The systematic position of Paraspathidium Noland - Ocean ...

European Journal of Protistology 46 (2010) 280–288

The systematic position of Paraspathidium Noland, 1937 (Ciliophora,

Litostomatea?) inferred from primary SSU rRNA gene sequences and

predicted secondary rRNA structure

Qianqian Zhang a , Zhenzhen Yi b , Weibo Song a,∗ , Khaled A.S. Al-Rasheid c , Alan Warren d

a Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China

b Laboratory of Protozoology, South China Normal University, Guangzhou 510631, China

c Zoology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

d Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK

Received 6 February 2010; received in revised form 13 May 2010; accepted 17 May 2010

Abstract

Traditionally the unusual ciliate Paraspathidium has been regarded as a gymnostome haptorid (Litostomatea) based on its

morphological features. In order to test this placement, the small-subunit (SSU) rRNA gene was sequenced for two isolates of

Paraspathidium apofuscum and phylogenetic trees were constructed. Furthermore, the putative structure of the variable regions

2 and 4 of the SSU rRNA gene were predicted and compared with those of other ciliates. Our analyses of SSU rRNA gene

sequences revealed (i) a clear separation of Paraspathidium from the haptorids and indeed the class Litostomatea, rejecting its

systematic position based on morphological characters and (ii) an equally clear association with the assemblage comprising

the classes Plagiopylea and Prostomatea. Putative secondary structures of the variable regions 2 and 4 of Paraspathidium are

similar to those of the plagiopyleans and prostomateans but differ from the hapotrids in Helix 10, Helix E10-1 and Helix E23-5.

Taken together, these results support the placement of Paraspathidium close to prostomateans and plagiopyleans, or even as a

distinct group possibly at ordinal rank, within the class Plagiopylea.

© 2010 Elsevier GmbH. All rights reserved.

Keywords: Paraspathidium; SSU rRNA; Secondary structure; Phylogeny; Plagiopylea

Introduction

Molecular methods, in particular the determination of

small-subunit rRNA (SSU rRNA) gene sequences, have

enabled the placement of a number of problematic genera

and higher taxa within the ciliates ,(e.g., Fan et al.

2009; Foissner and Stoeck 2008; Gao et al. 2008; Li et

al. 2009; Lynn and Strüder-Kypke 2002; Shin et al. 2000;

Yi et al. 2008). Such methods are especially useful for

∗ Corresponding author. Tel.: +86 532 8203 2283.

E-mail address: wsong@ouc.edu.cn (W. Song).

investigating the systematics of the early branching groups,

e.g., the prostomateans, plagiopyleans and litostomateans,

for which relatively few morphological and/or ontogenetic

characters are available. The genus Paraspathidium Noland,

1937 is an example of one such ciliate. Paraspathidium

is characterized by an apical, excavated oral opening surrounded

by dikinetids that form a conspicuous perioral

ciliary corona, a brosse and circumoral dikinetids bearing

unique, highly specialized cilia (Foissner 1997; Long et al.

2009; Figs. 1–10). The systematic position of Paraspathidium

has historically been uncertain (Foissner 1997). It was

initially assigned to the family Spathidiidae because of its

bilaterally flattened body shape and Spathidium-like oral

0932-4739/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.

doi:10.1016/j.ejop.2010.05.001

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Q. Zhang et al. / European Journal of Protistology 46 (2010) 280–288 281

Figs. 1–10. Paraspathidium apofuscum isolate 1 from life (1, 4, 7–9) and after silver carbonate (2, 3, 5), protargol (6), and silver nitrate (10)

impregnations (from Long et al. 2009). (1) Typical cell. (2) Anterior part of cell, to show the ciliature around the cytostome (arrows mark

dorsal brush kineties). (3 and 5) Lateral view of the buccal apparatus; arrow in Fig. 3 marks the brush kineties, arrowheads in Fig. 5 indicate

the dikinetids in the anterior part of the somatic kineties. (4 and 7–9) Different body shapes; arrow in Fig. 4 marks the brush, arrows in Figs.

