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Morphology of three new marine Frontonia species (Ciliophora; Peniculida) with note on the phylogeny of this genus
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European Journal of Protistology 49 (2013) 312–323
Morphology of three new marine Frontonia species (Ciliophora; Peniculida) with note on the phylogeny of this genus Xinpeng Fana,b , Xiaofeng Lina,∗ , Weiwei Liuc , Yuan Xuc , Saleh A. Al-Farrajd , Khaled A.S. Al-Rasheidd , Alan Warrene a
Laboratory of Protozoology, Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, South China Normal University, Guangzhou 510631, China b School of Life Science, East China Normal University, Shanghai 200062, China c Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China d Zoology Department, King Saud University, Riyadh 11451, Saudi Arabia e Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK Received 7 January 2012; received in revised form 14 June 2012; accepted 29 June 2012 Available online 21 July 2012
Abstract Members of the ciliate genus Frontonia are common colonizers of periphytic communities in aquatic biotopes. Recent studies indicate that their species diversity is higher than previously supposed. In this study the morphology and infraciliature of three new species, Frontonia sinica spec. nov., F. pusilla spec. nov., and F. elegans spec. nov., isolated from coastal waters of China, were investigated using live observation and silver impregnation methods. Frontonia sinica differs from its congeners by the following combination of characters: ellipsoidal body, about 116 somatic and five or six vestibular kineties, peniculi 1 and 2 four-rowed, peniculus 3 two-rowed, and a single contractile vacuole. Frontonia pusilla has about 72 somatic kineties, four-rowed peniculi 1 and 2, a two-rowed peniculus 3, and two contractile vacuoles. Frontonia elegans has 73 somatic kineties, four-rowed peniculi 1 and 2, a three-rowed peniculus 3, and two contractile vacuoles. In the present work, six new small-subunit rRNA gene sequences of six Frontonia species are used to construct the phylogenetic trees. Our phylogenetic analysis supports that the genus Frontonia may be paraphyletic. Meanwhile, no pattern of correlation could be found between the structures of peniculi and the phylogenetic relationships of Frontonia species in the present study. © 2012 Elsevier GmbH. All rights reserved. Keywords: Frontonia; Morphology; New species; Phylogeny; Small-subunit rRNA
Introduction The peniculid genus Frontonia was established by Ehrenberg (1838). Over 40 nominal species have since been described from aquatic biotopes worldwide (Borror 1963; Burkovsky 1970; Dragesco 1960; Dragesco 1972; Dragesco
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and Dragesco-Kernéis 1986; Foissner 1987; Foissner et al. 1994; Gil and Perez-Silva 1964a, b; Kahl 1931; Long et al. 2005, 2008; Petz et al. 1995; Roque 1961a, b; Roque and de Puytorac 1972; Song and Wilbert 1989). Frontonia can be easily separated from related genera by the following characters: (1) triangular buccal cavity (vs. bursiform or oval in Paramecium, Neobursaridium, Stokesia, Lembadion, and Urocentrum, and pyriform in Apofrontonia); (2) three similarly structured peniculi (vs. two peniculi and one quadrulus in Paramecium, Stokesia and Neobursaridium); (3) vestibular
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kineties at the right margin of the buccal cavity (vs. vestibular kineties absent in Paramecium, Lembadion, Urocentrum and Neobursaridium); (4) several postoral kineties (vs. postoral kineties absent in Apofrontonia); and (5) postoral suture terminating at the posterior end of the cell (vs. extending to the mid-body region of the dorsal side in Disematostoma) (Corliss 1979; Didier and Puytorac 1994; Foissner et al. 1994, 1999; Foissner and Song 2002; Kahl 1931; Serrano et al. 1990; Tuffrau and Savoie 1961). Species circumscription and delimitation among members of the genus Frontonia are, however, far from clear. This is mainly because accurate species identification is highly dependent on detailed knowledge of the infraciliature (e.g. the fine structure of the peniculi) but is lacking for about 20% of Frontonia species (Bullington 1939; Dragesco 1960; Fan et al. 2011; Long et al. 2008; Kahl 1931). Furthermore, although molecular data are playing an increasingly important role in the taxonomy and systematics of ciliates, such data are available for relatively few species of Frontonia (Fan et al. 2011; Fokin et al. 2006; Gao et al. 2008; Li et al. 2010; Pan et al. 2012; Zhang et al. 2010). Three previously unknown species of Frontonia were isolated during a survey of the ciliate fauna in coastal waters of northern and southern China. Careful investigations of specimens both in vivo and following silver impregnation, along with phylogenetic analyses based on small subunit (SSU) rRNA gene sequence data, confirm the validity of these three new species. In this paper we present detailed descriptions of the morphology of each species. Moreover, the phylogenetic trees were also constructed based on the small-subunit rRNA gene sequences, six of which were included firstly (Pan et al. 2012). The phylogeny of the genus Frontonia was analyzed.
Material and Methods Two populations of Frontonia sinica spec. nov. were collected. Pop-1 was isolated on 4 July 2002 from a shrimpculturing pond near Qingdao (36◦ 08 08 N; 120◦ 43 15 E), China, where the water temperature was 24 ◦ C, salinity 25‰, and pH 8.0. Pop-2 was isolated on 9 November 2008 from a mangrove wetland (22◦ 40 00 N; 114◦ 40 32 E) near Zhujiang River, Guangzhou, China, where the salinity was about 16‰. Frontonia pusilla spec. nov. was collected on 28 December 2008 from a mangrove wetland near Zhuhai (23◦ 07 48 E;113◦ 32 24 E), China, where the salinity was about 25‰. Frontonia elegans spec. nov. was collected on 28 December 2008 from a shrimp-culturing pond in the South China Normal University (23◦ 08 24 N; 113◦ 20 46 ), Guangzhou, China, where the salinity was about 9‰. Live ciliates were observed using differential interference contrast microscopy. 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. Drawings of impregnated specimens were made with the help of a camera lucida. Measurements were
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performed under magnifications of 100–1000×. Systematics and terminology are mainly according to Lynn (2008). Genomic DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing of the SSU rRNA gene were performed using a REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. The SSU rRNA gene was amplified by PCR with primers Euk A (5 -AAC CTG GTT GAT CCT GCC AGT-3 ) and Euk B (5 -TGA TCC TTC TGC AGG TTC ACC TAC-3 ) (Medlin et al. 1988). The SSU rRNA gene sequences of Frontonia subtropica, F. canadensis and F. magna were sequenced in Pan et al. (2012) but firstly used to construct phylogenetic trees in this work. Other additional sequences were downloaded from the GenBank database (for accession numbers, see Fig. 7). Sequences were aligned using Clustal W implemented in BioEdit 7.0 (Hall 1999). Ambiguously aligned regions and gaps were excluded before phylogenetic analyses. Colpoda inflata and Bresslaua vorax were used as the outgroup taxa. Maximum-likelihood bootstrapping analyses were carried out with 100 replicates using RAxML with the setting as described in Stamatakis et al. (2008). ML analyses were conducted online on the CIPRES Protal V 2.0 (http://www.phylo.org). A Bayesian inference (BI) analysis was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the Markov chain Monte Carlo algorithm. The program was run for 1,000,000 generations with a sample frequency of 100 and a burn-in of 2500 (Yi and Song 2011).
