Taxonomic Descriptions of Two Marine Ciliates, Euplotes dammamensis n. sp. and Euplotes balteatus (Dujardin, 1841) Kahl, 1932 (Ciliophora, Spirotrichea, Euplotida), Collected from the Arabian Gulf, Saudi Arabia

Abstract

The morphology, morphogenesis and infraciliature of two marine euplotid ciliates, Euplotes dammamensis n. sp. and Euplotes balteatus (Dujardin, 1841) Kahl, 1932, isolated from a sandy beach of the Arabian Gulf, Saudi Arabia, were investigated using observations in vivo and protargol-impregnation methods. Euplotes dammamensis n. sp. is characterized by a combination of features including its huge body size (100-170 x 80-120 jam), 10 conspicuous dorsal ridges, 10 normal-sized frontoventral and two marginal cirri, and 11 dorsal kineties. Euplotes balteatus is mainly characterized by 10 frontoventral, two caudal, and two left marginal cirri, 7-10 dorsal kineties and 5-7 prominent dorsal ridges as well as double-eurystomus silverline system. The small subunit rRNA (SSU-rRNA) gene sequences were determined for both species and phylogenetic analyses based on these data indicated that E. dammamensis is most closely related to E. parabalteatus Jiang et al., 2010, and E. balteatus clusters with E. plicatum Valbonesi et al., 1997, E. orientalis Jiang et al., 2010, and E. bisulcatus Kahl, 1932. [PUBLICATION ABSTRACT]

Headnote

Abstract.

The morphology, morphogenesis and infraciliature of two marine euplotid ciliates, Euplotes dammamensis n. sp. and Euplotes balteatus (Dujardin, 1841) Kahl, 1932, isolated from a sandy beach of the Arabian Gulf, Saudi Arabia, were investigated using observations in vivo and protargol-impregnation methods. Euplotes dammamensis n. sp. is characterized by a combination of features including its huge body size (100-170 x 80-120 jam), 10 conspicuous dorsal ridges, 10 normal-sized frontoventral and two marginal cirri, and 11 dorsal kineties. Euplotes balteatus is mainly characterized by 10 frontoventral, two caudal, and two left marginal cirri, 7-10 dorsal kineties and 5-7 prominent dorsal ridges as well as double-eurystomus silverline system. The small subunit rRNA (SSU-rRNA) gene sequences were determined for both species and phylogenetic analyses based on these data indicated that E. dammamensis is most closely related to E. parabalteatus Jiang et al., 2010, and E. balteatus clusters with E. plicatum Valbonesi et al., 1997, E. orientalis Jiang et al., 2010, and E. bisulcatus Kahl, 1932.

Key words: Marine ciliate, morphogenesis, morphology, new species, SSU-rRNA, taxonomy.

INTRODUCTION

Euplotes Ehrenberg, 1830 is a highly diversified and cosmopolitan genus, with a large number of species that have been observed and investigated in all kinds of biotopes. Traditionally, they are characterized by their body size and body shape, their dorsal and ventral ridges, and the cirral pattern on the ventral side. With the improvement in research methods during the last half century, some new specific features have been used to distinguish this kind of organism, such as: the numbers and arrangement of the cirri on the ventral surface; the shape of the adorai zone of membranelles and the number of membranelles; the shape of the macronucleus; the number of dorsal kineties; the silverline system pattern (Tuffrau 1960, Borror 1972, Carter 1972, Curds 1975). Based on these characteristics, more than 30 new morphospecies of Euplotes have been reported in the last three decades (Berger and Foissner 1989; Agatha eí al. 1990; Valbonesi and Luporini 1990, 1995; Petz et al. 1995; Coppellotti and Cisotto 1996; Song and Bradbury 1997; Song and Packroff 1997; Song and Wilbert 1997, 2002; Valbonesi et al. 1997; Lobban et al. 2005; Schwarz and Stoeck 2007; Schwarz et al. 2007; Wilbert and Song 2008; Jiang et al. 2010a, b; Shao et al. 2010; Pan et al. 2012).

