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ZOOTAXA

ISSN 1175-5326 (print edition)

ISSN 1175-5334 (online edition)Copyright © 2014 Magnolia Press

Zootaxa 3827 (3): 375–386

www.mapress.com/zootaxa/Article

http://dx.doi.org/10.11646/zootaxa.3827.3.7

http://zoobank.org/urn:lsid:zoobank.org:pub:14CD03DB-1613-4BCA-984E-58F5AD607A60

Morphology and Phylogeny of a New Frontonia Ciliate, F. paramagna spec. nov.

(Ciliophora, Peniculida) from Harbin, Northeast China

YING CHEN1§, YAN ZHAO2§, XUMING PAN2, WENQIAO DING1,

KHALED A. S. AL-RASHEID3 & ZIJIAN QIU1,4

1College of Life Science and Technology, Harbin Normal University, Harbin 150025, China2 Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China3Zoology Department, College of Science, King Saud University, P.O.Box 2455, Riyadh 11451, Saudi Arabia4Corresponding Author: Zijian Qiu, College of Life Science and Technology, Harbin Normal University, Harbin 150025, China.

E-mail: [email protected]§Co-first author

Abstract

This paper describes a new Frontonia ciliate, F. paramagna spec. nov., sampled from freshwater in Harbin, northeast Chi-

na, based on its morphology, infraciliature, ultrastructure and small subunit ribosomal RNA gene information. The new

species is defined by the following features: large sized freshwater form, 400–610 × 110–160 μm in vivo, about 179–201

somatic kineties, three peniculi, each with four kineties, three vestibular and six or seven postoral kineties, one elongated-

elliptical macronucleus, centrally-located, a single contractile vacuole, without canals, located right-dorsally in the poste-

rior half of the body. Sequence alignment and phylogenetic analyses based on the small subunit ribosomal RNA (SSU

rRNA) gene indicated that the new species has characters distinct from its known congeners. The ultrastructure of the

trichocyst and other extrusomes, and the subpellicular fibre system, were observed by both scanning electron microscopy

(SEM) and transmission electron microscopy (TEM). Much of the ultrastructure is here given for the first time by SEM,

and these features provide complementary data for taxonomic purposes.

Key words: Ciliates, Frontonia, new species, phylogeny, ultrastructure, taxonomy

Introduction

Frontonia ciliates are frequently found in marine, brackish and freshwater habitats (Al-Rasheid 1999; Carey 1992;

Fan et al. 2011a, 2012; Long et al. 2008) and many species have been described using live observation and silver

impregnation methods (Borror 1963; Burkovsky 1970; Bullington 1939; Foissner 1987; Foissner 1994; Gil &

Perez-Silva 1964a, b, c; Kahl 1931; Pan et al. 2013; Petz et al. 1995; Roque 1961a, b, c; Roque & de Puytorac

1972; Song & Wilbert 1989). These species have typically been distinguished from each other by the combination

of the following characteristics: their body shape and size, the number and location of their contractile vacuoles,

the morphology of their oral apparatus, and their general somatic ciliary pattern (Corliss 1979; Dragesco 1960;

Dragesco & Dragesco-Kernéis 1986; Foissner 1994; Roque & de Puytorac 1972). Only some species have been

well-described based upon both live observations and silver impregnation, however, while the description of

ultrastructure has been especially rare. Many Oligohymenophorea species are inadequately investigated in respect

to current taxonomic criteria; that is, they are poorly defined and described and, often, lack a statement of the type

material (Burkovsky 1970; Fan et al. 2011b, 2011c; Long et al. 2005; Pan et al. 2011, 2013a, 2013b, 2013c; Petz et

al. 1995; Roque & Puytorac 1972).

These organisms should ideally be identified morphologically by a combination of features coming from both

live and silver impregnation, like the buccal and somatic ciliature, position and character of the contractile

vacuoles, size and shape of the body and some ultrastructure features coming from SEM and TEM observations, as

well as their habitat (Foissner 1987; Foissner et al. 1994). Moreover, with the application of molecular techniques

Accepted by S. Adl: 6 Jun. 2014; published: 4 Jul. 2014 375

mailto:[email protected]

in taxonomy, species need to be compared not only at the morphological but also at the molecular level (Gao et al.

