner 2012; Foissner 2016). According to Vďačný are morphologically well adapted to the raptorial life- et al. (2011a), they are classified in two subclasses, the style by having toxicysts (also known as extrusomes) Rhynchostomatia Jankowski, 1980 and the Haptoria Corliss, 1974. The former subclass is characterized by Address for correspondence: Peter Vďačný, Department of Zool- a prominent proboscis and a ventrally located cytos- ogy, Faculty of Natural Sciences, Comenius University in Bratisla- va, Ilkovičova 6, 842 15 Bratislava, Slovak Republic; E-mail: peter. tome, and includes two orders, the Tracheliida Vďačný [email protected] et al., 2011b and the Dileptida Jankowski, 1978. The 256 Ľ. Rajter and P. Vďačný latter subclass contains five well-defined groups: (i) the ridae Berger et al., 1984 as redefined by Rajter and order Pleurostomatida Schewiakoff, 1896 with laterally Vďačný (2016) as well as its spathidiid phylogenetic flattened body and ventrally extending, slit-like cyto- home. Our rationale for these hypotheses was based stome; (ii) the order Didiniida Jankowski, 1978 with on the following assumptions. The Rhynchostomatia barrel-shaped to globular body carrying an anterior oral have maintained the most plesiomorphic morphology cone; (iii) the order Lacrymariida Lipscomb and Ri- among all litostomateans (i.e., ventrally located cyto- ordan, 1990 with teardrop-shaped cell whose anterior stome, preoral kineties corresponding to adoral orga- part is differentiated into a head-like structure; (iv) the nelles, and formation of anarchic fields during stoma- order Spathidiida Foissner and Foissner, 1988 with cy- togenesis) and therefore might be the best candidate lindroidal to spatulate body equipped with a typically for a sister group of the three haptorian orders studied. three-rowed dorsal brush; and finally (v) the order Hap- Members of the order Haptorida display some pleuro- torida Corliss, 1974 with bursiform body and usually stomatid (i.e., meridionally extending somatic kineties two-rowed brush. However, there are some free-living and reduced number of dorsal brush rows) as well as genera (e.g., Chaenea Quennerstedt, 1867; Helicopro- some spathidiid features (i.e., bursiform body and an- rodon Fauré-Fremiet, 1950; Homalozoon Stokes, 1890; teriorly localized oral bulge opening). Since somatic Mesodinium Stein, 1863; and Trachelotractus Foissner, ciliary structures are supposed to be phylogenetically 1997) whose systematic position remains enigmatic, more conserved than oral ones (Lynn 2008), haptorids mainly because of the long-branch artifacts in 18S might be more closely related to pleurostomatids than rRNA gene trees and/or because of phylogenetic unin- to spathidiids. Recently, we have recognized that the formativness of their 18S rRNA gene (Johnson et al. genus Pseudoholophrya Berger et al., 1984 clusters 2004; Vďačný et al. 2011a; Kwon et al. 2014; Vďačný within the order Spathidiida (Rajter and Vďačný 2016), and Rataj 2017). which was a rather surprising result because Pseudo- The majority of recent phylogenetic studies focused holophrya does not appear as a typical spathidiid. In mainly on the internal evolutionary relationships within the present study, we have obtained 18S rRNA gene as the subclass Rhynchostomatia (Vďačný et al. 2011b, well as ITS region sequences from a morphologically 2017; Vďačný and Foissner 2012; Jang et al. 2014; closely related genus, Paraenchelys Foissner, 1983, Vďačný and Rajter 2015), the order Pleurostomatida which enabled us to test the monophyly of the fam- (Lin et al. 2007, 2008; Gao et al. 2008; Pan et al. 2010, ily Pseudoholophryidae as well as its spathidiid phy- 2013; Vďačný et al. 2015; Wu et al. 2015, 2016), and logenetic home. Finally, we have carefully analyzed the orders Spathidiida and Haptorida (Vďačný et al. whether the secondary structure of the ITS2 molecule 2011a, 2012, 2014; Vďačný and Foissner 2013; Jang bears information about phylogenetic interrelation- et al. 2015, 2017; Rajter and Vďačný 2016). In spite of ships among free-living litostomateans and searched the great effort and considerable increase in taxon sam- for molecular signatures in the ITS2 region that could pling during the last decade, phylogenetic relationships address the segregation of the four main free-living li- among these main monophyletic groups have been left tostomatean lineages studied. almost unresolved. To cast more light onto this problem, in this study we have not only further increased the taxon and marker sampling, but we have also utilized MATERIALS AND METHODS phylogenetic information contained in the secondary structure of the litostomatean ITS2 molecules. Thus, with the considerably increased dataset and Sampling and sample processing complex phylogenetic approach, we obtained a pos- All newly sequenced species were collected in the Palearctic sibility to constrain evolutionary kinships between and the Nearctic realm from various terrestrial and semi-terrestrial four main monophyletic free-living litostomatean lin- habitats, except for a single pleurostomatid species, Litonotus crys- eages: Rhynchostomatia, Pleurostomatida, Haptorida tallinus, which was isolated from a freshwater habitat (for details, and Spathidiida. Specifically, we tested: (i) the sister see Supplementary Table S1). The non-flooded Petri dish method (Foissner et al. 2002) was used to cultivate soil and moss ciliates, group relationship between rhynchostomatians and the while the freshwater species was directly investigated after trans- three haptorian orders; (ii) the phylogenetic closeness portation of the aquatic sample to the laboratory. of the orders Haptorida and Pleurostomatida; and (iii) Isolated specimens were studied in detail under an optical mi- the monophyly of the peculiar family Pseudoholophy- croscope Leica DM2500 at low (50–400 ×) and high (1000 ×, Phylogenetic Interrelationships among Litostomateans 257 oil immersion) magnifications, using bright field and differen- The best evolutionary substitution models for maximum likeli- tial interference contrast optics. A special attention was paid to hood and Bayesian analyses were selected using jModelTest ver. taxonomically important features of litostomatean ciliates, as 0.1.1 under the Akaike information criterion (Guindon and Gascuel described by Rajter and Vďačný (2016). Species identification 2003; Posada 2008). Maximum likelihood analyses were performed was performed according to the following studies: Kahl (1930a, on the PhyML ver. 3.0 server (http://www.atgc-montpellier.fr/ 1930b, 1931), Foissner (1984, 1987), Foissner and Al-Rasheid phyml/) (Guindon et al. 2010), with SPR tree-rearrangement and (2007), Foissner and Xu (2007), Gabilondo and Foissner (2009), 1,000 non-parametric bootstrap replicates. Bayesian analyses were and Jang et al. (2017). conducted in the program MrBayes on XSEDE ver. 3.2.6 (Miller et al. 2010) on the CIPRES portal ver. 3.1 (http://www.phylo.org). Molecular methods Bayesian inferences were performed with four chains running si- After taxonomic identification, several specimens were picked multaneously for 5,000,000 generations and every 1000th tree be- from each isolated litostomatean species/population using a micro- ing sampled. The first 25% of the sampled trees were considered pipette. Obtained specimens were washed several times to remove as burn-in and discarded prior tree reconstruction. Consequently, contaminants and were directly transferred into the ATL tissue lyses a 50% majority rule consensus of the remaining trees was computed buffer. Subsequently, their genomic DNA was extracted with the and posterior probabilities of its branching pattern were estimated. DNeasy Tissue Kit (Qiagen, Hildesheim, Germany). For bootstrap values, we consider values < 70 as low, 70–94 as For the purposes of this study, we amplified two molecular moderate, and ≥ 95 as high following Hillis and Bull (1993). For markers, the 18S rRNA gene with the universal eukaryotic primers Bayesian posterior probabilities, we consider values < 0.94 as low, designed by Medlin et al. (1988) and the ITS1-5.8S-ITS2 region and ≥ 0.95 as high following Alfaro et al. (2003). with the forward primer ITS-F designed by Miao et al. (2008) and A super-network approach was applied to incorporate information from multiple alignments and trees as well as to depict their the reverse primer LO-R designed by Pawlowski (2000). Polymer- topological incongruence. A super-network was calculated from ase chain reaction (PCR) included 5 µl of the extracted template 80 randomly selected post-burn-in trees from the Bayesian infer- DNA, 0.4 µl of each primer (10 pmol/µl), and 10 µl of the multiplex ence of the 18S-A-D, ITSR-C and ITSR-D as well as the CON-1 PCR buffer (PCR multiplex Kit, Qiagen, Hildesheim, Germany). and CON-2 alignments, i.e., 10 trees were randomly chosen from The final volume was adjusted to 20 µl with deionized distilled wa- the posterior distribution of the Bayesian MCMC analyses of each ter. PCR conditions and quality check of the amplified DNA were of the eight alignments. The super-network was constructed in performed according to Vďačný et al. (2011a, 2012). Finally, the SplitsTree4 ver. 4.12.8 (Huson 1998), using the Z-closure option, resulting PCR products were purified using the NucleoSpin Gel and tree size weighted mean, ten runs, and the refined heuristic tech- PCR clean-up Kit (Macherey-Nagel, Düren, Germany). nique (Huson et al. 2004). The purified DNA fragments were cloned into a plasmid vector To avoid problems connected with uneven taxon sampling of using the pGEM®-T and the pGEM®-T Easy Vector Systems (Pro- the four main free-living litostomatean lineages studied, phyloge- mega, Fitchburg, Wisconsin, United States). After 12-hour incuba- netic interrelationships among them were examined with the likeli- tion of ligation mixtures, the created recombinant plasmids were hood-mapping method as implemented in the program Tree-Puzzle introduced into the host organism Escherichia coli (strain JM109). ver. 5.0 (Schmidt et al. 2002). This analysis was conducted on the Screening for clones with the desired DNA inserts was performed 18S-A and CON-1 alignments, whereby the program was employed with the blue-white selection. The recombinant plasmids were iso- to estimate transition/transversion parameters, nucleotide frequen- lated from the host bacteria using the extraction kit PureYield™ cies, and rate heterogeneity of the alignments (Strimmer and von Plasmid Miniprep System (Promega, Fitchburg, Wisconsin, United Haeseler 1997). To assess the support of an internal branch of the States) and sequenced on an ABI 3730 automatic sequencer (Mac- tested datasets, all possible quartets were calculated. For further de- rogen, Amsterdam, The Netherlands), using the universal M13 for- tails, see Vďačný et al. (2014) and Vďačný (2017). ward and reverse primers. Predictions of the putative secondary structure of the litostoma- Phylogenetic methods tean ITS2 molecules were performed using the free-energy minimi- zation approach on the Mfold webserver ver. 3.0 (http://unafold.rna. The obtained sequences were imported into Chromas ver. 2.33 albany.edu/?q=mfold/RNA-FoldingForm) (Zuker 2003). All folded (Technelysium Pty Ltd.) to check their quality. Subsequently, the ITS2 sequences showed the “ring model” with a similar pairing desired DNA sequences were trimmed at the 5’ and 3’ ends and pattern in helices II and III. Helix I was, however, present only in assembled into contigs using BioEdit ver. 7.2.5 (Hall 1999). Align- some taxa and its positional homology was ambiguous. Therefore ments of the trimmed DNA sequences were constructed on the we constructed consensus secondary structures only for the phy- GUIDANCE2 server (http://guidance.tau.ac.il/ver2/), using the logenetically conserved helices II and III in several higher litosto- MAFFT algorithm and 100 bootstrap repeats (Sela et al. 2015). matean taxonomic groups on the Alifold webserver (http://rna.tbi. Multiple alignments were generated for each marker: six 18S rRNA univie.ac.at/cgi-bin/RNAalifold.cgi) with default options (Bernhart gene, six ITS region and two concatenated datasets (for details, see et al. 2008; Gruber et al. 2008). Moreover, the base frequencies at Supplementary Table S2). Analyzed alignments included up to 64 each position and mutual information of the base-paired regions in free-living litostomatean taxa, for 56 of which both 18S rRNA gene helices II and III were calculated in the web program RNALogo and ITS region sequences were available. Trees were computed as (http://rnalogo.mbc.nctu.edu.tw) (Chang et al. 2008). The number unrooted and a posteriori were rooted with the midpoint method of conserved base pairs and unpaired bases in bulges and loops were implemented in FigTree ver. 1.2.3 (Andrew Rambaut, available at counted for each structural domain of the individual litostomatean http://tree.bio.ed.ac.uk/software/figtree/). ITS2 molecules as predicted on the Mfold webserver. 258 Ľ. Rajter and P. Vďačný