7 and 9 indicate the contractile vacuole. (6) Extrusomes (arrows). (10) Buccal area. Scale bars: 50 m.

structure (Noland 1937). Later, it was assigned to the family

Coelosomididae in the order Trichostomatida (Corliss

1961). This classification was accepted by Corliss (1979)

and Alekperov (2005), although Paraspathidium was treated

as incertae sedis in the family Coelosomididae by Corliss

(1979). Foissner (1997) established the family Paraspathidiidae,

as a subgroup of the Acropisthiina within the order

Haptorida, for this genus, a systematic placement that was

accepted by Alekperov et al. (2007). However, Foissner

(1997) noticed that some of the morphological features of

Paraspathidium, e.g., the complex contractile vacuole and

the dikinetid perioral ciliary corona, are reminiscent of the

prostomatids as described in Hiller (1993). In the most

recent schemes Paraspathidium was again placed in the family

Spathidiidae, order Haptorida (Jankowski 2007; Lynn

2008).

In order to re-evaluate the systematic position of Paraspathidium,

the SSU rRNA gene of two isolates of P.

apofuscum Long et al., 2009 was sequenced and phylogenetic

relationships with other ciliates were analyzed.

Materials and Methods

Collection and Identification of Specimens

Two isolates of Paraspathidium apofuscum were investigated.

The type isolate, described by Long et al. (2009),

was collected from a sandy beach at the Second Bathing

Bay of Qingdao (36 ◦ 08 ′ N; 120 ◦ 43 ′ E), China, on 22 April

2005. Isolate 2 was collected from the upper 0–4 cm sand

layer at Zhanqiao Bay, Qingdao, in October 2008. The dis-

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282 Q. Zhang et al. / European Journal of Protistology 46 (2010) 280–288

tance between the two collection sites is approximately 3 km

along the coastline. Specimens of the latter isolate were collected,

processed and identified after Long et al. (2009). For

a detailed description of the morphology of P. apofuscum see

Long et al. (2009) and Figs. 1–10. We found no difference

in the morphology of the two isolates analyzed. Terminology

and systematics follow Foissner (1997) and Lynn (2008).

Extraction and Sequencing of DNA

One or more cells of each isolate were isolated, transferred

into a drop of autoclaved seawater and washed in several

changes of sterile seawater in order to remove any other

protists that might be present (Miao et al. 2009). Washed

cells were collected after centrifuging for 5 min at 2400 × g

and removing excess water leaving the cells in a minimum

volume. Genomic DNA was extracted using a REDExtract-

N-Amp Tissue PCR Kit (Sigma, St. Louis, USA) according

to the manufacturer’s instructions (Yi et al. 2009).

PCR amplifications of the SSU rRNA gene were performed

using a TaKaRa ExTaq DNA Polymerase Kit (TaKaRa

Biomedicals, Japan). Primers used for SSU rRNA gene

amplification were Euk A (5 ′ -GAAACTGCGAATGGCTC-

3 ′ ) and Euk B (5 ′ -TGATCCTTCTGCAGGTTCACCTAC-3 ′ )

(Medlin et al. 1988). Polymerase chain reaction conditions

were according to Yi et al. (2009). The PCR products were

purified with a Gel Extraction Kit (Feijie, China), inserted

into the pUCm-T cloning vector (Shanghai Sangon Biological

Engineering & Technical Service Company, Shanghai,

China), and cultured in E. coli DH5 cells. Transformed

E. coli clones were detected by PCR amplifications using

the RV-M and M13-20 primers. The SSU rRNA genes were

sequenced in both directions on an ABI 3700 sequencer

(Invitrogen sequencing facility, Shanghai, China), using the

RV-M and M13-20 primers.

Phylogenetic Analyses

In addition to the two isolates of Paraspathidium apofuscum,

SSU rRNA sequences of 59 other ciliates from

the GenBank database were included in gene trees (see

GenBank accession numbers in Fig. 12). The flagellates

Prorocentrum micans and Symbiodinium pilosum (Dinoflagellata,

Dinophyceae) were chosen as the outgroup taxa

(Lynn 2003). The sequences were aligned using Clustal

W implemented in Bioedit Ver. 7.00 (Hall 2004). The

alignment was edited manually and the highly variable

regions in all ciliate classes were removed in Bioedit. The

resulting data matrix contained a total of 1698 characters

(nucleotide sites) categorized as follows: 638 constant characters;

215 variable, parsimony-uninformative characters;

845 parsimony-informative characters. Gaps were treated as

missing data.