Results and Discussion Order Peniculida Fauré-Fremiet in Corliss, 1956 Family Frontoniidae Kahl, 1926 Genus Frontonia Ehrenberg, 1838
Frontonia sinica spec. nov. (Figs 1A–H, 2A–J, Table 1) Diagnosis. Marine Frontonia about 100–200 m × 70–100 m in vivo, ellipsoidal body shape with ratio of length to width about 5:3. Five or six vestibular, 3–5 postoral, and 100–119 somatic kineties. Peniculi 1 and 2 each with four rows, peniculus 3 two-rowed. Macronucleus ellipsoidal, located in central region of body. Subterminal contractile vacuole located near dorsal side. Type locality. Shrimp-culturing water near Qingdao (36◦ 08 08 N; 120◦ 43 15 E), northern China. Type slides. A protargol slide with the holotype specimen is deposited in the collection of Laboratory of Protozoology, OUC, China, with registration number Lin-01-9-16-1. The holotype specimen is marked by an ink circle on the coverslip. A paratype slide with protargol-impregnated specimens is
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Fig. 1. (A–H) Frontonia sinica spec. nov. from life (A–D, G) and after protargol impregnation (E, F, H). (A) Ventral view of a typical specimen. (B) Ventral view to show the sutures and contractile vacuole. (C) Lateral view, showing the location of buccal cavity (arrow) and contractile vacuole. (D) Detail of pellicle, to show the resting extrusomes beneath the pellicle. (E, F) Ventral (E) and dorsal (F) views of holotype specimen showing the general infraciliature, arrowheads show the anterior and posterior sutures. (G) Extrusomes after extrusion. (H) Detailed structures of buccal region. Arrow mark the membranelle 3 which composed of two unequal kinety rows, arrowheads refer to the anteriorly shortened somatic kineties terminating along peniculus 1. AL, argentophilic line; AS, anterior suture; CV, contractile vacuole; Ma, macronucleus; P1–3, peniculi 1, 2 and 3; PK, postoral kineties; PM, paroral membrane; PS, posterior suture; VK, vestibular kineties. Scale bars = 50 m (A, E, F); 5 m (D); 10 m (G); 25 m (H).
deposited in the collection of Natural History Museum, London, UK, with registration number NHMUK 2012.3.14.1. Etymology. The species-group name sinica recalls the fact that this species was originally isolated in China Description. Body usually about 150 m × 90 m in vivo, ratio of length to width about 5:3, non-contractile, constantly elliptical in outline when viewed from ventral or dorsal side, anterior end broadly rounded, posterior portion tapered (Figs 1A, B, 2A, B), laterally compressed about 1.5–2:1 (Fig. 1C). Body densely ciliated, most somatic cilia about 10 m long, cilia in posterior region about 20 m long (Fig. 1A). Cytoplasm slightly grayish, often with many large (10–30 m across), yellowish food vacuoles that render the body more or less opaque and yellow-colored at lower magnifications (Figs 1A, 2A–C). Buccal cavity small and shallow,
elliptic to triangular in outline, about 25 m × 12 m in size, occupying about 15% of body length, with cilia about 15 m long (Figs 1A–C, 2B, C). Extrusomes spindle-shaped, about 8 m long in vivo, densely arranged beneath pellicle alongside somatic ciliary rows (Figs 1B, D, 2J); extruded extrusomes rod-shaped with one end curved, about 25 m long (Figs 1G, 2D, I). Single subterminal contractile vacuole located near dorsal margin of body, about 8–13 m in diameter, contracting at intervals of about 1 min (Figs 1A–C, 2C). Macronucleus flattened, ellipsoid in outline, located near body center and usually detectable in vivo with differential interference contrast microscopy, about 65 m × 40 m in size in vivo, 48–80 m × 23–50 m after protargol impregnation (Figs 1E, 2H). Single micronucleus, located near end of macronucleus, ovoid, about 5 m in diameter. Locomotion
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Fig. 2. (A–J) Frontonia sinica spec. nov from life (A–D, I, J), after protargol impregnation (E, F, H), and after Chatton–Lwoff silver nitrate impregnation (G). (A) Ventral view of a typical individual. (B) Ventral view of a slightly squeezed specimen, to show the oral field (arrow). (C) Lateral view of a slightly compressed specimen, showing the location of contractile vacuole (arrowhead), and oral field (arrow). (D, I) Extruded extrusomes (arrows). (E) Buccal area, arrows refer to the strong nematodesmata. (F) Oral apparatus, showing the peniculi 1–3. (G) Part of argyrome. (H) Macronucleus. (J) Arrows mark the resting extrusomes beneath the pellicle. Ma, macronucleus; P1–3, peniculi 1, 2 and 3. Scale bar = 100 m (A).
by revolving moderately rapidly on substrate or by swimming in water. Not sensitive to disturbance. Somatic ciliature as shown in Fig. 1E, F. About 114–118 longitudinal somatic kineties composed of densely spaced dikinetids, kineties progressively shortened anteriorly forming a conspicuous anterior suture (also called preoral suture) which extends from anterior end of buccal cavity, over apical end of cell and onto dorsal side; somatic kineties also progressively shortened posteriorly below oral region forming a posterior suture (Fig. 1B, E, F). Three to five postoral kineties consisting of densely spaced dikinetids, terminating anteriorly at buccal cavity and progressively shortened posteriorly, terminating along posterior suture (Fig. 1H). Nematodesmata highly developed, extending along posterior suture, each about 10–20 m long (Figs 1F, 2E).
Buccal apparatus genus typical (Figs 1F, H, 2F). Five or six (mostly six) short vestibular kineties composed of densely spaced dikinetids, running from anterior vertex of cavity and terminating along posterior suture. Three conspicuous peniculi located on left wall of buccal cavity: peniculi 1 and 2 about equal in length, positioned close to each other and parallel to left edge of vestibular wall, each composed of four rows of kinetosomes. Peniculus 3 slightly curved to right at both ends, composed of two kineties, right one of which extends entire length of buccal cavity, left one only extending to about 40% of buccal cavity length. Single-rowed paroral membrane running almost entire length of buccal cavity along its right edge. Several argentophilic lines to left of paroral membrane. Silverline system genus typical, i.e. as quadrangular cortical meshes that can be observed after silver nitrate impregnation (Fig. 2G).
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Table 1. Morphological characterizations of Frontonia sinica spec. nov. (Pop 1, first line; Pop 2, second line), F. pusilla spec. nov. (third line), and F. elegans spec. nov. (fourth line). Characteristica
Min
Max
M
SD
CV
n
Body length
120 100 58 70
180 122 75 92
157.4 111.7 65.3 82.4
14.7 6.6 4.8 6.7
9.3 5.9 7.3 8.1
16 15 15 15
Body width
80 80 21 25
140 98 30 50
102.2 90.0 26.1 39.2
14.6 5.8 2.8 7.3
14.3 6.5 10.7 18.6
16 15 15 15
Number of somatic kineties
114 100 70 69
118 119 77 78
116.1 107.0 72.3 73.5
1.1 5.8 2.0 3.2
0.9 5.4 2.8 4.4
16 15 15 11
Number of postoral kineties
3 4 3 3
5 5 3 3
3.9 4.7 – –
0.7 0.5 – –
17.9 9.9 – –
16 11 3 3
Number of vestibular kineties
5 5 3 4
6 5 3 4
5.9 – – –
0.3 – – –
5.1 – – –
16 11 3 4
Length of macronucleus
48 45 15 20
80 62 25 25
64.2 53.9 19.7 21.3
9.9 4.7 22.7 1.5
15.4 8.7 13.7 7.0
16 15 15 15
Width of macronucleus
23 30 10 14
50 44 20 20
32.0 34.5 14.1 15.6
7.9 4.5 2.4 1.5
24.7 12.7 17.3 9.9
16 15 15 15
Abbreviations: M, arithmetic mean; Max, maximum; Min, minimum; n, sample size; SD, standard deviation; CV, coefficient of variation (%). a Data from observations of protargol-impregnated specimens. All measurements in m.
