LIU An, YI Zhenzhen, LIN Xiaofeng HU Xiaozhong, Saleh A. AL-FARRAJ, and Khaled A. S. AL-RASHEID

1)Laboratory of Protozoology,Institute of Evolution & Marine Biodiversity,Ocean University of China,Qingdao266003,P. R. China

2)Laboratory of Protozoology,School of Life Science,South China Normal University,Guangzhou510631,P. R. China

3)Zoology Department,College of Science,King Saud University,Riyadh11451,Saudi Arabia

Molecular Phylogenetic Lineage of Plagiopogon and Askenasia (Protozoa, Ciliophora) Revealed by Their Gene Sequences

LIU An1), YI Zhenzhen2),*, LIN Xiaofeng2), HU Xiaozhong1), Saleh A. AL-FARRAJ3), and Khaled A. S. AL-RASHEID3)

1)Laboratory of Protozoology,Institute of Evolution & Marine Biodiversity,Ocean University of China,Qingdao266003,P. R. China

2)Laboratory of Protozoology,School of Life Science,South China Normal University,Guangzhou510631,P. R. China

3)Zoology Department,College of Science,King Saud University,Riyadh11451,Saudi Arabia

Prostomates and haptorians are two basal groups of ciliates with limited morphological characteristics available for taxonomy. Morphologically, the structures used to identify prostomates and haptorians are similar or even identical, which generate heavy taxonomic and phylogenetic confusion. In present work, phylogenetic positions lineage of two rare genera,PlagiopogonandAskenasia, were investigated. Three genes including small subunit ribosomal RNA gene (hereafter SSU rDNA), internal transcribed spacer region (ITS region), and large subunit ribosomal RNA gene (LSU rDNA) were analyzed, 10 new sequences five species each. Our findings included 1) class Prostomatea and order Haptorida are multiphyletic; 2) it may not be appropriate to place order Cyclotrichiida in subclass Haptoria, and the systematic lineage of order Cyclotrichiida needs to be verified further; 3) genusPlagiopogonbranches consistently within a clade covering most prostomes and is basal of clade Colepidae, implying its close lineage to Prostomatea; and 4)Askenasiais phylogenetically distant from the subclass Haptoria but close to classes Prostomatea, Plagiopylea and Oligohymenophorea. We supposed that the toxicyst ofAskenasiamay be close to taxa of prostomes instead of haptorians, and the dorsal brush is a more typical morphological characteristics of haptorians than toxicysts.

Plagiopogon;Askenasia; multi-gene phylogeny; SSU rDNA; ITS region; LSU rDNA

1 Introduction

The ciliated protozoa are an important group of protists with important significance in the microbial food web and an exceeding diversity of approximately 8000 described species (Lynn, 2008). Among ciliated protozoa, species of class Prostomatea and subclass Haptoria are often found in both terrestrial and marine habitats, and even in red tided seawaters (Dale and Dahl, 1987; Gustafsonet al., 2000; Hansen and Fenchel, 2006; Lynn, 2008; Müller, 1989). Morphologically, prostomates and haptorians are very simple and have relatively limited characteristics available for taxonomy (Lynn, 2008). Because of thesesimple structures, Prostomatea and Haptoria were once considered to be ancestral (Corliss, 1979; Lynn, 2008). However, the ultrastructural and molecular researches have shown that they are secondary species evolved from what (Bardele, 1989; Baroin-Tourancheauet al., 1992; Hiller, 1993; Lynnet al., 1999; Vd’acnyet al., 2010; Yiet al., 2010). There are still some biases in whether a par-ticular species should be placed in Prostomatea or Haptoria due to the discrepancy between morphological and molecular characteristics.

Plagiopogonhas been less described morphologically, thus being absent of detailed scales and ciliary patterns (Corliss, 1979; Foissneret al., 2008; Kahl, 1930; Lynn, 2008; Small and Lynn, 1985). Small and Lynn (1985) and Lynn (2008) placed it in family Colepidae (class Prostomatea, order Prorodontida) because bothPlagiopogonand other members of Colepidae have calcium carbonate plates and caudal cilia (Lipscomb and Riordan, 2012). In contrast, Kahl (1930) and Corliss (1979) assignedPlagiopogonto order Haptorida. These differences cannot be resolved because of limited morphological descriptions at present.