2012a, 2012b, 2013, 2014; Gao & Katz 2014; Seo et al. 2013; Zhang et al. 2014). In recent years, extensive work

at molecular level has been carried out on species from sandy sediment habitats in northern China (Fan et al. 2010;

Long et al. 2008; Pan et al. 2010; Zhang et al. 2011).

Recently, an unknown Frontonia was isolated from freshwater in Harbin, northeast China. After being

investigated using a wide range of methods, it is believed to be a new member of this genus, which is here named

Frontonia paramagna spec. nov.

Material and methods

Morphological and ultrastructural studies. Samples were collected from a puddle with a freshwater temperature

~14 °C, located in Harbin (N45°52′02.67″, E126°32′56.23″), northeast China, during May, 2010. Living cells were

observed using bright field and differential interference contrast microscopy at 100–1,000 × magnification. Silver

carbonate (Ma et al. 2003) and silver nitrate (Wilbert and Song 2008) impregnation methods were used to reveal

the infraciliature and argyrome. Drawings of impregnated specimens were made with the help of a camera lucida.

The preparation methods of the SEM samples followed Gao et al. (2011) and prepared samples were observed

using a Hitachi S-3400N SEM. The key to this method is first breaking up the cells with a certain concentration of

potassium permanganate, so that the protoplasm flows out and the cell membrane flips; thereafter the normal steps

can be followed. This new SEM operation can illustrate the inner part of a ciliate’s pellicle. The preparation of the

TEM samples followed Kual et al. (1982) and Lynn (1991), with a Hitachi H-7650 TEM being used for

observation. Terminology is mainly according to Lynn (2008) and Hausmann et al. (2003).

DNA extraction, amplification, purification and sequencing of the SSU rRNA gene: DNA was extracted

used the DNeasy & Tissue Kit (Shanghai, QIAGEN China, China) according to the modified protocol of the

manufacturer. DNA amplifications (PCR) were performed by using two distinct primers: 82 Forward (5’-GAA

ACT GCG AAT GGC TC-3’) and Eukaryotic B (5’-TGA TCC TTC TGC AGG TTC ACC TAC-3’) (Medlin et al.,

1988). The amplification cycles for the small subunit ribosomal RNA (SSU rRNA) extraction were as follows: 4

min at 94 °C followed by 35 cycles comprising 45 s at 94 °C, 1 min 15 s at 60 °C, 2 min at 72 °C. This was

followed by a final extension of 8 min at 72 °C.

The PCR products were purified using a SanPrep DNA Cleaning Kit (Sangon Biotech Co. Ltd., Shanghai,

China), inserted into the pMD®19-T simple vector (Takara Bio Technology Co. Ltd., Dalian, China), transformed

into Escherichia coli DH5α cells and sequenced in BGI (Beijing Genomics Institute, Qingdao, China) using a

pyrosequencing method.

Sequences and phylogenetic analyses. The SSU rRNA dataset for this study contained the newly sequenced

SSU rDNA and 76 related SSU rRNA gene sequences obtained from the NCBI database (http://

www.ncbi.nlm.nih.gov/). Alignments of SSU rRNA gene sequences (dataset I: Frontonia SSU rRNA gene

sequences only; dataset II: all the 77 SSU rRNA gene sequences investigated in this work) were carried out using

ClustalW implemented in BioEdit v7.1.3 (Hall 1999) and then aligned manually in MEGA v5.0 (Tamura et al.

2011). Furthermore, we also detected genetic similarity of the dataset of Frontonia species using ClustalW

implemented in BioEdit v7.1.3 (Table 3).