Comparison. Three species of Frontonia resemble F. sinica spec. nov. in terms of its body size and shape, namely F. acuminata Ehrenberg, 1838, F. depressa (Stokes, 1886) Kahl, 1931, and F. angusta Kahl, 1931. All three, however, differ from the latter by having: (1) more kinety rows in peniculi 1, 2 and 3 (5 or 6, 5 or 6, 3 or 4 in F. acuminata; 5, 5, 3 in F. depressa; 5, 5, 4 in F. angusta vs. 4, 4, 2 in F. sinica), and (2) fewer somatic kineties (55–60 in F. acuminata, 57–62 in F. depressa, 80–105 in F. angusta vs. 100–119 in F. sinica) (Foissner 1987; Foissner et al. 1994). Frontonia elongata Burkovsky, 1970 is the only species with the same number of penicular rows as F. sinica, i.e. 4, 4, 2 in peniculi 1, 2 and 3 respectively. However, the former can be separated from the new species by the following combination of characters: (1) two kineties of about equal length in peniculus 3 (vs. one row about half the length of the other in F. sinica); (2) body length in vivo (200–250 m vs. 100–180 m in F. sinica); (3) number of somatic kineties (40–45 vs. 100–119 in F. sinica); (4) number of postoral kineties (2 or 3 vs. 3–5 in F. sinica) (Burkovsky 1970).
Frontonia magna Fan et al., 2011 shows close relationship with F. sinica according to the phylogenetic tree. However, the former can be easily separated from the latter by its (1) larger cell size (200–450 m × 70–200 m vs. 100–200 m × 70–100 m), (2) higher number of somatic kineties (165–216 vs. 100–119), and (3) the different structure of peniculus 3 (four equal rows vs. two unequal rows) (Fan et al. 2011; Pan et al. 2012).
Frontonia pusilla spec. nov. (Figs 3A–F, 4A–O, Table 1) Diagnosis. Brackish water Frontonia about 70–100 m × 30–50 m in vivo, foot-shaped in outline. Three vestibular, three postoral and 70–77 somatic kineties. Peniculi 1 and 2 each with four kinety rows, peniculus 3 two-rowed. Macronucleus ellipsoidal, centrally located. Two contractile vacuoles.
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Fig. 3. (A–F) Frontonia pusilla spec. nov. from life (A–C) and after protargol impregnation (D–F). (A) Ventral view of a representative individual. (B) Variations in body shape. (C) Lateral view, to show the location of contractile vacuoles. (D) Detailed structure of buccal region, arrowhead shows the anteriorly shortened somatic kinety near peniculus 1. (E, F) Ventral (E) and dorsal (F) view of holotype specimen showing general infraciliature. AS, anterior suture; CV, contractile vacuole; P1–3, peniculi 1, 2 and 3; PK, postoral kineties; PM, paroral membrane; PS, posterior suture; VK, vestibular kineties. Scale bars = 30 m (A, B, E, F).
Type locality. A mangrove wetland near Zhuhai (23◦ 07 48 E; 113◦ 32 24 E), southern China. Type slides. A protargol slide with the holotype specimen is deposited in the collection of Laboratory of Protozoology, OUC, China, with registration number LWW-200811280501. The holotype specimen is marked by an ink circle on the coverslip. A paratype slide with protargol-impregnated specimens is deposited in the collection of Natural History Museum, London, UK, with registration number NHMUK 2012.3.14.2. Etymology. The species-group name pusilla (Latin, ‘small’) refers to the small size of this species. Description. Body about 70–100 m × 30–50 m in vivo, foot-shaped in outline with right side straight and left side slightly protruded at anterior third (Figs 3A, 4A, C). Dorsoventrally flattened about 3:2 (Figs 3C, 4B). Buccal cavity small, about 15 m × 8 m in size (Figs 3A, 4D, E). Cytoplasm containing numerous dark green granules, crystals in posterior region of body and food vacuoles (4–5 m across) often containing diatoms (Fig. 4F). Two contractile vacuoles located dorso-laterally, one in anterior the other in posterior third of body, each about 8 m in diameter (Figs 3A, 4C). Single ellipsoidal macronucleus about 20 m × 15 m (Fig. 4K). Extrusomes 4–5 m long, approximately 20 m long when extruded (Fig. 4G, H). Somatic cilia approximately 6 m long. Locomotion by crawling slowly on substrate or by swimming freely moderately fast. Infraciliature as shown in Figs 3D–F, 4L–N. Three postoral and 70–77 somatic kineties (Figs 3E, 4M). One shortened somatic kinety terminating anteriorly near mid-region of peniculus 1 (Fig. 3D, arrowhead). Peniculi closely spaced and parallel to each other. Peniculus 1 composed of four
kinety rows, outer two rows of which are shortened posteriorly; peniculus 2 composed of four rows of equal length. Peniculus 3 composed of two rows, outer row shortened posteriorly terminating at posterior third of inner row (Fig. 3D). Three vestibular kineties, inner one close to paroral membrane and shortened posteriorly compared to the other two (Figs 3E, 4M). Comparison. Frontonia pusilla can be distinguished from most congeners by its small body size and the number and location of the contractile vacuoles. Only two other species, namely F. ocularis Bullington, 1939 and F. fusca (Quennerstedt, 1869) Kahl, 1928, exhibit these characters and should therefore be compared with the new species. Frontonia pusilla differs from F. ocularis by the following combination of characters: (1) body foot-shaped in outline and with anterior part of body broader than posterior part (vs. body reniform, anterior part usually narrower than posterior part); (2) pigment-spot absent (vs. reddish brown pigment-spot present near right boarder of anterior extremity) (Bullington 1939). Frontonia pusilla can be distinguished from F. fusca by the absence of a pigment-spot (vs. dark-green spot present in the right anterior dorsal side of cell in F. fusca) and in having two (vs. three) kinety rows in peniculus 3 (Fokin 2008).
Frontonia elegans spec. nov. (Figs 5A–H, 6A–M, Table 1) Diagnosis. Brackish Frontonia about 75–90 m × 40–60 m in vivo. Three vestibular, four postoral, and 69–78 somatic kineties, seven of which are shortened anteriorly
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Fig. 4. (A–O) Frontonia pusilla spec. nov. from life (A–H) and after silver carbonate (I), silver nitrate (J), and protargol (K–O) impregnation. (A, C) Ventral view of two typical specimens, arrows in (C) indicate the contractile vacuoles. (B) Lateral view. (D) Buccal opening. (E) Buccal cavity, to show peniculi 1 and 2 (arrow). (F) Macronucleus and one of the two contractile vacuoles (arrow). (G) Arrowheads mark the resting extrusomes beneath the pellicle. (H) Extrusomes after extrusion. (I, J) Extrusomes in cells stained with silver carbonate (I) and silver nitrate (J). (K) Macronucleus. (L) Detailed structure of buccal area. (M) Vestibular and postoral kineties. (N) Anterior suture (arrowheads). (O) Lateral view of buccal region, arrows depict the cytopharyngeal rods. Ma, macronucleus; P1–3, peniculi 1, 2 and 3; PK, postoral kineties; PM, paroral membrane; VK, vestibular kineties. Scale bars = 30 m (A–C).
terminating along peniculus 1. Peniculi 1 and 2 each with four rows, peniculus 3 three-rowed. Macronucleus spherical, located in central region of body. Two contractile vacuoles. Type locality. A shrimp-culturing pond at South China Normal University, Guangzhou (23◦ 08 24 N; 113◦ 20 46 ), southern China. Type slides. A protargol slide with the holotype specimen is deposited in the collection of Laboratory of Protozoology,
OUC, China, with registration number LWW-200811280401. The holotype specimen is marked by an ink circle on the coverslip. A paratype slide with protargol-impregnated specimens is deposited in the collection of Natural History Museum, London, UK, with registration number NHMUK 2012.3.14.3. Etymology. The species–group name elegans (Latin, ‘elegant’) refers to the elegant body outline.
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Fig. 5. (A–H) Frontonia elegans spec. nov. (A–D, G, H) and F. fusca (E, F) from life (A–C, F) and after protargol (D, G, H) and silver nitrate (E) impregnation. (A) Ventral view of a typical individual. (B) Lateral views. (C) Variation in body shape. (D) Detailed structure of buccal region, arrowheads depict the anteriorly shortened somatic kineties. (E) Buccal apparatus of Frontonia fusca (Quennerstedt, 1869) Kahl, 1928 (from Fokin 2008). (F) Ventral view of F. fusca (from Fokin 2008), arrow depicts the pigment spot. (G, H) Ventral (G) and dorsal (H) view of general infraciliature. AS, anterior suture; P1–3, peniculi 1, 2 and 3; PK, postoral kineties; PM, paroral membrane; PS, posterior suture; VK, vestibular kineties. Scale bars = 40 m (A–C, F–H).
