Phylogenetic framework of the systematically confused Anteholosticha–Holosticha complex (Ciliophora, Hypotrichia) based on multigene analysis

Molecular Phylogenetics and Evolution 91 (2015) 238–247

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Phylogenetic framework of the systematically confused Anteholosticha– Holosticha complex (Ciliophora, Hypotrichia) based on multigene analysis q Xiaolu Zhao a,b,1, Shan Gao b,1, Yangbo Fan b, Michaela Strueder-Kypke c, Jie Huang a,b,⇑ a b c

Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

a r t i c l e

i n f o

Article history: Received 9 February 2015 Accepted 27 May 2015 Available online 4 June 2015 Keywords: Anteholosticha Arcuseries Holosticha Molecular phylogeny Multigene analysis

a b s t r a c t The Anteholosticha–Holosticha complex is an extremely divergent group within the urostylids, especially because the genus characterization lacks suitable synapomorphies. Previous studies have shown that morphological classification of species within this group often conflicts with SSU-rDNA data, that is this complex is not recovered as a monophyletic group and Anteholosticha spp. are widely dispersed throughout the urostylid assemblage in SSU-rDNA trees. In this study, we provided 38 new sequences (including the type species of Anteholosticha) of SSU-rDNA, ITS1-5.8S-ITS2 and LSU-rDNA genes to infer molecular phylogenies of all available taxa in the Anteholosticha–Holosticha complex. The results show that: (1) Holosticha is monophyletic in all trees, suggesting it is a well-defined genus; (2) Anteholosticha is polyphyletic and distinctly separated from Holosticha in all single-gene based and concatenated phylogenies; (3) the monophyly of Arcuseries, a recently established genus split from Anteholosticha, is strongly supported by all molecular data; (4) Anteholosticha multicirrata, Anteholosticha manca, Anteholosticha paramanca and Bakuella subtropica may share a most recent common ancestor; (5) multi-gene analyses receive higher support values than the single-gene analyses. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction The subclass Hypotrichia is characterized by the highly developed ciliature pattern, a diverse morphology and a very complicated division process (Adl et al., 2012; Berger, 2006, 2008; Corliss, 1979; Jiang et al., 2013; Lynn, 2008; Song et al., 2009; Vdˇacˇny´ et al., 2014). They inhabit wide range of marine, estuarine and fresh waters, sometimes with great abundance and biomass. Although numerous studies have been performed on the assignments and phylogenetic relationships of species within this group (Berger, 2006, 2008; Chen et al., 2013a; Corliss, 1979; Fan et al., 2013; Foissner and Stoeck, 2011; Gao et al., 2010, 2013; Kim et al., 2013; Lynn, 2008; Pan et al., 2013; de Puytorac, 1994; Singh and Kamra, 2013), incongruences between morphological and gene sequence data have always existed especially in q

This paper was edited by the Associate Editor J.B. Dacks.

⇑ Corresponding authors at: Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China (J. Huang). Fax: +86 27 68780816. E-mail address: [email protected] (J. Huang). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.ympev.2015.05.021 1055-7903/Ó 2015 Elsevier Inc. All rights reserved.

morphologically diverse taxa (Gentekaki et al., 2014; Huang et al., 2012; Lahr et al., 2013; Li et al., 2013; Moreira et al., 2007; Simpson et al., 2006; Zhang et al., 2014). According to Lynn’s system (2008), the order Urostylida comprises four families (Pseudokeronopsidae, Pseudourostylidae, Urostylidae, Epiclintidae). Yi et al. (2008) proposed a fifth family, Psammomitridae. Urostylida is considered as one of the most confounded groups of hypotrichs due to the lack of some important morphogenetic data and the convergent evolution of some diagnostic morphological features (Berger, 2006; Liu et al., 2010). Thus, the classification of the urostylids is difficult in practice, and phylogenetic relationships within this group are largely unresolved. The genus Holosticha Wrzes´niowski, 1877 originally included all hypotrichs with three frontal cirri, transverse cirri, and a midventral complex composed of cirri arranged in a zigzag pattern (Borror, 1972; Borror and Wicklow, 1983; Kahl, 1932). Following a detailed revision by Berger (2003), most species were transferred out of Holosticha and were tentatively assigned either to Caudiholosticha (with caudal cirri) or Anteholosticha (without caudal cirri). Berger (2003, 2006) inferred, however, that neither of