GTR+G+I was selected with AIC criteria in MrModeltest

v.2 (Nylander 2004), and was used for Bayesian Inference

(BI). The BI analysis was performed with MrBayes

3.1.2 (Ronquist and Huelsenbeck 2003). Markov chain

Monte Carlo (MCMC) simulations were then run with

two sets of four chains using the default settings, i.e.,

chain length 1,000,000 generations with trees sampled every

100 generations. The first 250,000 generations were discarded

as burn-in. A maximum likelihood (ML) tree was

constructed with PhyML V2.4.4 program (Guindon and

Gascuel 2003) using the GTR+G+I model selected under

the AIC criterion by Modeltest (Posada and Crandall 1998).

The reliability of internal branches was assessed using

a non-parametric bootstrap method with 1000 replicates.

The remaining trees were used to generate a consensus

tree and to calculate the posterior probabilities (PP) of

all branches using a majority-rule consensus approach.

Maximum-parsimony (MP) trees were constructed with

PAUP 4.0b 10 (Swofford 2002). Parameters for the MP

tree were as follows: heuristic search, 100 random-addition

sequences, and TBR branch-swapping. The reliability of

internal branches was estimated by bootstrapping with 1000

replicates. TREEVIEW v1.6.6 (Page 1996) and MEGA 4.0

(Tamura et al. 2007) were used to visualize tree topologies. In

order to further examine the relationships of Paraspathidium

with plagiopyleans, prostomateans and haptorids, the SSU

rRNA putative secondary structures of representative species

of these four groups were predicted. Default settings of

the mfold website

(Zuker 2003) were used to produce the

putative secondary structure of variable regions 2 (V2 region)

and 4 (V4 region), these regions having been selected following

inspection of the alignment. The structures were edited

with RnaViz 2.0 (Rijk and Wachter 1997) for aesthetic purposes.

Results

SSU rRNA Gene Sequences and Primary

Structure

The SSU rRNA gene sequences of the two isolates

of Paraspathidium apofuscum were deposited in

the NCBI/GenBank database with accession numbers of

FJ875139 (isolate 1) and FJ875140 (isolate 2). In both isolates

the SSU rRNA gene was 1679 nucleotides in length and

had a GC content of 44.85%, which is in the same range as

other ciliates. The sequences of the two isolates differed by

only three nucleotides.

Phylogenetic Position of Paraspathidium

In order to determine the phylogenetic position of Paraspathidium,

SSU rRNA gene trees were constructed using

multiple algorithms. The BI and ML trees were nearly identical

in topology and thus were combined into a single tree

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Q. Zhang et al. / European Journal of Protistology 46 (2010) 280–288 283

Fig. 11. Phylogenetic tree based on SSU rRNA gene computed with the Bayesian inference (BI) and maximum likelihood (ML) algorithms,

demonstrating the position of Paraspathidium apofuscum among the 11 classes of ciliated protozoa. Clades with posterior probabilities

(PP) = 100% and bootstrap (BP) support >98% are marked with solid circles at the nodes. Isolates newly sequenced are in bold. The scale bar

corresponds to 10 substitutions per 100 nucleotide positions.

(Fig. 11). As shown in Figs. 11, 12, the two isolates of P.

apofuscum clustered together with maximum support values

(1.00 BI, 100% ML, 100% MP). In BI analyses, Paraspathidium

branched basally from the plagiopylean clade at a very

deep level with moderate support (0.89 BI) and together

these formed a sister group to the class Prostomatea with

maximum support (1.00 BI). In the ML and MP analyses,

a cluster comprising Paraspathidium and the classes Plagiopylea

and Prostomatea was moderately to well-supported

(69% MP, 90% ML), although the relationships among the

three groups were unresolved. This cluster grouped with

the oligohymenophoreans, colpodeans and nassophoreans

with 1.00 posterior probabilities in BI analyses, over 95%

bootstrap support in the ML analysis (Fig. 11) and 97%

bootstrap support in the MP analysis (Fig. 12). Interestingly,

a close relationship between the genus Paraspathidium and

the Haptorida, or even between Paraspathidium and the class

Litostomatea, was not recovered in any of the trees.

SSU rRNA Putative Secondary Structure

Inspection of the alignment of SSU rRNA sequences

revealed some differences among Paraspathidium, plagiopyleans,

prostomateans and haptorids in the variable V2 and V4

regions. The putative secondary structures of these regions

were therefore predicted. The putative secondary structures

of V2 regions were based on the model of the interpretation

of the eukaryotic V2 region of Van de Peer et al. (2000), while

those of V4 regions were based on the model of Wuyts et al.