Description. Body 75–90 m × 40–60 m in vivo, ellipsoidal with right side straight and left side distinctly curved (Figs 5A, C, 6A, C). Dorsoventrally flattened about 3:2 (Fig. 5B). Buccal cavity about 20 m × 10 m occupying 15% of the body length; buccal area depressed in some specimens (Figs 5B, 6F). Cytoplasm containing numerous blue granules (Fig. 6G) and food vacuoles (4–5 m across) containing algae (Fig. 5A). Extrusomes spindle-shaped, 4–5 m long; when extruded about 15 m long. Single ellipsoidal macronucleus about 20 m × 15 m (Fig. 6M). Two contractile vacuoles 8–10 m in diameter located dorsolaterally, one in anterior third of cell the other in posterior third (Figs 5A, 6D); each contractile vacuole with a single excretory pore (Fig. 6E). Somatic cilia about 6 m long; in some individuals five or six longer (10 m) stiff cilia present at posterior end of body (Fig. 6H). Locomotion by crawling slowly on substrate or by swimming moderately fast. General infraciliature as shown in Fig. 5G, H. 69–78 somatic kineties that are progressively shortened both anteriorly and posteriorly forming conspicuous anterior and posterior sutures, both of which extend from ventral to dorsal side (Fig. 1G, H). Seven somatic kineties shortened anteriorly and terminate along peniculus 1 (Figs 5G, H, 6J). Buccal structure as shown in Figs 5D, 6I, K, L. Peniculi 1 and 2 each comprises four kinety rows of equal length. Peniculus 3 composed of three rows that are progressively shortened posteriorly. Three vestibular kineties each composed of densely
arranged dikinetids. Four postoral kineties, inner one usually shortened and comprising 3–5 dikinetids. Comparison. In terms of body shape, number of contractile vacuoles and the structure of the peniculi, Frontonia elegans most closely resembles F. fusca (Quennerstedt, 1869) Kahl, 1928. However, the former can be separated from the latter by the following combination of characters: (1) pigment spot absent (vs. present in F. fusca); (2) seven shortened somatic kineties that terminate anteriorly along peniculus 1 (vs. no shortened kineties in F. fusca); (3) one excretory pore (vs. 2 or 3 in F. fusca) per contractile vacuole (Fokin 2008; Fig. 5E, F). Frontonia elegans also resembles F. pusilla spec. nov. in terms of its body size, number of somatic kineties and the number and location of contractile vacuoles. However, the former can be differentiated from the latter by having a three-rowed (vs. two-rowed) peniculus 3 and about seven (vs. one) anteriorly shortened somatic kineties terminating along peniculus 1.
SSU rRNA gene sequence data The SSU rRNA gene sequences of the three new species are deposited in GenBank with accession numbers, length and G + C content as follows: F. sinica, FJ868197, 1748 bp, 44.51%; F. pusilla, FJ868201, 1702 bp, 44.59%; F. elegans, FJ868200, 1699 bp, 44.79%.
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Fig. 6. (A–M) Frontonia elegans spec. nov. from life (A–H) and after protargol (I, K–M) and silver carbonate (J) impregnation. (A–D) Different individuals, arrows in (D) mark the two contractile vacuoles. (E) Arrowheads depict the discharge pores of the two contractile vacuoles. (F) Buccal cavity. (G) Posterior portion, to show granules in the cytoplasm. (H) The cilia at caudal end (arrowheads). (I, K, L) Structure of buccal region, arrowheads in (I) depict the anteriorly shortened somatic kineties. (J) Dorsal infraciliature, arrow shows the posterior suture. (M) Macronucleus. Ma, macronucleus; P1–3, peniculi 1, 2 and 3; PK, postoral kineties; PM, paroral membrane; VK, vestibular kineties. Scale bars = 30 m (A–C, J, M).
Phylogenetic analyses Trees based on SSU rRNA gene sequence data and constructed using Bayesian inference and maximum-likelihood were almost identical, therefore only the ML tree is shown (Fig. 7). Five major groups were recovered within the class Oligohymenophorea. Four of these correspond to the four subclasses suggested in the classification system of Lynn (2008), namely Peniculia (1.00 BI, 100% ML),
Scuticociliatia (1.00 BI, 100% ML), Peritrichia (1.00 BI, 100% ML), and Hymenostomatia (1.00 BI, 86% ML). The other group (1.00 BI, 100% ML) corresponds to the subclass Mobilia that was established by Zhan et al. (2009). These findings are consistent with previous studies based on SSU rRNA gene sequence data (e.g. Fan et al. 2011; Fokin et al. 2006; Gao et al. 2008). Within the Peniculia clade, Lembadion bullinum is basal to the other peniculines included in the analyses, i.e. Paramecium (four species), Apofrontonia
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Fig. 7. Maximum-likelihood tree inferred from small subunit rRNA gene sequences. Nodal support for branches in the ML and Bayesian inference (BI) trees are marked in order. Black circles indicate full support in all analyses. All branches are drawn to scale. The scale bar corresponds to five substitutions per 100 nucleotide position. Newly sequenced species in this work and first included sequences are in bold font and marked by arrows.
(one species) and Frontonia (13 species). Frontonia species are divided into two assembles: F. pusilla, F. elegans and F. didieri cluster with Apofrontonia dohrni (0.83 BI, 62% ML), forming a sister clade with Paramecium; the other 11 Frontonia species including F. sinica, F. magna, F. canadensis and F. subtropica, group together with strong support (1.00 BI, 100% ML). In the latter assemblage, there are two well grouped clades, one containing F. leucas, F. vernalis and Frontonia sp., the other containing the remaining species. Within the latter group, the two populations of F. magna group well with each other and then cluster with F. sinica with moderate support (0.76 BI, 64% ML); F. canadensis associated with F. mengi also in a moderate supported clade (0.76 BI, 68% ML), which forms a sister group with the F. sinca-F. magna clade with the strongest support (1.00 BI, 100% ML); F. subtropica is basal to the clade containing F. lynni and F. tchibisovae with moderate support (0.88 BI, 71% ML).
Meanwhile, the phylogenetic analysis also supports the validity of our three new species: Frontonia pusilla spec. nov. and F. elegans spec. nov. being placed in one clade within the Frontonia assemblage, F. sinica spec. nov. in the other one including F. magna, F. canadensis and F. subtropica whose SSU rRNA gene sequences are firstly used to construct phylogenetic trees in this work (Pan et al. 2012). Previous studies have shown that, for morphospecies of the genus Frontonia, the structures of the peniculi (especially peniculus 3) are stable within a species but highly divergent among different species (Fan et al. 2011; Fokin 2008; Long et al. 2005, 2008; Pan et al. 2012). The peniculi would therefore appear to be potentially useful characters for assessing phylogenetic relationships within this genus. In the present study, however, no pattern of correlation could be found between the peniculi and the phylogenetic relationships recovered in the SSU rRNA gene tree.
Remarks on phylogeny
Acknowledgements
Previous studies on the molecular phylogeny of Frontonia have suggested that this genus may be paraphyletic (Gao et al. 2008). Our phylogenetic trees constructed including six new SSU rRNA gene sequences support this finding. Given the large number of Frontonia species that have yet to be sequenced (30 out of 43), it is not currently possible to draw any conclusion about the phylogeny of this genus.
This work was supported by the National Nature Science Foundation of China (project No.: 31071893), the research fund for the doctoral program of higher education (project No.: 20104407120006) and the research group project No. RGP-VPP-083, King Saud University Deanship of Scientific Research, Saudi Arabia. We greatly appreciate the comments of two anonymous reviewers and the associate editor (Dr. H. Berger). Our thanks are also due to Prof. Weibo Song and
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Ms. Feng Gao, OUC, for their comments and supply of references, respectively.