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these genera are monophyletic due to the lack of morphological synapomorphies. To date, about 40 species have been assigned to Anteholosticha, and new species are still being characterized and classified into this genus (Fan et al., 2014a; Park et al., 2013). The morphological hypothesis that Anteholosticha is not a monophyletic genus has been constantly supported by small subunit ribosomal RNA (SSU-rRNA) gene phylogenies (e.g., Chen et al., 2010; Park et al., 2013; Shao et al., 2011; Xu et al., 2011). As a first separation of the speciose genus Anteholosticha, three species (A. scutellum, A. petzi, A. warreni) with a roughly U-shaped pattern of transverse cirri, were transferred to a new genus Arcuseries by Huang et al. (2014). However, there are still six phylogenetically distinct branches with Anteholosticha species in SSU-rDNA trees (Fan et al., 2014a,b; Park et al., 2012, 2013), indicating that further splitting is needed. In order to infer more robust phylogenetic relationships, concatenated phylogenetic analyses of multiple genes have recently been used in different groups of ciliates and other protists (Gao et al., 2013; Gentekaki et al., 2014; Huang et al., 2012; Lahr et al., 2013; Li et al., 2013; Moreira et al., 2007; Simpson et al., 2006; Zhang et al., 2014). In the present study, we increased taxon representatives and sequenced gene markers other than the SSU-rDNA in order to perform more comprehensive phylogenetic analyses of the Anteholosticha–Holosticha complex and of the order Urostylida. In all, 38 new sequences of SSU-rDNA, ITS1-5.8S-ITS2 and LSU-rDNA genes from 19 species were characterized. The objectives of our study were: (1) to test previously proposed taxonomic classifications of urostylid species with data from three gene markers; (2) to determine the phylogenetic position of the genus Anteholosticha by characterizing the multigene data of its type species; (3) to test the validity of the recently established genus Arcuseries using additional molecular data; (4) to enhance our understanding of the evolutionary relationships within the order Urostylida with emphasis on the Anteholosticha–Holosticha complex. 2. Materials and methods 2.1. Ciliate collection and identification Nineteen species were collected and investigated in this study. The sampling information of each species and newly obtained GenBank Accession Numbers in this study are shown in Table 1. For species that can be cultured in the laboratory, several cells were isolated and cultured at room temperature in Petri dishes

with sterile filtered marine or fresh water and rice grains. For those species that could not be cultured, cells of the target species were isolated from the sample under the stereomicroscope and then were washed several times with sterile filtered marine water. Species identification was based on live observations and protargol-stained specimens (Chen et al., 2011b). General terminology and systematic classification are mainly according to Berger (2006) and Lynn (2008). 2.2. DNA extraction, PCR amplification and sequencing Genomic DNA was extracted from cleaned cells using the REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, USA) modified by Gong et al. (2007) or the DNeasy Blood & Tissue Kit (Qiagen, CA) with the modification that only 1/4 of the suggested volume for each solution was used. The PCR amplifications of SSU-rDNA, ITS1-5.8S-ITS2, and LSU-rDNA were performed using the TaKaRa ExTaq DNA Polymerase Kit (TaKaRa Biomedicals, Japan). PCR conditions and primers are used as previously described (Huang et al., 2014). Purified PCR product of the appropriate size was then inserted into the pMD™19-T vector (Takara Biotechnology, Dalian Co., Ltd.) and transformed into E. coli DH 5a cells. Genes were sequenced in both directions on an ABI-PRISM 3730 automatic sequencer (Applied Biosystems). The universal primers of the pMD™19-T vector (Code No. 6013, Takara Bio Inc.): M13-47 (50 -C GCCAGGGTTTTCCCAGTCACGAC-30 ) and RV-M (50 -GAGCGGATAAC AATTTCACACAGG-30 ) were used for sequencing. The internal sequencing primers were 900F (50 -CGATCAGATACCGTCCTAGT-30 ), 900R (50 -ACTAGGACGGTATCTGATCG-30 ) for SSU-rDNA and F2 (50 GGAGTGTGTAACAACTCACCTGC-30 ), R3 (50 -CATTCGGCAGGTGAGTT GTTACAC-30 ) for LSU-rDNA. 2.3. Datasets and alignments Four datasets (Table 2) based on SSU-rDNA, ITS1-5.8S-ITS2, LSU-rDNA and concatenated data were used to evaluate the phylogenetic relationships of the Anteholosticha–Holosticha complex. In total, 38 new sequences were determined, and other sequences were downloaded from the National Center for Biotechnology Information (NCBI) Database. Concatenation of the three genes was performed in SeaView v4 (Galtier et al., 1996; Gouy et al., 2010). Sequences were aligned on the web server Phylogeny. Fr (URL: http://www.phylogeny.fr) using MUSCLE as the alignment algorithm (Dereeper et al., 2008, 2010; Edgar, 2004) and the

Table 1 Species identified and newly sequenced in the present study. Species

Anteholosticha gracilis pop2 Anteholosticha manca Anteholosticha marimonilata pop1 Anteholosticha marimonilata pop2 Anteholosticha monilata Anteholosticha paramanca Anteholosticha pulchra pop2 Anteholosticha sp. Apoholosticha sinica Arcuseries petzi pop2 Arcuseries petzi pop3 Arcuseries warreni Heterokeronopsis pulchra Holosticha bradburyae pop1 Holosticha bradburyae pop2 Holosticha diademata pop1 Holosticha diademata pop2 Holosticha cf. heterofoissneri Psammomitra retractilis

