Morphology of three species of Amphileptus (Protozoa, Ciliophora, Pleurostomatida) from the South China Sea, with note on phylogeny of A. dragescoi sp. n.

Published by the International Society of Protistologists

The Journal of

Eukaryotic Microbiology

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

Morphology of Three Species of Amphileptus (Protozoa, Ciliophora, Pleurostomatida) from the South China Sea, with Note on Phylogeny of A. dragescoi sp. n. Hongbo Pana,b, Lifang Lic, Xiaofeng Lind, Jiqiu Lid, Saleh A. Al-Farraje & Khaled A. Al-Rasheide a Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, 201306, China b Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao, 266003, China c Marine College, Shandong University at Weihai, Weihai, 264209, China d Laboratory of Protozoology, Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, South China Normal University, Guangzhou, 510631, China e Zoology Department, King Saud University, PO Box 2455, Riyadh, 11451, Saudi Arabia

Keywords Ciliary pattern; ciliate; new species; SSU rDNA; taxonomy. Correspondence L. Li, Marine College, Shandong University at Weihai, Weihai 264209, China Telephone number: +86 631 5688303; FAX number: +86 631 5688303; e-mail: [email protected] Received: 9 February 2014; revised 3 June 2014; accepted June 12, 2014. doi:10.1111/jeu.12146

ABSTRACT Two new and one problematic species of pleurostomatids, Amphileptus dragescoi sp. n., A. wilberti sp. n., and A. marinus from coastal areas of the South China Sea, are described based on observations of live and protargol-impregnated specimens. Amphileptus dragescoi is different from its congeners by the presence of an apical group of extrusomes and the possession of 12–15 right and five left somatic kineties, two macronuclear nodules, and a single terminally positioned contractile vacuole. Amphileptus wilberti is diagnosed by oval or pyriform body, 15–19 right and seven or eight left somatic kineties, extrusomes arranged only in anterior portion of oral slit, usually three ventrally located contractile vacuoles, and two macronuclear nodules. Amphileptus marinus (Kahl, 1931) Song et al., 2004 is redescribed and its diagnosis is improved. One isolate which was misidentified as A. marinus by Song et al. (2004) is believed to represent an unknown form, named here as Amphileptus songi sp. n. Phylogenetic analyses of the SSU rDNA sequences indicate that the genus Amphileptus is paraphyletic, but its monophyly is not rejected by statistical tree topology tests.

AMPHILEPTUS Ehrenberg, 1830, the senior synonym of Hemiophrys Wrzesniowski, 1870, is characterized by (i) right ciliary rows forming one suture in the anterior portion of body; (ii) no sutures in the postoral region or posterior portion of the right side; (iii) no extrusomes attached to the dorsal margin; and (iv) the anterior end of the body not twisted to form a spoon-like apex (Canella 1960; Foissner 1984; Lin et al. 2007; Song 1991). Like other pleurostomatids, most congeners are very similar in body shape, and hence species identification has to depend on a series of various features, including cell size and shape, number and position of contractile vacuoles, shape and distribution of extrusomes, number of macronuclear nodules and ciliary rows, as well as on the presence/absence of cortical granules (Chen et al. 2011; Dragesco 1960; Dragesco and is 1986; Foissner et al. 1995; Lin et al. Dragesco-Kerne

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2005a). Hitherto, there are over 50 nominal species in this genus (Carey 1991; Song and Wilbert 1989; Song et al. 2009). However, the morphology of only around 20 of these has been studied sufficiently (Chen et al. 2011; Lin et al. 2005a,b, 2007; Song and Wilbert 1989; Sonntag and Foissner 2004); and only two small-subunit ribosomal DNA (SSU rDNA) sequences, one large-subunit ribosomal DNA sequence and one alpha-tubulin sequence were available in GenBank (Gao et al. 2008; Vd’acny et al. 2011; Zhang et al. 2012). Clearly, therefore, more taxonomic and/or molecular studies are needed in order to fully elucidate the diversity of this “old” genus in the order Pleurostomatida. During a recent survey of the ciliate fauna in southern China, the high diversity of pleurostomatids has been revealed and several novel species have been discovered