In the present study two Euplotes species were isolated from a sandy beach of the Arabian Gulf, Saudi Arabia, including one new species. These are described following examination of live and protargol-impregnated specimens, small subunit rRNA (SSU-rRNA) gene sequence homology and the phylogenetic relationship with their congeners.

MATERIALS AND METHODS

Morphological and morphogenetic studies

Euplotes dammamensis n. sp. and E. balteatus were collected on 16 December 2011 from the intertidal regions of a sandy beach on the Arabian Gulf (Dammam, Saudi Arabia, 26°33'07"N, 50°01'30"E) where the water temperature was 17°C and the salinity 65%o (In general, the salinity of the Arabian Gulf is about 38^4 l%o. The sampling location, however, is an intertidal sandy pool, and the salinity is, therefore, higher than in open waters). The upper 5 cm layer of sand was collected together with seawater from the site. Specimens were maintained in Petri dishes at room temperature (about 20°C) with added rice grains to enrich bacteria as food for the ciliates (Chen et al. 2012). Attempts to culture the two species failed; therefore all studies were carried out on freshly isolated specimens.

Isolated individuals were examined in vivo using bright field and Nomarski differential interference contrast microscopy. The protargol-impregnation method of Wilbert (1975) was used in order to reveal the infraciliature. Counts and measurements on stained specimens were performed at a magnification of 1,250 x. Drawings were made with the help of a camera lucida. Terminology is mainly according to Curds (1975).

SSU-rRNA gene sequence analysis

Genomic DNA extraction, PCR amplification, and SSU-rRNA gene amplification and sequencing of Euplotes dammamensis n. sp. and E. balteatus were performed according to Huang et al. (2012). The primers used for SSU-rRNA gene amplification were Eukaryotic universal A (5'-AAC CTG GTT GAT CCT GCC AGT-3 ') and Eukaryotic universal B (5'-TGA TCC TTC TGC AGG TTC ACC TAC-3') covering the full length of the gene by polymerase chain reaction (PCR) (Medlin et al. 1988). The new sequences have been deposited in the GenBank database (E. dammamensis: JX185743, E. balteatus: JX185744). The other nucleotide sequences used in this study and their GenBank/EMBL accession numbers are given after the names of the species in figure 91.

Alignment was performed using MUSCLE alignment implemented in Geneious v5.4 (Drummond et al. 2010). The resulting alignment was manually inspected using the software Mega v5.0 (Tamura et al. 2011). The alignment results of congeners morphologically similar to those in the present study have been set out in Table 3. The dataseis used for SSU-rRNA phylogenetic analyses included 1,582 positions. Loxodes striatus (U24248) was selected as the out-group species. A Bayesian inference (BI) analysis was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the GTR+I+G model selected by MrModeltest 2 (Nylander 2004) under the AIC criterion. Markov chain Monte Carlo (MCMC) simulations were run with two sets of four chains using the default settings: chain length 1,000,000 generations, with trees sampled every 100 generations. The first 2,500 trees were discarded as burnin. 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. A Maximum Likelihood (ML) analysis was performed with PhyML v3.0 (Guindon and Gascuel 2003) using the GTR+G+I model selected under the AIC criterion by Modeltest v.3.7 (Posada and Crandall 1998). BIONJ was used to obtain starting trees and BEST was selected for branch swapping. The reliability of internal branches was assessed using a non-parametric bootstrap method with 1,000 replicates. Finally, phylogenetic trees (BI and ML) were visualized and edited in Geneious v5.4 (Drummond et al. 2010).

RESULTS AND DISCUSSION

Euplotes dammamensis n. sp. (Figs 1-36; Tables 1, 2)

Diagnosis. Marine Euplotes with 10 conspicuous dorsal ridges, 100-170 x 80-120 \.im in vivo. Adorai zone comprising about three-quarters of the total cell length, with about 48 membranelles; consistently 10 frontoventral, five transverse and two marginal cirri, and, generally, two or three caudal cirri, with second cirrus prolonged significantly; 11 dorsal kineties with about 16 dikinetids in mid-dorsal row. Macronucleus variable in shape, anterior part typically C-shaped with posterior part distorted.

