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
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
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: firstname.lastname@example.org (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.
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
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-
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.
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
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.
SSU rRNA Gene Sequences and Primary
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
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
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
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
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.
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
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
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
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.
Q. Zhang et al. / European Journal of Protistology 46 (2010) 280–288 287
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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