RESULTS ria. As expected, new sequences from Litonotus crystallinus and L. muscorum were placed in the family Litonotidae, together with related taxa from the genus Phylogenetic analyses Loxophyllum (Figs 1–4). (ii) Monophyly of the order In this study, we obtained five new 18S rRNA gene Haptorida was also fully statistically supported by the sequences and ten new ITS1-5.8S-ITS2 region se- 18S rRNA gene, whereby the new 18S rRNA sequence quences of free-living litostomateans from the orders from Fuscheriides sp. clustered together with Fusche- Haptorida, Pleurostomatida and Spathidiida. Their ria terricola and Fuscheria sp. (Figs 1–4). Fuscheria length, GC content, and GenBank accession numbers nodosa was placed in a basal polytomy of the subclass were summarized, jointly with other sequences utilized Haptoria in the ITS1-5.8S-ITS2 trees, while this species in our phylogenetic analyses, in Supplementary Table was depicted in a sister position to the order Pleuros- S1. In total, we computed four 18S rRNA gene trees tomatida in the 18S-ITS trees with low statistical sup- from the 18S-A alignment (Fig. 1) and the 18S-B–D port. (iii) The order Spathidiida was distinguished only alignments (data not shown), four ITS1-5.8S-ITS2 in 18S rRNA gene and concatenated 18S-ITS Bayesian region trees from the ITSR-A dataset (Fig. 2) and the trees, but statistical support for its monophyletic origin ITSR-B–D datasets (data not shown) as well as two was low. Phylogenetic positions of Arcuospathidium 18S-ITS region trees from the CON-1 (Fig. 3) and the cultriforme scalpriforme, Spathidium amphoriforme CON-2 alignment (data not shown). To incorporate in- pop. 1, S. claviforme and S. terricola, as inferred from formation from all alignments and multiple trees, we the newly added ITS region sequences in the ITS and constructed a super-network (Fig. 4). concatenated 18S-ITS datasets, were basically congru- On the basis of the midpoint rooting method, ent with previous ITS and concatenated 18S-ITS trees, there was a deep bifurcation of the class Litostoma- respectively (Rajter and Vďačný 2016; Jang et al. 2017). tea into two main lineages, corresponding to the sub- The newly sequenced American population of Apo- class Rhynchostomatia and the subclass Haptoria, in bryophyllum schmidingeri (represented here by clones all 18S rRNA gene and concatenated 18S-ITS region 1 and 2) clustered together with German and Korean trees. Monophyletic origin of each subclass was fully populations of that species in a highly/fully statistically or highly statistically supported in both Bayesian and supported clade (Figs 1–3). Paraenchelys terricola was maximum likelihood phylogenies. Within the Rhyn- usually nested in the spathidiid cluster in the vicinity chostomatia, the orders Tracheliida and Dileptida were of Pseudoholophrya terricola and Acaryophrya sp., but uncovered with full or high statistical support only monophyletic origin of this clade was either poorly sta- in 18S rRNA gene and concatenated 18S-ITS region tistically supported or was left unsupported. Neverthe- trees. The branching pattern within the order Dileptida less, all three taxa formed the most distinct split within was congruent with results of our previous phyloge- the order Spathidiida in the super-network based on netic studies (Vďačný et al. 2011b, 2017; Jang et al. multiple post burn-in Bayesian trees (Fig. 4). 2014; Vďačný and Rajter 2015). The family Dilepti- All four main free-living litostomatean lineages dae was, however, revealed to be monophyletic with were also clearly recognizable in the super-network high statistical support only in Bayesian trees inferred based on 80 randomly selected post-burn-in trees from from the concatenated datasets, while the family Di- the Bayesian inference of eight alignments (Fig. 4). In- macrocaryonidae was depicted monophyletic with full terrelationships among these lineages were comparable or high support in all 18S rRNA gene and 18S-ITS re- with those found in the present 50% majority rule con- gion trees. sensus trees (Figs 1–3). However, the super-network Within the subclass Haptoria, three main phyloge- also revealed two conflicting relationships: the order netic lineages, corresponding to the orders Pleuros- Pleurostomatida is either a sister group of the Haptor- tomatida, Haptorida and Spathidiida, were recognized. ida or of the Spathidiida. To test these hypotheses, we (i) Monophyly of the order Pleurostomatida was corrob- performed quartet likelihood-mapping which favored orated with full statistical support in all alignments and the sister-group relationship between Pleurostomatida by all algorithms used, except for the ITS1-5.8S-ITS2 and Haptorida by 52.2% of data points for the 18S-A region trees, where the peculiar Protolitonotus magnus alignment and by 71.3% of data points for the CON-1 was placed in a basal polytomy of the subclass Hapto- dataset (Fig. 5). Phylogenetic Interrelationships among Litostomateans 259

Fig. 1. Phylogeny based on the 18S rRNA gene of 64 free-living litostomatean taxa (alignment 18S-A). Posterior probabilities for Bayesian inference and bootstrap values for maximum likelihood were mapped onto the 50% majority rule Bayesian consensus tree. Dashes indicate ML bootstrap values below 50%. Sequences in bold were obtained during this study. The scale bar indicates two substitutions per one hundred nucleotide positions. For details on taxa, evolutionary model used, and characteristics of the 18S-A alignment, see Supplementary Table S1 and S2. 260 Ľ. Rajter and P. Vďačný

Fig. 2. Phylogeny based on the ITS1-5.8S-ITS2 region of 60 free-living litostomatean taxa (alignment ITSR-A). Posterior probabilities for Bayesian inference and bootstrap values for maximum likelihood were mapped onto the best ML tree. Dashes indicate posterior probabilities below 0.50 and ML bootstrap values below 50%. Sequences in bold were obtained during this study. The scale bar indicates nine substitutions per one hundred nucleotide positions. For details on taxa, evolutionary model used, and characteristics of the ITSR-A alignment, see Supplementary Table S1 and S2. Phylogenetic Interrelationships among Litostomateans 261

