Studies on Three Diverse Frontonia Species (Ciliophora, Peniculida), with Brief Notes on 14 Marine or Brackish Congeners

Headnote

Abstract. Living observation and silver impregnation methods were used to investigate the morphology and infraciliature of three Frontonia ciliates (F. guangdongensis spec, nov., F ocularis Bullington, 1939 and F. schaefferi Bullington, 1939) that were isolated from coastal waters of the South China Sea. Frontonia guangdongensis spec. nov. may be recognized by the combination of the following characteristics: cells about 160 ? 35 urn in vivo; elongated body with right margin depressed in anterior third; length to width ratio 4: 1 to 5: 1; three or four vestibular and four or five postoral kineties; peniculi 1 and 2 each with four rows of kineties, peniculus 3 with two rows; one contractile vacuole in mid-body region right of cell median; brackish water habitat. A key based on morphological data for fourteen marine or brackish water Frontonia species found in China is also provided. In addition, the small subunit (SSU) rDNA gene was sequenced for F ocularis Bullington, 1939. Our phylogenetic analyses support the contention that the genus Frontonia is not monophyletic.

Key words: Frontonia guangdongensis, Frontonia ocularis, Frontonia schaefferi, new species, South China Sea.

INTRODUCTION

The class Oligohymenophora exhibits a much higher than expected level of morphospecies diversity, and it is undoubtedly the case that many new forms are still awaiting discovery (Fan et al. 2010, 2011b, c; Pan et al. 2010, 2011; Zhang et al. 2011; Gao et al. 2012a, b). The genus Frontonia was established by Ehrenberg (1838) and is commonly found in freshwater, brackish, and marine biotopes (Kahl 1931, Borror 1963, Burkovsky 1970, Song and Wilbert 1989, Petz et al. 1995, Long et al. 2005). Members of the genus can be distinguished by their body shape, the structure of their oral apparatus, and their general somatic ciliary pattern with its distinct preoral and postoral suture (Roque and de Puytorac 1972; Dragesco and Dragesco-Kernéis 1986; Foissner et al. 1994; Fan et al. 2011a, 2013).

Recent investigations have revealed a high diversity of Frontonia species in Chinese coastal waters, and the presence of new or poorly known taxa have highlighted the need to conduct further studies on this genus (Long et al 2005, 2008; Fan et al. 2011a, 2013; Pan et al. 2013). During the past seven years fourteen Frontonia species have been isolated from coastal waters of China seas, namely: F. didieri Long et al, 2008, F. elegans Fan et al, 2013, F. lynni Long et al., 2005, F. magna Fan et al. , 20 1 1 , F. mengi Fan et al. , 20 1 1 , F. guangdongensis spec, nov, F. multinucleata Long et al, 2008, F. cularis Bullington, 1939, F. pusilla Fan et al, 2013, F. canadensis Roque and Puytorac, 1972, F. schaefferi Bullington, 1939, iT sinica Fan et al, 2013, F. subtropica Pan et al. , 20 1 3 , and F. tchibisovae Burkovsky, 1 970. The great diversity of Frontonia species highlights the need for a key to the identification of these species that draws together the morphological data in an ordered form (Figs 5, 6; Table 1).

As part of an on-going faunistic study of marine ciliates in southern China, the morphology and infraciliature of three Frontonia species, F. guangdongensis spec, nov., F. ocularis Bullington, 1939, and/? schaefferi Bullington, 1939 were studied, along with the molecular phylogenetic assignment of F ocularis. In addition, a key to the marine and brackish water Frontonia species found in China is provided.

MATERIALS AND METHODS

Frontonia guangdongensis spec. nov. was collected from a coastal shrimp cultnring area in Nansha, Guangdong Province (23°43'N, 113°33?) on 9 November 2008, when the water temperature was 17°C, salinity 28%o, and pH 7.5. Frontonia ocularis Bullington, 1939 was collected from a mangrove wetland inZhuhai, Guangdong Province (21°48'N, 113°13?) on 28 November 2008, when the water temperature was 17°C, salinity 26%o, and pH 8.1. Frontonia schaefferi Bullington, 1939 was isolated from a mangrove wetland near Shenzhen, Guangdong Province (22°32'N, 1 13°40?) on 22 April 2009, when the water temperature was 25°C, salinity 21%o, and pH 7.8. In each case, sand (the top 5 cm layer) or sediment plus seawater were taken from the original sites.