Type location. Isolated from the Arabian Gulf (26°33'07"N, 50°01'30"E) on 16 December 2011. Salinity about 65%o and water temperature about 17°C.

Type specimens. The slide (registration number: CXR-20111216-20-01) containing the holotype specimen (Figs 8, 9, 27, 28) and one paratype slide (registration number: CXR-20111216-20-02) with protargol-impregnated specimens have been deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao.

Etymology. The species-group name dammamensis refers to the area (Dammam, Saudi Arabia) where the sample was collected.

Morphological description. Cells in vivo usually 130-150 jam long. Outline in frontal plane broadly elliptical to doliform (Figs 1, 19); left and right margins usually convex in well-fed individuals (Figs 2, 20); anterior end with a distinct projection on right side (Figs 1, 2, 19). Cell dorsoventrally flattened about 2:1 with ventral side straight or slightly convex and dorsal side strongly arched (Figs 4, 22). Ventral side with three conspicuous ridges, two right ridges extending posteriorly to transverse cirri, and leftmost ridge extending to marginal cirri (Figs 1,2, 19, arrowheads); ridges among transverse cirri short but prominent. Dorsal side usually with 10 longitudinal dorsal ridges extending over entire length of body, dominant in vivo and conspicuous even in stained specimens (Figs 3, 21, arrowheads). Dorsal cilia conspicuous, about 5 iam long; frontoventral cirri strong and about 35-40 iim long. Marginal and caudal cirri relatively fine, with second caudal cirrus longer than the others, and about 40 |jm in length (Figs 1, 2, arrows; 23, arrowhead).

Colourless ellipsoidal granules about 2x1 jam in size. On an apical view of the dorsal surface, these granules were packed together around the dorsal cilia in a rosette-pattern (Figs 6, 24, arrowheads); on a lateral view of the dorsal surface, the granules protruded out of the cortical surface and were packed like pyramids (Figs 7, 25, arrowheads). On the ventral surface, the granules were packed all around the cirri (Figs 1, 5, arrows). Cytoplasm colourless, transparent in the marginal area but opaque in the central part and packed with many shining globules (ca. 2-3 (xm across) and several food vacuoles with possibly flagellates or bacteria. Contractile vacuole not observed. Macronucleus variable in shape: anterior part typically C-shaped, and posterior part curved to distorted (Figs 9-12, 26, arrow). Micronucleus spherical, about 10 iim in diameter, and situated in a depression at the left anterior edge of the macronucleus (Figs 9-12, arrow).

Locomotion by crawling slowly on substrate while, occasionally, remaining stationary for rather long periods.

Infraciliature as shown in Figs 8, 9, 26-28. Adorai zone prominent, about three-quarters of total body length and composed of 44-51 membranelles. Paroral membrane about 20 iim in length, typically composed of many irregularly arranged kinetosomes, positioned beneath buccal lip, and generally revealed only by protargol impregnation (Fig. 8). Considering huge body size, all cirri relatively fine (with their bases usually .quite small relative to the ventral surface). Consistently 10 frontoventral cirri arranged in a normal pattern, five strong transverse cirri and two marginal cirri aligned with, usually, two caudal cirri (only five individuals out of 28 observed specimens with three caudal cirri). Always 11 dorsal kineties, extending over the entire length of the cell, except for the leftmost one which started at about the mid-body position and included about 11 dikinetids. Each middle row with about 16 dikinetids, while rightmost and second to rightmost rows with 17-22 and 17-24 dikinetids respectively (Figs 8, 9, 28, arrowheads). The silverline system was unfortunately not able to be obtained.