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Morphology of three new marine Frontonia species (Ciliophora; Peniculida) with note on the phylogeny of this genus Xinpeng Fana,b , Xiaofeng Lina,∗ , Weiwei Liuc , Yuan Xuc , Saleh A. Al-Farrajd , Khaled A.S. Al-Rasheidd , Alan Warrene a
Laboratory of Protozoology, Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, South China Normal University, Guangzhou 510631, China b School of Life Science, East China Normal University, Shanghai 200062, China c Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China d Zoology Department, King Saud University, Riyadh 11451, Saudi Arabia e Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK Received 7 January 2012; received in revised form 14 June 2012; accepted 29 June 2012 Available online 21 July 2012
Abstract Members of the ciliate genus Frontonia are common colonizers of periphytic communities in aquatic biotopes. Recent studies indicate that their species diversity is higher than previously supposed. In this study the morphology and infraciliature of three new species, Frontonia sinica spec. nov., F. pusilla spec. nov., and F. elegans spec. nov., isolated from coastal waters of China, were investigated using live observation and silver impregnation methods. Frontonia sinica differs from its congeners by the following combination of characters: ellipsoidal body, about 116 somatic and five or six vestibular kineties, peniculi 1 and 2 four-rowed, peniculus 3 two-rowed, and a single contractile vacuole. Frontonia pusilla has about 72 somatic kineties, four-rowed peniculi 1 and 2, a two-rowed peniculus 3, and two contractile vacuoles. Frontonia elegans has 73 somatic kineties, four-rowed peniculi 1 and 2, a three-rowed peniculus 3, and two contractile vacuoles. In the present work, six new small-subunit rRNA gene sequences of six Frontonia species are used to construct the phylogenetic trees. Our phylogenetic analysis supports that the genus Frontonia may be paraphyletic. Meanwhile, no pattern of correlation could be found between the structures of peniculi and the phylogenetic relationships of Frontonia species in the present study. © 2012 Elsevier GmbH. All rights reserved. Keywords: Frontonia; Morphology; New species; Phylogeny; Small-subunit rRNA
Introduction The peniculid genus Frontonia was established by Ehrenberg (1838). Over 40 nominal species have since been described from aquatic biotopes worldwide (Borror 1963; Burkovsky 1970; Dragesco 1960; Dragesco 1972; Dragesco
∗ Corresponding
author. Tel.: +86 20 85210644; fax: +86 20 85210644. E-mail address: [email protected] (X. Lin).
0932-4739/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ejop.2012.06.003
and Dragesco-Kernéis 1986; Foissner 1987; Foissner et al. 1994; Gil and Perez-Silva 1964a, b; Kahl 1931; Long et al. 2005, 2008; Petz et al. 1995; Roque 1961a, b; Roque and de Puytorac 1972; Song and Wilbert 1989). Frontonia can be easily separated from related genera by the following characters: (1) triangular buccal cavity (vs. bursiform or oval in Paramecium, Neobursaridium, Stokesia, Lembadion, and Urocentrum, and pyriform in Apofrontonia); (2) three similarly structured peniculi (vs. two peniculi and one quadrulus in Paramecium, Stokesia and Neobursaridium); (3) vestibular
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kineties at the right margin of the buccal cavity (vs. vestibular kineties absent in Paramecium, Lembadion, Urocentrum and Neobursaridium); (4) several postoral kineties (vs. postoral kineties absent in Apofrontonia); and (5) postoral suture terminating at the posterior end of the cell (vs. extending to the mid-body region of the dorsal side in Disematostoma) (Corliss 1979; Didier and Puytorac 1994; Foissner et al. 1994, 1999; Foissner and Song 2002; Kahl 1931; Serrano et al. 1990; Tuffrau and Savoie 1961). Species circumscription and delimitation among members of the genus Frontonia are, however, far from clear. This is mainly because accurate species identification is highly dependent on detailed knowledge of the infraciliature (e.g. the fine structure of the peniculi) but is lacking for about 20% of Frontonia species (Bullington 1939; Dragesco 1960; Fan et al. 2011; Long et al. 2008; Kahl 1931). Furthermore, although molecular data are playing an increasingly important role in the taxonomy and systematics of ciliates, such data are available for relatively few species of Frontonia (Fan et al. 2011; Fokin et al. 2006; Gao et al. 2008; Li et al. 2010; Pan et al. 2012; Zhang et al. 2010). Three previously unknown species of Frontonia were isolated during a survey of the ciliate fauna in coastal waters of northern and southern China. Careful investigations of specimens both in vivo and following silver impregnation, along with phylogenetic analyses based on small subunit (SSU) rRNA gene sequence data, confirm the validity of these three new species. In this paper we present detailed descriptions of the morphology of each species. Moreover, the phylogenetic trees were also constructed based on the small-subunit rRNA gene sequences, six of which were included firstly (Pan et al. 2012). The phylogeny of the genus Frontonia was analyzed.
Material and Methods Two populations of Frontonia sinica spec. nov. were collected. Pop-1 was isolated on 4 July 2002 from a shrimpculturing pond near Qingdao (36◦ 08 08 N; 120◦ 43 15 E), China, where the water temperature was 24 ◦ C, salinity 25‰, and pH 8.0. Pop-2 was isolated on 9 November 2008 from a mangrove wetland (22◦ 40 00 N; 114◦ 40 32 E) near Zhujiang River, Guangzhou, China, where the salinity was about 16‰. Frontonia pusilla spec. nov. was collected on 28 December 2008 from a mangrove wetland near Zhuhai (23◦ 07 48 E;113◦ 32 24 E), China, where the salinity was about 25‰. Frontonia elegans spec. nov. was collected on 28 December 2008 from a shrimp-culturing pond in the South China Normal University (23◦ 08 24 N; 113◦ 20 46 ), Guangzhou, China, where the salinity was about 9‰. Live ciliates were observed using differential interference contrast microscopy. 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. Drawings of impregnated specimens were made with the help of a camera lucida. Measurements were
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performed under magnifications of 100–1000×. Systematics and terminology are mainly according to Lynn (2008). Genomic DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing of the SSU rRNA gene were performed using a REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. The SSU rRNA gene was amplified by PCR with primers Euk A (5 -AAC CTG GTT GAT CCT GCC AGT-3 ) and Euk B (5 -TGA TCC TTC TGC AGG TTC ACC TAC-3 ) (Medlin et al. 1988). The SSU rRNA gene sequences of Frontonia subtropica, F. canadensis and F. magna were sequenced in Pan et al. (2012) but firstly used to construct phylogenetic trees in this work. Other additional sequences were downloaded from the GenBank database (for accession numbers, see Fig. 7). Sequences were aligned using Clustal W implemented in BioEdit 7.0 (Hall 1999). Ambiguously aligned regions and gaps were excluded before phylogenetic analyses. Colpoda inflata and Bresslaua vorax were used as the outgroup taxa. Maximum-likelihood bootstrapping analyses were carried out with 100 replicates using RAxML with the setting as described in Stamatakis et al. (2008). ML analyses were conducted online on the CIPRES Protal V 2.0 (http://www.phylo.org). A Bayesian inference (BI) analysis was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the Markov chain Monte Carlo algorithm. The program was run for 1,000,000 generations with a sample frequency of 100 and a burn-in of 2500 (Yi and Song 2011).