Askenasiawas previously assigned to Haptorida though it was placed in different families/orders in different systems (Corliss, 1979; Foissner and Foissner, 1988; Lynn, 2008). Based on small subunit ribosomal RNA gene (abbreviated as SSU rDNA) sequences; however, Zhanget al.(2012) revealed thatAskenasiawas related to Prostomatea and Plagiopylea instead of Litostomatea. This gene sequence (about 1200 bp in length) may not be appropriate for precisely assigning a speciesto its lineage. Furthermore, the assignment of Cyclotrichiida in Haptoria has always been questioned due to a set of conflicts between morphological characteristics and SSU rDNA phylogenetics (Johnsonet al., 2004; Lynn, 2008; Strüder-Kypkeet al., 2006; Vd’acnyet al., 2011; Zhanget al., 2012).

Phylogenetic analysis based on a set of genes and their secondary structures has evidenced the taxonomy of some controversial ciliate groups (Gaoet al., 2012a; Gaoet al., 2013; Gaoet al., 2012b; Huanget al., 2012; Liet al., 2013; Zhanget al., 2012). In the present study, we expanded sampling taxa with a focus on these two groups. Three DNA sequences,i.e., SSU rDNA, ITS region and large subunit ribosomal RNA gene (abbreviated as LSU rDNA) from five species were sequenced. The phylogenies inferred from concatenated gene sequences, in consideration of morphological and morphogenetic characters, will facilitate our understanding of the evolutionary history of the controversial ciliate groups.

2 Materials and Methods

2.1 Ciliate Collection and Identification

Askenasiasp. was collected from the coast seawater near Guangzhou (22°42′N, 114°32′E), China.Plagiopogon loricatus,Plagiopogonsp.,ProrodonovumandPlacussalinuswere collected from the coast seawater near Qingdao (36°08′N, 120°43′E), China. Ciliates were isolated under a dissecting microscope using glass micropipettes and were identified using live observations and silver impregnation techniques according to previous descriptions (Chenet al., 2012; Panet al., 2013). Terminology and systematic classification were according to Lynn (2008).

2.2 DNA Extraction, PCR Amplification and Sequencing

Ciliates were starved overnight in sterilized seawater at room temperature to minimize flagellate protozoacontamination. One or more individuals were isolated and transferred to a 1.5 mL microfuge tube with a minimum volume of water. Genomic DNA was extracted with DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following manufacturer’s instruction with the modification that one fourth of the volume suggested by the manufacturer’s instruction for each reagent solution. (Yiet al., 2012). Eukaryotic universal primer A (5’-AAC CTG GTT GAT CCT GCC AGT-3’) or 82F (5’-GAA ACT GCG AAT GGC TC-3’) and eukaryotic universal primer B (5’-TGAT CCT TCT GCA GGT TCA CCT AC-3’) bounding nearly the full length of SSU rDNAwere used to PCR amplification (Medlinet al., 1988). The reaction was denatured at 94℃ for 5 min, followed by 35 cycles of denaturing at 94℃ for 30 s, annealing at 56℃for 1 min and extending at 72℃ for 2 min, and a final extension at 72℃ for 7 min. The SSU rDNA sequences obtained were deposited in GenBank with accession numbers KC771342 (Plagiopogon loricatus) and KC771341 (Askenasiasp.) (Table 1). Primers for partial LSU rDNA amplification were 28S-F3： 5’-AC(C/G)-CGC-TG(A/G)-A(C/T)-TTA-AGC-AT-3’ and 28S-R2： 5’-AAC-CTTGGA-GAC-CTG-AT-3’. (Moreiraet al., 2007). A fragment of approximately 500 bp containing the ITS region was amplified using primers ITS-F： 5’-GTA-GGT-GAACCT-GCG-GAA-GGA-TCA-TTA-3’) and ITS-R： 5’-TAC-TGA-TAT-GCT-TAA-GTT-CAG-CGG-3’ (Yiet al., 2009). Sometimes, ITS region and partial LSU-rDNA were amplified together using the primers ITSF and 28SR2 with the following cycling conditions： denaturing at 94℃ for 5 min, followed by 40 cycles of denatuering at 94℃ for 15 s, annealing at 58℃ for 1 min, and extending at 72℃ 30 s, and a final extension at 72℃ for 7 min. The PCR product was purified using the TIANgel Midi Purification Kit and inserted into a pUCm-T vector (Shanghai Sangon Biological Engineering and Technical Service Company, China), amplified and sequenced bi-directionally (Invitrogen sequencing facility, Shanghai, China).

