Accepted Manuscript Phylogenomic Analyses Reveal Subclass Scuticociliatia as the Sister Group of Subclass Hymenostomatia within Class Oligohymenophorea Jin-Mei Feng, Chuan-Qi Jiang, Alan Warren, Miao Tian, Jun Cheng, Guanglong Liu, Jie Xiong, Wei Miao PII: DOI: Reference:

S1055-7903(15)00144-X http://dx.doi.org/10.1016/j.ympev.2015.05.007 YMPEV 5195

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

25 January 2015 26 April 2015 10 May 2015

Please cite this article as: Feng, J-M., Jiang, C-Q., Warren, A., Tian, M., Cheng, J., Liu, G-l., Xiong, J., Miao, W., Phylogenomic Analyses Reveal Subclass Scuticociliatia as the Sister Group of Subclass Hymenostomatia within Class Oligohymenophorea, Molecular Phylogenetics and Evolution (2015), doi: http://dx.doi.org/10.1016/j.ympev. 2015.05.007

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Phylogenomic Analyses Reveal Subclass Scuticociliatia as the Sister Group of Subclass Hymenostomatia within Class Oligohymenophorea Jin-Mei Feng1,

2*

, Chuan-Qi Jiang2*, Alan Warren3, Miao Tian2, Jun Cheng2,4,

Guang-long Liu2, Jie Xiong2, Wei Miao2§ 1

Department of Pathogenic Biology, School of Medicine, Jianghan University, Wuhan 430056, China

2

Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China

3

Department of Life Sciences, Natural History Museum, London SW7 5BD, UK

4

Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for

Aquatic Economic Animals, College of Fishery, Guangdong Ocean University, Zhanjiang 524088, China

§

Corresponding author:

Wei Miao (Email: [email protected], phone numbers: 86-27-68780050) *These authors contributed equally to this work.

E-mail addresses: Jin-Mei Feng: [email protected]; Chuan-Qi Jiang: [email protected]; Alan Warren: [email protected]; Miao Tian: [email protected]; Jun Cheng: [email protected]; Guang-Long Liu: [email protected]; Jie Xiong: [email protected]; Wei Miao: [email protected]

1 / 25

Abstract Scuticociliates

and

hymenostomes

are

two

groups

of

the

ciliate

class

Oligohymenophorea, a diverse clade that includes two model genera, Tetrahymena and Paramecium, which have been intensively studied due to their ease of culture and their amenability to a wide range of biochemical and genetic investigations. However, phylogenetic relationships among the subclasses of the Oligohymenophorea, and especially between the Scuticociliatia and Hymenostomatia, are not clearly resolved. Here, we investigate the phylogenetic relationship between the subclasses Scuticociliatia and Hymenostomatia based on omics data. The transcriptomes of five species, comprising four oligohymenophoreans and one colpodean, were sequenced. A supermatrix was constructed for phylogenomic analyses based on 113 genes encoding 43,528 amino acid residues from 26 taxa, including ten representatives of the class Oligohymenophorea. Our phylogenomic analyses revealed that the monophyletic Scuticociliatia is sister to the monophyletic Hymenostomatia, which together form the terminal branch within the monophyletic class Oligohymenophorea. Competing hypotheses for this relationship were rejected by topological tests. Our results provide corroborative evidence for the close relationship between the subclasses Scuticociliatia and Hymenostomatia, justifying the possible use of the model hymenostome T. thermophila as an effective experimental system to study the molecular and cellular biology of the scuticociliates.

Keywords: Oligohymenophorea, Scuticociliatia, Hymenostomatia, Phylogenomics analysis