(2000). Species or isolates from the same genus had nearly

identical structures, hence only one example from each genus

is shown (Figs. 13 and 14).

Structure of V2 Region

As shown in Fig. 13, a putative secondary structure model

consisting of a multi-branch loop and four paired regions

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284 Q. Zhang et al. / European Journal of Protistology 46 (2010) 280–288

Fig. 12. A maximum-parsimony tree inferred from complete SSU rRNA sequences, indicating the relationship of Paraspathidium with

prostomateans and plagiopyleans and the clear separation from the haptorids. The numbers at the forks represent the percentage of times the

group occurred out of 1000 trees. Nodes that are not supported by more than 40% bootstrap proportions are drawn as polytomies. GenBank

accession numbers are given after names of species. Newly sequenced isolates are in bold.

(helices 9, 10, E10-1 and 11) was predicted. A comparison

of the putative secondary structure of the V2 region

of Paraspathidium, plagiopyleans, prostomateans and haptorids

showed that the structures of helices 9 and 11 were

more conserved than helices 10 and 10-1, with Helix 9

consisting of two middle bulges and a terminal loop while

Helix 11 could potentially have a terminal loop. There was

more diversity in Helix 10 and Helix E10-1. The structure

of Helix 10 of Paraspathidium was similar to that of the

plagiopyleans and prostomateans, having a middle bulge

containing eight nucleotides (arrowed in Fig. 13), whereas

haptorids had no middle bulge in this helix. The structure

of Helix E10-1 showed more inter-class variation, particularly

with respect to the number of middle bulges, there

being two in Paraspathidium and plagiopyleans, two or three

in prostomateans, and zero or one in haptorids (arrowed in

Fig. 13).

Structure of V4 Region

The putative secondary structures of the V4 regions of

all ciliates investigated (Fig. 14), including Paraspathidium

apofuscum, were generally similar to those of other protists,

consisting of 11 helices and two pseudoknots (Wuyts et al.

2000). The typical deletions in the hyper variable V4 region

common to other ciliates, i.e., deletions of the entire E23-3,

E23-5 and E23-6, were also apparent in P. apofuscum. The

structure in this region was more variable than that of the V2

region with different patterns among the four groups, i.e., the

plagiopyleans, prostomateans, haptorids and Paraspathidium

(Fig. 14). Each group possessed a distinct predicted structure

with some differences in the number and position of bulges in

E23-1 and 2, the size of middle bulge in E23-11 and 12, and

size of middle bulge in E23-13 and 14. A comparison of this

region among the four groups showed the total length of Helix

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Q. Zhang et al. / European Journal of Protistology 46 (2010) 280–288 285

Fig. 13. Putative secondary structures of the small-subunit ribosomal RNA gene in the area of the variable region 2 comprising helices 9, 10,

E10-1 and 11 for 16 ciliate species belonging to the genus Paraspathidium, the order Haptorida, and the classes Plagiopylea and Prostomatea.

Note the diversity of the middle bulges in helices 10 and E10-1 (arrows).

E23-1 and 2 (or only Helix E23-1) was 39–43 bp in Paraspathidium

and of all plagiopyleans and prostomateans, but

was significantly shorter (28–34 bp) in the haptorid species

(Fig. 14). In the former three groups Helix E23-7 was present

(arrows in Fig. 14) but this was absent in all haptorids. The

loop between the 5 ′ end of Helix E23-13 and the 3 ′ end of

Helix E23-14 in prostomateans was longer than those in the

other groups (16 bases in prostomateans vs. 7–9 bases in

other groups; arrowheads in Fig. 14). The loop between the

5 ′ end of Helix E23-12 and the 3 ′ end of Helix E23-9 was

obviously shorter in Paraspathidium and one prostomatean

genus, Holophrya (1 base vs. 5–7 bases in other taxa; bold

arrows in Fig. 14). Finally, the link loop between the 3 ′ end of

Helix E23-11 and the 3 ′ end of Helix E23-8 of Paraspathidium

had a distinctive structure with an additional nucleotide

with base “A” (double arrowheads in Fig. 14) whereas this is

absent in all other groups.

