Eur. J. Protistol.

ARTICLE IN PRESS European Journal of

PROTISTOLOGY European Journal of Protistology 46 (2010) 121–132 www.elsevier.de/ejop

Morphology and SSU rRNA gene-based phylogeny of two marine Euplotes species, E. orientalis spec. nov. and E. raikovi Agamaliev, 1966 (Ciliophora, Euplotida) Jiamei Jianga, Qianqian Zhanga, Alan Warrenb, Khaled A.S. Al-Rasheidc, Weibo Songa, a

Laboratory of Protozoology, Ocean University of China, Qingdao 266003, China Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK c Zoology Department, King Saud University, Riyadh 11451, Saudi Arabia b

Received 27 September 2009; received in revised form 27 November 2009; accepted 30 November 2009

Abstract The living morphology, infraciliature and silverline system of two small marine Euplotes species, E. orientalis spec. nov. and E. raikovi Agamaliev, 1966, isolated from a sandy beach near Qingdao, China, were investigated. Euplotes orientalis is characterized by a combination of features including their small size (35-45 mm long), five or six conspicuous dorsal ridges, two cilia-free basal plaques, eight normal-sized frontoventral cirri (FVC), and a doublepatella-I type of silverline system. Euplotes raikovi is redescribed based on a Chinese population and includes the first detailed description of its morphology in vivo. It can be recognized by having one highly reduced and seven normalsized frontoventral cirri, a single marginal cirrus, and a double-patella-I type of silverline system. For both species the small subunit rRNA (SSU rRNA) gene sequence was determined. Phylogenetic analyses based on these data indicate that E. orientalis is most closely related to E. plicatum and E. bisulcatus, whereas E. raikovi clusters with its conspecific strains, close to E. nobilii and E. elegans. & 2009 Elsevier GmbH. All rights reserved. Keywords: Euplotids; Marine ciliates; Molecular systematics; Taxonomy

Introduction Ciliates belonging to the genus Euplotes Ehrenberg, 1830 are ubiquitously distributed in marine, freshwater, and terrestrial biotopes. Traditionally, species separation was based on body size and shape, the dorsal and ventral ridges, and the ciliary pattern on the ventral side (Curds 1975). Klein (1926) first discovered the silverline Corresponding author. Tel.: þ86 532 8203 2283.

E-mail address: [email protected] (W. Song). 0932-4739/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2009.11.003

system in Euplotes and just 34 years later Tuffrau (1960) established it as an important character for species circumscription. Tuffrau (1960) used the form of the macronucleus, the numbers of frontoventral cirri and dorsal kineties, and the silverline system in order to distinguish species. Borror (1972) and Carter (1972) revised the species checklist of the genus Euplotes that Tuffrau (1960) had proposed. Furthermore, Carter (1972) suggested that the number and shape of the adoral zone of membranelles are useful additional features for species separation. The most recent taxonomic

ARTICLE IN PRESS 122

J. Jiang et al. / European Journal of Protistology 46 (2010) 121–132

revision of Euplotes was that of Curds (1975) who recognized 51 species. In the meantime the description of further new species has increased this number to over 80 species (e.g. Berger and Foissner 1989; Lobban et al. 2005; Valbonesi et al. 1997). According to Berger (2001), almost 150 species and sub-species have been established or assigned to Euplotes. Thus, it is still one of the most confused ciliate genera in terms of species diversity and taxonomy even though many species have been investigated using a range of modern methods (e.g. Agatha et al. 1993; Burkovsky 1970; Carter 1972; Petz et al. 1995; Song and Wilbert 2002; Valbonesi and Luporini 1995). There are several reasons for this including: (1) in many early studies useful information for species circumscription (e.g. silverline system) was either insufficiently described or unreported, and; (2) most species share similarities in certain diagnostic features, e.g. body shape and size, numbers of cirri and dorsal kineties (Curds 1975; Kahl 1932). Recent surveys of the ciliate fauna of the northern China seas have so far revealed eight species of Euplotes including two that are new to science (Song and Packroff 1997; Song and Warren 2009; Song and Wilbert 1997). In the present paper we describe one new species and provide a redescription of the poorly known species E. raikovi Agamaliev, 1966 based on their living morphology and infraciliature. Small subunit ribosomal RNA (SSU rRNA) gene sequence data are also supplied for both species and their phylogenetic positions in the SSU rRNA gene tree are determined.

Genomic DNA extraction, PCR amplification, and SSU rRNA gene cloning and sequencing of Euplotes orientalis and E. raikovi were performed according to Yi et al. (2008a) and Gao et al. (2008). Primers used for SSU rRNA gene amplification were 16S-F (50 -AACCTGGTTGATCCTGCCAGT-30 ) and 16S-R (50 -TGATCCTTCTGCAGGTTCACCTAC-30 ) covering the full length of the gene. The new sequences have been deposited in the GenBank database with the following accession numbers: E. orientalis FJ875138, E. raikovi EU430745. The other nucleotide sequences used in this study and their GenBank/EMBL accession numbers are shown in Table 1. Loxodes striatus was selected as the out-group species. Phylogenetic trees based on the SSU rRNA sequences for the family Euplotidae were constructed using three different methods: Bayesian inference (BI), Maximum likelihood (ML), and Maximum parsimony (MP). Phylogenetic analyses were performed according to Yi et al. (2008b). An additional alignment for the sequences of E. orientalis, E. nobilii, E. rariseta, E. plicatum and E. bisulcatus were performed using Clustal W, ver. 1.80 (Thompson et al. 1994). The sequence of the Qingdao population of E. raikovi (abbreviated as pop CH) was also aligned with those of E. elegans and an E. raikovi population from USA (abbreviated as pop US). Similarities among sequences were calculated pairwise in MEGA (Kumar et al. 2004).