Cells were observed using bright field and differential interference contrast microscopy at 100 ? to 1,000 ? magnification. Protargol (Wilbert 1975), Chatton-Lwoff (Wilbert and Song 2008), and silver carbonate (Ma et al. 2003) methods were used to reveal the infraciliature and argyrome. Counts and measurements of stained specimens were performed at a magnification of 100-1,000 x Drawings were made with the help of a camera lucida. Terminology is mainly according to Corliss (1979).

Total genomic DNA was extracted from five cells for each species using the REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, USA) as described in Miao et al. (20 1 1). The universal primers Euk A (5 ' - AAC CTG GTT GAT CCT GCC AGT - 3 ') or 82 F (5' - GAA ACT GCG AAT GGC TC - 3') and Euk ? (5'- TGA TCC TTC TGC AGG TTC ACC TAC - 3 ') were used for PCR amplifications of the SSU rRNA gene (Medlin et al. 1988). Cloning and sequencing were performed according to Gao et al. (2012). Although three Frontonia species are described in this paper, we failed to extract DNA from either F guangdongensis or F schaefferi due to the low number of specimens of these species. The DNA analysis, therefore, is limited to F ocularis.

A total of 49 taxa were used for phylogenetic analysis, including the one newly sequenced and twelve previously sequenced Frontonia spp. (for Genbank accession numbers, see Fig. 7). Coleps nolandi Nitzsch, 1827 and C. hirtus Nitzsch, 1827 were used as the outgroup taxa. Sequences were aligned using Clustal W implemented in BioEdit 7.0 (Hall 1999). The final alignment of 1622 characters was used to construct phylogenetic trees according to the method described by Liu et al. (20 12). Briefly, the hierarchical nested likelihood ratio test implemented in MrModeltest (Nylander 2004) was used to select the best-fit model of nucleotide substitution, and this was then used for Bayesian inference (BI) analysis using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). The program was run for 1,000,000 generations with a sample frequency of 100 and a burn-in of 2,500 generations. PhyML version 2.4.4 (Guindon and Gascuel 2003) was used for the maximum likelihood (ML) analysis with the best model GTR + I (= 0.3301) + G (= 0.4754) selected by the program Modeltest version 3.4 (Posada and Crandall 1998). The reliability of internal branches was assessed using a non-parametric bootstrap method with 1,000 replicates.

RESULTS AND DISCUSSION

Frontonia guangdongensis spec. nov. (Fig. 1; Tables 1,2)

Diagnosis: Brackish water Frontonia, about 160 ? 35 µp? in vivo, elongated body shape with right margin depressed in anterior third of body; length to width ratio about 4:1 to 5:1; small buccal field about 10 to 12% of body length; 62-75 somatic kineties; three or four vestibular kineties, four or five postoral kineties; peniculi 1 and 2 each with four rows, peniculus 3 with two rows; macronucleus ellipsoidal and located in central region of body; one contractile vacuole in mid-body region right of cell median.

Type locality: A coastal shrimp-culturing area in Nansha (23°43'N, 113°33?), Guangdong province.

Deposition of type slides: One slide containing the holotype (registration no. LWW-08 110901-01) and several paratype slides (registration no. LWW- 08110901-02) with protargol-impregnated specimens are deposited in the Laboratory of Protozoology, Ocean University of China, China.

Etymology: The species name 'guangdongensis' refers to the location where this organism was first isolated.

Description: Cell in vivo distinctly elongate, usually about 150-170 * 35^0 um, with ratio of length to width about 4: 1 to 5: 1 (Fig. 1 A, H). Right margin slightly depressed in anterior third of body (Fig. 1 ?, ?, H, I). Dorsoventrally flattened about 5:4. Buccal cavity small and shallow, elliptical to triangular in outline, about 20 ? 11 µ?? in size, 10 to 12% of body length (Fig. IG). Cytoplasm grayish with many large (6-10 µp? across), black, polygonal crystal granules. Food vacuoles (1012 µp? across) and ingested algae distributed randomly in cytoplasm (Fig. ??, K). Macronucleus ellipsoidal, about 20 urn ? 15 um, located in mid-region of body (Fig. 1A, O). Single micronucleus located near end of macronucleus, spherical, about 5 µp? in diameter (Fig. 10). Single contractile vacuole in mid-body region right of cell median, about 7 urn in diameter, contracting at about one minute intervals (Fig. ??, K); no collecting canals observed; one contractile vacuole pore located on right-dorsal surface (Fig. IF). Two types of extrusomes, spindle (about 8 µp? long) and round (2 µp? across), densely arranged beneath pellicle (Fig. IC, D, M); somatic cilia generally about 6 µp? long, cilia in caudal region being longer than others at approximately 10 µp? long (Fig. IM). Locomotion by gliding on substrate or by swimming while rotating about long body axis.