Fig. 3. Phylogeny based on the 18S rRNA gene and the ITS1-5.8S-ITS2 region of 56 free-living litostomatean taxa (alignment CON-1). Posterior probabilities for the Bayesian inference and bootstrap values for maximum likelihood were mapped onto the 50% majority rule ML tree. Dashes indicate posterior probabilities below 0.50 and ML bootstrap values below 50%. The scale bar indicates five substitutions per ten nucleotide positions. For details on taxa, evolutionary model used, and characteristics of the CON-1 alignment, see Supplementary Table S1 and S2. 262 Ľ. Rajter and P. Vďačný

Fig. 4. Super-network of 66 free-living litostomatean taxa constructed from 80 randomly selected post-burn-in trees from the Bayesian inference of the 18S-A–D, ITSR-C and ITSR-D as well as the CON-1 and CON-2 alignments. The super-network was constructed in the program SplitsTree, using the Z-closure option, tree size weighted mean, ten runs, and the refined heuristic technique. For details on taxa and characteristics of the alignments analyzed, see Supplementary Table S1 and S2. Phylogenetic Interrelationships among Litostomateans 263

Fig. 5. Quartet likelihood-mapping showing distribution of phylogenetic signal in the 18S-A and the CON-1 alignment for three possible relationships among the four main free-living litostomatean lineages studied. The corners of the triangles show the percentage of fully resolved trees, i.e., phylogenetically informative signal. The rectangular areas show the percentage of trees that are in conflict. The central triangle shows the percentage of unresolved star-like trees, i.e., phylogenetically uninformative signal. Coding of free-living litostomatean lineages: H – Haptorida, P – Pleurostomatida, R – Rhynchostomatia, S – Spathidiida.

Putative secondary structure of the ITS2 molecule To summarize, the consensus structure of the lito- The consensus structure of the ITS2 molecule was stomatean ITS2 molecule revealed that: (i) helix II is predicted on the Alifold webserver from 63 free-living composed of eight base pairs in the stem and a terminal litostomatean taxa (Table 1). As shown in Fig. 6, the tetraloop; (ii) helix III includes 14 base pairs, an AG consensus structure consisted of a central loop bearing bulge on the stem and a terminal heptaloop; and (iii) two conservative helices corresponding to helix II and conservative nucleotide sites are positioned mainly in III of other eukaryotes (Schultz et al. 2005). In contrast, stems of both helices, while terminal loops show much the presence and structure of helix I were much more more variability (Figs 6 and 7). variable among the taxa analyzed, causing difficulties Furthermore, we separately predicted consensus in determination of its positional homology and pro- structures of helices II and III for both free-living litosto- posal of its consensus structure. matean subclasses (Fig. 7). The rhynchostomatian con- 264 Ľ. Rajter and P. Vďačný Δ G (37°C, kcal/mol) –20.89 –23.89 –23.89 –23.89 –20.71 –27.60 –24.13 –26.30 –26.45 –26.54 –26.83 –29.57 –25.12 –27.63 –21.23 –23.19 –28.79 –24.12 –24.89 –24.89 –26.44 –24.35 –25.30 –27.10 –29.19 –29.19 –28.75 –24.68 Central loop (nt) 52 52 52 52 51 53 49 52 52 51 51 54 47 48 54 49 54 48 51 51 47 50 51 55 55 55 55 60 Number of bulges 2 1 1 1 3 1 2 2 1 1 1 2 2 2 2 2 2 2 2 2 2 1 2 1 1 1 1 2 Number of nt in bulges 3 2 2 2 4 2 3 5 2 2 2 3 7 3 5 5 3 5 5 5 5 2 11 2 2 2 2 3

Helix III loop (nt) Terminal Terminal 4 4 4 4 4 4 5 4 3 3 6 4 4 4 4 4 4 6 3 3 6 4 4 4 4 4 4 4 Length (nt) 37 36 36 36 38 36 36 37 35 35 36 37 37 37 37 37 37 37 36 36 37 36 37 34 34 34 34 35 Terminal Terminal loop (nt) 4 4 4 4 4 4 4 4 4 4 4 6 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 Length (nt) Helix II 20 20 20 20 20 20 20 20 20 20 20 20 20 22 20 20 20 20 20 20 20 20 19 19 19 19 19 15 GC content (%) 38.53 25.00 25.00 25.00 32.11 32.11 41.90 37.61 35.51 34.91 33.64 34.23 36.54 41.12 33.33 35.85 31.53 38.10 38.32 38.32 32.69 28.30 32.71 40.74 37.96 37.96 39.81 45.45 Total length Total (nt) 109 108 108 108 109 109 105 109 107 106 107 111 104 107 111 106 111 105 107 107 104 106 107 108 108 108 108 110 pop. 1 pop. 2 (Jeju, Korea) (Jeju-do, Korea) (Ulsan, Korea) (Botswana) (Slovakia) pop. 1 pop. 2 (Korea) pop. 1 (Slovakia) pop. 2 (Slovakia) Characterization Characterization of ITS2 molecules of free-living litostomateans (arranged alphabetically within main lineages). For collection sites, GenBank entries and further details, Taxon Table 1. Table S1 Table see Supplementary Rhynchostomatia Apodileptus visscheri rhabdoplites Apotrachelius multinucleatus Apotrachelius multinucleatus Apotrachelius multinucleatus Dileptus costaricanus Dileptus costaricanus Dileptus jonesi Dileptus sp . Dimacrocaryon amphileptoides Dimacrocaryon Dimacrocaryon amphileptoides Dimacrocaryon Microdileptus breviproboscis Microdileptus Monomacrocaryon terrenum Monomacrocaryon Pseudomonilicaryon anguillula Pseudomonilicaryon brachyproboscis Pseudomonilicaryon fraterculum Rimaleptus binucleatus Rimaleptus mucronatus Rurikoplites armatus Rurikoplites armatus Rurikoplites armatus Rurikoplites longitrichus Trachelius ovum Trachelius Haptorida Fuscheria nodosa Pleurostomatida Amphileptus marinus Amphileptus salignus Amphileptus salignus Amphileptus spiculatus Kentrophyllum sp. Kentrophyllum Phylogenetic Interrelationships among Litostomateans 265 –21.81 –25.12 –24.92 –25.62 –23.47 –24.73 –19.37 –27.85 –27.85 –24.95 –24.09 –20.49 –21.68 –28.45 –19.47 –20.91 –24.99 –20.70 –22.48 –20.17 –26.49 –21.63 –17.32 –24.70 –17.95 –30.12 –27.92 –25.32 –22.55 –25.30 –20.69 –21.50 –21.50 –21.33 –26.72 54 54 54 53 51 53 53 55 55 51 54 54 56 50 55 54 54 52 54 55 54 56 54 49 57 52 54 50 53 53 53 54 54 54 54 1 1 1 1 1 1 1 2 2 2 2 1 2 2 2 1 1 1 1 1 1 2 1 1 1 1 1 1 2 2 2 1 1 1 1 2 2 2 2 2 2 2 4 4 4 4 2 5 2 4 2 2 4 3 4 2 7 3 2 4 2 2 2 7 6 7 2 2 2 2 6 4 4 4 4 4 5 6 6 6 6 6 5 6 4 6 6 4 4 4 6 5 6 4 4 6 6 4 5 4 6 6 6 4 6 34 34 34 34 34 31 38 38 38 38 36 34 38 38 36 36 36 35 36 36 34 37 32 36 36 36 36 34 28 35 36 36 36 36 36 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 5 5 5 5 5 5 5 19 19 19 21 19 21 19 19 19 19 19 19 21 19 19 19 19 19 19 19 19 19 19 20 21 19 19 19 19 19 19 19 19 19 21 41.12 39.25 36.79 37.74 37.74 30.48 29.46 29.46 29.63 30.63 35.78 31.19 35.78 31.25 35.78 38.53 27.10 39.81 23.64 36.70 34.86 24.55 47.00 24.78 36.70 36.70 36.19 33.96 49.00 27.10 33.03 33.94 32.11 35.78 31.19 107 107 106 106 106 105 112 112 108 111 109 109 109 112 109 109 107 108 110 109 109 110 100 113 109 109 105 106 100 107 109 109 109 109 109 clone 1 (USA ) clone 2 (USA) (Germany) (Korea) pop. 1 pop. 2 pop. 1 pop. 2 pop. 1 pop. 2 sp. sp. sp . Litonotus crystallinus Litonotus muscorum Loxophyllum chinense Loxophyllum helus Loxophyllum meridionale Protolitonotus magnus Protolitonotus Spathidiida Apobryophyllum schmidingeri Apobryophyllum schmidingeri Apobryophyllum schmidingeri Apobryophyllum schmidingeri Arcuospathidium cultriforme scalpriforme Arcuospathidium Arcuospathidium muscorum Arcuospathidium Bryophyllum Cultellothrix lionotiformis Enchelyodon Enchelys gasterosteus Enchelys megaspinata Paraenchelys terricola Protospathidium muscicola Protospathidium Semispathidium breviarmatum Spathidium amphoriforme pop. 1 Spathidium amphoriforme pop. 2 Spathidium ascendens Spathidium claviforme Spathidium muscicola Spathidium muscicola Spathidium papilliferum Spathidium papilliferum Spathidium polynucleatum Spathidium securiforme Spathidium simplinucleatum Spathidium simplinucleatum Spathidium sp . Spathidium terricola Trachelophyllum Trachelophyllum 266 Ľ. Rajter and P. Vďačný