2 / 25

1. Introduction Ciliated protozoa are single-cell eukaryotes with diverse morphologies, extensive distributions and are important trophic links in various microbial food webs (Lynn, 2008). The phylum Ciliophora is divided into 12 classes of which the Oligohymenophorea is arguably the most diverse and is consistently recovered as a monophyletic group in molecular phylogenies (Miao et al., 2004; Miao et al., 2001; Sanchez-Silva et al., 2003; Struder-Kypke et al., 2000). The Oligohymenophorea has long attracted the attention of researchers for a variety of reasons, two of the main ones being: (1) the inclusion within this group of two model genera, namely Tetrahymena and Paramecium, which include the first ciliates species to be successfully cultured axenically and to have their complete genome sequenced; (2) some oligohymenophoreans are obligate or facultative parasites of fishes, molluscs and crustaceans and cause significant economic losses to the global aquaculture industry. Examples include Ichthyophthirius multifiliis which is responsible for white spot disease in freshwater fishes (Abdel-Hafez et al., 2014; Forwood et al., 2014; Fu et al., 2014; Hoshino et al., 2014), and various species of scuticociliates that cause a range of diseases (collectively termed ‘scuticociliatosis’) in economically important host organisms (Harikrishnan et al., 2010; Jin et al., 2010; Zhan et al., 2014). In traditional classification systems of ciliates based on morphostatic morphology the class Oligohymenophorea was divided into two subclasses - Hymenostomatia and Peritrichia (Corliss, 1979). In these systems the scuticociliates were an order (Scuticociliatida) within the subclass Hymenostomatia, thus reflecting the similarities of their morphology, infraciliature, and ultrastructure with those of the hymenostomes. Puytorac (1994) elevated the Scuticociliatida to rank of subclass thus separating the scuticociliates from the subclass Hymenostomatia. The recognition of the Scuticociliatia and Hymenostomatia as separate subclasses was accepted in all subsequent classification systems of ciliates (Lynn, 2008; Lynn and Small, 1997; Lynn and Small, 2002). In the most recent classification scheme of the phylum Ciliophora, Lynn (2008) recognized

six

subclasses

within

the 3 / 25

class

Oligohymenophorea,

namely

Hymenostomatia, Peniculia, Scuticociliatia, Peritrichia, Apostomatia, and Astomatia. However, even with the application of molecular analyses, phylogenetic relationships among these subclasses are poorly understood (Lynn, 2003). Furthermore, because of the limited availability of molecular data for representatives of the Apostomatia and Astomatia, investigations are mainly focused on the other four subclasses. The most commonly used gene marker for phylogenetic analyses in ciliates is the small subunit rRNA (SSU rRNA) which was first used to demonstrate divergences among ciliates over 25 years ago (Lynn and Sogin, 1988). Several phylogenetic analyses derived from SSU rRNA gene sequences have subsequently placed the hymenostomes and peritrichs as sister lineages (Li et al., 2006; Miao et al., 2004; Miao et al., 2001), indicating that the subclass Hymenostomatia is most closely related to the subclass Peritrichia rather than Scuticociliatia. However, a more recent analysis based on three concatenated rDNA sequences revealed that Scuticociliatia is more closely related to Hymenostomatia than to either Peniculia or Peritrichia (Gao et al., 2013). Therefore, in order to resolve these relationships more clearly, an analysis based on genomic data was carried out. Phylogenomic analyses that used omics data have generally improved the robustness of molecular phylogenetic reconstructions and untangled many previously debated phylogenies among major eukaryotic lineages (Chiari et al., 2012; Hampl et al., 2009; Liang et al., 2013; Philippe et al., 2009; Philippe et al., 2005; Philippe and Telford, 2006; Ryan et al., 2013; Struck and Fisse, 2008). With respect to the phylum Ciliophora, the first phylogenomic analysis was carried out to explore the phylogenetic position of the ambiguous taxon Protocruzia (Gentekaki et al., 2014). Phylogenomic analysis focusing on the phylogenetic relationships of the subclasses within the class Oligohymenophorea has yet to be performed. In order to elucidate the confused phylogenetic relationship between the subclasses Hymenostomatia and Scuticociliatia, we expanded taxonomic sampling by sequencing the transcriptome of five ciliate taxa, i.e. one representative of the class Colpodea (Colpoda aspera) and four representatives of the class Oligohymenophorea 4 / 25

(Pseudocohnilembus persalinus, Paralembus digitiformis, Campanella umbellaria, and Carchesium polypinum). The expanded data set included 113 genes encoding 43,528 amino acid residues from 26 taxa. Phylogenomic analyses of the expanded data set were carried in order to elucidate evolutionary relationships among subclasses within the Oligohymenophorea. 2. Materials and methods 2.1. Ciliate collection, identification and culture Pseudocohnilembus persalinus was supplied by Prof. Weibo Song, Institute of Evolution and Marine Biodiversity, Ocean University of China. It was cultured at 30°C in autoclaved seawater (salinity 20‰) and was fed with E. coli. Paralembus digitiformis was supplied by Prof. Kuidong Xu, Marine Organism Taxonomy and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences. It was cultured at room temperature in autoclaved seawater (salinity 20‰) and was fed with flesh of the crustacean Metapenaeus ensis that had been minced and cooked in boiling water for 5 minutes. Colpoda aspera was supplied by Prof. Tuanyuan Shi, Zhejiang Academy of Agricultural Science. It was cultured in the laboratory following the method of Shi et al. (2014). Basically, ciliates were cultured in autoclaved PBS medium (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·12H2O, 2 mM KH2PO4) mixed with LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0) in a proportion of 1% (v/v), and adding 1% LB medium every 24h. Cells were maintained at 30°C and harvested after 3 days. Campanella umbellaria was obtained from the surface of Characeae plants, and Carchesium polypinum was obtained from the surface of branches and leaves submerged in water. Both species were collected from the East Lake (30°32’N; 114°22’E), Wuhan, China, from November, 2012 to January, 2013. Each population was isolated in the laboratory using ophthalmological scissors and tweezers, and then picked out using dissecting needles and glass micropipettes. Campanella umbellaria and Carchesium polypinum were morphologically identified according to previous 5 / 25