Results Materials and methods Euplotes orientalis spec. nov. (Figs. 1 and 2; Table 2) Euplotes orientalis spec. nov. was collected on 25 October 2006 from the top 10 cm of sandy littoral sediments from a coastal habitat near Qingdao (Tsingtao, 1201180 E; 361040 N) where the water temperature was 19 1C and the salinity 28%. Euplotes raikovi was collected on 19 November 2006 from the same beach, water temperature 17 1C, salinity 29 %. In each case the sand with water from the sampling site were maintained in Petri dishes at room temperature (ca. 23 1C) with rice grains as food source to enrich the growth of bacterial food for the ciliates (Wang et al. 2008). The specimens were examined in vivo using bright field and Nomarski differential interference contrast microscopy (Song et al. 2009). The protargol-impregnation method of Wilbert (1975) was used in order to reveal the infraciliature. The Chatton-Lwoff silver nitrate method was used to reveal the silverline systems (Wilbert and Song 2008). Counts and measurements on stained specimens were performed at a magnification of 1250. Drawings were made with the help of camera lucida. Terminology is mainly according to Curds (1975).

Diagnosis. Small marine Euplotes, in vivo about 40 mm long; five or six conspicuous dorsal ridges. Buccal field about 65% of cell length and with about 22 membranelles; eight nomal-sized frontoventral cirri and two highly reduced basal plaques; two marginal and two caudal cirri; six or seven dorsal kineties with about seven dikinetids in mid-dorsal row. Macronucleus C-shaped. Dorsal silverline system double-patella-I type. Type location. Sandy beach near Laoshan, Qingdao, China. Water temperature about 19 1C, salinity about 28%. Type specimens. Two hapantotype slides (registration numbers 2009:5:11:1 and 2009:5:11:2) with protargoland silver nitrate-impregnated specimens respectively are deposited in the Natural History Museum, London, UK. A third hapantotype slide is deposited in the Laboratory of Protozoology, OUC, China (No. JJM2006102501) Etymology. The species-group name orientalis recalls the fact that this species was first described from oriental (Chinese) coastal waters.

ARTICLE IN PRESS J. Jiang et al. / European Journal of Protistology 46 (2010) 121–132

Table 1.

123

Small subunit rRNA sequences from Genbank used in this study. New sequences are in bold.

Species name

GB number

Species name

GB number

Species name

GB number

Amphisiella annulata Aspidisca steini Aspidisca aculeata Certesia quadrinucleata Diophrys appendiculata Diophrys oligothrix Diophrys scutum Diophryopsis hystrix Euplotes aediculatus Euplotes bisulcatus Euplotes sp. Euplotes crassus Euplotes daidaleos Euplotes elegans Euplotes encysticus Euplotes euryhalinus Euplotes eurystomus Euplotes focardii

DQ832260 AF305625 EF123704 DQ059581 AY004773 DQ353850 DQ353851 EF486861 M14590 EF094965 AF492705 AY361895 EF690811 DQ309868 EF535728 EF094968 AJ310491 EF094960

Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes Euplotes

AJ305252 AJ549210 AY361900 AJ305254 EF094970 EF094963 FJ875138 FJ346568 AF452708 AJ305247 EF094964 EF094966 EF094974 EU430745 FJ423449 FJ423448 EF690810 AY004772

Euplotes woodruffi Euplotidium arenarium Gastrocirrhus monilifer Laboea strobila Loxodes striatus Onychodromopsis flexilis Phacodinium metchnikoffi Prodiscocephalus borrori Protocruzia adherens Protocruzia contrax Paradiophrys irmgard Paradiophrys sp. Strombidium apolatum Strombidinopsis jeokjo Tintinnidium mucicola Uronychia binucleata Uronychia setigera Uronychia transfuga

AF492707 Y19166 DQ864734 AF399151 U24248 AY498652 AJ277877 DQ646880 AY217727 DQ190467 EU189070 EU189071 DQ662848 AJ628250 AY143563 EF198667 EF198669 AF260120

harpa magnicirratus minuta muscicola nobilii octocarinatus orientalis parabalteatus parawoodruffi parkei patella plicatum raikovi pop US raikovipop CH rariseta sinicus trisulcatus vannus

Fig. 1. Euplotes orientalis spec. nov. in vivo (A – C), after silver nitrate (D, E) and protargol (F, G) impregnation, and E. pseudoraikovi Alekperov, 2005 (H, I, from Alekperov 2005). (A) Ventral view of a representative specimen. (B) Dorsal view, showing the granules beneath the pellicle. (C) Dorsal view, to show ridges. (D, E) Silverline system on ventral and dorsal side, note the reduced frontoventral cirri (arrowheads). (F, G) Ventral and dorsal view of the same specimen, showing infraciliature and nuclear apparatus, arrowheads point to the reduced frontoventral cirri. (H, I) Silverline system and infraciliature on ventral and dorsal side, arrowheads show the reduced frontoventral cirri. AZM, adoral zone of membranelles; CC, caudal cirri; MC, marginal cirri; PM, paroral membrane; TC, transverse cirri. Bars, 20 mm.