Morphogenesis. Three specimens were observed in the early and middle stages of morphogenesis and based on this, we can provide some of the morphogenetic features of this new species: 1) the oral primordium in the opisthe developed de novo in a subcortical pouch; 2) nine frontoventral and five transverse cirri developed from the five frontoventral transverse anlagen which were formed apokinetally in both daughter cells and then fragmented in a "3:3:3:3:2" pattern; 3) the migratory cirrus in the proter developed de novo; 4) the left marginal cirri developed from the marginal anlagen which were formed de novo; and 5) the origin of the dorsal kineties anlagen in both daughter cells was in a primary mode, i.e. anlagen for proter and opisthe appeared as one set of densely packed dikinetids (Figs 13-18,29-36).

Remarks and comparison. This new species can be identified by the characteristics of its cells in both living and protargol-impregnated specimens although, unfortunately, no silverline system could be revealed. Nonetheless, the species can be unambiguously identified by the combination of the following features: its huge body size, the presence of 10 dominant dorsal ridges, the shape and distribution of the cortical granules, its curved C-shaped macronucleus and, probably, the high salinity of its habitat. It differs from the morphologically closely-related congener, Euplotes charon (Müller, 1786) Ehrenberg, 1830, in having a bigger body size (100-170 x 80-120 [im vs. 70-110 x 65-95 [xm), more conspicuous dorsal ridges (10 vs. seven) and dorsal kineties (11 vs. 9-10), fewer membranelles (44-51 vs. 51-60) and less dikinetids in the mid-dorsal kinety (ca. 16 vs. ca. 22). It can also be distinguished by the shape of its macronucleus (the anterior part being a typical C-shape but the posterior part being curved to distorted compared to a typical C-shape in E. charon), as well as by the shape of its adorai zone, which is even, compared to the proximal portion being conspicuously broad in is. charon (Table 2; Song and Packroff 1997). The separation of these two species is also strongly supported by molecular data, since their SSU-rRNA gene sequences differ by 340 nucleotides and exhibit just 80.6% similarity [E. charon GenBank accession number: AF492705; submitted by Song (2002)] (Li and Song 2006).

Hitherto, about 16 morphotypes possessing several dorsal ridges, 10 frontoventral, and two left marginal cirri have been reported (Table 2). Besides Euplotes charon and E. dammamensis, E. antarcticus Fenchel and Lee, 1972, E. petzi Wilbert and Song, 2008, E. harpa Stein, 1859, E. focardii Valbonesi and Luporini, 1990 and the Arabian Gulf population of E. balteatus also have a big size and a large number of adorai membranelles. We will provide a detailed comparison between E. dammamensis and E. balteatus, in the following section, here we compare E. dammamensis with the other four species listed.

Euplotes antarcticus differs from E. dammamensis in its body shape, which is very elongated and almost rectangular in outline except for the pointed posterior region, whereas E. dammamensis is approximately rectangular with two rounded ends. E. antarcticus also has a smaller body size (85 x 30 [xm vs. 100-170 x 100 jam), fewer membranelles (ca. 30 vs. 44-51), fewer dorsal kineties (eight vs. 11) and dorsal ridges (six vs. 10) (Table 2; Fenchel and Lee 1972).

Euplotes petzi has been found, to date, only in the Antarctic area. It differs from E. dammamensis in having a smaller body size (50-80 x 30-50 [jm vs. 100-170 x 80-120 iam), fewer dorsal kineties (six vs. 11) and dorsal ridges (five vs. 10), and has a sausage shaped or slightly curved macronucleus with closely packed marginal cirri (Table 2; Agatha et al. 1993; Petz et al. 1995; Wilbert and Song 2008).

Euplotes harpa is also a huge (150-160 |jm long) marine Euplotes with 10 dominant dorsal ridges. It can be easily distinguished from E. dammamensis, however, by the number of its adorai membranelles (65-70 vs. 44-51) and dorsal kineties (13 vs. 11), as well as in having more dikinetids in the mid-dorsal kinety (40-45 vs. ca. 22) (Table 2; Tuffrau 1960). In addition, the SSUrRNA gene sequences of E. harpa and E. dammamensis differ in 373 nucleotides and exhibit 78% similarity [E. harpa GenBank accession number: AJ305252; submitted by Petroni (2001)] (Petroni et al. 2002).