reports (Dias et al., 2010; Foissner, 1992; Shi et al., 2004). The infraciliatures of the two species were revealed by protargol staining according to the method of Shi and Frankel (1990). Photomicrographs of Campanella umbellaria and Carchesium polypinum from life and infraciliatures of them were made with camera OLYMPUS (BX51), and they were shown in Supplementary Fig. S1. Additionally, though Campanella umbellaria and Carchesium polypinum are uncultured peritrichs, both of them are common species in East Lake (30°32’N; 114°22’E), Wuhan, China, and can be collected almost all year round, especially in winter. We have collected and isolated them from the environmental samples, and freeze-stored multiple DNA and RNA samples of them in our lab. We state that if anyone needs the biological materials of them, we will supply DNA and RNA samples of these two species to them upon request, or one can also collect the environmental samples by themselves and isolate the two species following our methods. Ciliates were checked by light microscopy to inspect their viability and for contaminants, especially for the two uncultured peritrichs which were collected from the environment. Potential rDNA sequences were extracted from the transcriptome assembly (see below), and species identifications were verified by BLAST searching the Genbank database using the rDNA sequences. 2.2. RNA extraction, library construction and Illumina sequencing The RNA of isolated cells was stabilized using the RNA Protect Cell Reagent (Qiagen, Cat. # 76526). Total RNA was extracted using the RNeasy Protect Cell Mini Kit

(Qiagen,

Cat.

#

74624)

according

to

protocol

in

TetraFGD

(http://tfgd.ihb.ac.cn/index/smphelp) (Xiong et al., 2013). Total RNA concentrations were determined by the e-Spect ES-2 spectrophotometer (MALCOM, Japan) and RNA integrity was verified using gel electrophoresis in denaturing 1.2% agarose. Poly-A mRNAs were isolated using Sera-mag Magnetic Oligo (dT) beads from Illumina, and then fragmented using divalent cations at 94°C for 5 minutes. Double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, Camarillo, CA) with random hexamer primers from 6 / 25

Illumina. The synthesized cDNA was subjected to end-repair, phosphorylation, 3' adenylation and adapter ligation in sequential. After these steps, cDNA fragments with an insert size of about 200bp were purified by gel electrophoresis. The purified cDNA template was enriched by PCR amplification and the quality of the RNA library was validated in a LightCycler480 (Roche Diagnostics) using an Illumina PhiX174

Control.

Libraries of Pseudocohnilembus persalinus,

Campanella

umbellaria and Carchesium polypinum were sequenced for 81 bp at both ends (paired-end) using an Illumina Genome Analyzer IIx sequencer, the others Colpoda aspera and Paralembus digitiformis for 100bp and 101bp at both ends (paired-end) by using an Illumina HiSeq2000 sequencer, respectively. Low quality reads were filtered by the fastq_quality_filter in FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Transcriptomes were then assembled de novo using Trinity software with default parameters (Grabherr et al., 2011). Protein sequences were obtained by translating the assembled transcripts (or transcript fragments) using the ciliate codon table by the getorf program in EMBOSS package (Rice et al., 2011). All the raw sequencing reads were deposited into the NCBI Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/Traces/sra/) under accession number as follows: Pseudocohnilembus persalinus SRR1768438, Paralembus digitiformis SRR1768439, Colpoda aspera SRR1768440, Campanella umbellaria SRR1768423, and Carchesium polypinum SRR1768437. 2.3 Data sources Omics data and SSU rRNA gene sequences used in the present work are listed in Supplementary Table S1. Genomic data of four Tetrahymena spp. (T. borealis, T. elliotti, T. malaccensis, and T. thermophila) were downloaded from the Tetrahymena GENOME

DATABASE

(TGD)

(http://ciliate.org/index.php/home/welcome),

Ichthyophthirius multifiliis from the Ichthyophthirius GENOME DATABASE (IchDB) (http://ich.ciliate.org/index.php/home/welcome), Oxytricha