Fig. 2. Euplotes orientalis spec. nov. in vivo (A – G), after silver nitrate (H, I) and protargol (J) impregnation, and other morphologically similar small, marine Euplotes species (K-R) with a double-patella-I silverline pattern and 10 frontoventral cirri. (A) Ventral view of a representative specimen, arrows show the ridges on ventral side. (B, D, E) Ventral view of other specimens, arrows point to the contractile vacuole (B) and the position of the reduced frontoventral cirri (E). (C) Cells of different size and shape, arrow indicates the projection on the right anterior margin. (F) Dorsal view, to show the dorsal ridges (arrows). (G) Detailed view, arrows point to the granules around the dorsal cilia. (H, I) Ventral (H) and dorsal (I) silverline system, arrowheads show the reduced frontoventral cirri. (J) Infraciliature of ventral side, arrowheads point to the reduced frontoventral cirri. (K, L) E. algivora (from Agatha et al. 1990). (M, N) E. rariseta (from Song and Packroff 1997). (O, P) E. margherensis (from Coppellotti and Cisotto 1996). (Q, R) E. petzi (from Petz et al. 1995). Bars, 20 mm (A, B, J), 30 mm (C; K, also applies to L, O-R), 5 mm (G), 15 mm (M, N).

Description. Cell in vivo usually 35-45 mm long, generally oval in outline (Figs. 1A, 2A-E). Both left and right margins convex; anterior end narrowly rounded with a distinct projection at right side (Figs 1A; 2C, arrow). Buccal field approximately 65% of body length. Three conspicuous ventral ridges extending posteriorly to transverse cirri (Figs 1A; 2A, arrows, 2D, E) with some shorter ridges between them. Five or six conspicuous dorsal ridges (Figs 1C; 2F, arrows). Several ellipsoid granules, possibly mitochondria, ca. 0.8 mm long, densely packed around dorsal cilia beneath pellicle (Figs 1B; 2G, arrows). Cytoplasm colorless, highly transparent in cell margin, opaque in central part where different-sized lipid droplets and a few food vacuoles are usually present. Contractile

vacuole about 3 mm in diameter, located posterior of the rightmost transverse cirrus (Fig. 2B, arrow). Macronucleus inverted U-shaped (Fig. 1G). Micronucleus not observed. Locomotion typically by moderately fast crawling or jerking movements. Infraciliature as shown in Fig. 1D-G. Paroral membrane small and wide, composed of many irregularly arranged kinetosomes, positioned below buccal lip (Fig. 1F). Adoral zone prominent, composed of 18-25 membranelles. Invariably eight frontoventral cirri, basal plaques IV/2 and V/2 uniquely composed of single dikinetids (Fig. 1D, F, 2H, J, arrowheads); five close-set transverse cirri; two marginal and two caudal cirri. Six or seven dorsal kineties, all sparsely ciliated, mid-dorsal kinety with six to eight dikinetids (Fig. 1E, G); in addition there are one or two


Table 2. Morphometric data of Euplotes orientalis spec. nov. (upper row, in bold), E. raikovi (middle row) and E. elegans (from Schwarz et al. 2007; bottom row). Data are based on protargol-impregnated specimens. AM, adoral membranelles; CC, caudal cirri; CV, coefficient of variation in %; DK, dorsal kineties; FVC, frontoventral cirri; Max, maximum; MC, marginal cirrus; Min, minimum; n, number of cells measured; RC, reduced cirrus (basal plaques); SD, standard deviation; TC, transverse cirri.

125

ilarities among E. orientalis, E. nobilii and E. raikovi are firmly supported by the SSU rRNA gene sequence data. The sequence of E. orientalis differs in 294 nucleotides from E. nobilii and has a structural similarity of only 84.3%, while it differs in 214 nucleotides from E. raikovi, with a structural similarity of 93%. The sequences of E. orientalis and E. bisulcatus differ in 51 nucleotides and have a structural similarity of 97.2% while those of E. orientalis and E. plicatum differ in only 6 nucleotides and have a structural similarity of 99.7% (Fig. 5G).

Characteristics

Min Max Mean SD CV n

Body length in mm

32 50 69

42 64 96

36.5 56.8 83.4

2.90 7.9 4.50 7.9 6.50 7.8

Body width in mm

23 41 38

31 48 63

26.1 44.4 52.3

2.26 8.7 15 1.93 4.3 16 5.90 11.3 43

Qingdao Population of Euplotes raikoviAgamaliev, 1966 (Figs. 3 and 4; Table 2)

No. of AM

18 22 47

25 29 64

21.5 26.7 54.9

2.00 9.3 1.89 7.1 4.60 8.3

15 16 29

No. of FVC

10 7 9

10 7 9

10 7 9

0 0 0

0 0 0

25 25 25

No. of RC

2 1 1n

2 1 1n

2 1 1n

0 0 0n

0 0 0n

25 25 ?

No. of TC

5 5 5

5 5 5

5 5 5

0 0 0

0 0 0

25 25 25

No. of MC

2 1 1

2 1 1

2 1 1

0 0 0

0 0 0

25 25 25

No. of CC

2 2 2

2 2 2

2 2 2

0 0 0

0 0 0

25 25 25

No. of DK

6 6 9

7 7 10

6.6 6.9 9.4

0.51 7.7 0.34 5.0 0.50 5.2

15 16 25

No. of dikinetids in middorsal kinety

6 10 15

8 13 20

7.3 11.4 17.7

0.62 8.4 0.89 7.8 1.40 8.1

15 16 25

2 5 ?

1.9 4.1 ?

0.26 13.4 15 0.68 16.7 16 ? ? ?