Euplotes focardii was discovered by Valbonesi and Luporini (1990) and is highly variable, both in its size (38-110 x 30-92 jam) and in the number of dikinetids in the mid-dorsal kinety (13-22). It resembles E. dammamensis in the number and arrangement of the cirri on the ventral surface and in the ratio of the length of the adorai zone of membranelles to the total body length (50-75% vs. 75%). It also resembles E. dammamensis in the number of dorsal kineties (10 vs. 11), dikinetids in the mid-dorsal kinety (13-22 vs. 14-18), and adorai membranelles (45-65 vs. 44-51). Euplotes focardii can be distinguished from E. dammamensis, however, in the features of its dorsal surface (only the leftmost two ridges are rather pronounced and six other ridges are inconspicuous vs. ten prominent and conspicuous dorsal ridges), the shape of its macronucleus (a typical horseshoe-shaped vs. the anterior part being C-shape but the posterior part being curved to distorted) (Table 2; Valbonesi and Luporini 1990). The divergence of these two forms is supported by SSU-rRNA gene sequence data, as they differ in 351 nucleotides and exhibit 81.3% similarity [E. focardii GenBank accession number: EF094960; submitted by Giuseppe and Dini (2006)] (Vallesi et al. 2008).

Although Euplotes neapolitanus Wichterman, 1964 is also a large marine organism (130 x 70 (xm) with 10 frontoventral, two caudal, two left marginal cirri, 11 dorsal kineties, and about 18 dikinetids in the mid-dorsal kinety, it can be easily distinguished from E. dammamensis through the absence of dorsal ridges and through the proximal portion of the adorai zone of membranelles being curved at about 90° angle to the right, and being composed of more membranelles (Wichterman 1964).

Euplotes balteatus (Dujardin, 1841) Kahl, 1932 (Figs 37-66; Tables 1, 2)

Some new data, especially in respect to the molecular information, have been obtained from the population studied here and we therefore present an updated description and an improved diagnosis based on present and previous works (Dujardin 1841, Kahl 1932, Tuffrau 1964, Pan et al. 2012).

Improved diagnosis. Marine Euplotes with 3-5 dorsal ridges, 30-150 x 30-105 iam in vivo. Adorai zone about 67-75% of cell length, with 25-80 membranelles. 10 frontoventral, five transverse, 2-3 caudal and two marginal cirri, 7-8 dorsal kineties bearing 10-16 dikinetids in mid-dorsal row. Macronucleus C-shaped or horseshoe-shaped. Micronucleus ellipsoidal, and attached to left arm of macronucleus. Dorsal silverline system in a double-eurystomus pattern.

Voucher slides. Two voucher slides of protargolstained cells have been deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao (registration number CXR-20111216-21-01, 02).

Morphological description of the Saudi population. Cells in vivo usually 70-100 x 50-75 [im in size. Body outline ellipsoidal with anterior and posterior ends slightly narrowed, as shown in Figs 37, 54. Left margin of anterior end truncated in some individuals (Figs 38, 55). Buccal field approximately three-quarters of total cell length. Three conspicuous ridges on ventral side, two right ridges extending posteriorly to transverse cirri, leftmost ridge interrupted by paroral membrane (Figs 37, 38, 55, 57). Five dominant ridges on dorsal side extending over entire cell length (Figs 39, 56, arrowheads).

Cytoplasm colourless, highly transparent at posterior part and oral area, but opaque in central part where it is packed with many different-sized gray granules and a few food vacuoles. Contractile vacuole under the fourth right transverse cirrus (Figs 37, 38,54, arrow). Macronucleus variable in shape: usually typical horseshoe-shaped (Fig. 44); occasionally, approximately ring-shape, with two ends expanded and almost connected to each other (Fig. 59). Micronucleus spherical, and attached to left arm of macronucleus (Figs 48, 58, arrowhead).