GENOME

Oxytricha DATABASE

trifallax

from

the

(OxyDB)

(http://oxy.ciliate.org/index.php/home/welcome), Paramecium tetraurelia from the 7 / 25

ParameciumDB (http://paramecium.cgm.cnrs-gif.fr/), and Stylonychia lemnae from the

GENOME

Stylonychia

DATABASE

(StyloDB)

(http://stylo.ciliate.org/index.php/home/welcome/) (Aeschlimann et al., 2014). Two Plasmodium spp. (P. falciparum and P. yoelii) were selected as outgroup taxa for all analyses, and their genomic data were downloaded from the PlasmoDB (http://plasmodb.org/plasmo/). All transcriptomic data of the 11 ciliate species released by Gentekaki et al. (2014) were retrieved from the CAMERA portal (https://portal.camera.calit2.net/gridsphere/grid-sphere) MMETSP0125-20120918 (Condylostoma

(Aristerostoma

magnum),

under

sp.),

accession

samples

MMETSP0210-20121227

MMETSP0205-20121125

(Euplotes

focardii),

MMETSP0213-20121227 (Euplotes harpa), MMETSP0209-20121228 (Litonotus sp.), MMETSP0127-20121128

(Platyophrya

(Protocruzia

MMETSP0211-20121228

adherens),

MMETSP0123-20130129 (Strombidinopsis

macrostoma), MMETSP0216-20120918

(Schmidingerella

acuminatum)

and

(Pseudokeronopsis

arcuata),

riccii),

MMETSP0126-20121128

MMETSP0208-20121228

(Strombidium

inclinatum). Transcriptome assembly was performed with the program suite Trinity (Grabherr et al., 2011). Putative ORF prediction was implemented by the getorf program of the EMBOSS site (-table 6, -minsize 600) (Rice et al., 2011). We used the Reciprocal Best Hits (RBH) approach in Basic Local Alignment Search Tool (BLAST) to obtain genes that are putatively orthologous between two species. Only hits with an E value less than 10 -20 were retained and the RBH lists were filtered to discard putative paralogs. The RBH-pair was retained only if the score of the second-best hit of either gene in the other genome was less than half of the score of the best hit. Only gene sets including more than 70% (at least 18 different species of our 26 species) of all species were retained for further analyses. All selected genes are described in Supplementary Table S2. The SSU rRNA gene sequences of the above 26 species were retrieved from GenBank

and

the

CAMERA

portal

(https://portal.camera.calit2.net/gridsphere/grid-sphere). Additionally, SSU rRNA 8 / 25

gene sequences of the five transcriptome-sequenced ciliates in this study, Colpoda aspera, Pseudocohnilembus persalinus, Paralembus digitiformis, Campanella umbellaria, and Carchesium polypinum, were obtained by BLAST searching against the corresponding transcriptome. Altogether, 31 SSU rRNA genes were used for phylogenetic analyses. 2.4. Sequence alignment and phylogenetic analyses Sequence alignments for the SSU rRNA gene and for the 113 protein coding genes were implemented by MUSCLE version 3.6 software with the default parameters (Edgar, 2004). Ambiguously aligned regions were detected and trimmed using Gblocks version 0.91b (Castresana, 2000) (-b2=0.65, -b3=10, -b4=5, -b5=a). The 113 individual alignments of the 26 taxa were concatenated into a supermatrix by software SCaFoS version 4.42 using the data set assembling panel (Roure et al., 2007). The maximum-likelihood (ML) analysis for the concatenated data set was conducted in RAxML version 7.2.6 (Stamatakis, 2006) with an LG amino acid substitution matrix, frequencies empirically estimated (+F), and a Γ model of site heterogeneity with four categories (LG+F+Γ4). Bootstrap support of the ML analysis was evaluated with 100 replicates. Bayesian inference (BI) analysis for the concatenated data set was implemented by the software PhyloBayes 3.3 (Lartillot et al., 2009) under a mixture model of CAT+POI+Γ4 with two independent Markov chain Monte Carlo (MCMC) runs. The detailed parameters used to run PhyloBayes were as follows: a discrete gamma distribution of rate variation with four rate categories (Γ4), the relative exchange rates modeled by the Poisson process (POI), and the amino acid profiles estimated using the CAT model (CAT). In order to evaluate the convergence of the two independent MCMC runs, the bpcomp program within PhyloBayes was used. The bpcomp program was used to compare the discrepancy of bipartition frequencies between the two runs and to output a consensus tree (PhyloBayes 3.3 manual). According to the PhyloBayes manual, a “good run” and adequate convergence of the two runs occurs when the largest discrepancy 9 / 25