Euplotes raikovi was originally reported by Agamaliev (1966) based on silver nitrate-impregnated specimens (Fig. 5A, B) and reinvestigated by Washburn and Borror (1972), Miceli et al. (1981), Alekperov and Asadullayeva (1999), Alekperov (2005) and Jiang et al. (2008). However, none of these reports included a detailed description of the morphology in vivo. Therefore we here give a supplementary redescription based on the Qingdao population. Cell in vivo about 40-56 mm in length; body shape broadly oval (Figs. 3A, 4A, C); dorsoventrally flattened about 1:3 (Figs. 3B, 4D). Adoral zone about 65% of cell length (Fig. 3A) and composed of 22-29 membranelles. Three conspicuous ventral ridges (Figs 3A; 4A, arrowheads) and about five conspicuous dorsal ridges extending almost entire length of body (Figs 3I; 4B, arrowheads). Several bar-shaped granules, possibly mitochondria, ca. 2.5 0.8 mm, beneath pellicle packed around dorsal dikinetids (Figs. 3J, 4F, arrows). Contractile vacuole positioned posterior and right of midcell, typical of genus. Macronucleus C-shaped containing irregular-shaped nucleoli. Locomotion typically by moderately fast crawling or jerking movements. Invariably seven frontoventral cirri, basal plaque V/2 composed of a single dikinetid (Fig. 3C, G, arrowheads; 4E, H, arrowheads), five transverse, and two caudal cirri. Single marginal cirrus located posterior of buccal field (Fig. 4C, arrow). Six or seven dorsal kineties extending almost entire length of body with additional ca. 3-5 dikinetids ventrally on left margin; mid-dorsal row with about 10-13 dikinetids (Figs. 3D, 4G). Dorsal silverline system of double-patella-I type (Figs. 3H, 4I). SSU rRNA gene sequence analysis. The complete SSU rRNA gene sequences of E. raikovi pop CH is 1897 bp in length and has a GC content of 45.6%. The sequences of E. raikovi pop CH and E. raikovi pop US differ by only three nucleotides and have a similarity of 99.8%. Furthermore, sequences of these two populations differ from E. elegans in 200 nucleotides and 197

No. of dikinetids in leftmost 1 kinety on ventral side 3 ?

15 16 43

? not available. n Data from the description and the illustrator.

dikinetids ventrally on left margin. Dorsal silverline system double-patella-I type (Figs. 1E, 2I). SSU rRNA gene sequence analysis. The complete SSU rRNA gene sequence of Euplotes orientalis is 1850 bp in length and has a GC content of 48.1%. The dissim-



Fig. 3. Euplotes raikovi Agamaliev, 1966in vivo (A – B, F, I, J), after protargol (C, D, E) and silver nitrate (G, H) impregnation. (A) Ventral view of representative cell. (B) Right lateral view, to show the ridges. (C, D) Ventral (C) and dorsal (D) view, showing the infraciliature and nuclear apparatus, arrowhead shows the reduced frontoventral cirrus. (E) Different shapes of macronucleus. (F) Ventral views of different cells. (G, H) silverline system on ventral and dorsal side, note the reduced frontoventral cirrus (arrowhead). (I) Dorsal view, to show the ridges. (J) Dorsal view of posterior portion of cell, showing the sub-pellicular granules around the dorsal cilia. AZM, adoral zone of membranelles; CC, caudal cirri; FVC, frontoventral cirri; MC, marginal cirrus; TC, transverse cirri. Bars, 30 mm (A, also applies to C, D, G, H; F).

nucleotides respectively and have sequence similarities of 89.8% and 90% respectively (Fig. 5H).

Discussion The dargyrome of Euplotes Curds (1975) defined five types of dargyrome, i.e. patterns of dorsal silverline system, in Euplotes: (1) single vannus (single cross-connectives), (2) double eurystomus (two equal rows of polygons between ciliary rows), (3) double patella-I, II patterns (two unequal rows of polygons), (4) multiple (three or four rows), and (5) complex (with an irregular network of polygons). In a later revision, the number of types of dargyrome was reduced to three, namely single, double, and multiple (Gates and Curds 1979), although Borror and Hill (1995) erroneously suggested that this revised scheme

continued to recognize a distinction between the ‘multiple’ and ‘complex’ types. The 3-pattern scheme was accepted by Valbonesi and Luporini (1995) whose work showed that the type of dargyrome can, to a large extent, be attributed to cell cortical ridges. Other workers, however, have continued to use the original scheme (Agatha et al. 1990; Song and Bradbury 1997). The present study supports the view that the double types of dargyrome are species-dependent and, for some species, highly reliable. Thus, we believe that the ‘double’ types are useful for species circumscription and accept Curds (1975) original scheme of five dargyrome types.

Euplotes orientalis Only one congener of E. orientalis, namely E. pseudoraikovi, has been reported with two reduced frontoventral cirri and a double-patella-I type of


127

Fig. 4. Photomicrographs of Euplotes raikoviAgamaliev, 1966in vivo (A-D, F), after protargol (E, G) and silver nitrate (H, I) impregnation (A-E, G, I, from Jiang et al. 2008). (A) Ventral view, arrowheads point to the ventral ridges. (B) Dorsal view, to show the ridges (arrowheads). (C) Ventral view of representative individual, arrow shows the single marginal cirrus. (D) Lateral view. (E) Ventral view of infraciliature, arrowhead points to the basal bodies of the reduced frontoventral cirrus. (F) Detailed view, arrows point to the granules around the dorsal cilia. (G) Dorsal side, to show the dorsal kineties. (H, I) Ventral (H) and dorsal (I) views of silverline system, arrowhead points to the reduced cirrus, arrow shows the single marginal cirrus. Bars, 30 mm (A, also applies to H, I; D).