The final alignment of dataset II, representing 77 taxa, was used to construct phylogenetic trees by Bayesian

inference (BI) and Maximum Likelihood (ML) analyses. The species Trimyema minutum and Plagiopyla frontata

were selected as the outgroup taxa. The BI tree was constructed with MrBayes v3.1.2 (Huelsenbeck & Ronquist

2001; Guindon & Gascuel 2003; Ronquist & Huelsenbeck 2003), with the best models being selected by the

MrModeltest v2.2 program (n = 6, rates = invgamma) (Nylander 2004). Likewise, PhyML v3.0 (Guindon et al.

2010) was employed to construct a ML tree and Modeltest v3.7 (Posada & Crandal 1998; Posada & Buckley 2004)

was used to find the models (GTR+I+G) of DNA substitution that best fitted the SSU rRNA data in the Maximum

Likelihood analyses. The same model was used to estimate the site likelihoods for those trees prior to doing the

approximately unbiased (AU) test. The score of the constraint tree was compared with the unconstrained ML result

using the AU test option implemented in CONSEL (Shimodaira & Hasegawa 2001).

CHEN ET AL.376 · Zootaxa 3827 (3) © 2014 Magnolia Press

http://www.ncbi.nlm.nih.gov/



Results

Subclass Peniculia Fauré-Fremiet in Corliss, 1956

Genus Frontonia Ehrenberg, 1838

Frontonia paramagna spec. nov.

Diagnosis. Freshwater Frontonia about 400–610 × 110–160 μm in vivo; dorsoventrally slightly flattened with

conspicuously small buccal cavity; about 179–201 somatic kineties; three vestibular and six to seven postoral

kineties; peniculi 1–3 each with four kinetid rows; single ellipsoidal macronucleus; one contractile vacuole located

equatorially on the right margin; extrusomes spindle-shaped.

Type slides. A silver carbonate-impregnated slide containing the holotype specimen is deposited in the

Laboratory of Protozoology, Ocean University of China (CY20111229-1). Three silver nitrate slides containing

paratype specimens are deposited in the Laboratory of Protozoology, Ocean University of China (CY20111229-2,

3, 4).

Type locality. A puddle in a freshwater wetland in Harbin (N45°52′02.67″, E126°32′56.23″), Heilongjiang

Province, China.

Etymology. The prefix “para-” refers to the species being similar to Frontonia magna.

Morphological and Ultrastructural description. Table 1 and Figures 1–3: Size huge and varied, in vivo

about 400–610 × 110–160 μm, but mostly 500–550 μm in length; ratio of length: width about 3.5:1. Body shape

constant, long ellipsoidal in outline; left margin straight while right margin slightly convex (Figs. 1A, 1B, 1C, 1D

2A,2B). Both ends rounded with the front end wider and slightly bent to the right. Dorsal-ventral side slightly

flattened. Single ellipsoidal macronucleus, positioned at body centre (Figs. 1G, 2K). Single contractile vacuole,

about 100 μm in diameter, positioned in mid-body right of cell median; one contractile vacuole pore located on

right dorsal surface (Figs. 1A, 1D, 1G, 2A, 2C). Most of the extrusomes spindle-shaped, about 5 μm long, densely

arranged beneath cortex (Figs. 1E, 2I, 2J, 3C, 3F). The other two types of extrusomes were observed for the first

time (Figs. 1E, 3D, 3E). Cytoplasm is transparent, colourless to slightly grey. Cytoplasm filled by many yellow and

grey food vacuoles, with different diameters, as well as dark granules and crystals containing bacteria, diatoms and

other small ciliates (Figs. 1A, 2A, 2B). 180–199 somatic kineties. somatic cilia about 8 μm long (Figs. 1F; 1G).