Somatic ciliature as shown in Fig. IE, F, G, P-T. About 52 to 65 longitudinal somatic kineties, commencing at anterior end of cell, forming a conspicuous anterior suture that extends from anterior end of buccal cavity to dorsal side (Fig. IE, F, P, S); posterior part of somatic kineties terminating below posterior region of oral apparatus forming the postoral suture (Fig. IG, Q, T). Three to four vestibular kineties with close-set dikinetids (Fig. IG, R). Four or five postoral kineties left of postoral suture, beginning anteriorly below buccal cavity and gradually shortening from left to right (Fig. IG, R).

Buccal apparatus as shown in Fig. IG, N, R. Three conspicuous peniculi (Pl-3) located on left wall of shallow buccal cavity, slightly curved to right at anterior end. Peniculi 1 and 2 about equally long, parallel to each other, and each composed of four rows of kinetosomes. Peniculus 3 composed of two kineties, right one of which extends entire length of buccal cavity, left one extending only to about anterior 4/5 of cavity length (Fig. IG, N). Double-rowed paroral membrane on right side of buccal cavity: inner row composed of densely arranged monokinetids, outer row composed of loosely arranged dikinetids (Fig. IG, R).

Comparison and remarks: There are three spe- cies that closely resemble Frontonia guangdongensis spec. nov. in terms of the conspicuously elongated body shape: F. mengi Fan, 2011, F. pallida Czapik, 1979 and F microstoma Kahl, 1931.

Frontonia mengi differs from F guangdongensis in three respects. Firstly, its body size in vivo is larger (250 ? 45 urn vs. 160 ? 35 urn); secondly, it has more kinety rows in peniculi 1 and 2 (each row has five in F mengi vs. four in F. guangdongensis): thirdly, the contractile vacuole of F. mengi is located in the posterior third of the cell, whereas in F guangdongensis it is located in the mid-body region (Fan et al. 20 Ila).

Frontonia pallida can be clearly distinguished from F guangdongensis by the following combina- tion of characteristics: F pallida has three kinety rows in peniculus 2, whereas F guangdongensis has four; F pallida has a funnel-shaped cytopharynx (vs. absent in F guangongensis); F pallida's contractile vacuole is to the left of the cell median, whereas it is to the right in F guangdongensis (Czapik 1979, Dragesco and Drag- esco-Kernéis 1986).

Frontonia guangdongensis spec. nov. can also be easily separated from F microstoma Kahl, 193 1 in hav- ing only a single contractile vacuole with no canals whereas the latter has two contractile vacuoles with associated canals. Frontonia guangdongensis also has fewer somatic kineties (52-65 vs. 110-120 in F microstoma) and fewer kinety rows in peniculus 3 (2 vs. 3 or 4) (Roque 1961, Carey 1992).

Frontonia ocularis Bull in «ton, 1939 (Figs 2, 3; Tables 1, 2)

The original definition of this organism was based only on live specimens and no information about its infraciliature was previously available (Bullington 1939). Based on data from both the present and previous studies, an improved diagnosis is presented here, including a description of its infraciliature.

Improved diagnosis: Marine or brackish Frontonia, about 115-140 ? 50-75 um in vivo, reniform with anterior end broad and posterior end slightly narrowed, right margin depressed in anterior 1/3 of body; length to width ratio about 2:1; small buccal field about 15 to 20% of body length; a prominent pigment on right side of anterior extremity; about 93-107 somatic kineties; three vestibular and three or four postoral kineties; peniculi 1 and 2 each with four rows, peniculus 3 with two rows; macronucleus ellipsoidal, located in central region of body; two contractile vacuoles located rightventral, one in anterior half and the other in posterior half of body.