silverline system (Fig. 1H, I). According to the original description by Alekperov (2005), this form differs from our new species in having more membranelles (40-45 vs. 18-25), fewer frontoventral cirri (6 vs. 8) and fewer dorsal kineties (about 5 vs. 6 or 7). However, 40-45 membranelles is too many for such a small species (3045 mm in length). The photomicrograph in Alekperov (2005) is clearly that of another species and does not match the drawing and description. Euplotes pseudoraikovi needs to be carefully re-investigated in order to verify its diagnostic characters. Furthermore, the photomicrograph in Alekperov (2005) of E. raikovi (Plate 24-3) shows two small basal plaques. Since the photomicrograph clearly shows the presence of one marginal cirrus, this taxon cannot be E. orientalis. Euplotes parkei Curds, 1974 is the only small marine Euplotes reported as having eight normal-sized, and no reduced frontoventral cirri. Furthermore, compared to E. orientalis, E. parkei has relatively inconspicuous ridges, more dikinetids in the middorsal kinety (8-11 vs. 6-8) and a double-eurystomus (vs. double-patella-I) type of silverline system (Curds 1974).

Five small, marine species of Euplotes possessing 10 frontoventral cirri and a double-patella-I silverline pattern have been reported, namely E. algivora, E. nobilii, E. margherensis, E. rariseta and E. petzi (Agatha et al. 1990; Coppellotti and Cisotto 1996; Curds 1974; Petz et al. 1995; Song and Packroff 1997; Valbonesi and Luporini 1990; Wilbert and Song 2008). It should be noted, however, that in each case none of the frontoventral cirri is reduced. The Antarctic form E. algivora differs from E. orientalis in having more membranelles (28-37 vs. 18-25), one extremely long (vs. two short and fine) marginal cirrus and two (vs. five) dorsal ridges (Agatha et al. 1990; Fig. 2K, L; Table 3). Valbonesi and Luporini (1990) reported E. nobilii from the Antarctic. It is separated from E. orientalis by having only one (vs. two) marginal cirrus (Table 3). Euplotes petzi, another Antarctic form, differs from E. orientalis in being larger (51-72 38-47 mm vs. 3242 23-32 mm after silver impregnation), having more adoral membranelles (28-33 vs. 18-25) and two closely spaced (vs. well-separated) marginal cirri (Wilbert and Song 2008; Fig. 2Q, R; Table 3).



Fig. 5. Infraciliature of some marine Euplotes with one reduced frontoventral cirrus (A – F) and the alignments of SSU rRNA sequences of morphologically similar Euplotes species (G, H). Arrowheads point to cirrus V/2. (A, B, C) E. raikovi (A, B, from Agamaliev 1966; C, from Agamaliev 1967). (D) E. strelkovi (from Agamaliev 1967). (E, F) E. elegans (from Schwarz et al. 2007). (G) Seven regions of the alignment of nuclear small subunit rRNA sequences of Euplotes orientalis, E. plicatum, E. bisulcatus, E. nobilii, and E. rariseta. (H) Four regions of the alignment of nuclear small subunit rRNA sequences of E. raikovi pop CH, E. raikovi pop US and E. elegans, showing the high diversity among the sequences. Regions are separated by double lines, and positions of nucleotides at the beginning and end of each region are given at the top of each column. The differences in sequence length were compensated for by introducing alignment gaps (-) in the sequences. Matched sites are marked with dots. Bars, 20 mm (A, also applies to B – D) and 40 mm (E, F).

Table 3. Comparison of E. orientalis with some related marine congeners having a double-patella-I type of silverline system and 10 frontoventral cirri. AM, adoral membranelles; DK, dorsal kineties; FVC, frontoventral cirri; RC, reduced cirri (basal plaques). Characteristics

E. orientalis

E. algivora

E. nobilii

E. margherensis

E. rariseta

E. petzi

Body size in mmn No. of membranelles No. of FVC No. of RC No. of marginal cirri No. of dorsal kineties No. of dikinetids in mid-dorsal kinety Other features

32-42 23-32 18-25 8 2 2 6-7 6-8

40-59 24-40 28-37 10 0 1 6 7-12

29-43 18-29 18-22 10 0 1 8 9

43-64 34-45 35-45 10 0 1 6 9-11

22-30 17-23 17-21 10 0 1 7 5-7

51-72 38-47 28-33 10 0 2 6 8-10

5-6 dorsal ridges

NF

NF

NF

Data source

present work

2 dorsal ridges; single marginal cirrus very long Agatha et al. 1990

Valbonesi and Luporini 1990

Coppellotti and Cisotto 1996

Song and Packroff 1997

2 fine marginal cirri closely spaced Wilbert and Song 2008

NF: no special feature. n Measurement after silver impregnation.

Euplotes margherensis can be clearly distinguished from E. orientalis by having more membranelles (35-45 vs. 18-25), one (vs. two) marginal cirrus, and

more dikinetids (9-11 vs. 6-8) in the mid-dorsal kinety (Coppellotti and Cisotto 1996; Fig. 2O, P; Table 3).