Results and Discussion Order Peniculida Fauré-Fremiet in Corliss, 1956 Family Frontoniidae Kahl, 1926 Genus Frontonia Ehrenberg, 1838
Frontonia sinica spec. nov. (Figs 1A–H, 2A–J, Table 1) Diagnosis. Marine Frontonia about 100–200 m × 70–100 m in vivo, ellipsoidal body shape with ratio of length to width about 5:3. Five or six vestibular, 3–5 postoral, and 100–119 somatic kineties. Peniculi 1 and 2 each with four rows, peniculus 3 two-rowed. Macronucleus ellipsoidal, located in central region of body. Subterminal contractile vacuole located near dorsal side. Type locality. Shrimp-culturing water near Qingdao (36◦ 08 08 N; 120◦ 43 15 E), northern China. Type slides. A protargol slide with the holotype specimen is deposited in the collection of Laboratory of Protozoology, OUC, China, with registration number Lin-01-9-16-1. The holotype specimen is marked by an ink circle on the coverslip. A paratype slide with protargol-impregnated specimens is
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Fig. 1. (A–H) Frontonia sinica spec. nov. from life (A–D, G) and after protargol impregnation (E, F, H). (A) Ventral view of a typical specimen. (B) Ventral view to show the sutures and contractile vacuole. (C) Lateral view, showing the location of buccal cavity (arrow) and contractile vacuole. (D) Detail of pellicle, to show the resting extrusomes beneath the pellicle. (E, F) Ventral (E) and dorsal (F) views of holotype specimen showing the general infraciliature, arrowheads show the anterior and posterior sutures. (G) Extrusomes after extrusion. (H) Detailed structures of buccal region. Arrow mark the membranelle 3 which composed of two unequal kinety rows, arrowheads refer to the anteriorly shortened somatic kineties terminating along peniculus 1. AL, argentophilic line; AS, anterior suture; CV, contractile vacuole; Ma, macronucleus; P1–3, peniculi 1, 2 and 3; PK, postoral kineties; PM, paroral membrane; PS, posterior suture; VK, vestibular kineties. Scale bars = 50 m (A, E, F); 5 m (D); 10 m (G); 25 m (H).
deposited in the collection of Natural History Museum, London, UK, with registration number NHMUK 2012.3.14.1. Etymology. The species-group name sinica recalls the fact that this species was originally isolated in China Description. Body usually about 150 m × 90 m in vivo, ratio of length to width about 5:3, non-contractile, constantly elliptical in outline when viewed from ventral or dorsal side, anterior end broadly rounded, posterior portion tapered (Figs 1A, B, 2A, B), laterally compressed about 1.5–2:1 (Fig. 1C). Body densely ciliated, most somatic cilia about 10 m long, cilia in posterior region about 20 m long (Fig. 1A). Cytoplasm slightly grayish, often with many large (10–30 m across), yellowish food vacuoles that render the body more or less opaque and yellow-colored at lower magnifications (Figs 1A, 2A–C). Buccal cavity small and shallow,
elliptic to triangular in outline, about 25 m × 12 m in size, occupying about 15% of body length, with cilia about 15 m long (Figs 1A–C, 2B, C). Extrusomes spindle-shaped, about 8 m long in vivo, densely arranged beneath pellicle alongside somatic ciliary rows (Figs 1B, D, 2J); extruded extrusomes rod-shaped with one end curved, about 25 m long (Figs 1G, 2D, I). Single subterminal contractile vacuole located near dorsal margin of body, about 8–13 m in diameter, contracting at intervals of about 1 min (Figs 1A–C, 2C). Macronucleus flattened, ellipsoid in outline, located near body center and usually detectable in vivo with differential interference contrast microscopy, about 65 m × 40 m in size in vivo, 48–80 m × 23–50 m after protargol impregnation (Figs 1E, 2H). Single micronucleus, located near end of macronucleus, ovoid, about 5 m in diameter. Locomotion
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Fig. 2. (A–J) Frontonia sinica spec. nov from life (A–D, I, J), after protargol impregnation (E, F, H), and after Chatton–Lwoff silver nitrate impregnation (G). (A) Ventral view of a typical individual. (B) Ventral view of a slightly squeezed specimen, to show the oral field (arrow). (C) Lateral view of a slightly compressed specimen, showing the location of contractile vacuole (arrowhead), and oral field (arrow). (D, I) Extruded extrusomes (arrows). (E) Buccal area, arrows refer to the strong nematodesmata. (F) Oral apparatus, showing the peniculi 1–3. (G) Part of argyrome. (H) Macronucleus. (J) Arrows mark the resting extrusomes beneath the pellicle. Ma, macronucleus; P1–3, peniculi 1, 2 and 3. Scale bar = 100 m (A).
by revolving moderately rapidly on substrate or by swimming in water. Not sensitive to disturbance. Somatic ciliature as shown in Fig. 1E, F. About 114–118 longitudinal somatic kineties composed of densely spaced dikinetids, kineties progressively shortened anteriorly forming a conspicuous anterior suture (also called preoral suture) which extends from anterior end of buccal cavity, over apical end of cell and onto dorsal side; somatic kineties also progressively shortened posteriorly below oral region forming a posterior suture (Fig. 1B, E, F). Three to five postoral kineties consisting of densely spaced dikinetids, terminating anteriorly at buccal cavity and progressively shortened posteriorly, terminating along posterior suture (Fig. 1H). Nematodesmata highly developed, extending along posterior suture, each about 10–20 m long (Figs 1F, 2E).
Buccal apparatus genus typical (Figs 1F, H, 2F). Five or six (mostly six) short vestibular kineties composed of densely spaced dikinetids, running from anterior vertex of cavity and terminating along posterior suture. Three conspicuous peniculi located on left wall of buccal cavity: peniculi 1 and 2 about equal in length, positioned close to each other and parallel to left edge of vestibular wall, each composed of four rows of kinetosomes. Peniculus 3 slightly curved to right at both ends, composed of two kineties, right one of which extends entire length of buccal cavity, left one only extending to about 40% of buccal cavity length. Single-rowed paroral membrane running almost entire length of buccal cavity along its right edge. Several argentophilic lines to left of paroral membrane. Silverline system genus typical, i.e. as quadrangular cortical meshes that can be observed after silver nitrate impregnation (Fig. 2G).
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Table 1. Morphological characterizations of Frontonia sinica spec. nov. (Pop 1, first line; Pop 2, second line), F. pusilla spec. nov. (third line), and F. elegans spec. nov. (fourth line). Characteristica
Min
Max
M
SD
CV
n
Body length
120 100 58 70
180 122 75 92
157.4 111.7 65.3 82.4
14.7 6.6 4.8 6.7
9.3 5.9 7.3 8.1
16 15 15 15
Body width
80 80 21 25
140 98 30 50
102.2 90.0 26.1 39.2
14.6 5.8 2.8 7.3
14.3 6.5 10.7 18.6
16 15 15 15
Number of somatic kineties
114 100 70 69
118 119 77 78
116.1 107.0 72.3 73.5
1.1 5.8 2.0 3.2
0.9 5.4 2.8 4.4
16 15 15 11
Number of postoral kineties
3 4 3 3
5 5 3 3
3.9 4.7 – –
0.7 0.5 – –
17.9 9.9 – –
16 11 3 3
Number of vestibular kineties
5 5 3 4
6 5 3 4
5.9 – – –
0.3 – – –
5.1 – – –
16 11 3 4
Length of macronucleus
48 45 15 20
80 62 25 25
64.2 53.9 19.7 21.3
9.9 4.7 22.7 1.5
15.4 8.7 13.7 7.0
16 15 15 15
Width of macronucleus
23 30 10 14
50 44 20 20
32.0 34.5 14.1 15.6
7.9 4.5 2.4 1.5
24.7 12.7 17.3 9.9
16 15 15 15
Abbreviations: M, arithmetic mean; Max, maximum; Min, minimum; n, sample size; SD, standard deviation; CV, coefficient of variation (%). a Data from observations of protargol-impregnated specimens. All measurements in m.