Fig. 6. Consensus secondary structure of the ITS2 molecule of free-living litostomateans. The ITS2 molecule shows an internal loop, radiat- ing two helices. There is an A-G bulge in the stem of helix III.

sensus structure of helix II contained eight base pairs in gle ITS2 sequence was available from Fuscheria no- the stem and a terminal tetraloop in contrast to the hapto- dosa. The length of stems and terminal loops in helix rian helix II which included seven base pairs in the stem II were the same among all consensus structures, while and a terminal pentaloop. The consensus structure of the numbers of nucleotides in stems and loops of helix III rhynchostomatian helix III displayed 15 base pairs in the were different. For instance, the consensus structure of stem and a terminal tetraloop, whereas 14 base pairs and helix III in the orders Pleurostomatida and Haptorida a terminal hexaloop were found in the haptorian helix exhibited a terminal tetraloop, whereas there was a hex- III. Thus, the consensus stems of helices II and III were aloop in the order Spathidiida. On the other hand, the longer by one base pair in the subclass Rhynchostoma- number of base pairs in the consensus helix III stem was tia, while the terminal loops of helices II and III were the same in the orders Spathidiida and Pleurostomatida, longer by one or two nucleotide/-s in the subclass Hapto- i.e., there were 14 pairing nucleotides. However, there ria. Interestingly, the more conservative helix II showed were 11 pairing nucleotides and nine mismatch nucleo- a similar structural pattern between consensuses of the tides, forming an asymmetrical internal loop, in the or- class Litostomatea and the subclass Rhynchostomatia der Haptorida represented by F. nodosa. (eight base pairs in the stem and a terminal tetraloop). Conserved and variable nucleotide positions of heli- This indicates a plesiomorphic nature of the rhynchos- ces of the ITS2 molecule tomatian helix II on one hand and an apomorphic character of the haptorian helix II on the other one. According to RNA logo analyses, the litostomatean As concerns the subclass Haptoria, consensus sec- helix II displays a conserved motif 5’-GU–GAGA-3’ ondary structures of helices II and III were separately with an antiparallel RNA sequence 5’-UCUC–AU-3’ at predicted for the orders Haptorida, Pleurostomatida and its stem (Figs 6–8). Dashes in the motif represent varia- Spathidiida. However, in case of haptorids, only a sin- ble nucleotide sites at the class level, but there might be Phylogenetic Interrelationships among Litostomateans 267

Fig. 7. Consensus secondary structure of ITS2 helices II and III in various higher litostomatean taxa.

present a certain pattern at lower taxonomic levels. For II stem are 5’-GA-3’ and 5’-AG-3’ with antiparallel se- instance, within the subclass Rhynchostomatia, the var- quences being 5’-UC-3’ and 5’-CU-3’, respectively. The iable sites are always 5’-AA-3’ with the opposite strand orders Haptorida and Pleurostomatida show a 5’-AG-3’ carrying 5’-UU-3’ in tracheliids, while 5’-AA-3’ and vs. 5’-CU-3’ variant, while the order Spathidiida exhib- 5’-GG-3’ with the opposite strand bearing 5’-UU-3’ and its, on the contrary, a 5’-GA-3’ vs. 5’-UC-3’ variant. 5’-CC-3’, respectively, in dileptids. The invariable pat- This molecular signature may further support the close- tern of tracheliids may indicate their plesiomorphic na- ness of haptorids and pleurostomatids. Interestingly, ture within the Rhynchostomatia, while the nucleotide helix II of pleurostomatids displays a rather variable diversity of dileptids might reflect their derived nature. nucleotide composition in the stem, while nucleotides In the subclass Haptoria, the variable sites of the helix of the terminal loop are absolutely conserved. On the 268 Ľ. Rajter and P. Vďačný

Fig. 8. Structure logo of ITS2 helices II and III in various higher litostomatean taxa. The height of a base is proportional to its frequency in multiple sequence alignments.

other hand, although spathidiids are a highly diverse particular pattern was detected either in the variable or group, the nucleotide composition of their helix II stem the conservative part of helix III for the three haptorian is most strongly conserved among all higher free-living orders studied. litostomatean taxa, supporting the monophyletic origin of spathidiids (Fig. 7). DISCUSSION The basal part of the litostomatean helix III stem is more variable than the second part that shows a highly conservative motif 5’-AGCA-UCACA vs. UGU- Phylogenetic interrelationships between free-living GAGCU-3’ (Figs 6–8). Interestingly, the subclass litostomateans Rhynchostomatia has a more conservative RNA logo According to Vďačný et al. (2011a), free-living cili- for helix III than the subclass Haptoria does (Fig. 8). No ates of the class Litostomatea are classified into two Phylogenetic Interrelationships among Litostomateans 269 subclasses altogether having seven main lineages/or- corresponds to paroral membrane and preoral kineties ders. Specifically, the first subclass Rhynchostomatia to adoral membranelles of other ciliates, the ventral cy- includes the orders Tracheliida and Dileptida, while the tostome located at the base of the proboscis, and an- subclass Haptoria contains the orders Haptorida, Pleu- archic fields produced during stomatogenesis (Vďačný rostomatida, Didiniida, Lacrymariida, and Spathidiida. and Foissner 2009; Vďačný et al. 2010, 2011a, 2011b, Monophylies of all these orders, except for spathidiids, 2012). In contrast, haptorians display several derived are well supported by morphological apomorphies and morphological and morphogenetical features: a simple 18S rRNA gene sequences (e.g. Vďačný et al. 2011a, oral ciliature, an apical cytostome, and a non-complex 2014). However, the evolutionary interrelationships stomatogenesis without formation of anarchic fields among the haptorian orders are very poorly resolved (Foissner 1996). This suite of characters led to a hy- and understood (e.g. Zhang et al. 2012; Vďačný et al. pothesis that haptorians became secondarily simplified 2014; Gao et al. 2016; Vďačný and Rataj 2017). due to body polarization (Vďačný et al. 2011a, 2012). A comparatively broad sampling of four molecular The derived nature of haptorians is indicated also by markers (18S and 5.8S rRNA genes as well as internal the present secondary structure analyses of the ITS2 transcribed spacers 1 and 2) is now available for four molecule. Thus, the more conservative helix II shows free-living litostomatean groups, i.e., rhynchostoma- a similar structural pattern between consensuses of the tians, haptorids, pleurostomatids, and spathidiids. All class Litostomatea and the subclass Rhynchostomatia, present phylogenetic analyses and the mid-point root- which in turn indicates the apomorphic character of the ing method consistently indicate that there is a deep haptorian helix II (see above). split between rhynchostomatians and the three hapto- As concerns the subclass Haptoria, evolutionary rian orders studied. This result corresponds well with relationships among its orders are considered unclear some recent molecular studies based on the 18S rRNA in both morphological and molecular phylogenies (e.g. gene (e.g. Strüder-Kypke et al. 2006; Gao et al. 2008; Strüder-Kypke et al. 2006; Gao et al. 2008; Zhang et al. Vďačný et al. 2010, 2011a, 2011b, 2014; Rajter and 2012; Vďačný et al. 2014; Kwon et al. 2014; Rajter and Vďačný 2016) and is also corroborated by the specific Vďačný 2016; Vďačný and Rataj 2017). However, the length of stems and loops of helices II and III of the present study cast more light onto this problem. Spathi- ITS2 molecule. Specifically, there are eight and 15 nu- diids are shown as a distinct and independent lineage, cleotide pairs in helix II and III in rhynchostomatians, while pleurostomatids and haptorids are depicted as sis- while seven and 14 nucleotide pairs in helix II and III in ter groups (Figs 1–4). This evolutionary scenario is also haptorians. Moreover, rhynchostomatians exhibit a ter- favored by quartet likelihood-mapping (Fig. 5) and is minal tetraloop both in helix II and III, while haptorians supported by the secondary structure of the ITS2 mol- very likely had ancestrally a pentaloop in helix II and ecule (Figs 7 and 8). Specifically, haptorids and pleu- a hexaloop in helix III (Fig. 9). rostomatids share a terminal tetraloop in helix III as Rhynchostomatians also differ conspicuously from well as a 5’-AG vs. CU-3’ motif in the variable part of haptorians morphologically in having a proboscis and helix II. On the contrary, members of the order Spathi- a complex oral ciliature composed of a circumoral and diida maintained the plesiomorphic terminal hexaloop a perioral kinety as well as of multiple preoral kineties in helix II and evolved a unique 5’-GA vs. UC-3’ vari- (Vďačný and Foissner 2012). In spite of this, rhynchos- ant in the variable part of helix II (Fig. 9). Interestingly, tomatians were traditionally assigned to various hap- pleurostomatids and haptorids also display meridionally torian groups in morphology-based frameworks (e.g. extending somatic kineties and a non-three-rowed dor- Kahl 1931; Corliss 1974; Foissner and Foissner 1988; sal brush (Fig. 9). On the contrary, spathidiids typically Lipsomb and Riordan 1990; Lynn 2008). However, al- exhibit anteriorly curved somatic kineties and a three- ready the first molecular studies about litostomatean rowed dorsal brush with anterior monokinetidal tails. In phylogeny have uncovered rhynchostomatians and re- accordance with structural conservatism hypothesis, we maining free-living litostomateans (i.e. haptorians) to be suppose that somatic kineties are evolutionary conserv- independent lineages (Strüder-Kypke et al. 2006; Gao ative structures, reflecting closeness of pleurostoma- et al. 2008; Vďačný et al. 2010, 2011a, 2011b). Indeed, tids and haptorids. Indeed, dorsal brush seems to carry members of the subclass Rhynchostomatia exhibit some evolutionary information about segregation of the main important ciliate plesiomorphic features: the aforemen- free-living litostomatean lineages (Kwon et al. 2014). tioned complex oral ciliature where circumoral kinety In this light, didiniids, lacrymariids and chaeneids 270 Ľ. Rajter and P. Vďačný