129

Euplotes rariseta differs from E. orientalis by being smaller (22-30 mm vs. 32-42 mm after silver impregnation) and having only one (vs. two) marginal cirrus (Curds et al. 1974; Song and Packroff 1997; Fig. 2M, N; Table 3). Two other forms that resemble E. orientalis in general morphology are E. plicatum Valbonesi et al., 1997 and E. bisulcatus Kahl, 1932. Euplotes plicatum differs from E. orientalis in that none of its frontoventral cirri is reduced (vs. two reduced cirri in the latter), having more dikinetids (14 vs. 6-8) in the mid-dorsal kinety, and a double-eurystomus (vs. double-patella-I) type of silverline system (Valbonesi et al. 1997). Interestingly, based on SSU rRNA gene sequence data, E. orientalis is more closely related to E. plicatum than to any other species of Euplotes, despite their differences in morphology. Euplotes bisulcatus is also a small form, ca. 40 mm in length in vivo. It can be easily distinguished from E. orientalis by the prominent double-edged ridges separated by shallow grooves, the number of frontoventral cirri (9 vs. 8) and the number of marginal cirri (1 vs. 2) (Kahl 1932; Borror 1968). It is also noteworthy that the dorsal silverline system of E. bisulcatus is of a special double-eurystomus type as a result of its unique dorsal structure (Valbonesi and Luporini 1995).

the published photomicographs. Curds (1975) overlooked this difference and described E. strelkovi as having a double-patella-II, rather than double-patella-I, type of silverline system. Nevertheless, E. strelkovi can be separated from E. raikovi by the number of frontoventral (8 vs. 7) and, uniquely, in having six (vs. five) transverse cirri (Fig. 5D) (Agamaliev 1967). Euplotes elegans was originally reported by Kahl (1932) who supplied a brief description based on observations in vivo. It was redescribed by Tuffrau (1960) who used silver impregnation to reveal the silverline system, however this was believed by Schwarz et al. (2007) to be a misidentification. Euplotes elegans sensu Tuffrau (1960) was described as new species (E. pseudoelegans) by Schwarz and Stoeck (2007) based on isolates collected in eastern Denmark. Euplotes elegans is a medium-sized marine species with a double-patella-I type of silverline system. Compared with E. raikovi, E. elegans has more membranelles (47-64 vs. 22-29), frontoventral cirri (9 vs. 7), dorsal kineties (9-10 vs. 6-7), and dikinetids in the mid-dorsal row (15-20 vs. 10-13) (Schwarz et al. 2007).

Euplotes raikovi

Trees constructed using different algorithms showed similar topologies in our target group and thus were combined (Fig. 6). The analyses provide full posterior probability (PP) and bootstrap support for the monophyly of Euplotidae (1.00BI/100ML/100MP, data not shown). As shown in Fig. 6, six major groups are resolved excluding E. rariseta, E. sinicus and E. parabalteatus. These clades were previously identified by Yi et al. (2009). The newly sequenced E. orientalis clusters with E. plicatum with high support value (0.98BI/100ML/95MP, data not shown) and, together with E. bisulcatus, form one of the major clades with full support (1.00BI/100ML/100MP, data not shown). The two populations of E. raikovi are closely related (1.00BI/100ML/100MP, data not shown) and placed in another major clade (clade III) together with E. nobilii and E. elegans, with maximum PP and bootstrap values (1.00BI/100ML/100MP, data not shown). The addition of E. raikovi and E. orientalis increased the resolution of the phylogenetic tree of Euplotes compared to those in previous studies (Achilles-Day et al. 2008; Petroni et al. 2002; Schwarz et al. 2007; Yi et al. 2009). In particular, the position of E. raikovi is more stable and better supported than in previous trees. Euplotes orientalis grouped with E. plicatum and E. bisulcatus in a clade whose position is rather unstable according to Achilles-Day et al. (2008) and Yi et al. (2009) although its position in our tree is more

In terms of its morphology in vivo and infraciliature, the Qingdao population of E. raikovi corresponds closely with the original description especially in having the small basal plaque V/2 (Agamaliev 1966; Fig. 5A, B). Agamaliev (1967) described a Caspian Sea population, some individuals of which had a normal-sized cirrus V/2 (Fig. 5C, arrowhead). Later, E. raikovi populations with a stable small basal plaque V/2 were recorded from the Atlantic coast of the USA (Washburn and Borror 1972), the Mediterranean Sea (Miceli et al. 1981), the coast of the Caspian Sea again (Alekperov and Asadullayeva 1999; Alekperov 2005), and the east coast of China (Jiang et al. 2008). Considering cirrus V/2 is always reduced in all other populations, we suggest that those individuals in the Caspian Sea population reported by Agamaliev (1967) with eight normal-sized frontoventral cirri may represent a separate form. Furthermore, E. raikovi sensu Alekperov and Asadullayeva 1999, and E. raikovi sensu Agamaliev (2005) have more membranelles than other populations (45-55 vs. 22-32). Of all the marine Euplotes species only two, E. strelkovi Agamaliev, 1967 and E. elegans Kahl, 1932, have one reduced frontoventral cirrus (Agamaliev 1967; Schwarz et al. 2007). In the original description of E. strelkovi, Agamaliev (1967) wrongly depicted the silverline system in his line diagram when compared to

Phylogenetic analyses of E. orientalis and E. raikovi based on SSU rRNA gene sequence data



Fig. 6. Phylogenetic tree based on SSU rRNA sequences showing the positions of Euplotes orientalis and E. raikovi pop CH (arrows), by Bayesian inference (BI), Maximum likelihood (ML), and Maximum parsimony (MP). Numbers near branches are as follows: BI posterior probability value, ML and MP bootstrap values. ‘‘n’’ reflects disagreement among phylogenies. The wellsupported (40.95 BI, 490% ML, 490% MP) branches are marked with solid circles. Family Euplotidae is highlighted in gray. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. Euplotid clades I-VI were designated according to Yi et al. (2009).

consistent with the latter. Though these three species grouped together, they are of different dorsal silverline system types as mentioned above. The overall topology of our tree is most similar to that of Yi et al. (2009) although support for several of the nodes is rather low, especially based on ML and MP analyses (Fig. 6), and probably insufficient to resolve the interrelationships within Euplotes. Nevertheless, the phylogenetic analyses presented here (Fig. 6), along with the comparisons of the nucleotide sequences of the SSU rRNA gene regions (Fig. 5G, H), support the validity of both E. orientalis and E. raikovi as distinct species.