Description of Zhuhai population: Body 115-120 ? 50-60 µp? in vivo, reniform with anterior end broad and posterior end slightly narrowed, right margin depressed in anterior 1/3 of body (Figs 2A, C, 3A, C). Ratio of length to width between 2: 1 and 3:1 (Figs 2A, 3 A). Dorsoventrally flattened about 3:2. Buccal cavity about 20 ? 10 µp?, occupying 15 to 20% of body length (Fig. 3D, F). Cytoplasm slightly grayish, often with many large (8-10 µp? across), black granules at posterior end of body (Fig. 2A). Many dark-green food vacuoles and small, blue crystal granules (1-2 µp?) distributed randomly in cytoplasm (Fig. 2A). Brown-black pigment spot usually present on extreme right border near anterior end of body (Figs 2A, 3 ?, ?, K). Extrusomes spindle-shaped, about 4 µp? long but, when extruded, about 15 µp? long and rod-shaped with one end curved (Figs 2D, 3K). Macronucleus ellipsoidal, about 25 ? 15 urn and located in mid-region of body (Fig. 2A). No micronucleus observed. Two contractile vacuoles 7-10 µp? in diameter, located right-ventrally, one each in anterior and posterior 1/3 of body respectively (Figs 2B, 3B). Somatic cilia about 6 µp? long (Fig. 3E); cilia in caudal end longer than others, about 8 µ?? long. Locomotion by revolving moderately rapidly on substrate or by swimming in water while rotating clockwise about the long body axis.

Somatic ciliature as shown in Figs 2E, F, G, 3G, I, J, M, O. About 93 to 107 somatic kineties shortened progressively both anteriorly and posteriorly from lateral side to oral area, forming conspicuous anterior and posterior sutures that extend from anterior end of buccal cavity over apical end of cell and onto dorsal side. Buccal structure as shown in Figs 2G, 3H, O. Three conspicuous peniculi located on left wall of buccal cavity: peniculi 1 and 2 about equal in length, positioned close to each other parallel to edge of left vestibular wall, slightly curved to right at anterior ends and each composed of four rows of kinetosomes (Figs 2G, 3H). Peniculus 3 composed of two kinety rows which are about equal in length. Paroral membrane double-rowed, located on right side of buccal cavity (Figs 2G, 3H). Three vestibular kineties extending along the paroral membrane, each composed of densely arranged dikinetids. Three or four postoral kineties (Fig. 2G). Several argentophilic lines left of paroral membrane (Fig. 3N).

Comparison and remarks: The population studied here corresponds very well with the original description (Bullington 1939) in terms of body shape, the prominent pigment spot in the anterior border, the number and location of the contractile vacuoles, and the manner of spiralling when swimming forwards. Hence, the identification of this species is not in doubt. Compared with previous descriptions of F ocularis, our population has a smaller body size (about 120 ? 55 µ?? vs. about 141 ? 76 µ??) and the prominent pigment spot is brown-black, whereas it is cited as being reddish-brown in earlier descriptions (Bullington 1939). However, we consider these dissimilarities to be population-dependent.

SSU rRNA gene sequence: The SSU rRNA gene sequence of Frontonia ocularis Bullington, 1939 has been deposited in the GenBank database with accession number, length, and G+C content as follows: FJ868198, 1746 bp, 44.73%.

Frontonia schaefferi Bullington, 1939 (Fig. 4; Tables 1,2)

When this species was first described by Bullington (1939), neither the details of its living morphology, nor a clearly-outlined diagnosis was given. Hence, an improved diagnosis based on both previous and present studies is supplied here.

Improved diagnosis: Marine or brackish Frontonia, about 95-120 ? 50-65 um in vivo, elliptical or reniform with anterior end broad and posterior end slightly narrowed; small buccal field about 20% of body length; about 59-80 somatic kineties; three vestibular and five postoral kineties; peniculi 1 and 2 each with four rows, peniculus 3 with two rows; macronucleus ellipsoidal, located in central region of body; one contractile vacuole, positioned on right-dorsal side at about 3/5 distance down body length and with about eight, long collecting canals.