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 61, 644–654

Three Amphileptus spp. from China

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(Chen et al. 2011; Pan et al. 2010, 2013; Wu et al. 2013, 2014). As a part of this work, this paper describes three Amphileptus species. MATERIALS AND METHODS Sample collection, observation, and identification Both Amphileptus dragescoi sp. n. and Amphileptus marinus were isolated from Donghai Island (22°550 18″N, 110°310 00″E), Zhanjiang, China, on December 15, 2009. The water temperature was 23.8 °C, pH 7.4 and the salinity 24.8&. Amphileptus wilberti sp. n. was collected from the mangrove wetland in Techeng Island (21°080 52″N, 110°260 20″E), Zhanjiang, China on April 7, 2010. The water temperature was 24.6 °C, pH 12.53 and the salinity 13.6&. The location and habitats of Donghai Island and Techeng Island are shown in Fig. 1. Cells were observed in vivo with bright-field and differential interference contrast microscopy. The protargol staining method was used to reveal the ciliary pattern and nuclear apparatus (Wilbert 1975). Counts, measurements, and drawings of stained specimens were carried out with the help of a camera lucida. Terminology and systematics follow Lynn (2008) and Lin et al. (2005a). DNA extraction, sequencing, and comparison The REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO) was used to extract the genomic DNA as described by Jiang et al. (2013). The universal oligonucleotide primers (Euk A: 50 -AACCTGGTTGATCCTGCCAGT-30 ; Euk B 50 -TGATCCTTCTGCAGGTTCACCTAC-30 ) designed by Medlin et al. (1988) were used for PCR amplifications of SSU rDNA. The method of cloning and sequencing were performed as described in Gao et al. (2013).

Phylogenetic analyses The accession numbers of all sequences used in the present analyses are as presented in Fig. 5. Sequences were aligned using the GUIDANCE algorithm (Penn et al. 2010a) in the GUIDANCE web server (Penn et al. 2010b) firstly and further modified with BioEdit 7.0 manually (Hall 1999). The final alignment of 1,509 characters and 21 taxa was used to construct phylogenetic trees under the GTR + I + G evolutionary model that was selected as the most appropriate model for phylogenetic analyses by Modeltest v.3.4 (Posada and Crandall 1998) and MrModeltest v.2.2 (Nylander 2004). Then the Maximum likelihood (ML) analyses were performed using RAxML-HPC2 v7.2.8 (Stamatakis 2006; Stamatakis et al. 2008) on the CIPRES Science Gateway (Miller et al. 2010). The Bayesian inference (BI) analyses was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using GTR + I + G as the best model. Four simulations Markov Chain Monte Carlo algorithms (MCMC) were run for 2,000,000 generations, sampling every 100th generation. The first 5,000 trees were discarded as burn-in. The remaining trees were used to calculate the posterior probabilities of the majority rule consensus tree. To test the monophyly of the family Amphileptidae against competing phylogenetic hypotheses, constrained ML trees were generated based on the SSU rRNA gene alignment. For all constraints, internal relationships within the constrained groups and among the remaining taxa were left unspecified. The site-wise likelihoods for the resulting constrained topologies and the non-constrained ML topology were calculated using PAUP (Swofford 2002) and were then subjected to the AU test (Shimodaira 2002) as implemented in CONSEL (Shinmodaira and Hasegawa 2001). Statistical analyses Two-tailed t-test (Qinn and Keough 2002) was conducted to determine whether body length differences between different species were statistically significant. RESULTS Amphileptus dragescoi sp. n. (Table 1; Fig. 2)

Figure 1 Location and the habitats of sampling sites. A, B. The mangrove wetlands of Techeng Island at low tide (A) and at high tide (B). C. The coast of Donghai Island.