Table 1 Clarification of the species with the gene sequences obtained in this study

2.3 Phylogenetic Analysis

Our phylogenetic analysis was done as the described by Huanget al.(2012). Sequences were aligned using Muscle 3.7 (Edgar, 2004) with default parameters, and then modified manually by Bioedit 7.0 (Hall, 1999). Modeltest 3.7 (Posada and Crandall, 1998) and Mr-Modeltest v2.0 (Nylander, 2004) using the AIC criterion were selected with the GTR+I+G model, which was then used for maximum likelihood (ML) and Bayesian inference (BI) analyses. ML tree was constructed using PhyML v2.4.4 (Guindon and Gascuel, 2003) and the reliability of internal branches was assessed using a nonparametric bootstrap method with 1000 replicates. The Bayesian analysis was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The program was run for 1000000 generations at a sampling frequency of 100 and a burn-in of 2500 trees. All remaining trees after discarding the burn-in were used in the calculation of posterior probabilities using a majority rule consensus. Topologies were visualized with Treeview v1.6.6 (Page, 1996) and MEGA 5.0 (Tamuraet al., 2011).

3 Results

3.1 Phylogenetic Analyses Inferred from SSU rDNA

Fig.1 Maximun likelihood (ML) and Bayesian inference (BI) phylogenetic trees inferred from SSU rRNA gene sequences from all ciliate classes, with emphasis on classes Litostomatea, Prostomatea and Plagiopylea. Two species from Dinophyceae were selected as outgroup taxa. Sequences newly obtained are in bold (arrow). Numbers near nodes represent ML non-parametric bootstrap values and BI posterior probabilities. Black circles indicate full support (100 ML, 1.00 BI).‘-’ indicates disagreement between ML and BI analyses. The scale bar corresponds to 5 substitutions per 100 nucleotide positions.

As shown in Table 1, we obtained 10 new sequences from five species (Plagiopogon loricatus,Askenasiasp.,Plagiocampasp.,ProrodonovumandPlacussalinus). In our SSU rDNA tree (Fig.1), the Karyorelictea-Heterotrichea clade branches basally following the outgroup Dinophyceae. The clade containing Spirotrichea, Armophorea and Litostomatea clusters with the sister clade forming by species in Nassophorea, Colpodea, Phyllopharyngea, Oligohymenophorea, Plagiopylea and Prostomatea, respectively. Neither tree demonstrates the monophyly of class Protostomatea.Balanionmasanensisis placed near Plagiopylea.Urotrichasp. has an unstable cluster relationship in our repeated phylogenetic analyses, falling into clade Prostomatea or clustering together withParaspathidiumsp. (data not shown). The main clade of prostomes comprises nine genera includingApocoleps,Nolandia,Tiarina,Coleps,Plagiopogon,Pelagothrix,Prorodon,SpathidiopsisandPlacus.Plagiopogon loricatusis always at the base of Colepidae (ML 76, BI 0.72). Three previously released SSUr DNAsequences (about 1200 bp) and our nearly complete sequence (1678 bp) ofAskenasiaform a highly supported clade, along withBalanion masanensis(ML 98, BI 1.00).