Description of Shenzhen population: Size in vivo about 95-100 ? 50-55 um. Elliptical or reniform in outline with anterior end broad and posterior end slightly narrowed (Fig. 4A, B, H, I, K). Dorsoventrally flattened about 3 :2 (Fig. 4M). Buccal cavity small and shallow, elliptical to triangular in outline, about 20 µ?? ? 10 µ??, which corresponds to about 20% of body length (Fig. 4J). Cytoplasm colourless to slightly grayish, often with many small black granules (3 - 4 µp? across) at anterior end of body (Fig. 4A, H, I). A few small blue crystal granules (1-3 µ?? in diameter) distributed randomly in cytoplasm (Fig. 4A, H, I). Macronucleus ellipsoidal, about 25 ? 15 µp?, located in body centre (Fig. 4A). No micronucleus observed. One contractile vacuole, about 10 µp? in diameter, positioned on right-dorsal side at about three-fifths distance down body length, with about eight long collecting canals (Fig. 4A, H): one contractile vacuole pore located on right-ventral surface (Fig. 4F). Extrusomes spindle-shaped, about 5 µp? long, densely arranged beneath pellicle (Fig. 4L), approximately 20 µp? long when extruded (Fig. 4N). Somatic cilia approximately 6 µp? long. Locomotion by crawling slowly on substrate or by swimming in water with clockwise rotation about long body axis.

General infraciliature as shown in Fig. 4E-G, O-Q. Anterior and postoral sutures conspicuous, both extending onto dorsal side (Fig. 4E, F). About 59 to 80 somatic kineties. Five postoral kineties with dikinetids, beginning below the oral and terminating on the postoral suture (Fig. 4E, F). Three vestibular kineties with densely arranged kinetosomes extending from anterior vertex of buccal cavity and terminating at postoral suture (Fig. 4G, O). Three conspicuous peniculi, about half of the length of the buccal cavity: peniculi 1 and 2 about equal in length, each composed of four rows of kinetosomes, about half length of buccal cavity; peniculus 3 slightly curved to right at anterior end, composed of two shorter kineties which are about equal in length (Fig. 4G, O). Single-rowed paroral membrane on right edge of buccal cavity running from anterior to posterior edge of buccal overture, with well-developed oral pharyngeal fibres in the buccal area (Fig. 4G, Q).

Comparison and remarks: Frontonia schaefferi Bullington, 1939 was originally reported by Bullington (1939) with only its infraciliature described. The Shenzhen population closely resembles that described by Bullington (1939) except for a minor difference in the body size in vivo (120 ? 65 µp? vs. 100 x 55 µp? in Shenzhen population) which we believe is population-dependent.

Phylogenetic analysis

The topologies of the SSU rRNA gene trees constructed using Bayesian inference and maximum-likelihood analyses were similar, therefore only the BI tree is shown (Fig. 7). The fourteen Frontonia species are divided into two distinct clades, supporting the viewpoint that Frontonia is not monophyletic (Fokin et al. 2006; Gao étal. 2008; Fan étal. 2011, 2013). The clade containing F ocularis, F pusilla, F. elegans and F. didieri clusters with members of the genus Paramecium and Apofrontonia dohrni, and then groups with a highly supported clade (1.00 BI, 87% ML) that includes the other ten Frontonia species. Number of contractile vacuoles and kinety rows in peniculus 3 could generally match the molecular cluster patterns within the genus Frontonia. F ocularis, F pusilla and F. elegans, having two contractile vacuoles and two or three kinety rows in peniculus 3 (Fan et al. 2013), fall into a clade. Similarly, the members with four or five kinety rows in peniculus 3 and one contractile vacuole (Bullington 1939; Fan et al. 201 la; Foissner et al. 1 994; Long et al. 2005, 2008; Pan et al. 2013), i.e. F magna, F subtropica, F canadensis, F. leucas, F. vernalis, F. mengi, F. tchibisovae, and F. lynni, are sister to each other. However, F didieri and F sinica, with one contractile vacuole and two rows in peniculus 3 (Fan et al. 2013, Long et al. 2008), do not cluster together.

Acknowledgements. This work was supported by the Natural Science Foundation of China (project numbers: 31201696, 31201708, 41006098, 31222050), specialized Research Fund for the Doctoral Program of Higher Education (project number: 20104407120006) and Research Group Project (No. RGP-VPP-083), King Saud University Deanship of Scientific Research. Many thanks are due to Prof. Weibo Song (OUC) and Dr. Alan Warren (Natural History Museum, London), who kindly read the draft and gave some helpful suggestions.