Acknowledgements This work was supported by the Natural Science Foundation of China (project No. 30870264), the Darwin Initiative Programme (project no. 14-015), which is funded by the UK Department for Environment, Food and Rural Affairs, and a joint grant from the Center of Excellence in Biodiversity, King Saud University. We are grateful to Mr. Hongbo Pan, a graduate student at the Laboratory of Protozoology, Ocean University of China (OUC), for help with sample

collection. Thanks are also due to Prof. Xiaozhong Hu and Dr. Chen Shao, Laboratory of Protozoology, OUC, for their valuable comments and kindly reading the first version of the MS.

References Achilles-Day, U.E.M., Pro¨schold, T., Day, J.G., 2008. Phylogenetic position of the freshwater ciliate Euplotes daidaleos within the family of Euplotidae, obtained from small subunit rDNA gene sequence. Denisia 23, 411–416. Agamaliev, F.G., 1966. New species of psammobiotic ciliates of the western coast of the Caspian Sea. Acta Protozool. 4, 169–183. Agamaliev, F.G., 1967. Faune des cilie´s me´sopsammiques de la coˆte ouest de la Mer Caspienne. Cah. Biol. Mar. 8, 359–402. Agatha, S., Spindler, M., Wilbert, N., 1993. Ciliated protozoa (Ciliophora) from Arctic sea ice. Acta Protozool. 32, 261–268. Agatha, S., Wilbert, M., Spindler, M., Elbra¨chter, M., 1990. Euplotide ciliates in sea ice of the Weddell Sea (Antarctica). Acta Protozool. 29, 221–228.


Alekperov, I., 2005. In: An Atlas of the Free-living Ciliates (Classes Kinetofragminophora, Colpodea, Oligohymenophora, Polyhymenophora). ‘‘Borchali’’ Publ. House, Baku. Alekperov, I., Asadullayeva, E.S., 1999. New, little-known and characteristic ciliate species from the Apsheron coast of the Caspian Sea. Tr. J. Zool. 23, 215–225. Berger, H., 2001. In: Catalogue of Ciliate Names 1. Hypotrichs. Verlag Helmut Berger, Salzburg. Berger, H., Foissner, W., 1989. Morphology and biometry of some soil hypotrichs (Protozoa, Ciliophora) from Europe and Japan. Bull. Br. Mus. Nat. Hist. (Zool.) 55, 19–46. Borror, A.C., 1968. Systematics of Euplotes (Ciliophora, Hypotrichia); toward union of the old and the new. J. Protozool. 15, 802–808. Borror, A.C., 1972. Revision of the order Hypotrichida (Ciliophora, Protozoa). J. Protozool. 19, 1–23. Borror, A.C., Hill, B.F., 1995. The order Euplotida (Ciliophora): taxonomy, with division of Euplotes into several genera. J. Eukaryot. Microbiol. 42, 457–466. Burkovsky, I.V., 1970. The ciliates of the mesopsammon of the Kandalaksha Gulf (White sea) I. Acta Protozool. 7, 475–499. Carter, H.P., 1972. Infraciliature of eleven species of the genus Euplotes. Trans. Am. Microsc. Soc. 91, 466–492. Coppellotti, O., Cisotto, P., 1996. Description of Euplotes margherensis nov. spec. (Ciliophora, Hypotrichida) from the Lagoon of Venice. Ital. J. Zool. 63, 163–167. Curds, C.R., 1974. Descriptions of three species of Euplotes (Protozoa: Ciliatea). Bull. Br. Mus. Nat. Hist. (Zool.) 27, 113–125. Curds, C.R., West, B.J., Dorahy, J.E., 1974. Euplotes rariseta sp. n. (Protozoa, Ciliatea), a new small marine hypotrich. Bull. Br. Mus. Nat. Hist. (Zool.) 27, 95–102. Curds, C.R., 1975. A guide to the species of the genus Euplotes (Hypotrichida, Ciliatea). Bull. Br. Mus. Nat. Hist. (Zool.) 28, 3–61. Gao, S., Chen, Z., Shao, C., Long, H., Al-Rasheid, K.A.S., Song, W., 2008. Reconsideration of the phylogenetic position of Frontonia-related Peniculia (Ciliophora, Protozoa) inferred from the small subunit ribosomal RNA gene sequences. Acta Protozool. 47, 47–54. Gates, M.A., Curds, C.R., 1979. The dargyrome of the genus Euplotes (Hypotrichida, Ciliophora). Bull. Br. Mus. Nat. Hist. (Zool.) 35, 127–134. Jiang, J., Shao, C., Li, L., Ma, H., Gong, J., Song, W., 2008. Morphological studies on seven hypotrichous ciliates (Protozoa, Ciliophora) from coastal waters off Qingdao, China. Acta Zootaxon. Sin. 33, 115–119 (in Chinese with English summary). Kahl, A., 1932. Urtiere oder Protozoa. I: Wimpertiere oder Ciliata (Infusoria) 3. Spirotricha. Tierwelt Dtl. 25, 399–650. Klein, B.M., 1926. Ergebnisse mit einer Silbermethode bei Ciliaten. Arch. Protistenk. 56, 243. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinfor. 5, 150–163. Lobban, C.S., Modeo, L., Verni, F., Rosati, G., 2005. Euplotes uncinatus (Ciliophora, Hypotrichia), a new species with zooxanthellae. Mar. Biol. 147, 1055–1061.