3.2 Phylogenetic Analyses Inferred from LSU rDNA

The LSU rDNA trees show similar topologies with SSU rDNA (Fig.2). Litostomatea, Heterotrichea, Spirotrichea, Colpodea and Plagiopylea form independent clades, respectively.Plagiopogon loricatusandProrodon ovum, which are two species with newly sequenced SSU rDNA, cluster together withProrodonsp., forming a distinct clade with high support values (ML 96, BI 1.00). The clade comprisingAskenasiasp.,Paraspathidium apofuscumandCyclotrichium cyclokaryonis supported by moderate to high values (ML 76, BI 1.00), and is sister to Prostomatea (ML 94, BI 1.00), which is not congruent with the result of the SSU rDNA analysis.

Fig.2 Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees based on LSU rDNA sequences.Sarcocystis rileyiandProrocentrum consutumwere selected as outgroup taxa. New sequences in our work are shown in bold and arrowed. Numbers near nodes represent ML non-parametric bootstrap values and BI posterior probabilities. Black dots indicate full supports (100 ML, 1.00 BI). ‘-’ indicates disagreement between ML and BI analyses. The scale bar corresponds to 5 substitutions per 100 nucleotide positions.

3.3 Phylogenetic Analyses Inferred from ITS Region

The topologies inferred from ITS region are more similar to the LSU rDNA than to the SSU rDNA (Fig.3). Since there is no species of Plagiopylea sequenced in the ITS region, Plagiopylea is absent from our ITS analyses. Five species,Plagiocampasp.,Plagiopogonloricatus,Prorodonovum,PlacussalinusandAskenasiasp., form a sister clade with Oligohymenophorea with moderate to high support values (ML 65, BI 0.97).Askenasiasp. groups together withP. salinus(ML 61, BI 0.73), forming a sister clade to the other three prostomes.

3.4 Phylogenetic Analysis Based on the Whole Transcribed Unit of Ribosomal RNA Genes

Our phylogenetic trees based on the whole transcribed unit of ribosomal RNA genes is shown as Fig.4, in which 14 concatenated sequences of six classes of Ciliophora are included. Plagiopylea is absent in our combined analysis due to the lack of ITS region. The revealed topologies are similar to those from LSU rDNA. Each class is placed in a single branch, andAskenasiasp.,PlagiopogonlaricatusandProrodonform a strongly supportedclade (ML 99, BI 1.00) adjacent to Oligohymenophorea and Colpodea.

Fig.3 Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees based on ITS region sequences.Symbiodiniumsp. was selected as the outgroup taxon. New sequences are shown in bold and arrowed. Numbers near nodes represent ML non-parametric bootstrap values and BI posterior probabilities. Black circles indicate full support (100 ML, 1.00 BI). ‘-’ indicates disagreement between ML and BI analyses. The scale bar corresponds to 10 substitutions per 100 nucleotide positions.

Fig.4 Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees based on the transcribed unit of ribosomal RNA genes (SSU rDNA, ITS region and LSU rDNA).Symbiodiniumsp. was selected as outgroup taxon. New sequences obtained in our work are shown in bold and arrowed. Numbers near nodes represent ML non-pa- rametric bootstrap values and BI posterior probabilities. Black dots indicate full support (100 ML, 1.00 BI). The scale bar corresponds to 5 substitutions per 100 nucleotide positions.

4 Discussion

4.1 Classification of Prostomes and Haptorians

Considering thatUrotricha(Prorodontida, Urotrichidae) andBalanion(Prorodontida, Balanionidae) branch independent of the core prostome clade, the monophyly of Prostomatea is not supported. Further research is needed to resolve the phylogenetic positions ofUrotrichaandBalanion. Furthermore, our ITS region (Fig.3) and LSU rDNA (Fig.2) phylogenetic analyses also supported the contention that Prostomatea is a secondarily derived group of ciliates, which is congruent with previous works based on SSU rDNA analyses (Baroin-Tourancheauet al., 1992; Lynnet al., 1999).

The monophyly of haptorians is also rejected by ourwork.Paraspathidium,CyclotrichiumandAskenasiaare more related with Prostomatea and Plagiopylea, questioning their high-level taxonomic assignments based on their overall morphology (Zhanget al., 2012). The new sequences will allow us to further discuss early phylogenetic assignment ofAskenasia.