(maxdiff) in the bipartition frequencies between the two runs is less than 0.1. In this study, the maxdiff was 0.046. For the SSU rRNA gene, the general time reversible model taking into account a gamma-distributed rate heterogeneity among sites (GTR+I+Γ) was selected using jModelTest 0.1.1 (Posada, 2008) under the Akaike Information Criterion (AIC). ML analysis was conducted in RAxML version 7.2.6 (Stamatakis, 2006) and computed using 1,000 bootstrap replicates. BI analysis was performed by MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003), with four incrementally heated Markov chains running for 1,000,000 generations sampling every 100 generations. Two separate runs were performed to confirm the convergence of the chains. The potential scale reduction factor convergence diagnostic, and the average standard deviation of split frequencies, were used to assess the convergence of the two runs. The initial 25% of the sampled trees were discarded as burn-in. In order to assess differences in tree topologies of alternative hypotheses, topology tests were performed for the expanded data set. We manually created constraint tree topologies with reference to the alternative hypotheses using Mesquite version 2.75 software (Maddison and Maddison, 2011), and then performed the RAxML analysis with each constraint using the -g option and LG+F+Γ4 model. The best ML tree was considered the constrained ML tree. The site likelihood values were computed for both the constrained and unconstrained ML trees by the software RAxML with the same model mentioned above. The likelihood values obtained were analyzed using the approximately unbiased (AU) and Shimodaira-Hasegawa (SH) tests with CONSEL v.0.1j software (Shimodaira and Hasegawa, 2001). 2.5. Estimation of the mutational saturation and removal of the saturated sites Generally, mutational saturation of the compared sequences is the major potential phylogenetic artifact. We used the method previously described by Philippe et al. (1994) to estimate the saturation level of our concatenated gene data set (Philippe et al., 1994). The inferred number of substitutions (patristic distance) between each 10 / 25

couple of species was computed from branch lengths of the best ML tree (using a LG+F+Γ model) using the program Mesquite version 2.75 software (Maddison and Maddison, 2011). The number of observed differences (uncorrected pairwise distance) was calculated by the program MEGA6 (Tamura et al., 2013). The saturation plot was drawn to estimate the saturation level by showing all the pairs of species with an abscissa value equal to the patristic distances and an ordinate equal to the uncorrected pairwise distances. The mutational saturation was revealed by the presence of a plateau within which the patristic distances increased, whereas the pairwise distances remained constant. If mutational saturations were observed after drawing the saturation plot, we split the original data set into a saturated and an unsaturated amino acid substitution and removed the saturated one by selecting the 'cut-off' value with the program ASaturA (Van de Peer et al., 2002). The neighbor-joining (NJ) tree was then calculated based only on the unsaturated amino acid substitutions under the WAG substitution matrix by the program ASaturA (Van de Peer et al., 2002). 3. Results 3.1. Phylogenomic analyses In the present study, we expanded the taxonomic sampling of the ciliate clades by integrating data from five newly obtained ciliates transcriptomes with the 11 available ciliate transcriptomes released by Gentekaki et al. (2014) and eight pre-existing ciliate genomic data from TGD, IchDB, OxyDB, ParameciumDB and StyloDB. Altogether, omics data of 24 species were used in our analyses, including ten representatives of the class Oligohymenophorea. Additionally, two species of Plasmodium were used as outgroup taxa. We assembled a data set comprising 113 nuclear genes encoding 43,528 aligned amino acid residues from the 26 taxa by analyzing available genome and transcriptome data. The assembled phylogenomic data set in the present study contains the highest number of oligohymenophorean ciliates to date. The tree topologies yielded by using two algorithms (ML and BI analyses) were consistent, therefore a single tree topology is presented with support values from both 11 / 25

analyses indicated on branches (Fig. 1). Generally, our phylogenomic analyses, which were performed both by ML calculated with RAxML under the LG+F+Γ4 model and by Bayesian inference with the CAT mixture model, provide full statistical support (100% ML/1.0 BI) for the monophyly of the class Oligohymenophorea based on the concatenated data set (Fig. 1). Within the class Oligohymenophorea, phylogenetic trees based on our expanded data set reveal that representatives of the subclasses Hymenostomatia, Scuticociliatia, and Peritrichia cluster within their respective groups with maximal support (100% ML/1.0 BI). More interestingly, the subclass Scuticociliatia is sister to the subclass Hymenostomatia, and together these form a terminal clade within the class Oligohymenophorea with maximal support (100% ML/1.0 BI) (Fig. 1). Our results, therefore, indicate a strong phylogenetic affinity between

the

subclasses

Scuticociliatia

and

Hymenostomatia.