References

REFERENCES

Borror A. C. (1963) Morphology and ecology of the benthic ciliated protozoa of Alligator Harbor, Florida. Arch. Protistenk. 106: 465-534

Bullington W. E. (1939) A study of spiralling in the ciliate Frontonia with a review of the genus and description of two new species. Arch. Protistenk. 92: 10-66

Burkovsky I. V. (1970) The ciliates of the mesopsammon of the Kandalaksha Gulf (White Sea) I. Acta Protozool. 7: 475^189

Carey P. G. (1992) Marine Interstitial Ciliates. An Illustrated Key. Chapman and Hall, London

Corliss J. O. (1979) The ciliated protozoa: characterization, classification and guide to the literature. 2nd ed. Oxford, New York, Toronto, Sydney, Paris, Frankfurt

Czapik A. (1979) Frontonia pallida sp. n. un nouveau cilié psammophile (Hymenostomata, Peniculina). Acta Protozool. 18: 527-530

Dragesco J., Dragesco-Kernéis A. (1986) Ciliés libres de l'Afrique intertropicale. Faune Tropicale 26: 1-559

Fan X., Chen X., Song W., Al-Rasheid ? A. S., Warren A. (2010) Two new marine scuticociliates, Sathrophilus planus ?. sp. and Pseudoplatynematum dengin. sp., with improved definition of Pseudoplatynematum (Ciliophora, Oligohymenophora). Eur. J. Protistol. 46:212-220

Fan X., Chen X., Song W, Al-Rasheid K. A. S., Warren A. (201 la) Two novel marine Frontonia species, F. mengi spec. nov. and F magna spec. nov. (Protozoa, Ciliophora), with notes on their phylogeny based on small-subunit rRNA gene sequence data. Int. J. Syst. Evol. Microbiol. 61: 1476-1486

Fan X., Hu X., Al-Farraj S., Clamp J. C, Song W. (2011b) Morphological description of three marine ciliates (Ciliophora, Scuticociliatia), with establishment of a new genus and two new species. Eur. J. Protistol. 47: 186-196

Fan X., Lin X., Al-Rasheid ? A. S., Warren ?., Song W. (201 lc) The diversity of scuticociliates (Protozoa, Ciliophora): a report on eight marine forms found in coastal waters of China, with a description of one new species. Acta Protozool. 50: 219-234

Fan X., Lin X., Liu W, Xu Y., Al-Farraj S., Al-Rasheid ? A. S., Warren A. (2013) Morphology and phylogeny of three new marine Frontonia species (Ciliophora, Peniculia) from coastal waters of China. Eur. J. Protistol. (in press)

Foissner W., Berger H., Kohmann F. (1994) Taxonomische und ökologische Revision der Ciliaten des Saprobiensystems - Band III: Hymenostomata, Prostomatida, Nassulida. Informationsberichte des Bayerischen: Landesamtes für Wasserwirtschaft 1/94: 1-548

Fokin S., Andreoli I., Verni F., Petroni G. (2006) Apofrontonia dohrni sp. n. and the phylogenetic relationships within Peniculia (Protista, Ciliophora, Oligohymenophorea). J. Eukaryot. Microbiol. 35: 289-300

Gao F., Strüder-Kypke M., Yi Z., Miao M., Al-Farraj S., Song W. (2012a) Phylogenetic analysis and taxonomic distinction of six genera of pathogenic scuticociliates (Protozoa, Ciliophora) inferred from small-subunit rRNA gene sequences. Int. J. Syst. Evol. Microbiol. 62: 246-256

Gao F., Laura ?., Song W. (2012b) Insights into the phylogenetic and taxonomy of Philasterid ciliates (Protozoa, Ciliophora, Scutiocociliatia) based on analyses of multiple molecular markers. Mol. Phylog. Evol. 64: 308-317

Gao S., Chen ?., Shao G, Long H., Al-Rasheid ?. ?. S., Song W. (2008) Reconsideration of the phylogenetic position of Frontom'a-related Peniculia (Ciliophora, Protozoa) inferred from the small subunit ribosomal RNA gene sequences. Acta Protozool. 47: 47-54

Guindon S., Gascuel O. (2003) A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 52: 696-704

Hall ? A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT Nucleic Acids Symp. Ser. 41: 95-98

Kahl ?. (1931) Urtiere order Protozoa I: Wimpertiere order Ciliata (Infusoria) 2. Holotricha. Tierwelt. Dtl. 21: 181-398