Locomotion by revolving moderately rapidly on substrate or by swimming in water about long body axis. Buccal

cavity small and shallow, approximately triangular in outline, occupying about 1/10 of body length (Figs. 1A, 1D,

1F; 1H, 2F, 2G, 2H, 2L, 3A, 3B). Three short vestibular kineties (VK1–3) on the right of the buccal cavity running

from the anterior vertex to the postoral suture of the cavity, with dense kinetosomes (Figs. 1H; 2F, 2H). Three

peniculi deeply located on the left wall of cavity: peniculi 1 and 2 (P1, 2) about equally long and close to each

other, peniculus 3 (P3) slightly separated from P1, 2 and curved to right. Each peniculus composed of four rows of

kinetosomes (Figs. 1F, 1H; 2H, 2L, 2M). Peniculus 3 composed of four shorter kineties, of which the right two are

complete and the left two shortened (Figs. 1H; 2H, 2L). Single paroral membrane (PM) on right edge of the buccal

cavity runs from anterior of the buccal overture to the posterior edge. Argentophilic line (AL) positioned parallel to

VK (Figs. 1H; 2F, 2H).

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were combined to study

the subpellicular ultrastructure of F. paramagna spec. nov. Three types of extrusomes were evident, including a

spindle trichocyst, another new type of trichocyst and a mucocyst. A large number of spindle trichocysts with a

cap-like structure were distributed densely in the specimens (Figs. 3C, 3F). The other two types of extrusomes,

however, were rare and could be observed only by TEM. The mucocysts were an elongated oval-shape, about 2 μm

long and with transverse microfilaments on their surface, and can emit micro-mesh (Fig. 3D). The new type of

trichocyst were about 5 μm long, in the shape of a long rod with a dark, flat, front end, and appeared mainly around

the oral apparatus (Fig. 3E).

This investigation also revealed a three layered fibre system beneath the pellicle. The first layer of fibre was

close to the pellicle, with a diameter of about 0.12 μm. The fibres of the second layer were densely wrapped around

the extrusomes (Figs. 3F, 3G); while the fibres of the third layer were loose, covering most of the subpellicular

structures and adjoined the cytoplasm.

Zootaxa 3827 (3) © 2014 Magnolia Press · 377A NEW CILIATE FRONTONIA PARAMAGNA

TABLE 1. Morphometric data of Frontonia paramagna spec. nov. Data based on randomly selected specimens with

silver impregnation. Measurements in μm. CV, coefficient of variation in %; Max, maximum; Mean, arithmetic mean;

Min, minimum; SD, standard deviation.

FIGURE 1. Frontonia paramagna spec. nov. from life (A, B, C, E) and after staining with silver carbonate (D, F–H). A.

Ventral view of a typical individual, arrow refers to contractile vacuole. B. Lateral view. C. Different body shapes. D. Ventral

views showing arrangement of extrusomes, sutures and contractile vacuole. E. Three types of extrusomes. F, G. Infraciliature of

ventral and dorsal side. H. Structure of oral apparatus. Scale bars in (A, B, F, G) = 200 µm. AS, anterior suture; CV, contractile

vacuole; CVP, contractile vacuole pore; Ma, macronucleus; P1–3, peniculus 1, 2, 3; PK, postoral kineties; PM, paroral

membrane; PS, postoral suture; VK, vestibular kineties.

Finally, the VK in the oral apparatus were observed by SEM. This showed that a membranous structure was

wrapped around the root of each VK to form a coarse columnar structure, with interwoven fibres linking these

columnar structures to the VK (Fig. 3H).

SSU rRNA gene sequence and phylogeny of Frontonia paramagna spec. nov. (Table 3 and Figure 4). The

newly sequenced SSU rRNA gene of Frontonia paramagna spec. nov., with a length of 1,667 bp, has been

deposited in the GenBank database with accession number JQ868786. The genetic similarity and number of

nucleotide differences between the new species and its congeners has been set out in Table 3, showing details of the

molecular differences between this new species and other comparable species. Each of the SSU rRNA gene

sequences of the species available from GenBank for genus Frontonia have similarities with this new species