Movement slow, involving crawling on substrate and remaining stationary for long periods.

Cirri and adorai membranelles fine and long. Adorai membranelles, frontoventral, and left marginal cirri approximately 25 iam long, caudal cirri about 20 (im, and transverse cirri 30-35 iam.

Infraciliature as shown in Figs 47,48, 58-62. Paroral membrane small, typically composed of many irregularly arranged kinetosomes, positioned close to surface of buccal lip and easily revealed in vivo (Figs 37, 38, 57, double-arrowheads). Adorai zone of membranelles resembling that of Euplotes vannus, proximal portion curved at about 90° angle to right, and composed of 31-39 membranelles. Consistently, 10 frontoventral and five strong transverse cirri forming a normal pattern, two left marginal cirri separated and aligned evenly with two caudal cirri. Seven dorsal kineties extending over almost entire length of cell, with middle row containing 10-13 dikinetids, but leftmost row including only 4-7 dikinetids (Figs 47, 60). Dorsal silverline system in a double-eurystomus pattern (Figs 40, 62).

Morphogenesis. Only one specimen in the middle stage of morphogenesis was obtained. The morphogenesis of this species can be summarized as follows: 1) the oral primordium form in a subcortical pouch; 2) nine frontoventral and five transverse cirri develop from the five frontoventral transverse anlagen which fragment in a "3:3:3:3:2" pattern; 3) the leftmost frontal cirrus develops de novo on the cell surface in both dividers; and 4) the left marginal cirri develop from the marginal anlagen which are formed de novo (Figs 49, 50, 63-66).

Comparison with different populations. This species was first described by Dujardin (1841) and subsequently redescribed several times (Kahl 1932, Tuffrau 1964, Pan et al. 2012). We identify our form by the basic ciliature on the ventral and dorsal surfaces, the marine habitat, the five dorsal ridges, and the double-eurystomus silverline system pattern. Nonetheless, there are some small differences between the Arabian Gulf population and historic populations, such as cell size, the number of dorsal kineties and dikineties in the mid-dorsal kinety, as well as the shape of the macronucleus: for details see Table 2.

The original description was brief and incomplete: oval shaped body with the anterior end slightly narrowed, five inconspicuous dorsal ridges (Dujardin 1841). About a century later, Kahl (1932) gave a good succinct description: cell length about 60-80 iam in vivo, dorsoventrally flattened, three to five slight dorsal ridges, ten frontoventral, five transverse, four caudal cirri (Figs 43, 44; Table 2). The morphology of the silverline system and infraciliature were supplied by Tuffrau (1964) (Figs 41, 42, 45; Table 2) and Pan et al. (2012) (Figs 46, 51-53; Table 2). In the present Arabian Gulf population, it is noticeable that the five dorsal ridges are dominant. Because this diagnostic trait usually varies in relation with the cell's nutritive conditions, it is regarded as a population-level difference.

Supposed synonym. In the original description (Kahl 1932), Euplotes alatus is a medium marine species (75-90 (im long) with 10 frontoventral, two caudal, and two marginal cirri, and five conspicuous dorsal ridges (the leftmost 2nd ridge being huge and prominent). In Borror's redescription, it has eight dorsal kineties, 10-12 dikinetids in the mid-dorsal kinety, about 26 adorai membranelles, and the double-eurystomus silverline system pattern (Table 2; Figs 67-72; Borror 1968). We incline to consider these characters as inner-specific differences and agree with Song and Wilbert (2002) that E. alatus is possibly a junior synonym of E. balteatus. For the same reason, E. quinquecarinatus Borror, 1968 may also be a junior synonym of E. balteatus (Table 2; Figs 80-83; Borror 1968, Song and Wilbert 2002).