131

Miceli, C., Luporini, P., Bracchi, P., 1981. Morphological description, breeding system, and nuclear changes during conjugation of Euplotes raikovi Agamaliev from Mediterranean Sea. Acta Protozool. 31, 215–224. Petroni, G., Dini, F., Verni, F., Rosati, G., 2002. A molecular approach to the tangled intrageneric relationships underlying phylogeny in Euplotes (Ciliophora, Spirotrichea). Mol. Phylogenet. Evol. 22, 118–130. 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. Stapfia 40, 1–223. Schwarz, M.V.J., Stoeck, T., 2007. Euplotes pseudoelegans n. sp. (Hypotrichida; Euplotidae): description of a new species previously misidentified as Euplotes elegans Kahl, 1932. Acta Protozool. 46, 193–200. Schwarz, M.V.J., Zuendorf, A., Stoeck, T., 2007. Morphology, ultrastructure, molecular phylogeny, and autecology of Euplotes elegans Kahl, 1932 (Hypotrichida; Euplotidae) isolated from the anoxic Mariager Fjord, Denmark. J. Eukaryot. Microbiol. 54, 125–136. Song, W., Bradbury, P., 1997. Comparative studies on a new brackish water Euplotes, E. parawoodruffi n. sp., and a redescription of Euplote woodruffi GAW, 1939 (Ciliophora; Hypotrichida). Arch. Protistenkd. 148, 399–412. Song, W., Packroff, G., 1997. Taxonomische Untersuchungen an marinen Ciliaten aus China mit Beschreibungen von zwei neuen Arten, Strombidium globosaneum nov. spec. und S. platum nov. spec. (Protozoa, Ciliophora). Arch. Protistenkd. 147, 331–360. Song, W., Shao, C., Yi, Z., Li, L., Warren, A., Al-Rasheid, K.A.S., Yang, J., 2009. The morphology, morphogenesis and SSrRNA gene sequence of a new marine ciliate, Diophrys apoligothrix spec. nov. (Ciliophora; Euplotida). Eur. J. Protistol. 45, 38–50. Song, W., Warren, A., 2009. Protohypotrichids, Discocephalids and Euplotids. In: Song, W., Warren, A., Hu, X. (Eds.), Free-Living Ciliates of the Bohai and Yellow Seas, China. Science Press, Beijing, pp. 415–459. Song, W., Wilbert, N., 1997. Morphological investigations on some free living ciliates (Protozoa, Ciliophora) from China Sea with description of a new hypotrichous genus, Hemigastrostyla nov. gen. Arch. Protistenkd. 148, 413–444. Song, W., Wilbert, N., 2002. Faunistic studies on marine ciliates form the Antarctic benthic area, including descriptions of one epizoic form, 6 new species and 2 new genera (Protozoa; Ciliophora). Acta Protozool. 41, 23–61. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionsspecific gap penalties and weight matrix choice. Nucl. Acid Res. 22, 4673–4680. Tuffrau, M., 1960. Re´vision du genre Euplotes, fondeé sur la comparaison des structures superficielles. Hydrobiologia 15, 1–77. Valbonesi, A., Luporini, P., 1990. Description of two new species of Euplotes and Euplotes rariseta from Antarctica. Polar Biol. 11, 47–53.



Valbonesi, A., Luporini, P., 1995. Euplotes bisulcatus, a key species for a better understanding of the organization and evolution of Euplotes argyrome patterns. Eur. J. Protistol. 31, 32–37. Valbonesi, A., Apone, F., Luporini, P., 1997. Morphology and biology of a new species of Euplotes, Euplotes plicatum sp. n. (Ciliophora: Euplotidae). Acta Protozool. 36, 287–294. Wang, Y., Miao, M., Zhang, Q., Gao, S., Song, W., Al-Rasheid, K., Warren, A., Ma, H., 2008. Three marine interstitial Scuticociliates, Schizocalyptra similis sp. n., S. sinica sp. n. and Hippocomos salinus Small and Lynn, 1985 (Ciliophora: Scuticociliatida), isolated from Chinese costal waters. Acta Protozool. 47, 377–387. Washburn, E.S., Borror, A.C., 1972. Euplotes raikovi Agamaliev, 1966 (Ciliophora, Hypotrichida) from New Hampshire: description and morphogenesis. J. Protozool. 19, 604–608. Wilbert, N., 1975. Eine verbesserte Technik der Protargolimpra¨gnation fu¨r 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. Hist. 42, 979–1012.

Yi, Z., Song, W., Warren, A., Roberts, D., Al-Rasheid, K.A.S., Chen, Z., Al-Farraj, S.A., Hu, X., 2008a. A molecular phylogenetic investigation of Pseudoamphisiella and Parabirojimia (Protozoa, Ciliophora, Spirotrichea), two genera with ambiguous systematic positions. Eur. J. Protistol. 44, 45–53. Yi, Z., Song, W., Shao, C., Warren, A., Al-Rasheid, K.A.S., Roberts, D., Miao, M., Al-Quraisy, S.A., Chen, Z., 2008b. Phylogeny of some systematically uncertain urostyloids Apokeronopsis, Metaurostylopsis, Thigmokeronopsis (Ciliophora, Stichotrichia) estimated with small subunit rRNA gene sequence information: discrepancies and agreements with morphological data. Eur. J. Protistol. 44, 254–262. Yi, Z., Song, W., Clamp, J., Chen, Z., Gao, S., Zhang, Q., 2009. Reconsideration of systematic relationships within the order Euplotida (Protista, Ciliophora) using new sequences of the gene coding for small-subunit rRNA and testing the use of combined data sets to construct phylogenies of the Diophrys-like species. Mol. Phylogen. Evol. 50, 599–607.