Sampling location

Jiaozhou Bay, Qingdao, northern China Jiaozhou Bay, Qingdao, northern China Mollusc-farming waters of the Yellow Sea, Qingdao, northern China Jiaozhou Bay, Qingdao, northern China Aquaculture farm of Qinling Mountains, Xi0 an Mangrove wetland, Shenzhen, southern China Jiaozhou Bay, Qingdao, northern China Jiaozhou Bay, Qingdao, northern China Mangrove wetland, Shenzhen, southern China Jiaozhou Bay, Qingdao, northern China Jiaozhou Bay, Qingdao, northern China Jiaozhou Bay, Qingdao, northern China Mangrove wetland, Shenzhen, southern China Jiaozhou Bay, Qingdao, northern China Qingshui Bay, Hong Kong Mangrove wetland, Shenzhen, southern China Mangrove wetland, Shenzhen, southern China Daya Bay, Huizhou, southern China Jiaozhou Bay, Qingdao, northern China

Accession numbers SSU

LSU

ITS-5.8S

KF306397 – – – – – KF306393 – – KF306398 KF306394 – – – – – KF306396 – –

KF306413 KF306399 KF306408 KF306409 KJ697762 KF306415 KF306401 KF306402 KJ697763 KF306414 KF306406 KF306410 KJ697763 KF306403 KF306400 KF306412 KF306411 KF306404 KF306405

KF306392 – KF306387 KF306388 KJ697762 KF306389 KF306382 KF306383 KJ697763 – KF306385 KF306390 KJ697763 – KF306381 – KF306391 KF306384 –

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Table 2 Dataset assembled in the present work. Dataset

Description

1

SSU-rDNA sequences including all available species of the Anteholosticha–Holosticha complex; 71 taxa with 5 new sequences LSU-rDNA sequences including all available species of the Anteholosticha–Holosticha complex; 49 taxa with 19 new sequences ITS1-5.8S-ITS2 sequences including all available species of the Anteholosticha–Holosticha complex; 52 taxa with 14 new sequences Concatenation of SSU-rDNA, ITS1-5.8S-ITS2, and LSU-rDNA sequences including all taxa in Dataset 2; 49 taxa with 38 new sequences

2 3 4

default parameters. Both ends of the alignments were trimmed before further analysis. The final alignments (available from the authors upon request) of the four datasets for subsequent phylogenetic analyses were as follows: 1767 characters for Dataset 1 (SSU-rDNA), 560 characters for Dataset 2 (ITS1-5.8S-ITS2), 1882 characters for Dataset 3 (LSU-rDNA), and 4209 characters for Dataset 4 (Three-gene combined). Sequence similarities of the three molecular markers (SSU-rDNA, ITS1-5.8S-ITS2, partial LSU-rDNA) within and between the genera Anteholosticha and Holosticha were performed using the Sequence Identity Matrix in BioEdit 7.0.9 (Hall, 1999). 2.4. Phylogenetic analyses Phylogenetic trees were constructed using each of the above four datasets. Four species (Uronychia multicirrus, Apodiophrys ovalis, Paradiophrys zhangi, Diophrys scutum) in Uronychiidae were chosen as outgroup taxa. Maximum Likelihood (ML) analyses, with 1000 bootstrap replicates to estimate the reliability of internal branches, were carried out using RAxML-HPC2 on XSEDE v 7.6.3 (Stamatakis, 2006; Stamatakis et al., 2008) with the GTRGAMMA model provided on the online server CIPRES Science Gateway (Miller et al., 2010). Bayesian Inference (BI) analysis was performed using Mrbayes on XSEDE v 3.2.2 (Ronquist and Huelsenbeck, 2003) on CIPRES Science Gateway with the best-fit model GTR+I+G selected by Akaike Information Criterion (AIC) in MrModeltest v2 (Nylander, 2004). Markov chain Monte Carlo (MCMC) simulations were then run with two sets of four chains using the default settings: chain length of 1,000,000 generations and trees sampled every 100 generation and the first 25% of sampled trees were discarded as burn-in. The remaining trees were used to calculate the posterior probabilities (PP) with a majority rule consensus. Tree topologies were visualized with MEGA 5 (Tamura et al., 2011). 2.5. Topology testing To assess the possibility of morphologically-based taxa as monophyletic groups, ML trees with enforced topological constraints (Table 3) were generated using RAxML (Stamatakis et al., 2008). Internal relationships within the constrained group and among the remaining taxa were unspecified. The resulting constrained topologies were then compared to the unconstrained ML topologies using the Approximately Unbiased (AU) test (Shimodaira, 2002) implemented in CONSEL v 0.1 (Shimodaira and Hasegawa, 2001). 3. Results 3.1. Sequence analyses In this study, we newly characterized five SSU-rDNA sequences, 19 LSU-rDNA sequences and 14 ITS1-5.8S-ITS2 sequences from 19

Table 3 Approximately unbiased test results. Significant differences (P-value < 0.05) between the best maximum likelihood trees and the best constrained topologies are shown in bold. Datasets

Topology constraints

-Ln likelihood

AU (p)

SSU-rDNA

Unconstrained Anteholosticha + Holosticha Anteholosticha + Arcuseries Anteholosticha

13170.59 13652.70 13640.19 13618.28

0.962 2e078 3e068 0.001

ITS1-5.8S-ITS2

Unconstrained Anteholosticha + Holosticha Anteholosticha + Arcuseries Anteholosticha