4.2 Phylogeny ofPlagiopogon

In all our phylogenetic trees,P.loricatusconsistently clusters with prostomes. Especially in SSU rDNA trees, which recover relatively more species in Prostomatea, Colepidae form a moderately supported clade withP.loricatus.This strongly supports the viewpoint of Small and Lynn (1985) and Lynn (2008),Plagiopogonshould be placed in family Colepidae. Undoubtedly, the classification ofPlagiopogonas a member of either family Enchelyidae or order Haptorida, should be rejected. All the members of Colepidae, includingPlagiopogon, have calcium carbonate plates and caudal cilia (Lipscomb and Riordan, 2012). The calcium carbonate plates ofPlagiopogon, however, are small and uniquely shaped (Czapik and Jordan, 1976). In addition, evenPlagiopogonis similar toColepsin form, it lacks the latter’s armour and posterior spines. Given thatPlagiopogonbranches basally in Colepidae, the calcium carbonate plates and the armour and posterior spines in Colepidae may be derived morphological characteristics forColeps, indicating thatPlagiopogonis in an ancestral order to Colepidae.

4.3 Phylogeny ofAskenasia

In terms of SSU rDNA topologies,Askenasiais closer to plagiopyleans than to prostomateans. The LSU rDNA analyses, however, showed thatAskenasiais more related to Prostomatea than to Plagiopylea, and similar results are also obtained from ITS region analyses and the whole transcribed unit analyses. Such relationship may also result from the lack of a plagiopylean ITS region. Although there is no reliable result for us to get a precise relationship betweenAskenasiaand other groups, the classification in whichAskenasiais assigned to haptorians is definitely not supported by our transcribed unit analyses (Corliss, 1979; Lynn, 2008). Although Lynn (2008) consideredAskenasiaas a haptorian because of the presence of toxicysts, some important haptorian characteristics, such as distinct rhabdos, dorsal brush (DB) and tightly meshed silverline system, are absent in the genus (Corliss, 1979; Foissner and Foissner, 1988; Krainer and Foissner, 1990; Vd’acnyet al., 2011). In fact, toxicyst is also a characteristics of Prorodontida (Prostomatea). Considering the relationship ofAskenasiato prostomes inferred from our transcribed unit analyses, we suggested that the toxicysts inAskenasiaare related to those of prostomes instead of haptorians, and that the dorsal brush is a more important characteristic of Haptoria than toxicysts.

4.4 Cyclotrichiida

Cyclotrichiida comprises four genera,Mesodinium,Myrionecta,AskenasiaandRhabdoaskenasia(Lynn, 2008). There are a lot of morphological and molecular evidences distinguishing Cyclotrichiida from Haptoria. The dorsal brush is regarded as a synapomorphy for Litostomatea, and one of the ancestral features among typical haptorians (Foissner and Foissner, 1988; Vd’acnyet al., 2010, 2011), while it is absent in Cyclotrichiida. Notwithstanding the morphological distinction with haptorians mentioned above, Lynn (2008) placed Cyclotrichiida in Haptoria, considering the presence of toxicysts and the litostomes to be related SSU rDNA secondary structures. Our analyses ofAskenasiaprovided additional evidence for the phylogeny of Cyclotrichiids and rejected the assignment of Cyclotrichiida to Haptoria. As discussed above, the toxicysts of cyclotrichiids are possibly related to those of prostomes. Because of the limited secondary structure and gene information, we could not make an unambiguous conclusion about the taxonomy of cyclotrichiids. Further research will be performed to test our suppositions.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 31222050, 41376141, 3112041, and 31471973), and by the international research projects from King Saud University (Nos. RGPVPP-083, IRG14-22).

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(Edited by Qiu Yantao)

(Received December 10, 2013; revised January 21, 2014; accepted April 27, 2015)

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

* Corresponding author. Tel： 0086-20-85210644 E-mail： zyi@scnu.edu.cn