Additionally,

phylogenetic analyses based on the SSU rRNA gene sequences of the 31 populations revealed that the subclass Scuticociliatia is basal within the class Oligohymenophorea, and the subclass Hymenostomatia is sister to Peritrichia (Supplementary Fig. S2). In order to evaluate saturation level of our phylogenomic data set, a saturation plot was drawn. The slope of the regression line (unsaturated data have slope = 1) of our saturation plot is 0.4085 (Fig. 2), indicating very little substitution saturation in our concatenated data set. After removal of the saturated sites, the neighbor-joining (NJ) tree based on the unsaturated sequences also supports the hypothesis that the subclass Scuticociliatia is sister to the subclass Hymenostomatia with maximal support (data not shown). The three representatives of the class Colpodea, i.e. Aristerostoma sp., Platyophrya macrostoma and the newly sequenced Colpoda aspera, cluster together, and are sister to the class Oligohymenophorea with maximal support. Additionally, the tree topologies derived from both the ML and BI analyses show that representative species of the class Spirotrichea group into a clade. Furthermore, the ambiguous taxon Protocruzia adherens forms an independent lineage branching before the class Spirotrichea and after heterotrich C. magnum, indicating that P. adherens is not a 12 / 25

spirotrich and is separated from both classes Heterotrichea and Spirotrichea. 3.2. Topology testing The phylogenetic affinities of the subclasses within the class Oligohymenophorea derived from our phylogenomic analyses conflict with those based on SSU rRNA gene sequence data. In order to assess these differences, the approximately unbiased (AU) and Shimodaira-Hasegawa (SH) tests were performed on the concatenated data set in the present study (Table 1). Results of both the AU test and SH test reject the hypothesis that the subclass Hymenostomatia is sister to Peritrichia (p-value equal 7e-062 and 0, respectively). Additionally, the competing hypotheses that the subclass Scuticociliatia is at the base of the oligohymenophorean assemblage is also rejected by both the AU and SH tests (p-value equal 9e-006 and 0, respectively). 4.

Discussion Although members of the class Oligohymenophorea typically have a paoral and

three oral polykinetids, there is no strong morphological or ultrastructural synapomorphy for this group (Lynn, 2008). Nevertheless, phylogenetic analyses based on SSU rRNA gene sequences generally recover the Oligohymenophorea as a monophyletic group, albeit often with only moderate bootstrap support (Lynn, 2008; Miao et al., 2004). In the present study based on omics data, the monophyly of the class Oligohymenophorea was recovered with maximal support in both the ML and BI

trees.

However,

the

phylogenetic

relationships

among

the

four

oligohymenophorean subclasses for which molecular data are available are noticeably different from those derived from the SSU rRNA gene sequences (Fig. 3). Specifically, in studies based on SSU rRNA gene sequences, the subclass Hymenostomatia is most closely related to the Peritrichia (Li et al., 2006; Miao et al., 2004; Miao et al., 2001), whereas in the present study based on phylogenomic analyses Hymenostomatia is sister to Scuticociliatia, and Peniculia is sister to Peritrichia. Although our finding that the subclass Scuticociliatia is sister to the subclass Hymenostomatia conflicts with that derived from the SSU rRNA gene sequence data 13 / 25

(Supplementary Fig. S2), other lines of evidence from previous studies support our results. The scuticociliates were first established as an order within the subclass Hymenostomatia in the traditional classification system of ciliates proposed by Corliss (Corliss, 1979). With respect to their life history, the scuticociliates and the hymenostomes share similar macrostome-microstome transformations in some species, with the microstome form feeding on bacteria and the macrostome form feeding on their smaller conspecifics (Corliss, 1973; Small et al., 1986). Scuticociliates and hymenostomes also have: a similar oral apparatus, typically with a dikinetid paroral and three oral polykinetids (Lynn, 2008); supraepiplasmic microtubules extending the length of the cell throughout their cell cycle (Lynn, 2008); the same pattern of somatic kinetids (Small and Lynn, 1981; Small and Lynn, 1985); and similar particle array patterns within the ciliary membranes (Bardele, 1981). Furthermore, phylogenetic studies based on SSU rRNA gene sequence data have revealed a group of loxocephalids, namely Paratetrahymena, Cardiostomatella and Dexiotrichides, that appear to be intermediate forms with affinities to both subclasses (Li et al., 2010; Yi et al., 2009; Zhang et al., 2010). In these studies, the genera Paratetrahymena and Cardiostomatella clustered together in a group that was basal within the Scuticociliatia clade, whereas the genus Dexiotrichides clustered with Urocentrum turbo forming a group that was more closely related to the subclass Hymenostomatia. Furthermore, based on morphological and morphogenetic data, the loxocephalids are most similar to the tetrahymenids, suggesting a close evolutionary relationship between these two groups. For example: (1) the oral ciliature of Cardiostomatella vermiformis is arranged in a typical Tetrahymena-like pattern, although it lacks both an apical plate and a scutica (Al-Rasheid, 2001; Li et al., 2006; Zhang et al., 2014); (2) Dexiotrichides pangi has a postoral intercalary kinety which is a typical tetrahymenid character (Song et al., 2005; Song et al., 2003); (3) Paratetrahymena wassi has a monoparakinetal pattern of morphogenesis, similar to that of the hymenostomes (Li et al., 2010). Thus, there is molecular, morphological and morphogenetic support for the placement of the above three taxa in an intermediate position between the Scuticociliatia and Hymenostomatia, further 14 / 25