8837.04 9086.82 9088.55 9066.42

0.961 4e005 5e055 2e006

LSU-rDNA

Unconstrained Anteholosticha + Holosticha Anteholosticha + Arcuseries Anteholosticha

18798.70 19484.08 19482.54 19370.38

0.840 3e006 6e005 1e004

Concatenated three genes

Unconstrained Anteholosticha + Holosticha Anteholosticha + Arcuseries Anteholosticha

38846.12 40224.68 40220.58 40054.34

0.926 1e007 2e072 9e045

species, including data from the type species Anteholosticha monilata (Table 1). Sequence similarities of these three marker genes were compared within and between Anteholosticha and Holosticha, respectively (Supplementary Tables 1–3). On average, the SSU-rRNA gene is the most conserved among the three markers and species within the genus Holosticha always obtain the highest similarity for all the three genes.

3.2. SSU-rDNA phylogeny (Dataset 1, Table 2, and Fig.1) Phylogenetic trees using two different methods (ML and BI) generated nearly congruent relationships. We therefore present only the ML topology with support values from both algorithms at the nodes. As shown in Fig.1, the basal position of the family Pseudoamphisiellidae to the urostylid-oxytrichid clade is strongly supported in the SSU-rDNA tree (96% ML, 1.00 BI). Within the urostylid-oxytrichid clade, the well-supported cluster of Holosticha and Psammomitra (97% ML, 1.00 BI) occupies the basal position, followed by the recently established genus Arcuseries (Huang et al., 2014). The order Urostylida is not monophyletic, since species of the family Oxytrichidae nest within it and form a sister group to the remaining urostylids with high support values (81% ML, 1.00 BI). The monophyly of Holosticha is fully supported (100% ML, 1.00 BI) while species in Anteholosticha are widely dispersed among the urostylids, clustering in several divergent subgroups in the SSU-rDNA tree (marked as A–F, Fig. 1). The recently described species Anteholosticha paramanca, A. multicirrata and Bakuella subtropica, group together with A. manca (subgroup A), although the support values are very low (55% ML, 0.65 BI). This cluster groups with Neobakuella, Apobakuella and Diaxonella with full support values (100% ML, 1.00 BI). Interestingly, Bakuella subtropica nests within three Anteholosticha species, closely related to A. paramanca, and this relationship is highly supported (92% ML, 1.00 BI). Two populations of Anteholosticha gracilis (subgroup B) are placed in a fully supported clade consisting of Monocoronella carnea, Neourostylopsis flavicana and Bergeriella ovata, while the internal relationships within this clade are very poorly resolved (i.e., 61% ML, 0.55 BI). Anteholosticha pulchra (subgroup C) is placed within the family Pseudokeronopsidae, and basal to this branch is a fully supported cluster of two Anteholosticha species (A. pseudomonilata and A. marimonilata) (subgroup D). The type species Anteholosticha

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Fig. 1. Maximum likelihood (ML) tree inferred from SSU-rRNA gene sequences of representative taxa and species newly sequenced (bold) in this study (71 taxa and 1767 nucleotide characters). Numbers at nodes represent the bootstrap values of ML and the posterior probabilities of Bayesian analysis (BI), respectively. Fully supported branches are marked with solid circles at the nodes. ‘‘–’’ indicates the disagreement between BI tree and the reference ML tree. The scale bar corresponds to two substitutions per 100 nucleotide positions. A–F represents the different subgroups of Anteholosticha species.

monilata (subgroup E) shows a close relationship to Pseudourostyla cristata (92% ML, 1.00 BI). Moreover, the basal position of Anteholosticha multistilata (subgroup F) to the well-supported core group of urostylids (referred to as ‘‘core urostylids’’) is highly supported by both ML and BI analyses (90% ML, 1.00 BI). 3.3. LSU-rDNA (Dataset 2, Table 2, Fig. 2) and ITS1-5.8S-ITS2 (Dataset 3, Table 2, Fig. 3) phylogeny In this study, we added 14 new sequences of ITS1-5.8S-ITS2 and 19 of LSU-rDNA, expanding the total taxa in our phylogenetic analyses to 52 and 49, respectively. Analyses inferred from LSU-rDNA sequences showed similar topologies to that of the SSU-rDNA tree.

The sister relationship between oxytrichids and the core group of urostylids is well supported by BI analysis (0.91), but only poorly supported by ML analysis (66%). Basal to this large group is a strongly supported monophyletic cluster of Arcuseries (99% ML, 1.00 BI). Similarly, the close relationship of Holosticha and Psammomitra received maximal support (100% ML, 1.00 BI), occupying the basal position of the in-group in the phylogenetic tree. The position of Anteholosticha pulchra differed from that in the SSU-rDNA tree: it clusters with the Nothoholosticha-Apoholosticha clade instead of the Pseudokeronopsis-Uroleptopsis clade. However, this relationship may vary when more LSU-rDNA data are available as the support values from both methods are poor (47% ML, 0.58 BI).