Characters Min Max Mean SD CV n

Body length 318 362 340.8 13.0 3.7 15

Body width 96 124 113.0 7.6 6.8 15

Number of somatic kineties 180 200 189.7 6.3 3.3 15

Number of vestibular kineties 3 3 3.0 0 0 15

Number of postoral kineties 6 7 6.5 0 0 15

Number of ciliary row in peniculus 1 4 4 4.0 0 0 15




ranging from 91.1% to 98.6% with 22 to 147 nucleotides difference from other congeneric species (Table 3). The

phylogenetic trees generated through both Bayesian Inference (BI) and Maximum Likelihood (ML) methods had

similar topologies, therefore, they were combined into a single consensus tree (Fig. 4). The new species Frontonia

spec. nov. clustered together with Frontonia sp. / vernalis with high support (100% ML, 1.00 BI) and then formed

a clade with F. leucas (100% ML, 1.00 BI). The clade became the sister group with the clusters of F. tchibisovae /

lynni / mengi / magna, also with high support (100% ML, 1.00 BI). But genus Frontonia did not form a

monophyletic group since Frontonia sp. (FN667825) and F. didieri were separated and clustered with Paramecium

and Apofrontonia species instead of with their congeners (68% ML, 0.99 BI).

FIGURE 2. Frontonia paramagna spec. nov. from life (A, B, F, G, I) and after staining with silver carbonate (D, E, H, J, K, M)

and silver nitrate (C, L). A. Ventral view of a typical individual; arrow marks the contractile vacuole. B. Dorsal view of a

typical individual. C. Arrow indicates the contractile vacuole pore. D. The postoral kineties. E. Arrows mark the somatic

kineties. F-G. Photomicrographs of buccal field (arrows), showing vestibular kineties, paroral membrane and peniculi 1, 2, 3.

H. To show the argentophilic line (the arrow below) and the paroral membrane (the arrow above). I, J. The spindle-shaped

trichocyst. K. The macronucleus. L. Arrow indicates the peniculus 3. M. Photomicrograph of peniculi1, 2, 3 (P1, P2, P3). Scale

bars in (B, C) = 200 µm; PM, paroral membrane; VK, vestibular kineties; PK, postoral kineties; Ma, macronucleus


FIGURE 3. Frontonia paramagna spec. nov. from scanning electron microscopy (SEM) (A, B, F, G, H) and transmission

electron microscopy (TEM) (C–E). A. SEM photograph of a typical individual. B. Arrowhead shows the buccal field. C, F.

Arrowhead shows the spindle-shaped trichocyst. D. Arrowhead shows the mucocyst. E. Arrowhead shows another type of

trichocyst. G. Arrowheads show the fibre layers. H. Arrowheads show the vestibular kineties (VK) and the columnar structure.

Scale bars in (A) =20µm; in (B) =6µm; in Fig (C, D) = 0.3µm; in Fig. (E, F) =1µm; in Fig. G = 2µm; in Fig. H = 4µm.

Discussion

Morphology and Ultrastructure. Since the genus Frontonia was established (Ehrenberg 1838), about 30 nominal

species have been recognized and described (Kahl 1931; Borror, 1963; Burkovsky 1970; Dragesco and Dragesco-

Kernéis 1986; Foissner 1987; Long et al. 2005, 2008; Fan et al. 2011a, 2012; Pan et al. 2012). Recent

investigations using silver staining methods have demonstrated that the location of the contractile vacuole, the

infraciliature, and especially the structure of the oral apparatus, are the most reliable criteria for species

identification (Foissner 1994; Petz et al. 1995). Considering the structure of the oral apparatus, the body shape and

size, and the habitat, at least four species should be compared with this new species, namely: F. magna Fan et al.,

2011, F. leucas Ehrenberg, 1838, F. pallida Czapik, 1979 and F. terricola Foissner, 1987 (Table 2).


Frontonia paramagna spec. nov. appears similar to F. magna in having three peniculi each with four rows of

kinetosomes, a large body size and one contractile vacuole. The differences, however, are as follows: F. paramagna

spec. nov. has a long ellipsoidal shape and is about 400–610 × 110–160 μm in vivo: it is longer than F. magna

whose ellipsoidal dimensions are about 300–450 × 100–200 μm in vivo. Additionally, F. magna has more

vestibular kineties (five or six vs. three in F. paramagna) and fewer postoral kineties (four vs. six or seven in F.

paramagna). Similarly, F. magna has one contractile vacuole located on its left margin at the level of the posterior

third of the cell, but F. paramagna spec. nov. has its contractile vacuole located on its right margin at the level of

the posterior half of the cell. Most pertinently, however, the habitat of F. magna is brackish water, whereas that of F.

paramagna spec. nov. is freshwater. On the basis of these differences we can confidently classify them as being

different species (Fan et al. 2011a; Pan et al. 2012).