Comparison. Three species of Frontonia resemble F. sinica spec. nov. in terms of its body size and shape, namely F. acuminata Ehrenberg, 1838, F. depressa (Stokes, 1886) Kahl, 1931, and F. angusta Kahl, 1931. All three, however, differ from the latter by having: (1) more kinety rows in peniculi 1, 2 and 3 (5 or 6, 5 or 6, 3 or 4 in F. acuminata; 5, 5, 3 in F. depressa; 5, 5, 4 in F. angusta vs. 4, 4, 2 in F. sinica), and (2) fewer somatic kineties (55–60 in F. acuminata, 57–62 in F. depressa, 80–105 in F. angusta vs. 100–119 in F. sinica) (Foissner 1987; Foissner et al. 1994). Frontonia elongata Burkovsky, 1970 is the only species with the same number of penicular rows as F. sinica, i.e. 4, 4, 2 in peniculi 1, 2 and 3 respectively. However, the former can be separated from the new species by the following combination of characters: (1) two kineties of about equal length in peniculus 3 (vs. one row about half the length of the other in F. sinica); (2) body length in vivo (200–250 m vs. 100–180 m in F. sinica); (3) number of somatic kineties (40–45 vs. 100–119 in F. sinica); (4) number of postoral kineties (2 or 3 vs. 3–5 in F. sinica) (Burkovsky 1970).
Frontonia magna Fan et al., 2011 shows close relationship with F. sinica according to the phylogenetic tree. However, the former can be easily separated from the latter by its (1) larger cell size (200–450 m × 70–200 m vs. 100–200 m × 70–100 m), (2) higher number of somatic kineties (165–216 vs. 100–119), and (3) the different structure of peniculus 3 (four equal rows vs. two unequal rows) (Fan et al. 2011; Pan et al. 2012).
Frontonia pusilla spec. nov. (Figs 3A–F, 4A–O, Table 1) Diagnosis. Brackish water Frontonia about 70–100 m × 30–50 m in vivo, foot-shaped in outline. Three vestibular, three postoral and 70–77 somatic kineties. Peniculi 1 and 2 each with four kinety rows, peniculus 3 two-rowed. Macronucleus ellipsoidal, centrally located. Two contractile vacuoles.
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Fig. 3. (A–F) Frontonia pusilla spec. nov. from life (A–C) and after protargol impregnation (D–F). (A) Ventral view of a representative individual. (B) Variations in body shape. (C) Lateral view, to show the location of contractile vacuoles. (D) Detailed structure of buccal region, arrowhead shows the anteriorly shortened somatic kinety near peniculus 1. (E, F) Ventral (E) and dorsal (F) view of holotype specimen showing general infraciliature. AS, anterior suture; CV, contractile vacuole; P1–3, peniculi 1, 2 and 3; PK, postoral kineties; PM, paroral membrane; PS, posterior suture; VK, vestibular kineties. Scale bars = 30 m (A, B, E, F).
Type locality. A mangrove wetland near Zhuhai (23◦ 07 48 E; 113◦ 32 24 E), southern China. Type slides. A protargol slide with the holotype specimen is deposited in the collection of Laboratory of Protozoology, OUC, China, with registration number LWW-200811280501. The holotype specimen is marked by an ink circle on the coverslip. A paratype slide with protargol-impregnated specimens is deposited in the collection of Natural History Museum, London, UK, with registration number NHMUK 2012.3.14.2. Etymology. The species-group name pusilla (Latin, ‘small’) refers to the small size of this species. Description. Body about 70–100 m × 30–50 m in vivo, foot-shaped in outline with right side straight and left side slightly protruded at anterior third (Figs 3A, 4A, C). Dorsoventrally flattened about 3:2 (Figs 3C, 4B). Buccal cavity small, about 15 m × 8 m in size (Figs 3A, 4D, E). Cytoplasm containing numerous dark green granules, crystals in posterior region of body and food vacuoles (4–5 m across) often containing diatoms (Fig. 4F). Two contractile vacuoles located dorso-laterally, one in anterior the other in posterior third of body, each about 8 m in diameter (Figs 3A, 4C). Single ellipsoidal macronucleus about 20 m × 15 m (Fig. 4K). Extrusomes 4–5 m long, approximately 20 m long when extruded (Fig. 4G, H). Somatic cilia approximately 6 m long. Locomotion by crawling slowly on substrate or by swimming freely moderately fast. Infraciliature as shown in Figs 3D–F, 4L–N. Three postoral and 70–77 somatic kineties (Figs 3E, 4M). One shortened somatic kinety terminating anteriorly near mid-region of peniculus 1 (Fig. 3D, arrowhead). Peniculi closely spaced and parallel to each other. Peniculus 1 composed of four
kinety rows, outer two rows of which are shortened posteriorly; peniculus 2 composed of four rows of equal length. Peniculus 3 composed of two rows, outer row shortened posteriorly terminating at posterior third of inner row (Fig. 3D). Three vestibular kineties, inner one close to paroral membrane and shortened posteriorly compared to the other two (Figs 3E, 4M). Comparison. Frontonia pusilla can be distinguished from most congeners by its small body size and the number and location of the contractile vacuoles. Only two other species, namely F. ocularis Bullington, 1939 and F. fusca (Quennerstedt, 1869) Kahl, 1928, exhibit these characters and should therefore be compared with the new species. Frontonia pusilla differs from F. ocularis by the following combination of characters: (1) body foot-shaped in outline and with anterior part of body broader than posterior part (vs. body reniform, anterior part usually narrower than posterior part); (2) pigment-spot absent (vs. reddish brown pigment-spot present near right boarder of anterior extremity) (Bullington 1939). Frontonia pusilla can be distinguished from F. fusca by the absence of a pigment-spot (vs. dark-green spot present in the right anterior dorsal side of cell in F. fusca) and in having two (vs. three) kinety rows in peniculus 3 (Fokin 2008).
Frontonia elegans spec. nov. (Figs 5A–H, 6A–M, Table 1) Diagnosis. Brackish Frontonia about 75–90 m × 40–60 m in vivo. Three vestibular, four postoral, and 69–78 somatic kineties, seven of which are shortened anteriorly
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Fig. 4. (A–O) Frontonia pusilla spec. nov. from life (A–H) and after silver carbonate (I), silver nitrate (J), and protargol (K–O) impregnation. (A, C) Ventral view of two typical specimens, arrows in (C) indicate the contractile vacuoles. (B) Lateral view. (D) Buccal opening. (E) Buccal cavity, to show peniculi 1 and 2 (arrow). (F) Macronucleus and one of the two contractile vacuoles (arrow). (G) Arrowheads mark the resting extrusomes beneath the pellicle. (H) Extrusomes after extrusion. (I, J) Extrusomes in cells stained with silver carbonate (I) and silver nitrate (J). (K) Macronucleus. (L) Detailed structure of buccal area. (M) Vestibular and postoral kineties. (N) Anterior suture (arrowheads). (O) Lateral view of buccal region, arrows depict the cytopharyngeal rods. Ma, macronucleus; P1–3, peniculi 1, 2 and 3; PK, postoral kineties; PM, paroral membrane; VK, vestibular kineties. Scale bars = 30 m (A–C).
terminating along peniculus 1. Peniculi 1 and 2 each with four rows, peniculus 3 three-rowed. Macronucleus spherical, located in central region of body. Two contractile vacuoles. Type locality. A shrimp-culturing pond at South China Normal University, Guangzhou (23◦ 08 24 N; 113◦ 20 46 ), southern China. Type slides. A protargol slide with the holotype specimen is deposited in the collection of Laboratory of Protozoology,
OUC, China, with registration number LWW-200811280401. The holotype specimen is marked by an ink circle on the coverslip. A paratype slide with protargol-impregnated specimens is deposited in the collection of Natural History Museum, London, UK, with registration number NHMUK 2012.3.14.3. Etymology. The species–group name elegans (Latin, ‘elegant’) refers to the elegant body outline.
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Fig. 5. (A–H) Frontonia elegans spec. nov. (A–D, G, H) and F. fusca (E, F) from life (A–C, F) and after protargol (D, G, H) and silver nitrate (E) impregnation. (A) Ventral view of a typical individual. (B) Lateral views. (C) Variation in body shape. (D) Detailed structure of buccal region, arrowheads depict the anteriorly shortened somatic kineties. (E) Buccal apparatus of Frontonia fusca (Quennerstedt, 1869) Kahl, 1928 (from Fokin 2008). (F) Ventral view of F. fusca (from Fokin 2008), arrow depicts the pigment spot. (G, H) Ventral (G) and dorsal (H) view of general infraciliature. AS, anterior suture; P1–3, peniculi 1, 2 and 3; PK, postoral kineties; PM, paroral membrane; PS, posterior suture; VK, vestibular kineties. Scale bars = 40 m (A–C, F–H).