Discussion

Morphologically, Paraspathidium is highly divergent from

typical haptorids in possessing an atypical dorsal brush,

a flattened body, (very likely) an open circumoral kinety,

and an oralized somatic ciliature comprising densely ciliated

dikinetids around the dominant cytostome (Foissner

1997; Long et al. 2009). Nevertheless, Paraspathidium

has usually been treated as a haptorid within the class

Litostomatea, which is a “melting pot” for several (possibly

paraphyletic) groups with an apically positioned

cytostome, uniform somatic cilia and an undifferentiated

oral apparatus (Corliss 1979; Foissner 1997; Lynn

2008).

This is the first analysis of the phylogeny of Paraspathidium

based on molecular data. Significantly, analysis

of SSU rRNA nucleotide sequences rejects placement of

Paraspathidium in the order Haptorida or even in the

class Litostomatea. Rather, it falls into a moderately wellsupported

clade with plagiopyleans and prostomateans

(Figs. 11 and 12). Furthermore, the predicted secondary

structures of the V2 and V4 regions of the SSU rRNA

gene are consistent with this finding. The close relationship

between Paraspathidium and prostomateans was suggested

by Foissner (1997) based on certain morphological features

such as their complex contractile vacuole and dikinetid

perioral ciliary corona. According to the definition of the

class Plagiopylea in Lynn (2008), Paraspathidium is also

morphologically similar to plagiopyleans (with exception

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286 Q. Zhang et al. / European Journal of Protistology 46 (2010) 280–288

Fig. 14. Putative secondary structures of the small-subunit ribosomal RNA molecule in the area of variable region 4, comprising helices E23-1,

(2), 4, 7, 8, 9, 10, 11, 12, 13, 14 and two pseudoknots formed by helices E23-9 to 12, helices E23-13 and 14 for 16 ciliate species. The number

of nucleotides in Helix E23-1and 2 (or only E23-1) for each species is given above the long arrow. Helix E23-7 is found only in Paraspathidium

and the classes Plagiopylea and Prostomatea (arrows). Note the distinct structure of Helix E23-14 in prostomateans (arrowheads), of Helix

E23-9 in Paraspathidium and Holophrya (bold arrows), and of Helix E23-8 in Paraspathidium (double arrowheads).

of odontostomatids) in terms of its variable body shape,

uniform holotrichous somatic ciliation, and kinetosomes

encircling the cytostome. The present findings are also consistent

with the taxonomic scheme of Corliss (1979), in

which Paraspathidium is considered to be closely related to

the plagiopyleans. Paraspathidium has a unique combination

of characters. For instance, it has an Acropisthiina-like

apical cytostome (Fig. 2), and its dorsal brush consists of

kineties with irregularly distributed kinetosomes, and is thus

obviously different from the spathidiids (Long et al. 2009;

Figs. 3, 5). However, in terms if its general appearance, it

strongly resembles the spathidiids (Foissner 1997; Fig. 1).

These features suggest that Paraspathidium should be separated

at a higher taxonomic level than previously supposed

,(e.g., Foissner 1997; Long et al. 2009). Its position on SSU

rRNA gene trees (Figs. 11, 12) is also consistent with this

finding and provides further support for the distinctness of

Paraspathidium.

It is widely acknowledged that taxon sampling may significantly

influence branching positions of taxa in gene trees,

especially if the representation of taxa is low, as in the present

case (Poe 1998). Therefore, gene sequences of additional taxa

within the Paraspathidiidae, Plagiopylea and Prostomatea are

urgently needed in order to provide a definitive resolution of

the phylogenetic position of Paraspathidium. In the meantime

we suggest that the family Paraspathidiidae should be

treated as incertae sedis within the class Plagiopylea, and

close to Prostomatea. It is likely that it should be ranked at

the ordinal level, but further investigations will be needed in

order to support this hypothesis.

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Q. Zhang et al. / European Journal of Protistology 46 (2010) 280–288 287

Acknowledgements

This work was supported by the Natural Science Foundation

of China (project No. 30870264), the Darwin Initiative

Programme (project number: 14-015) which is funded by the

UK Department for Environment, Food and Rural Affairs,

and the Centre of Excellence in Biodiversity, King Saud

University. Thanks are due to Mr. Hongan Long and Mr Yangang

Wang, Laboratory of Protozoology, OUC, for sample

collection and draft reading, respectively.

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