Fig. 9. Evolutionary hypothesis of interrelationships among the four free-living litostomatean lineages studied. This scenario was suggested on the basis of morphology and the consensus secondary structure of the ITS2 molecules. CK – circumoral kinety, DB – dorsal brush, OB – oral bulge, OO – oral bulge opening, P – proboscis, PE – perioral kinety, PR – preoral kineties, SK – somatic kineties. Phylogenetic Interrelationships among Litostomateans 271 appear to be more closely related to spathidiids than to state of knowledge, the best diagnostic characteristics pleurostomatids and haptorids. Interestingly, didiniids, separating spathidiids from other orders of the subclass lacrymariids and chaeneids exhibit anteriorly curved or Haptoria. even spiraling somatic kineties and their dorsal brush Statistical support for the monophyletic origin of begins with anterior monokinetidal tails as well. A re- spathidiids is, however, low in molecular analyses. The latedness of spathidiids, didiniids, lacrymariids and spathidiid clade was usually recognized only in 18S chaeneids is also indicated by genes coding for the 28S rRNA gene trees but with very poor statistical support. rRNA molecule and alpha-tubulin (Zhang et al. 2012). In the present ITS region trees, it was left unsupported, but in the Bayesian trees based on the concatenated 18S Monophyly of the order Spathidiida rRNA gene and ITS region dataset, statistical support The highly diverse order Spathidiida was estab- for spathidiid monophyly achieved a value of 0.94, lished by Foissner and Foissner (1988) on the basis of which is only slightly below the level of significance. oral infraciliature as well as localization and shape of Therefore, we assume that addition of further molecu- the cytostome. Later on, spathidiids were redefined in lar markers would increase statistical support for mono- the light of molecular analyses that indicated didiniids phyletic origin of the order Spathidiida. to be excluded from the order Spathidiida and sev- Phylogenetic home of the family Pseudoholophryi- eral ‘traditional’ haptorids (e.g., Acropisthiidae Foiss- dae ner and Foissner, 1988 and Enchelyidae Ehrenberg, 1838) to be included among spathidiids (Vďačný et al. The family Pseudoholophryidae was founded by 2011a). The ancestrally tree-rowed dorsal brush with Berger et al. (1984) on the basis of the absence of dor- anterior monokinetidal tails and the anteriorly curved sal brush. Later on, Foissner and Foissner (1988) im- somatic kineties became the best diagnostic features proved diagnostic features of pseudoholophryids after of the order Spathidiida (Vďačný and Foissner 2013; reinvestigation of several Pseudoholophrya terricola Rajter and Vďačný 2016). However, the apomorphic/ populations. They recognized that this species car- plesiomorphic nature of these diagnostic characters re- ries a diffuse dorsal brush composed of many clavate mains questionable and is discussed below. bristles alternating with typical somatic cilia. Besides A three-rowed dorsal brush was very likely a prop- the genus Pseudoholophrya Foissner, 1984, the genera erty of the last common ancestor (LCA) of the subclass Ovalorhabdos Foissner, 1984 and Paraenchelys Foiss- Haptoria (Kwon et al. 2014), suggesting its plesiomor- ner, 1983 also exhibit the same unique brush pattern phic condition in spathidiids. However, at the present and were therefore included into the family Pseudohol- state of knowledge, we cannot exclude that the three- ophryidae. Foissner et al. (2002) elevated pseudohol- rowed brush was not a property of the LCA of the Hap- ophryids from the family to the order rank, because of toria and evolved convergently in Trachelius ovum from the diffuse dorsal brush, the spiraling somatic kineties, the subclass Rhynchostomatia and the LCA of spathi- and the small size of the cytostome. Recently, based diids. The anteriorly curved somatic kineties were very on the 18S rRNA gene, we discovered that pseudohol- likely present both in the LCA of the spathidiids and the ophryids might represent a spathidiid lineage that is LCA of the subclass Haptoria (Vďačný and Foissner closely related to the genus Acaryophrya André, 1915 2013). Thus, spathidiids could directly inherit this pat- (Rajter and Vďačný 2016). Therefore, we suppressed tern from the LCA of the subclass Haptoria, suggesting the order Pseudoholophryida, transferred the family its plesiomorphic character, or might have evolved it in- Pseudoholophryidae into the order Spathidiida, and as- dependently, indicating its apomorphic but homoplastic signed the genus Acaryophrya to the family Pseudohol- nature. Interestingly, some ‘traditional’ haptorids (e.g. ophryidae. For the first time, we have the possibility to Enchelyodon and Lagynophrya), with meridionally ex- more robustly test the monophyly of this family by mo- tending ciliary rows, were also assigned to the spathi- lecular methods. According to the present phylogenetic diid cluster (Vďačný and Foissner 2013). This ciliary trees, the 18S rRNA gene sequence of Paraenchelys pattern, however, apparently evolved convergently terricola clusters together with/near to Pseudohol- between haptorids and these ‘traditional’ haptorids ophrya terricola and Acaryophrya sp. But monophylet- (Vďačný et al. 2011a). Nonetheless, the combination of ic origin of Pseudoholophrya, Paraenchelys, and Acar- the ancestrally three-rowed dorsal brush and the anteri- yophrya is either poorly statistically supported or is left orly curved somatic kineties seems to be, at the present unsupported. However, since these three genera form 272 Ľ. Rajter and P. Vďačný a distinct clade in multiple post burn-in Bayesian trees, to three helices in litostomateans (Ponce-Gordo et al. the family Pseudoholophryidae became the best recog- 2011; present study) and spirotricheans (Weisse et al. nizable lineage within the spathidiid cluster in the pre- 2008; Yi et al. 2008, Li et al. 2013, 2017), and three to sent super-network analyses (Fig. 4). Therefore, we be- four helices in scuticociliates (Miao et al. 2008). In the lieve that addition of further molecular markers might hairpin model, the common loop is started and closed lead to strong statistical support for pseudoholophryids, by helix I and radiates helices II and III in peritrichs as redefined by Rajter and Vďačný (2016). (Sun et al. 2010) and litostomateans (Vďačný et al. Putative secondary structure of the free-living litos- 2012). Interestingly, both ITS2 models were detected in litostomateans, the ring model by Ponce-Gordo et al. tomatean ITS2 molecule (2011) and the hairpin model by Vďačný et al. (2012). The ITS2 region is located between the 5.8S rRNA The existence of two models could be explained by the and the 28S rRNA gene in the rRNA locus. The ITS2 dynamic conformational model proposed by Côté et al. molecule participates in maturation processes of both (2002). The ring structure forms during early events aforementioned rRNA molecules. The presence of of rRNA maturation, while the hairpin structure dur- deletions or mutations within ITS2 leads to failure in ing the subsequent processing events. Regardless of the production of mature rRNA molecules, as evidenced model used, the structure and motifs of helices II and by many experimental studies (e.g. van Nues et al. III are conservative among various ciliate groups. Thus, 1994; Côté et al. 2002; Ferreira-Cerca 2008 and refer- there is a pyrimidine-pyrimidine mismatch in helix II of ences therein). Thus, ITS2 plays an important role in heterotricheans (Shazib et al. 2016) and oligohymeno- transcript processing and hence remains rather con- phoreans (Coleman 2005; Miao et al. 2008; Sun et al. servative in its primary and secondary structure among 2010, 2013), as typical for eukaryotes (Coleman 2007; many eukaryotes (Coleman 2003; Schultz et al. 2005; Schultz et al. 2005). By contrast, this mismatch is not Wolf et al. 2005). Therefore, this molecule has become present in litostomateans and spirotricheans (Weisse a popular tool in many phylogenetic studies, including et al. 2008; Yi et al. 2008; Li et al. 2013, 2017). This those on ciliates (e.g. Coleman 2005; Miao et al. 2008; indicates that the loss of the pyrimidine-pyrimidine Weisse et al. 2008; Yi et al. 2008; Sun et al. 2010, 2013; mismatch is a molecular synapomorphy of litostomate- Ponce-Gordo et al. 2011; Vďačný et al. 2012; Li et al. ans and spirotricheans, providing a further support for 2013; Shazib et al. 2016; Li et al. 2017). the SAL hypothesis based on extensive molecular data The ITS2 region has a comparatively similar length (Gentekaki et al. 2014, 2017). Since both models match in all free-living litostomatean taxa, ranging from 100 very well in the structure and motifs of helices II and to 112 