Description. Body 75–90 m × 40–60 m in vivo, ellipsoidal with right side straight and left side distinctly curved (Figs 5A, C, 6A, C). Dorsoventrally flattened about 3:2 (Fig. 5B). Buccal cavity about 20 m × 10 m occupying 15% of the body length; buccal area depressed in some specimens (Figs 5B, 6F). Cytoplasm containing numerous blue granules (Fig. 6G) and food vacuoles (4–5 m across) containing algae (Fig. 5A). Extrusomes spindle-shaped, 4–5 m long; when extruded about 15 m long. Single ellipsoidal macronucleus about 20 m × 15 m (Fig. 6M). Two contractile vacuoles 8–10 m in diameter located dorsolaterally, one in anterior third of cell the other in posterior third (Figs 5A, 6D); each contractile vacuole with a single excretory pore (Fig. 6E). Somatic cilia about 6 m long; in some individuals five or six longer (10 m) stiff cilia present at posterior end of body (Fig. 6H). Locomotion by crawling slowly on substrate or by swimming moderately fast. General infraciliature as shown in Fig. 5G, H. 69–78 somatic kineties that are progressively shortened both anteriorly and posteriorly forming conspicuous anterior and posterior sutures, both of which extend from ventral to dorsal side (Fig. 1G, H). Seven somatic kineties shortened anteriorly and terminate along peniculus 1 (Figs 5G, H, 6J). Buccal structure as shown in Figs 5D, 6I, K, L. Peniculi 1 and 2 each comprises four kinety rows of equal length. Peniculus 3 composed of three rows that are progressively shortened posteriorly. Three vestibular kineties each composed of densely
arranged dikinetids. Four postoral kineties, inner one usually shortened and comprising 3–5 dikinetids. Comparison. In terms of body shape, number of contractile vacuoles and the structure of the peniculi, Frontonia elegans most closely resembles F. fusca (Quennerstedt, 1869) Kahl, 1928. However, the former can be separated from the latter by the following combination of characters: (1) pigment spot absent (vs. present in F. fusca); (2) seven shortened somatic kineties that terminate anteriorly along peniculus 1 (vs. no shortened kineties in F. fusca); (3) one excretory pore (vs. 2 or 3 in F. fusca) per contractile vacuole (Fokin 2008; Fig. 5E, F). Frontonia elegans also resembles F. pusilla spec. nov. in terms of its body size, number of somatic kineties and the number and location of contractile vacuoles. However, the former can be differentiated from the latter by having a three-rowed (vs. two-rowed) peniculus 3 and about seven (vs. one) anteriorly shortened somatic kineties terminating along peniculus 1.
SSU rRNA gene sequence data The SSU rRNA gene sequences of the three new species are deposited in GenBank with accession numbers, length and G + C content as follows: F. sinica, FJ868197, 1748 bp, 44.51%; F. pusilla, FJ868201, 1702 bp, 44.59%; F. elegans, FJ868200, 1699 bp, 44.79%.
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Fig. 6. (A–M) Frontonia elegans spec. nov. from life (A–H) and after protargol (I, K–M) and silver carbonate (J) impregnation. (A–D) Different individuals, arrows in (D) mark the two contractile vacuoles. (E) Arrowheads depict the discharge pores of the two contractile vacuoles. (F) Buccal cavity. (G) Posterior portion, to show granules in the cytoplasm. (H) The cilia at caudal end (arrowheads). (I, K, L) Structure of buccal region, arrowheads in (I) depict the anteriorly shortened somatic kineties. (J) Dorsal infraciliature, arrow shows the posterior suture. (M) Macronucleus. Ma, macronucleus; P1–3, peniculi 1, 2 and 3; PK, postoral kineties; PM, paroral membrane; VK, vestibular kineties. Scale bars = 30 m (A–C, J, M).
Phylogenetic analyses Trees based on SSU rRNA gene sequence data and constructed using Bayesian inference and maximum-likelihood were almost identical, therefore only the ML tree is shown (Fig. 7). Five major groups were recovered within the class Oligohymenophorea. Four of these correspond to the four subclasses suggested in the classification system of Lynn (2008), namely Peniculia (1.00 BI, 100% ML),
Scuticociliatia (1.00 BI, 100% ML), Peritrichia (1.00 BI, 100% ML), and Hymenostomatia (1.00 BI, 86% ML). The other group (1.00 BI, 100% ML) corresponds to the subclass Mobilia that was established by Zhan et al. (2009). These findings are consistent with previous studies based on SSU rRNA gene sequence data (e.g. Fan et al. 2011; Fokin et al. 2006; Gao et al. 2008). Within the Peniculia clade, Lembadion bullinum is basal to the other peniculines included in the analyses, i.e. Paramecium (four species), Apofrontonia
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Fig. 7. Maximum-likelihood tree inferred from small subunit rRNA gene sequences. Nodal support for branches in the ML and Bayesian inference (BI) trees are marked in order. Black circles indicate full support in all analyses. All branches are drawn to scale. The scale bar corresponds to five substitutions per 100 nucleotide position. Newly sequenced species in this work and first included sequences are in bold font and marked by arrows.
(one species) and Frontonia (13 species). Frontonia species are divided into two assembles: F. pusilla, F. elegans and F. didieri cluster with Apofrontonia dohrni (0.83 BI, 62% ML), forming a sister clade with Paramecium; the other 11 Frontonia species including F. sinica, F. magna, F. canadensis and F. subtropica, group together with strong support (1.00 BI, 100% ML). In the latter assemblage, there are two well grouped clades, one containing F. leucas, F. vernalis and Frontonia sp., the other containing the remaining species. Within the latter group, the two populations of F. magna group well with each other and then cluster with F. sinica with moderate support (0.76 BI, 64% ML); F. canadensis associated with F. mengi also in a moderate supported clade (0.76 BI, 68% ML), which forms a sister group with the F. sinca-F. magna clade with the strongest support (1.00 BI, 100% ML); F. subtropica is basal to the clade containing F. lynni and F. tchibisovae with moderate support (0.88 BI, 71% ML).
Meanwhile, the phylogenetic analysis also supports the validity of our three new species: Frontonia pusilla spec. nov. and F. elegans spec. nov. being placed in one clade within the Frontonia assemblage, F. sinica spec. nov. in the other one including F. magna, F. canadensis and F. subtropica whose SSU rRNA gene sequences are firstly used to construct phylogenetic trees in this work (Pan et al. 2012). Previous studies have shown that, for morphospecies of the genus Frontonia, the structures of the peniculi (especially peniculus 3) are stable within a species but highly divergent among different species (Fan et al. 2011; Fokin 2008; Long et al. 2005, 2008; Pan et al. 2012). The peniculi would therefore appear to be potentially useful characters for assessing phylogenetic relationships within this genus. In the present study, however, no pattern of correlation could be found between the peniculi and the phylogenetic relationships recovered in the SSU rRNA gene tree.
Remarks on phylogeny
Acknowledgements
Previous studies on the molecular phylogeny of Frontonia have suggested that this genus may be paraphyletic (Gao et al. 2008). Our phylogenetic trees constructed including six new SSU rRNA gene sequences support this finding. Given the large number of Frontonia species that have yet to be sequenced (30 out of 43), it is not currently possible to draw any conclusion about the phylogeny of this genus.
This work was supported by the National Nature Science Foundation of China (project No.: 31071893), the research fund for the doctoral program of higher education (project No.: 20104407120006) and the research group project No. RGP-VPP-083, King Saud University Deanship of Scientific Research, Saudi Arabia. We greatly appreciate the comments of two anonymous reviewers and the associate editor (Dr. H. Berger). Our thanks are also due to Prof. Weibo Song and
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Ms. Feng Gao, OUC, for their comments and supply of references, respectively.
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