FIGURE 4. ML and BI trees based on SSU rRNA gene sequences (Dataset II) computed with PHYML and MrBayes. The first

and second values at the nodes represent bootstrap values for ML and BI analyses, respectively; a ‘*’ indicates a disagreement

in topology that could not be represented on the consensus tree. The scale bar represents 5 changes per 100 positions. The

sequences of Trimyema munutum and Plagiopyla frontata were used to root the tree. The position of the new species in the

phylogenetic trees is marked with an arrow.

Turning to F. leucas, F. pallida and F. terricola, these species, like F. paramagna spec. nov., inhabit freshwater

and have only one contractile vacuole. There remain, however, conspicuous differences between them (Dragesco &

Dragesco-Kernéis 1986; Foissner et al. 1987; Foissner et al. 1994).

d b d ( ) d i h


First, F. leucas is more slender in size, at 120–360 × 110–120 μm in vivo, while F. pallida (150–160 × 60–66

μm) and F. terricola (70–110 × 60–73 μm) are much smaller than F. paramagna spec. nov.

(400–610 × 110–160 μm).

Second, the last two species have different kinety rows in peniculi 1–3. F. pallida’s peniculi 1–3 contain four,

three and two kinety rows respectively, F. terricola’s peniculi 1 and 2 have four kinety rows, while peniculus 3 has

three kinety rows (comparedto four rows in peniculi 1–3 in F. paramagna spec. nov.).

Third, F. paramagna differs from the other species in the number of vestibular and postoral kineties. The

number of postoral kineties in F. leucas is four or five, in F. pallida, five, and F. terricola, six (compared to six or

seven in F. paramagna spec. nov.). Meanwhile, the number of vestibular kineties in F. pallida is four (compared to

three in F. paramagna spec. nov. and the other species). Hence, F. paramagna spec. nov. can be clearly

distinguished from F. leucas, F. pallida and F. terricola.

In addition, SSU rRNA analysis results revealed that F. paramagna spec. nov. is close to F. vernalis

Bullington, 1939, however, there are clear differences between them in morphology. F. vernalis is smaller (197 ×

108 μm compared to 400–610 × 110–160 μm) and has two to three contractile vacuoles (compared to only one in F.

paramagna spec. nov.). Besides, F. vernalis is a marine species while F. paramagna spec. nov. is a freshwater

species.

Sequences comparison and phylogenetic analyses. Trees constructed using different algorithms showed

similar topologies in this investigation. The unstable topologies for the order Peniculida were mainly caused by the

position instability of the species Urocentrum turbo. All of the species of genus Frontonia were positioned in the

order Peniculida, although they did not form a monophyletic clade due to the split from F. didieri (Fig. 4). The

clade formed by Frontonia paramagna spec. nov. /Frontonia sp./ F. vernalis cluster with F. leucas with full

support, although even these four species have very different morphological characters (Table 2) and SSU rRNA

sequences (Fig. 4, Table 3). Furthermore, we also carried out an Approximately Unbiased (AU) test on the SSU-

rDNA dataset to assess the monophyly of Frontonia species. The test could not significantly reject the constrained

topology where all the Frontonia species were forced to be monophyletic (AU=0.889, AU>0.05). And more

molecular information may be needed in future work for a better understanding of this genus.

Acknowledgements

This work was supported by the National Science Foundation of China (No.31101613 and 30970311), HNU Youth

Elite Fund (10KXQ-01), and research Group Project (No. RGP- 083), King Saud University Deanship of Scientific

Research. Many thanks are due to Prof. Weibo Song, and graduate students of the Laboratory of Protozoology,

Ocean University of China for their technical and institutional help.

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