supporting the close relationship between these two subclasses. There is growing evidence that the phylogenetic signal from SSU rRNA gene sequences does not always accurately reflect ciliate phylogeny (Evans et al., 2010; Feng et al., 2014; Gentekaki et al., 2014). Phylogenomic analyses that use omics data have generally improved the robustness of molecular phylogenetic reconstructions and untangled many previously debated phylogenies in various taxa (Brown et al., 2013; Burki et al., 2009; Burki et al., 2012; Burki et al., 2007; Feng et al., 2014). Phylogenetic analyses based on SSU rRNA gene sequences have usually revealed that the subclass Hymenostomatia is the most closely related to the Peritrichia (Li et al., 2006; Miao et al., 2004; Miao et al., 2001). However, the subclass Scuticociliatia was found to be more closely related to the Hymenostomatia than to either the Peniculia or the Peritrichia in analyses based on three concatenated rDNA gene sequences (Gao et al., 2013), casting further doubt on the reliability of SSU rRNA gene data alone for revealing phylogenetic relationships among the oligohymenophorean subclasses. Omics data are available for relatively few ciliate taxa (Abernathy et al., 2007; Aeschlimann et al., 2014; Aury et al., 2006; Gentekaki et al., 2014; Ricard et al., 2008; Swart et al., 2013) and no omics data of scuticociliates were available hitherto. In the present study, the transcriptomes of five ciliates, including two representatives (Pseudocohnilembus persalinus and Paralembus digitiformis) of the subclass Scuticociliatia, were sequenced and phylogenetically analysed with 24 other taxa. Our results robustly support that the monophyletic Scuticociliatia is sister to the monophyletic Hymenostomatia, and that these group together as a terminal branch within the monophyletic Oligohymenophorea. Furthermore, topology tests reject previously proposed postulations derived from SSU rRNA gene sequence data that the subclass Hymenostomatia is sister to Peritrichia, and that Scuticociliatia is basal to the rest of the Oligohymenophorea. It is also noteworthy that few saturated characters were detected in our aligned sequences (Fig. 2), however NJ analysis based on unsaturated characters of our data set also robustly supports the sister relationship between the Scuticociliatia and Hymenostomatia and their terminal position within 15 / 25

the oligohymenophorean assemblage (data not shown). Tetrahymena thermophila, a representative of the subclass Hymenostomatia, is a well-known model organism because: (1) it can be axenically cultured in the laboratory, growing rapidly to high densities in a variety of media; (2) its life cycle allows the use of conventional tools of genetic analyses, and; (3) molecular genetics approaches for sequence-enabled experimental analyses of gene function have been developed (Asai and Forney, 2000.; Turkewitz et al., 2002). Compared to T. thermophila, scuticociliates have received relatively little attention and studies on their molecular and cellular biology are lacking. However, some scuticociliates cause a variety of refractory or lethal diseases among aquaculture animals, and their management and control remains a challenging problem to the aquaculture industry (Harikrishnan et al., 2010; Jin et al., 2010; Munday et al., 1997). Thus, there is a pressing need to increase knowledge and understanding of the molecular and cellular biology of scuticociliates. In the present study, a close relationship between the subclasses Scuticociliatia and Hymenostomatia is revealed, opening the possibility of using T. thermophila as an experimental system to study the molecular and cellular biology of scuticociliates.

Acknowledgements The authors are indebted to Prof. Weibo Song (Ocean University of China), Prof. Kuidong Xu (Institute of Oceanology, Chinese Academy of Sciences), and Prof. Tuanyuan Shi (Zhejiang Academy of Agricultural Science) for kindly providing the Pseudocohnilembus persalinus, Paralembus digitiformis, and Colpoda aspera, respectively. This work is supported by the Natural Science Foundation of China (Project No. 31372168).