Liu W, Yi Z., Lin X., Warren ?., Song W (2012) Phylogeny of three choreotrich genera (Protozoa, Ciliophora, Spirotrichea), with morphological, morphogenetic and molecular investigations on three strobilidiid species. Zool. Scr. 41: 417-434

Long H., Song W, Gong J., Hu X., Ma H., Zhu M., Wang M. (2005) Frontonia lynni n. sp., a new marine ciliate (Protozoa, Ciliophora, Hymenostomatida) from Qingdao, China. Zootaxa 1003: 57-64

Long H., Song W, Al-Rasheid ? A. S., Wang Y, Yi Z., Al-Quraishy S., Lin X., Al-Farraj S. (2008) Taxonomic studies on three marine species of Frontonia from northern China: F. didieri n. sp., F multinucleata n. sp. and F. tchibisovae Burkovsky, 1970 (Ciliophora: Peniculida). Zootaxa 1687: 35-50

Ma H., Choi J., Song W. (2003) An improved silver carbonate impregnation for marine ciliated protozoa. Acta Protozool. 42: 161-164

Medlin L., Elwood H. J., Stickel S., Sogin M. L. (1988) The characterization of enzymatically amplified eukaryotic 16s-like rRNA-coding regions. Gene 71: 491^199

Miao M.., Shao G, Chen X., Song W (201 1) Evolution of discocephalid ciliates: Molecular, morpho logical and ontogenetic data support a sister group of discocephalids and pseudoamphisiellids (Protozoa, Ciliophora) with establishment of a new suborder Pseudoamphisiellina subord. n. Sci. China (Life Sci.) 54: 634-641

Nylander J. A. (2004) MrModeltest version 2. Distributed by the author. Department of Systematic Zoology, Evolutionary Biology Centre, Uppsala University

Pan H., Huang J., Hu X., Fan X., Al-Rasheid ? A. S., Song W (2010) Morphology and SSU rRNA gene sequences of three marine ciliates from Yellow Sea, China, including one new species, Uronema heteromarinum nov. spec. (Ciliophora, Scuticociliatida). Acta Protozool. 49: 45-59

Pan X., Gao F., Liu W, Fan X., Warren ?., Song W (2013) Morphology and SSU rRNA gene sequences of three Frontonia species, including a description of F subtropica spec. nov. (Ciliophora, Peniculida). Eur. J. Protistol. (in press)

Pan X., Shao C, Ma H., Fan X., Al-Rasheid K. A. S., Al-Farraj S., Hu X. (201 1) Redescriptions of two marine scuticociliates from China, with notes on stomatogenesis in Parauronema longum (Ciliophora, Scuticociliatida). Acta Protozool. 50: 301-310

Petz W, Song W, Wilbert N. (1995) Taxonomy and ecology of the ciliate fauna (Protozoa, Ciliophora) in the endopagial and pelagial of the Weddell Sea, Antarctica. Protistologica 40: 1-223

Posada D., Crandall ? A. (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14: 8 17-818

Ronquist F, Huelsenbeck J. P. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574

Roque M. (1961) Frontonia microstoma, Kahl. J. Protozool. 8: 334-341

Roque M., de Puytorac P. (1972) Frontonia canadensis sp. nov. (Cilié Hyménostome Péniculien). Natural. Cañad. 99: 41 1 - 416

Song W., Wilbert N. (1989) Taxonomische Untersuchungen an Aufwuchsciliaten (Protozoa, Ciliophora) im Poppelsdorfer Weiher. Bonn. Lauterbornia 3: 2-221

Wilbert N. (1975) Eine verbesserte Technik der Protargolimprägnation für Ciliaten. Mikrokosmos 64: 171-179

Wilbert N., Song W. (2008) A further study on littoral ciliates (Protozoa, Ciliophora) near King George Island, Antarctica, with description of a new genus and seven new species. J. Nat. His. 42: 979-1012

Zhang Q., Miao M., Strüder-Kypke M. C, Al-Rasheid ? A. S., AlFarraj S., Song W. (201 1) Molecular evolution of Cinetochilum and Sathrophilus (Protozoa, Ciliophora, Oligohymenophorea), two genera of ciliates with morphological affinities to scuticociliates. Zool. Scr. 40: 317-325

Received on 22nd October, 2012; revised on 5* December, 2012; accepted on 15* December, 2012