Comparison with related congeners. In terms of the 10 frontoventral, two caudal, two marginal cirri, and several dorsal ridges, there are ten potentially related species that should be compared with E. balteatus: E. trisulcatus Kahl, 1932; E. cristatus Kahl, 1932, sensu Tuffrau (1960), E. magnicirratus Carter, 1972, E. euryhalinus Valbonesi and Luporini, 1990, E. plicatum Valbonesi et al., 1997, E. petzi Wilbert and Song, 2008, E. orientalis Jiang et al., 2010, E. wilberti Pan et al., 2012, E. parabalteatus Jiang et al., 2010, E. dammamensis n. sp. (Table 2). Of these, E. cristatus differs from E. balteatus in its single-vannus silverline system pattern (Tuffrau 1960). For the same reason, E. orientalis and E. petzi can be easily distinguished from E. balteatus through their double-patella silverline system pattern (Petz et al. 1995, Wilbert and Song 2008, Jiang et al. 2010b).

Euplotes magnicirratus is similar to E. balteatus in its cell size, the basic ciliature on both ventral and dorsal sides, the marine habitat, and the features of its dorsal ridges. The former differs from the latter, however, in having more adorai membranelles (49-52 vs. generally 25-39) and, particularly, stronger cirri on the ventral side (Table 2; Figs 73-75; Carter 1972). In addition, the SSU-rRNA gene sequences of E. magnicirratus and E. balteatus differ in 165 nucleotides and exhibit 91.1% similarity [E. magnicirratus GenBank accession number: AJ549210; submitted by Petroni (2003)] (Table 3; Petroni et al. 2002).

Euplotes plicatum is a small (42-55 x 24-40 iim) organism collected from the harbour of Lyttelton (Christchurch, New Zealand) whose diagnosis closely resembles that ofis. balteatus: marine habitat, 10 frontoventral, five transverse, two caudal, and two marginal cirri, 22-25 adorai membranelles, double-eurystomus silverline system pattern (Table 2; Valbonesi et al. 1997). It can be distinguished from E. balteatus, however, through several morphological differences, i.e. the number of dorsal ridges (7-8 vs. 3-5) and dorsal kineties (10 vs. 7-8). Also, the SSU-rRNA gene sequences of the two species differ in 14 nucleotides and exhibit 99.2% similarity [E. plicatum GenBank accession number: EF094966; submitted by Giuseppen and Dini (2006)] (Table 3; Vallesi et al. 2008).

Euplotes euryhalinus differs from E. balteatus in having more dorsal kineties (11 vs. generally 7-8), and more dikinetids in the mid-dorsal kinety (18 vs. 10-16) (Table 2; Valbonesi and Luporini 1990). The two species can also be easily distinguished by their molecular information, with 228 different nucleotides between their SSU-rRNA gene sequences and a similarity of 87.8% [E. euryhalinus GenBank accession number: EF094968; submitted by Giuseppen and Dini (2006)] (Table 3; Vallesi et al. 2008).

Euplotes parabalteatus is similar to E. balteatus in cell shape and size, cirral pattern and silverline system. The former, however, can be separated from the latter by the feature of dorsal ridges (absent vs. 3-5 conspicuous), having fewer adorai membranelles (19-23 vs. 25-80) and dorsal kineties (6-7 vs. 7-8), the shape of macronucleus (slightly curved-bar-shaped vs. inverted C-shaped) (Figs 87-90; Jiang et al. 2010a). The divergence of these two forms is supported by SSU-rRNA gene sequence data, as they differ in 317 nucleotides and exhibit 82.3% similarity [E. parabalteatus Gen-Bank accession number: FJ346568; submitted by Li and Song (2008)] (Table 3; Jiang et al. 2010a).

Euplotes wilberti was collected from King George Island, Antarctica and originally reported by Song and Wilbert (2002) as another population of E. balteatus. The Antarctica population differs from E. balteatus in several respects, however, including: the number of dorsal ridges (6-7 vs. 3-5) and dorsal kineties (8-10 vs. 7-8) and the character of the marginal cirri (densely ranged vs. widely separated). Based on these differences, Pan et al. (2012) identified it as a new species, E. wilberti (Figs 84-86; Table 2; Song and Wilbert 2002, Pan et al. 2012).