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version. 16 / 25

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Figure Captions

Fig. 1. Phylogenomic relationship of ciliates. Maximum likelihood tree topology from analysis of a phylogenomic matrix comprising 43,528 unambiguously aligned amino acid residues for 26 taxa inferred using RAxML software under an LG+F+Γ4 model, with two Plasmodium spp. as outgroups. New sequences are shown in bold. Numbers at nodes are ML bootstrap values followed by BI posterior probability. Bootstrap values from 100 replicates are given on the nodes. The scale bar corresponds to 0.1 expected substitutions per site.

Fig. 2. Saturation plot of the 113-gene data set. X-axis: the inferred number of substitutions between the same two sequences determined using a maximum likelihood method. Y-axis: the observed number of differences between pairs of species sequences. Each black dot represents a species-pair.

Fig. 3. A. Phylogenetic relationships within class Oligohymenophorea derived from phylogenomic tree and SSU rDNA tree. B. Additional stylized drawings or morphotypes of representatives of hymenostomes, scuticociliates, and the loxocephalids which are intermediate taxa between the hymenostomes and scuticociliates. Representative of the subclass Hymenostomatia - Tetrahymena - after Lynn (2008). Representative of the subclass Scuticociliatia - Pseudocohnilembus persalinus - after Song (2000a). Morphotypes of Cardiostomatella vermiformis after Wang (2007). Dotted box shows representatives of Tetrahymena and intermediate

22 / 25

taxa

that share

some similar

morphological characters.

Solid box shows

representatives of scuticociliates including the loxocephalids.

Table 1. Topology test based on the 113-gene data set. Tree topologies can be rejected

when

the

p-values

of

the

approximately

unbiased

(AU)

and

Shimodaira-Hasegawa (SH) tests are lower than 0.05. Rank

Phylogenetic hypothesis

likelihood

∆likelihood AU

SH

T0

ML tree

-847,636

0

1.000

1.000

T1

Hymenostomatia

to -848,302

666

7e-062

0

Subclass Scuticociliatia at the base -848,304

668

9e-006

0

sister

group

Peritrichia T2

of class Oligohymenophorea

23 / 25

100 /1.0

Tetrahymena thermophila

100 /1.0 Tetrahymena malaccensis 100 /1.0 100 /1.0

Tetrahymena elliotti

Hymenostomatia

Tetrahymena borealis Ichthyophthirius multifiliis

100 /1.0

Pseudocohnilembus persalinus

100 /1.0

Scuticociliatia

100 /1.0

Oligohymenophorea

Paralembus digitiformis Paramecium tetraurelia

96/0.96 100 /1.0

100 /1.0

Peniculia

Carchesium polypinum Campanella umbellaria

Peritrichia

Platyophrya macrostoma 100 /1.0 70 /0.58

Aristerostoma sp.

100 /1.0

Colpodea

Colpoda aspera Litonotus sp. 86 /0.99

Litostomatea

Euplotes harpa

100 /1.0

Euplotes focardii 100/1.0

Strombidinopsis acuminatum

78/0.90

Schmidingerella arcuata

94/1.0 100/1.0

Strombidium inclinatum

100/1.0

Spirotrichea

Pseudokeronopsis riccii 100 /1.0

Oxytricha trifallax

100 /1.0

Stylonychia lemnae

Protocruzia adherens Condylostoma magnum 100/1.0

Plasmodium yoelii Apicomplexa Plasmodium falciparum

0.1

Postciliodesmatophora

Uncorrected pairwise distance

1.8 1.6 1.4 1.2 1.0 0.8 y = 0.4085x 0.6 0.4 0.2 0.0 0

0.2

0.4

0.6

0.8

1

Patristic distance

1.2

1.4

1.6

1.8

Graphical abstract 100 /1.0 100 /1.0 100 /1.0 100 /1.0 100 /1.0

Scuticociliatia

Oligohymenophorea

Peniculia 100 /1.0

100 /1.0

70/0.58 78/0.90

96/0.96

Hymenostomatia

Peritrichia

Colpodea Litostomatea

86 /0.99 100/1.0 Protocruzia adherens

Spirotrichea

Condylostoma magnum Postciliodesmatophora 100/1.0

Apicomplexa

0.1

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Highlights 1. We obtained five newly sequenced transcriptome data of ciliates. 2. Phylogenomic analyses revealed that Scuticociliatia is sister to Hymenostomatia. 3. T. thermophila might be used as an experimental system to study scuticociliates.

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