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Fig. 2. Maximum likelihood (ML) tree inferred from partial LSU-rDNA gene sequences of representative taxa and species newly sequenced (bold) in this study (49 taxa and 1882 nucleotide characters). Numbers at nodes represent the bootstrap values of ML and the posterior probabilities of Bayesian analysis (BI), respectively. Fully supported branches are marked with solid circles at the nodes. ‘‘–’’ indicates the disagreement between BI tree and the reference ML tree. The scale bar corresponds to five substitutions per 100 nucleotide positions. A–E represents the different subgroups of Anteholosticha species.

The major difference between the ITS1-5.8S-ITS2 tree and the other topologies is that the Oxytrichidae clade groups with Arcuseries, Holosticha and Psammomitra instead of clustering with the core urostylids, although the support values from the two analyses are variable (55% ML, 0.93 BI). The second incongruence is that the type species, Anteholosticha monilata, is closely related to A. marimonilata although this is only poorly supported (65% ML, 0.71 BI). Moreover, the groupings of different populations of the same species are all fully supported while node supports of different genera are relatively low. 3.4. Concatenated phylogenetic analyses (Dataset 4, Table 2, Fig. 4) The phylogenetic tree based on the concatenated dataset of all three genes share a highly similar topology with the LSU-rDNA tree. Overall, the concatenated tree displays higher support values (all nodes but six show over 90% ML bootstrap support) across the tree than any of the trees of the single gene analyses. As also revealed in the LSU-rDNA tree, analyses inferred from concatenated data indicate: (1) Holosticha is monophyletic, clearly separated from species of Anteholosticha and closely related to Psammomitra; (2) the monophyly of Arcuseries, separated from

Anteholosticha, is fully supported; (3) the genus Anteholosticha is polyphyletic and its species are distributed all over the core urostylids clade; (4) the type species Anteholosticha monilata has a close relationship to Pseudourostyla cristata. 3.5. Hypothesis testing (Table 3) Statistical tests (AU tests) were carried out on all single genes and the concatenated datasets to assess the hypotheses of morphologically-based taxa as monophyletic lineages (i.e., Anteholosticha, Anteholosticha–Holosticha and Anteholosticha– Arcuseries). At the 5% significance level, the hypothesis that Anteholosticha and Holosticha form a monophyletic group was rejected by all datasets. The hypothesized monophyly of the genus Anteholosticha and the forced grouping of Anteholosticha and Arcuseries were also rejected. The rejection of the monophyly of Anteholosticha suggests that this genus needs to be further split. 4. Discussion In the current study, we provide comprehensive tree constructions based on SSU-rDNA, ITS1-5.8S-ITS2, LSU-rDNA, and

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Fig. 3. Maximum likelihood (ML) tree inferred from ITS1-5.8S-ITS2 sequences of representative taxa and species newly sequenced (bold) in this study (52 taxa and 560 nucleotide characters). Numbers at nodes represent the bootstrap values of ML and the posterior probabilities of Bayesian analysis (BI), respectively. Fully supported branches are marked with solid circles at the nodes. ‘‘–’’ indicates the disagreement between BI tree and the reference ML tree. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. A–D represents the different subgroups of Anteholosticha species.

concatenated data, respectively, including all available sequences of species in the Anteholosticha–Holosticha complex. We perform single gene and concatenated analyses to test whether the polyphyly of Anteholosticha is also supported by other marker genes. We also test the validity of the previously proposed genus Arcuseries using additional molecular data. In addition, the characterization of multiple genes of the type species of the genus Anteholosticha (A. monilata) enables us to determine the phylogenetic position of Anteholosticha. The SSU-rDNA tree (Fig. 1) constructed in this work is consistent with those published in previous studies (Park et al., 2013; Shao et al., 2011; Xu et al., 2011) and shows that Holosticha has a close relationship with Psammomitra and that species assigned to the genus Anteholosticha are distributed among several clades within the core urostylids assemblage. Our multigene analyses also confirm the polyphyly of Anteholosticha with high support values, which strengthens the morphological hypothesis that the diagnosis of Anteholosticha is only a combination of plesiomorphies, lacking good synapomorphies (Berger, 2003). The distinct phylogenetic positions of Anteholosticha species demonstrate that they will

require additional research and possible re-assignment to other, or even new, genera. 4.1. Phylogenetic position of Holosticha The monophyly of the genus Holosticha is well supported in all the phylogenetic trees (Figs 1–4), confirming the monophyletic status based on morphology (Berger, 2003, 2006): the distinctly rightwards curved left marginal row and the widened proximalmost adoral membranelles are good synapormophies to unify this genus, strongly supporting the redefined diagnosis of Holosticha by Berger (2003). Based on the available gene sequence data, Holosticha is distinctly separated from its morphologically similar genus Anteholosticha in previous (Fan et al., 2014a,b; Huang et al., 2014) and in our well-sampled phylogenetic trees (Figs 1–4). This separation is further supported both by the low sequence similarities between species of these two genera and by the AU test results (Table 3 and Supplementary Tables 1–3), which confirms that the shared morphological characters of Holosticha and Anteholosticha are plesiomorphies (e.g., the three