nucleotides (Ponce-Gordo et al. 2011; Vďačný III but differ in helix I, this structure is very likely the et al. 2012; Jang et al. 2014; present study). Interest- dynamic constituent of the ITS2 transcripts, enabling ingly, this is the shortest ITS2 region among all already switching from the ring to the hairpin pattern (Vďačný investigated ciliates from the subphylum Intramacronu- et al. 2012). cleata. Specifically, the length of ITS2 sequences var- ies from 168 to 169 nt within the genus Paramecium Comparison of ITS2 molecules of free-living and en- (Coleman 2005), from 168 to 217 nt in scuticociliates dosymbiotic litostomateans (Miao et al. 2008), and from 165 to 175 nt in peritrichs The secondary structure of the ITS2 molecule was (Sun et al. 2010, 2013). Spirotrichean ciliates exhibit studied only in three species of endosymbiotic litos- the longest ITS2 sequences typically exceeding 200 tomateans from the subclass Trichostomatia, i.e., in nucleotides (Weisse et al. 2008; Yi et al. 2008; Li et Balantidium coli, Isotricha prostoma and Troglodytel- al. 2013, 2017). On the other hand, the genera Spiro- la abrassarti (Ponce-Gordo et al. 2011). The trichos- stomum and Anigsteinia from the class Heterotrichea tomatian ITS2 primary and secondary structures match of the subphylum Postciliodesmatophora display the very well those of free-living litostomateans. Specifi- shortest ITS2 sequences, being only 79–81 nt long, cally, the trichostomatian ITS2 molecule (i) exhibits among all ciliates hitherto studied (Shazib et al. 2016). three helices, with helix I being the shortest and helix Two alternative secondary structures of the ITS2 III being the longest; (ii) lacks the pyrimidin-pyrimidin molecule were proposed for ciliates: a ring and a hair- mismatch in helix II; and (iii) displays the conserva- pin model. In the ring model, a common loop bears tive motif 5’-GU–GAGA vs. UCUC–AU-3’ in helix II. two helices in heterotricheans (Shazib et al. 2016), two As in free-living haptorians, there are seven pairs and Phylogenetic Interrelationships among Litostomateans 273 a pentaloop in the trichostomatian helix II. However, Coleman A. W. (2003) ITS2 is a double-edged tool for eukaryote trichostomatians display a motif 5’-AA vs. UU-3’ or evolutionary comparisons. Trends Genet. 19: 370–375 Coleman A. W. (2005) Paramecium aurelia revisited. J. Eukaryot. 5’-AU vs. AU-3’ in the variable part of helix II. The first Microbiol. 52: 68–77 motif occurs also in tracheliids and dipletids, which in- Coleman A. W. (2007) Pan-eukaryote ITS2 homologies revealed by dicates its homoplastic nature, while the second motif RNA secondary structure. Nucleic Acids Res. 35: 3322–3329 is unique to trichostomatians. Corliss J. O. (1974) The changing world of ciliate systematics: his- torical analysis of past efforts and a newly proposed phylogenetic scheme of classification for the protistan phylum Ciliophora. Syst. Zool. 23: 91–138 CONCLUSIONS Côté C. A., Greer C. L., Peculis B. A. (2002) Dynamic conformational model for the role of ITS2 in pre-rRNA processing in yeast. RNA 8: 786–797 Utilization of the 18S rRNA gene along with the Ehrenberg C. G. (1838) Die Infusionsthierchen als vollkommene ITS region is revealed to be beneficial for phylogenet- Organismen. 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Familien analyses of the 18S rRNA gene and the ITS region har- Holophryidae, Prorodontidae, Plagiocampidae, Colepidae, Enchelyidae und Lacrymariidae nov. fam. Ann. Nat. Hist. monize well with assumptions based on the structural Mus. Wien Ser. B Bot. Zool. 84: 49–85 conservatism hypothesis, postulating that somatic cili- Foissner W. (1984) Infraciliatur, Silberliniensystem und Biometrie ary structures are phylogenetically more informative einiger neuer und wenig bekannter terrestrischer, limnischer than oral patterns. und mariner Ciliaten (Protozoa: Ciliophora) aus den Klassen Kinetofragminophora, Colpodea und Polyhymenophora. Stap- Acknowledgements. We are very grateful to two anonymous fia 12: 1–165 Foissner W. (1987) Neue terrestrische und limnische Ciliaten (Pro- reviewers for their constructive criticism and thoughtful com- tozoa, Ciliophora) aus Österreich und Deutschland. Sber. Ös- ments. This work was supported by the Slovak Research and Devel- terr. Akad. 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Vďačný P., Rajter Ľ., Shazib S. U. A, Jang S. W., Kim J. H., Shin Wu L., Jiao X., Shen Z., Yi Z., Li J., Warren A., Lin X. (2016) New M. K. (2015) Reconstruction of evolutionary history of pleuro- taxa refresh the phylogeny and classification of pleurostomatid stomatid ciliates (Ciliophora, Litostomatea, Haptoria): interplay ciliates (Ciliophora, Litostomatea). Zool. Scr. 46: 245–253 of morphology and molecules. Acta Protozool. 54: 9–29 Yi Z., Chen Z., Warren A., Roberts D., Al-Rasheid K. A. S., Miao Vďačný P., Rajter Ľ., Shazib S. U. A, Jang S. W., Shin M. K. (2017) M., Gao S., Shao C., Song W. (2008) Molecular phylogeny of Diversification dynamics of rhynchostomatian ciliates: the Pseudokeronopsis (Protozoa, Ciliophora, Urostylida), with re- impact of seven intrinsic traits on speciation and extinction in consideration of three closely related species at inter- and intra- a microbial group. Sci. Rep. 7: 9918 specific levels inferred from the small subunit ribosomal RNA Weisse T., Strüder-Kypke M. C., Berger H., Foissner W. (2008) gene and the ITS1-5.8S-ITS2 region sequences. J. Zool. 275: Genetic, morphological, and ecological diversity of spatially 268–275 separated clones of Meseres corlissi Petz & Foissner, 1992 Zhang Q., Simpson A., Song W. (2012) Insights into the phylogeny (Ciliophora, Spirotrichea). J. Eukaryot. Microbiol. 55: 257–270 of systematically controversial haptorian ciliates (Ciliophora, Wolf M., Achtziger M. C., Schultz J., Dandekar T., Müller T. (2005) Litostomatea) based on multigene analyses. Proc. R. Soc. Lond. Homology modeling revealed more than 20,000 rRNA inter- B Biol. Sci. 279: 2625–2635 nal transcribed spacer 2 (ITS2) secondary structures. RNA 11: Zuker M. (2003) Mfold web server for nucleic acid folding and hy- 1616–1623 bridization prediction. Nucleic Acids Res. 31: 3406–3415 Wu L., Clamp J. C., Yi Z., Li J., Lin X. (2015) Phylogenetic and taxonomic revision of an enigmatic group of haptorian ciliates, with establishment of the Kentrophyllidae fam. n. (Protozoa, Ciliophora, Litostomatea, Pleurostomatida). PLoS One 10: Received on 11th August, 2017; revised on 31st October, 2017; e0123720 accepted on 2nd November, 2017 Phylogenetic Interrelationships among Litostomateans 277 MF288137 JX070867 KP868777 MF288136 JX070864 KP868776 KP868775 KP868774 MF288135 JX070868 KP868773 MF288142 MF288141 MF288134 JX070869 GenBank entry 36.71 33.61 41.08 37.53 35.05 38.11 38.78 38.39 39.88 34.52 34.74 30.83 30.83 30.83 37.02 GC (%) 365 363 331 365 368 349 330 349 351 365 354 360 360 360 362 Length (nt) KJ680552 HM581677 KP868769 KJ680551 HM581674 KP868768 KP868767 KP868766 MF288144 HM581679 KP868765 MF288147 KJ680554 MF288143 HM581678 GenBank entry 42.31 42.32 43.02 42.49 42.46 42.75 42.50 42.74 42.40 41.74 41.70 42.41 42.33 42.41 42.32 ITS1-5.8S-ITS2 region GC (%) 1534 1640 1548 1525 1639 1558 1553 1521 1592 1641 1554 1587 1538 1582 1640 18S rRNA 18S rRNA gene Length (nt) - - - - (Hedw.) (Hedw.) Hedw.) Hedw.) Hypnum cupressiforme Brachythecium rutabulum Upper-soil layer covered with leaf-litter from Un Upper-soil hwa-ri, Onyang-eup, Ulju-gun, Ulsan, Korea - Transporta Soil from a puddle in front of the Idaho tion Department Building, Boise, Idaho, USA Terrestrial moss from the Devínska Kobyla Hill, Terrestrial Bratislava, Slovakia Decaying wood mass from the Hwangseong park, Hwangseong-dong, Gyeongju-si, Gyeongsangbuk- do, Korea Soil from the outskirts of the town of Selker, Upper Soil from the outskirts of town Selker, Austria, Austria Terrestrial moss ( Terrestrial from the surroundings of a student dormitory in Mlynská dolina, Bratislava, Slovakia Leaf litter from a park in the village of Mikšová, Bytča District, Slovakia Leaf litter from the town of Levoča, Slovakia Terrestrial moss [ Terrestrial Leaf litter and soil from a wetland near the Ham beak mountain, Gohan-eup, Jeongseon-gun, Gang won-do, Korea Soil from the Chobe River floodplain, Kabolebole Peninsula, Botswana Schimp.] from a floodplain forest in the surround Slovakia ings of the village Pusté Úľany, Freshwater from the Galti pond in Ulsan Grand Park, Ulsan, Korea Freshwater from the Meonmulkkak wetland, Seonheul-ri, Jocheon-eup, Jeju-si, Jeju-do, Korea Freshwater from the Seonsaemi pond, Seonheul-ri, Jocheon-eup, Jeju, Korea Ephemeral pond in the Austrian Central Alps, Austrian Central Ephemeral pond in the surroundings of Gross-glockner-Hochalpenstrasse, the Austria Hochtor, Collection site