Euplotes trisulcatus is a "slim" organism, with a ratio of body length to width of about 2:1. It differs from E. balteatus in two respects: the number of dorsal ridges (stable three vs. 3-5) and the shape of the macronucleus (slightly curved vs. inverted C-shaped) (Figs 76-79; Table 2; Kahl 1932, Tuffrau 1960). Based on these morphological features alone it should be synonymised with E. balteatus. The SSU-rRNA gene sequences of the two species, however, differ in 196 nucleotides and exhibit 89.5% similarity [E. trisulcatus GenBank accession number: EF690810; submitted by Schwarz and Stoeck (2007); unpublished] (Table 3). In view of this, detailed redescription of E. trisulcatus is necessary, especially to ensure that the morphometric and molecular data are based on the same population.

Euplotes dammamensis, meanwhile, the new species described in this work, can be clearly distinguished from the present population of E. balteatus by its body size (100-170 [im vs. 60-100 pm), the number of dorsal ridges (10 vs. 3-5), and dorsal kineties (11 vs. 7-8) (Table 2). The dissimilarity between them is also supported by the molecular data, since their SSU-rRNA gene sequences differ by 337 nucleotides and exhibit 80.3% similarity (Table 3).

Phylogenetic analyses of two Euplotes species based on SSU-rRNA gene sequences

The ML and BI trees showed an identical topological structure (Fig. 91). Euplotes spp. formed a well-supported clade (ML 100%, BI 1.00). As shown in Fig. 91, this group includes six well-supported clades and several species for which relationships remain unresolved. This is consistent with results presented in previous SSU-rRNA trees (Yi et al. 2009; Jiang et al. 2010a, b). The new species, E. dammamensis n. sp., did not cluster with the morphologically similar species E. charon. In fact, it clustered with a small-sized E. parabalteatus without any dorsal ridges, together with E. sinicus branching independently at the basal position for all other Euplotes species. The phylogenetic analyses presented here, along with the comparisons of the nucleotide sequences of the SSU-rRNA gene regions, support the validity of E. dammamensis, E. charon, E. harpa, and E. focardii as distinct species. The newly sequenced Euplotes balteatus clusters with E. plicatum and E. orientalis with high support value (ML 100%, BI 1.00) and, together with E. bisulcatus, form one of the major clade with full support (ML 100%, BI 1.00). Although these four species grouped together, E. bisulcatus has a different cirral pattern (nine frontoventral cirri and single marginal cirrus), and E. orientalis has a different dorsal silverline system type (double-patella), as mentioned above. It is noteworthy that five morphologically similar species, E. balteatus, E. plicatum, E. magnicirratus, E. euryhalinus, and E. parabalteatus did not cluster in a group, which tends to support the opinion of Petroni et al. (2002), Schwarz et al. (2007), and Yi et al. (2009) that phylogenetic reconstructions of the Euplotes species based on molecular evidence in some cases disagree with taxonomic classifications and phylogenetic reconstructions based on morphological characters (e.g. dorsal argyrome pattern).

Acknowledgements. Our deep thanks are expressed to the anonymous reviewers and editors for their helpful criticisms and comments on the first version of this manuscript. The research was sponsored by the Natural Science Foundation of China (project Nos. 41006086 to Chen; 31172041 to Shao), King Saud University, Deanship of Scientific Research, Research Group Project (No. RGP-VPP-083), the Discipline Project of Ningbo University (Nos. xkcl 1006; xkll 1102) and K. C. Wong Magna Fund in Ningbo University. We also thank Prof. Weibo Song (Ocean University of China) and Dr. Zhenzhen Yi (South China Normal University) for their constructive suggestions and modifications to the manuscript.

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Submitted on 1st November, 2012; revised on 28th January, 2012; accepted on 11th March, 2013