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Fig. 4. Maximum likelihood (ML) tree inferred from concatenated three genes (SSU-ITS1-5.8S-ITS2-LSU) of representative taxa and species newly sequenced (bold) in this study (49 taxa and 4209 nucleotide characters). Numbers at nodes represent the bootstrap values of ML and the posterior probabilities of Bayesian analysis (BI), respectively. Fully supported branches are marked with solid circles at the nodes. The scale bar corresponds to five substitutions per 100 nucleotide positions. A–E represents the different subgroups of Anteholosticha species.

enlarged frontal cirri and the presence of frontoterminal cirri) or derived through convergent evolution (e.g., midventral complex composed of midventral pairs only), which was discussed in detail by Berger (2006). In Berger’s system (2006), Holosticha and Psammomitra are placed within the Holostichidae because their midventral complex is composed of cirral pairs only and by the presence of three enlarged frontal cirri. Moreover, Psammomitra has been separated from Holostichidae and assigned to a new family, Psammomitridae, since it displays an extremely contractile, elongated tripartite body and distinctly shortened midventral rows (Yi et al., 2009). In accordance with previous studies (Huang et al., 2014; Yi and Song, 2011), all phylogenetic trees in this work show that Psammomitra is separated by a relatively long branch from its closest relative, Holosticha. The highly supported cluster of Holosticha and Psammomitra is located outside of the oxytrichid-core urostylids clade in all trees except for the ITS1-5.8S-ITS2 tree. Considering the morphological similarities and the close phylogenetic relationship of these two genera, we suppose that they share a most recent common ancestor. In addition, the deeply branched phylogenetic position of Holosticha indicates that members of Holostichidae (sensu Berger, 2006) such as Diaxonella, Pseudoamphisiella and Anteholosticha should be transferred out of this family.

4.2. Concatenated analyses strongly support the establishment of Arcuseries Arcuseries is a recently established genus which includes those species previously assigned to Anteholosticha that have a U-shaped or bow-shaped pattern of the transverse cirri (Huang et al., 2014). Arcuseries currently comprises three species, namely A. petzi, A. scutellum and A. warreni. The SSU-rDNA sequence of A. warreni was not sampled in the previously constructed tree (Huang et al., 2014). In the present study, we have added this SSU-rDNA sequence as well as multiple gene sequences of A. warreni and A. petzi (Fig. 4) in order to verify their taxonomic assignment. All analyses strongly support the monophyly of Arcuseries and its separation from Anteholosticha and other urostylids. This relationship was also supported by the AU tests, which rejected the hypothesis that Anteholosticha and Arcuseries form a monophyletic lineage (Table 3). 4.3. Subgroup A of Anteholosticha and Bakuella Bakuella subtropica nests among three Anteholosticha species, namely A. multicirrata, A. manca and A. paramanca, however this clade is poorly supported in the SSU-rDNA tree (Fig. 1). Within this clade, Bakuella groups with A. paramanca with high support rather

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than clustering with the morphologically similar genera Neobakuella and Apobakuella. This raises the question whether B. subtropica is likely to be a congener of A. paramanca. It is noteworthy that the main morphological feature separating B. subtropica from Anteholosticha is the presence of one or two midventral rows which comprise only three to five cirri (Chen et al., 2013b). Interestingly, the short midventral row may also be present in A. manca (Berger, 2006) which, to some extent, indicates that the short midventral row may not be a valid character to separate taxa at genus level. Since only one species with atypical short midventral row is known in Bakuella, and LSU-rDNA and ITS1-5.8S-ITS2 data are lacking, we refrain from transferring B. subtropica to Anteholosticha in this study. Multigene analyses including more sequences are needed to confirm its phylogenetic position and facilitate our understanding on the evolution of the midventral row. In the absence of LSU-rDNA and ITS1-5.8S-ITS2 sequence data for Anteholosticha multicirrata and Bakuella subtropica, only two species (A. manca and A. paramanca) represent subgroup A in all trees except the SSU-rDNA tree (Figs 2–4). The well-supported clade of A. manca and A. paramanca was clearly separated from other Anteholosticha species in both ML and BI analyses (Figs 2– 4). The four species of subgroup A are similar in terms of body shape and size, the presence of numerous macronuclear nodules, the distinctly shortened midventral complex and the brackish/ marine habitat, thus supporting the separation from their congeners in the SSU-rDNA phylogenetic tree. In addition, from the morphogenetic point of view, these four species share the feature that the parental adoral zone of membranelles is renewed completely by the primordium. These findings suggest that the members of subgroup A may share a most recent common ancestor. However, given that the support values of this group are not high in SSU-rDNA tree, and that LSU-rDNA and ITS1-5.8S-ITS2 data are available for only two of its members, it is too early to determine whether they are congeners. Thus, phylogenetic analyses based on multi-gene data with more taxa are needed to provide a more robust relationship for this group. 4.4. Phylogenetic positions of other Anteholosticha species (subgroups B to F) Hitherto the phylogenetic position of Anteholosticha gracilis (subgroup B) has remained unresolved as it clustered with Monocoronella carnea with poor ML bootstrap values and variable posterior probabilities in previous SSU-rDNA and ITS1-5.8S-ITS2 trees (Chen et al., 2011a; Huang et al., 2014). In the present study, we obtained another population of A. gracilis, which enabled us to perform phylogenetic analyses based on LSU-rDNA data. The grouping of A. gracilis with Monocoronella is maximally supported both in the LSU-rDNA tree and in the concatenated phylogeny. Morphologically, however, these two species show quite different ciliary patterns. For example, A. gracilis has four frontal cirri whereas M. carnea has five to seven frontal cirri forming an arched row (Chen et al., 2011a). Consequently, their apparent close relationship in gene trees might be due to the poor taxon sampling, given that molecular data are available for less than 10% of known hypotrich species. Anteholosticha pulchra (subgroup C), groups within the family Pseudokeronopsidae, clustering with Pseudokeronopsis and Uroleptopsis with varying support values in SSU-rDNA and ITS1-5.8S-ITS2 trees, whereas it groups with another clade of the Pseudokeronopsidae (Nothoholosticha–Heterokeronopsis– Apoholosticha) in the LSU-rDNA tree, albeit with low support. Although the phylogenetic position of A. pulchra is not stable within the Pseudokeronopsidae, this placement either challenges the monophyly of Pseudokeronopsidae in all phylogenetic trees