Vďačný and (Kahl, 1931) Vďačný et al. in Vďačný et al. in Vďačný et al. in (Kahl, 1931) Foissner, 1984 (Kahl, 1931) Foissner, Foissner, 1995 [Botswana] Foissner, Foissner, 1995 [Slovakia] Foissner, Dragesco, 1963 Rimaleptus binucleatus Vďačný and Pseudomonilicaryon fraterculum 2012 Foissner, Pseudomonilicaryon brachyproboscis Vďačný and Pseudomonilicaryon brachyproboscis 2008 Foissner, Pseudomonilicaryon anguillula Vďačný and Foissner, 2012 Vďačný and Foissner, (Foissner, 1981) (Foissner, terrenum Monomacrocaryon Vďačný et al., 2011b (Foissner, 1981) (Foissner, breviproboscis Microdileptus 2012 Vďačný and Foissner, Dimacrocaryon amphileptoides Dimacrocaryon Kahl, 1931 [pop. 2] Dimacrocaryon amphileptoides Dimacrocaryon Kahl, 1931 [pop. 1] Dileptus costaricanus Dileptus jonesi Dileptus costaricanus Vďačný and Foissner, 2012 [SKS-175, Ulsan, Vďačný and Foissner, Korea] Apotrachelius multinucleatus Vďačný and Foissner, 2012 [SKS-60, Jeju-do, Vďačný and Foissner, Korea] Apotrachelius multinucleatus Vďačný and Foissner, 2012 [SKS-536, Jeju, Korea] Vďačný and Foissner, Apotrachelius multinucleatus Rhynchostomatia Apodileptus visscheri rhabdoplites Foissner, 2012 Foissner, Characterization of 18S rRNA gene and ITS region sequences of the litostomatean ciliates used in phylogenetic analyses. Newly obtained are bold face S1. Characterization of 18S rRNA Table Taxon 278 Ľ. Rajter and P. Vďačný a a a MG264149 MG264150 – KU925883 MF288140 KU925878 MF288139 KU925882 KM222058 KP868779 – KP868778 – MF288138 – JX070865 – MG264151 43.68 45.09 – 39.81 31.56 38.39 35.91 39.12 39.48 39.06 – 39.36 – 37.85 – 35.69 – 44.03 1273 1262 – 412 358 409 362 409 385 343 – 343 – 354 – 367 – 1265 MG264143 – U80313 KM025129 KJ680553 – MF288146 – – KP868772 MG264144 KP868771 KF733753 MF288145 JQ723965 HM581675 JF263448 – 42.15 – 42.39 42.00 42.06 – 42.85 – – 42.71 41.58 42.63 42.02 42.52 42.12 42.52 41.9 – 1637 – 1637 1534 1536 – 1580 – – 1550 1633 1546 1592 1592 1593 1639 1638 – - - - (Hedw.) (Hedw.) Populus alba L .) in Brachythecium rutabulum Mnium ) from a municipal park in [Brachythecium rutabulum Leaf litter from forest of the Dilijan National Park Province, Re Tavush located in the north-eastern public of Armenia Benthos from a hypertrophic shallow water reser voir in the city of Modra, Slovakia Not available Futian mangrove wetland, Shenzhen, China Freshwater from the Jumgol reservoir, Mugeo- Freshwater from the Jumgol reservoir, dong, Nam-gu, Ulsan, Korea Mangrove wetland, Daya Bay, China Mangrove wetland, Daya Bay, Futian mangrove wetland, Shenzhen, China Leaf litter and soil from the Sangnasan mountain, Bi-ri, Heuksan-myeon, Sinan-gun, Jeollanam-do, Korea Circulating water system of a fish culture, Qingdao, China Terrestrial moss Terrestrial Schimp.] from the surroundings of (Hedw.) Comenius University in Mlynská dolina, Bratislava, Slovakia moss [ Terrestrial Schimp.] from the National Bioreserve Jurské jaz Slovakia ero in the Malé Karpaty Mts., Svätý Júr, Leaf litter from a poplar tree ( the surroundings of Comenius University in Mlynská dolina, Bratislava, Slovakia Ayers Mud and soil from an ephemeral pool on the Rock, Australia Bark of elm-like tree from the Haeinsa temple, Gaya-myeon, Hapcheon-gun, Gyeongsangnam-do, Korea Bromeliad litter from Botanical Garden, Rio de Janeiro, Brazil Floodplain soil from Boise, Idaho, USA Ephemeral puddle from the city of Boise, Idaho, USA moss ( Terrestrial the village of Mikšová, Bytča District, Slovakia (Vďačný and Foissner, (Vďačný and Foissner, Wu et al., 2015 Wu (Vuxanovici, 1960) Foissner (Vuxanovici, (Foissner and Schade in pop. 2 pop. 1 (Foissner and Schade in (Foissner and Schade in Foiss - Berger et al., 1983 Berger sp. sp. Foissner, 1983 Fuscheria nodosa Foissner, Litonotus crystallinus et al., 1995 Haptorida Enchelyodon Amphileptus spiculatus (Ehrenberg, 1831) Ehrenberg, 1831) Ehrenberg, ovum (Ehrenberg, Trachelius 1833 Amphileptus salignus Amphileptus salignus 2008) Vďačný and Rajter, 2015 Vďačný and Rajter, 2008) Rurikoplites longitrichus Pleurostomatida Amphileptus marinus Rurikoplites armatus 2015 [pop. 2, Vďačný and Rajter, 2000) Foissner, Slovakia] Fuscheriides Rurikoplites armatus 2015 [pop. 1, Vďačný and Rajter, 2000) Foissner, Slovakia] Fuscheria uluruensis Foissner and Gabilondo, 2009 2009 in Gabilondo and Foissner, Rurikoplites armatus 2015 [Korea] Vďačný and Rajter, 2000) ner, Fuscheria terricola (Penard, 1922) Vďačný et (Penard, 1922) Rimaleptus mucronatus al., 2011b Fuscheria sp . Litonotus muscorum (Kahl, 1931) Blatterer and 1988 Foissner, Phylogenetic Interrelationships among Litostomateans 279 a a a a MG264155 MG264152 KT246094 KY556655 – JX070875 KU925877 JX070874 KU925881 JX070879 MG264154 KT246095 KY556653 KT246092 JX070880 KU925880 MG264153 JX070870 KT246092 40.92 42.29 43.95 31.81 – 37.77 36.51 36.64 40.29 34.41 43.36 42.63 32.69 43.63 35.60 40.20 42.37 32.43 42.34 1283 1291 810 371 – 368 430 363 417 372 1287 929 364 1052 368 408 1291 367 1103 MG264147 MG264145 KT246081 KY556648 KF733758 JF263447 – JF263446 KC469985 JF263445 KT246076 KT246084 KY556646 KT246078 JF263444 JN974455 MG264146 JF263441 KT246077 42.40 43.02 43.20 42.83 42.82 43.34 – 43.45 42.39 43.28 43.50 42.41 43.06 43.04 43.33 42.24 42.99 42.99 42.34 1639 1625 1546 1590 1595 1636 – 1641 1604 1638 1547 1549 1593 1559 1634 1636 1640 1640 1558 - - - - (Hedw.) (Hedw.) (Hedw.) (Hedw.) Populus alba L. ) in Brachythecium rutabulum Brachythecium rutabulum Terrestrial moss from the Nature Park Biokovo, Terrestrial Dalmatia, Croatia Leaf litter from a forest near O’Leary Road, Port USA Georgia, Wentworth, Leaf litter from a floodplain forest in the surround Slovakia ings of the village Pusté Úľany, Leaf litter and soil from the surroundings of Mugeo-dong, Nam-gu, Ulsan, Junggol reservoir, Korea Soil from the Chobe River, Kasane, Botswana Soil from the Chobe River, Bromeliad tank, Jamaica Coastal waters of the South China Sea, Nansan Is land, Zhanjiang, Guangdong, China Floodplain soil from the city of Boise, Idaho, USA Zhanjiang mangrove wetland, Shenzhen, China moss [ Terrestrial Terrestrial mosses from the Pyhätunturi mountain, Terrestrial Pyhä-Luosto National Park in the Lapland region, Finland Terrestrial moss [ Terrestrial Schimp.] from the surroundings of Comenius University in Mlynská dolina, Bratislava, Slovakia voir in the city of Modra, Slovakia Lichens and music bryopsida from the Hwaamsa Goseong-gun, Gangwon- Toseong-myeon, temple, do, Korea Schimp.] from the National Bioreserve Jurské jaz Slovakia ero in the Malé Karpaty Mts., Svätý Júr, Benthos from a hypertrophic shallow water reser Garden water tank from the city of Boise, Idaho, USA Leaf litter from a poplar tree ( the surroundings of Comenius University in Mlynská dolina, Bratislava, Slovakia Water from a shrimp farm in Changyi, Shandong Water Province, China Leaf litter from a forest near O’Leary Road, Port USA Georgia, Wentworth, mosses, Germany Terrestrial Foissner