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(Figs 1–4), or the genus assignment of A. pulchra. Morphologically, A. pulchra differs from Pseudokeronopsis rubra mainly in details of the cortical granulation according to Kahl (1932) and then was transferred to Pseudokeronopsis by Borror and Wicklow (1983). However, this was not accepted by Berger (2006) because of the obvious difference in their frontal cirri pattern (four frontal cirri vs. bicorona). Nevertheless, it seems reasonable to remove both A. pulchra and A. gracilis from the genus Anteholosticha as they both have four frontal cirri whereas most other Anteholosticha spp. only have three (Berger, 2006). Furthermore, the inferred phylogenetic relationships may be artifacts caused by taxon undersampling in the molecular trees. Thus, further research of this group, including more taxa with multi-gene data, are needed in order to determine the correct generic identity of putative species of Anteholosticha. Anteholosticha marimonilata and A. pseudomonilata, which were recently described by Xu et al. (2011) and Li et al. (2011) respectively, form a fully supported subgroup (D), a sister clade to the Pseudokeronopsidae in our SSU-rDNA tree. Morphologically, these two species resemble each other in several features (e.g. body shape, position of contractile vacuole, ciliary arrangement), supporting their close relationship. Likewise, A. marimonilata occupies a similar position both in the LSU-rDNA and the concatenated trees whereas it groups with A. monilata (subgroup E), the type species of Anteholosticha, in the ITS1-5.8S-ITS2 tree. In all other phylogenetic trees, A. monilata is closely related to Pseudourostyla cristata with strong support values. However, A. monilata is clearly distinguishable from Pseudourostyla in the arrangement of its frontal cirri (3-4 enlarged FC vs. bicorona) and the number of marginal rows (one LMR and RMR vs. more than one LMR and RMR). Morphogenetically, A. monilata also differs from Pseudourostyla by only partial replacement of the parental adoral zone (only the proximal portion of the parental adoral zone being replaced vs. completely replaced). Given their morphological and morphogenetic differences, the apparent close phylogenetic relationship of A. monilata and Pseudourostyla is very likely an artifact resulting from undersampling of relevant taxa in gene trees. Thus, it would be wiser to await a more comprehensive concatenated phylogeny before drawing any conclusions as to the correct position of A. monilata and other members of subgroup E. Anteholosticha multistilata, originally described under the name Holosticha multistilata (Kahl, 1928), was transferred to Anteholosticha by Berger (2003). Shin et al. (2000) identified a population with four frontal cirri as ‘‘Holosticha multistylata’’ sensu Shin and Kim (1993) and sequenced the SSU-rRNA gene (GenBank Accession No. AJ277876). However, this species was classified as Anteholosticha intermedia in Berger’s monograph (2006) since Anteholosticha multistilata has a high number (8–10) of enlarged frontal cirri based on the original description. Thus, the SSU-rDNA sequence of ‘‘Holosticha multistylata’’ submitted to NCBI (GenBank Accession No. AJ277876, subgroup F, Fig. 1) actually belongs to Anteholosticha intermedia. Moreover, ‘‘multistilata’’ was misspelled as ‘‘multistylata’’ in GenBank. Consequently, we chose to use the name Anteholosticha intermedia to refer to this sequence in our SSU-rDNA tree. It branches off first among all Anteholosticha spp. represented in our SSU-rDNA tree, however, its phylogenetic position was unstable, varying according to which taxa are included in the analysis (Huang et al., 2014). Due to the great systematic problems with A. intermedia (for further details, see Berger, 2006), and the unavailability of the multigene data, we have to await sequences of additional genes of this taxon in order to determine its correct phylogenetic position. In conclusion, we provide 38 new sequences of SSU-rDNA, ITS1-5.8S-ITS2 and LSU-rDNA of 19 urostylids in this study, which enabled us to perform phylogenetic analyses of the Anteholosticha– Holosticha complex and the order Urostylida based on expanded