and Foissner and Foissner and Foissner and (Kahl, 1930) Wu, 2013 Wu, Eberhard, 1862 Foissner, 1984 Foissner, Pan et al., 2013 Jang et al., 2017 Stokes, 1884 sp. sp. sp. Paraenchelys terricola Epispathidium Apobryophyllum schmidingeri Al-Rasheid, 2007 [clone 1, USA] Enchelys megaspinata Spathidiida Acaryophrya sp. Kahl, 1926 Enchelys gasterosteus Wu et al., 2016 Wu magnus Protolitonotus Enchelyodon Loxophyllum meridionale cultriforme scalpriforme Arcuospathidium 2003 (Kahl, 1930) Foissner, Cultellothrix lionotiformis Foissner, 2003 Foissner, Bryophyllum Loxophyllum helus Apobryophyllum schmidingeri Al-Rasheid, 2007 [South Korea] Balantidion pellucidum Loxophyllum chinense Apobryophyllum schmidingeri Apobryophyllum schmidingeri muscorum (Dragesco and Arcuospathidium 1984 Dragesco-Kernéis, 1979) Foissner, Al-Rasheid, 2007 [clone 2, USA] Al-Rasheid, 2007 [Germany] 280 Ľ. Rajter and P. Vďačný a a a MG264158 JX070877 KT246099 KT246098 MG264156 KY556650 JX070873 KY556654 MG264157 – KY556656 KY556651 KY556652 JX070876 KT246093 KT246097 KT246096 JX070878 43.80 34.88 40.19 41.78 43.85 30.40 36.78 42.86 41.05 – 36.97 42.29 35.25 29.64 38.79 43.48 42.53 33.43 1283 367 729 943 1293 375 367 350 1296 – 357 350 366 361 856 1058 917 362 KT246082 JF263451 KT246090 KT246089 KT246079 KY556642 JF263450 KY556647 KT246086 KT246085 KY556649 KY556643 KY556645 JF263449 KT246080 KT246088 KT246087 JF263452 43.38 43.27 42.64 42.77 43.01 43.01 43.45 43.40 42.73 42.45 43.31 43.75 43.22 43.32 42.81 43.09 42.99 43.33 1549 1641 1555 1550 1553 1581 1641 1546 1561 1550 1593 1593 1592 1639 1551 1557 1556 1641 - - - - Populus alba L. ) in Hedw.) Hedw.) Hypnum cupressiforme Decaying wood mass from the National Bioreserve Jurské jazero in the Malé Karpaty Mts., Svätý Jur, Slovakia Floodplain soil from the city of Boise, Idaho, USA Terrestrial moss from a municipal park in the vil Terrestrial lage of Mikšová, Bytča District, Slovakia Decaying wood mass from the surroundings of Comenius University in Mlynská dolina, Bratislava, Slovakia Leaf litter from the Devínska Kobyla Hill, Bratisla va, Slovakia Leaf litter and soil from the Daewangam Park, Ilsan-dong, Dong-gu, Ulsan, Korea Floodplain soil from the Krüger National Park, Africa Limpopo and Mpumalanga provinces, South Soil from the surroundings of a maple tree in Daehak-ro, Gyeongsan-si, University, Yeungnam Gyeongsangbuk-do, Korea Leaf litter from a municipal park in the village of Mikšová, Bytča District, Slovakia Leaf litter and soil from the Ssanggyesa temple, Sacheon-ri, Uisin-myeon, Jeollanam-do, Korea Terrestrial moss ( Terrestrial from the surroundings of a student dormitory in Mlynská dolina, Bratislava, Slovakia Leal litter and soil from the Daewangam Park, Ilsan-dong, Dong-gu, Ulsan, Korea Leaf litter and soil from the Jungjok mountain, Joil- ri, Samdong-myeon, Ulju-gun, Ulsan, Korea Floodplain soil, Botswana moss from a municipal park in the vil Terrestrial lage of Mikšová, Bytča District, Slovakia Leaf litter from a poplar tree ( the surroundings of Comenius University in Mlynská dolina, Bratislava, Slovakia Leaf litter from the Devínska Kobyla Hill, Bratisla va, Slovakia Floodplain soil from the city of Boise, Idaho, USA Kahl, 1930 [pop. 1] Kahl, 1930 [pop. 2] Dragesco and Berger et al., 1984 Berger (Foissner et al., 2002) Kahl, 1930 [pop. 2] Kahl, 1930 [pop. 1] Kahl, 1930 Kahl, 1930 Wenzel, 1955 Wenzel, Kahl, 1930 [pop. 2] Kahl, 1930 [pop. 1] (Foissner, 1987) Jang et al., (Foissner, sp. These new sequences also contain a variably long 5’-end of the 28S rRNA gene. These new sequences also contain a variably long 5’-end of the 28S rRNA Spathidium terricola 2017 Spathidium sp. 2 Spathidium simplinucleatum Spathidium simplinucleatum Greeff, 1888 [pop. 1] Spathidium amphoriforme Greeff, Spathidium securiforme Semispathidium breviarmatum Vďačný and Semispathidium breviarmatum 2013 Foissner, Spathidium polynucleatum Jang et al., 2017 Spathidium claviforme Spathidium papilliferum Pseudoholophrya terricola Spathidium ascendens Spathidium papilliferum Spathidium muscicola Protospathidium muscicola Protospathidium Dragesco-Kernéis, 1979 1888 [pop. 2] Spathidium amphoriforme Greeff, Spathidium muscicola Trachelophyllum a Phylogenetic Interrelationships among Litostomateans 281 - d 0.4030 – 1.0000 5.1596 1.0000 1.7822 3.8379 1.7822 0.2860 0.2334 0.1822 0.2985 1566 TIM2 56 – CON-2 c 0.3780 0.6310 1.0000 7.6729 0.7590 3.5419 3.9416 2.3292 0.2810 0.2267 0.1889 0.3034 1747 GTR 56 – CON-1 0.5310 0.4810 1.0000 7.7023 0.6040 4.6972 7.7023 2.0078 – – – – 196 TVMef 56 0.930 ITSR-F 0.5740 0.3070 1.0000 5.1519 0.3612 2.2547 2.2840 1.6267 0.2817 0.1613 0.1920 0.3650 307 GTR 56 0.306 ITSR-E 0.5060 0.2640 1.0000 6.6706 1.0000 2.4893 2.9721 2.4893 0.3110 0.1611 0.1692 0.3587 324 TIM2 59 0.420 ITSR-D 0.5510 0.2850 1.0000 6.5530 1.0000 2.6761 2.9741 2.6761 0.3118 0.1619 0.1717 0.3546 333 TIM2 59 0.341 ITSR-C 0.5330 0.3530 1.0000 7.9820 1.0000 2.6889 4.1822 2.6889 0.2982 0.1474 0.1919 0.3625 268 TIM2 60 0.533 ITSR-B Alignment 0.2620 – 1.0000 7.3407 1.0000 2.3434 3.1266 2.3434 0.2890 0.1436 0.1882 0.3792 277 TIM2 60 0.345 ITSR-A 0.3640 0.6840 1.0000 7.1934 0.8177 2.4957 5.5015 1.4579 0.2857 0.2350 0.1839 0.2955 1370 GTR 56 0.995 18S-F 0.3230 0.6280 1.0000 9.0867 1.1197 3.2729 6.0259 1.8985 0.2831 0.2438 0.1820 0.2911 1440 GTR 56 0.930 18S-E 0.3030 0.6030 1.0000 7.5256 0.8458 3.0527 5.7122 1.6774 0.2813 0.2377 0.1848 0.2962 1383 GTR 63 0.990 18S-D 0.3280 0.6290 1.0000 6.6950 0.8277 3.1400 6.6950 1.6929 0.2868 0.2366 0.1932 0.2834 1377 TVM 64 0.990 18S-C 0.3170 0.5940 1.0000 8.1041 0.9563 2.9671 5.1060 1.9716 0.2817 0.2517 0.1811 0.2854 1463 GTR 63 0.877 18S-B 0.2950 0.5770 1.0000 8.1540 0.8848 3.1495 5.3410 1.9434 0.2822 0.2503 0.1806 0.2868 1459 GTR 64 0.880 18S-A a b Unreliably aligned columns were removed from the alignment at the given cutoff values. These were chosen on the basis of the calclated condifence scores on the Guidance2 server. These were chosen on the basis of calclated condifence scores values. Unreliably aligned columns were removed from the alignment at given cutoff The CON-2 dataset was created by combining the 18S-F and ITSR-F alignments. The best fitting evolutionary models were selected for each dataset under the Akaike information criterion in jModelTest. A, C, G, T, base frequencies; [AC], [AG], [AT], [CG], [CT], [GT], rate substi [AG], [AT], T, base frequencies; [AC], A, C, G, Akaike information criterion in jModelTest. The best fitting evolutionary models were selected for each dataset under the The CON-1 dataset was created by combining the 18S-E and ITSR-E alignments. Γ I [GT] [CT] [CG] [AT] [AG] [AC] T G C Cutoff value Cutoff A No. of char. Model Table S2. Characterization and evolutionary models of the alignments analyzed Table Characteristic No. of taxa a b c d tution matrices; I, proportion of invariable sites; Γ, gamma distribution shape parameter. tution matrices; I, proportion of invariable sites; Γ,