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datasets. For the first time, multigene sequences of the type species of Anteholosticha, A. monilata, are used to determine the phylogenetic position of the genus Anteholosticha. However, additional taxon sampling is needed to perform more comprehensive and robust concatenated analyses, which will provide further insights into the phylogeny and reclassification of species in the Anteholosticha–Holosticha complex. Acknowledgements The work is supported by the Natural Science Foundation of China (Project Nos. 31430077, 41406135, 31401963) and International Research Coordination Network for Biodiversity of Ciliates from NSF China (31111120437). We are grateful to Prof. Weibo Song, Dr. Yan Zhao, Ms. Jiamei Li and An Liu, colleagues in Ocean University of China, for their kind help with species identification and gene sequencing. We gratefully acknowledge Dr. Alan Warren of Natural History Museum for his suggestions and help to improve this manuscript. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2015.05. 021. References Adl, S., Simpson, A., Lane, C., Lukeš, J., Bass, D., Bowser, S., Brown, M., Burki, F., Dunthorn, M., Hampl, V., Heiss, A., Hoppenrath, M., Lara, E., Gall, L., Lynn, D., Mcmanus, H., Mitchell, E., Mozley-Stanridge, S., Parfrey, L., Pawlowski, J., Rueckert, S., Shadwick, L., Schoch, C., Smirnov, A., Spiegel, F., 2012. The revised classification of eukaryotes. J. Eukaryot. Microbiol. 59, 429–493. Berger, H., 2003. Redefinition of Holosticha Wrzesniowski, 1877 (Ciliophora, Hypotricha). Eur. J. Protistol. 39, 373–379. Berger, H., 2006. Monograph of the Urostyloidea (Ciliophora, Hypotricha). Monogr. Biol. 85 (1–1304), i–xvi. Berger, H., 2008. Monograph of the Amphisiellidae and Trachelostylidae (Ciliophora, Hypotricha). Monogr. Biol. 88 (1–737), i–xvi. Borror, A., 1972. Revision of the order Hypotrichida (Ciliophora, Protozoa). J. Protozool. 19, 1–23. Borror, A., Wicklow, B., 1983. The suborder Urostylina Jankowski (Ciliophora, Hypotrichida): morphology, systematics and identification of species. Acta. Protozool. 22, 97–126. Chen, X., Gao, S., Song, W., Al-Rasheid, K.A.S., Warren, A., Gong, J., Lin, X., 2010. Parabirojimia multinucleata spec. nov. and Anteholosticha scutellum (Cohn, 1866) Berger, 2003, two marine ciliates (Ciliophora, Hypotrichida) from tropical waters in southern China, with note on their SSU rRNA gene sequences. Inter. J. Syst. Evol. Microbiol. 60, 234–243. Chen, X., Dong, J., Lin, X., Al-Rasheid, K.A.S., 2011a. Morphology and phylogeny of a new urostylid ciliate, Monocoronella carnea n. gen., n. sp. (Ciliophora, Hypotricha) from Daya Bay, southern China. J. Eukaryot. Microbiol. 58, 497–503. Chen, X., Clamp, J., Song, W., 2011b. Phylogeny and systematic revision of the family Pseudokeronopsidae (Protista, Ciliophora, Hypotricha), with description of a new estuarine species of Pseudokeronopsis. Zool. Scr. 40, 659–671. Chen, X., Miao, M., Ma, H., Shao, C., Al-Rasheid, K.A.S., 2013a. Morphology, morphogenesis and small subunit (SSU) rRNA gene sequence of the new brackish water ciliate Strongylidium orientale sp. nov. (Ciliophora, Stichotrichia) from Hong Kong, southern China. Int. J. Syst. Evol. Microbiol. 63, 1155–1164. Chen, X., Hu, X., Lin, X., Al-Rasheid, K.A.S., Ma, H., Miao, M., 2013b. Morphology, ontogeny and molecular phylogeny of a new brackish water ciliate Bakuella subtropica sp. n. (Ciliophora, Hypotricha) from southern China. Eur. J. Protistol. 49, 611–622. Corliss, J., 1979. The Ciliated Protozoa: Characterization, Classification and Guide to the Literature. Pergamon Press, Oxford. de Puytorac, P., 1994. Phylum Ciliophora Doflein, 1901. In: de Puytorac, P. (Ed.), Traité de zoologie, infusoires ciliés. Masson, Paris. Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J.F., Guindon, S., Lefort, V., Lescot, M., Claverie, J.M., Gascuel, O., 2008. Phylogeny. fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465–W469. Dereeper, A., Audic, S., Claverie, J.M., Blanc, G., 2010. BLAST-EXPLORER helps you building datasets for phylogenetic analysis. BMC Evol. Biol. 10, 8. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Fan, Y., Warren, A., Al-Farraj, S.A., Chen, X., Shao, C., 2013. Morphology and SSU rRNA gene-based phylogeny of two Diophrys-like ciliates from northern China,

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