The cell size is about 90–140 9 10–15 lm in vivo. The body is elongated or lanceolate, only slightly contractile, with the posterior end pointed; the neck region is significant, about 30–50% of the cell length (Fig. 2A, C, F, G, J). The pellicle is smooth and thin, beneath which numerous dot-like cortical granules (< 0.5 lm) are densely arranged (Fig. 2I). The right side is densely ciliated, with cilia ca. 8 lm long, while the left side is sparsely ciliated, with cilia ca. 3 lm long. The cytoplasm is somewhat greyish, with several greasily shining globules (1–2 lm across). The extrusomes are clavate, straight or slightly curved, about 10 lm long in vivo. Some (usually three or four) of them are clustered at the anterior end to form a conspicuous apical group; others are scattered in the main body


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Table 1. Morphometric characteristics of Amphileptus dragescoi sp. n. (upper line), A. marinus (middle line) and A. wilberti sp. n. (lower line) from protargol-impregnated specimens

netids in the anterior 1/3 to 1/2 and monokinetids in the rest (Fig. 2D, L).

Characters

Min

Max

Mean

SD

CV

n

Body length

72 123 112 22 30 54 12 13 15 5 5 7 1 2 2 19 15 21 15 10 17 6 6 10

131 285 200 30 78 80 15 21 19 5 8 8 2 2 2 45 30 30 38 20 25 11 11 12

103.5 200.2 152.6 25.7 53.0 67.6 13.2 15.4 16.4 5.0 6.6 7.5 2.0 2.0 2.0 30.7 20.3 25.1 21.2 13.7 21.1 7.6 8.3 11.0

15.06 42.74 24.77 2.05 2.38 9.17 0.93 2.34 1.08 0 0.88 0.52 0.20 0 0 6.27 3.15 2.95 4.86 2.10 2.74 1.40 1.42 0.63

14.5 21.3 16.2 8.0 22.0 13.6 7.1 15.1 6.6 0 13.2 6.9 10.4 0 0 20.4 15.6 11.8 23.0 15.3 13.0 18.5 17.2 5.7

24 24 14 24 24 14 24 24 14 24 24 14 24 24 14 23 24 14 23 24 14 22 23 6

Amphileptus marinus (Kahl 1931) Song et al. 2004 (Table 1; Fig. 3)

Body width

Number of right somatic kinetiesa Number of left somatic kinetiesb Number of macronuclear nodules Length of macronuclear nodules Width of macronuclear nodules Length of extrusomes

CV = coefficient of variation in %; Max = maximum; Min = minimum; n = number of specimens; SD = standard deviation. All measurements in µm. a Perioral kinety 2 included. b Perioral kinety 1 and dorsal brush kinety included.

(Fig. 2B, C, H, K, L). A single contractile vacuole, about 8–10 lm in diam., is terminally positioned and (Fig. 2A, G) there are two oval macronuclear nodules about 9–15 lm long in vivo, centrally positioned (Fig. 2J). The single micronucleus is located between the two macronuclear nodules. The movement is by swimming with a slow anticlockwise rotation around the longitudinal axis, seldom gliding on the substrate. The ciliary pattern is shown in Fig. 2D, E. There are 12– 15 right somatic kineties, consisting of closely spaced basal bodies; the anterior intermediate kineties on the right side are shortened anteriorly and form a distinct suture (Fig. 2E, arrowhead). Consistently, there are five left somatic kineties, including the dorsal brush kinety and the first perioral kinety. The dorsal brush kinety is composed of closely spaced dikinetids in the anterior 1/3–1/2 of body length and sparsely spaced monokinetids in the posterior portion (Fig. 2D, L). There are two perioral kineties (PK1, 2) around the oral slit: PK1, at left of the oral slit, consists of closely spaced dikinetids in the anterior half and monokinetids in the posterior part; PK2 extends along the right margin of the oral slit and is similar to PK1 in structure, i.e., comprises diki-

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The cell size is about 150–300 9 30–80 lm in vivo. The body shape is variable, usually elongated, or slenderly lanceolate with a bluntly rounded posterior end in outline. Conspicuous neck region occupies nearly half of the cell length (Fig. 3A, B, G–I). The pellicle is thin and smooth, with numerous tiny colourless cortical granules (about 0.5 lm across) closely arranged beneath the cell surface (Fig. 3J). The cilia on the right side are densely spaced, about 10 lm long, but sparsely distributed and difficult to detect in vivo on the left side. The cytoplasm is colourless or somewhat greyish and contains many tiny granules (1–2 lm across), which render the main part of the body more or less opaque (Fig. 3I, L). The about 6–11 lm long fusiform extrusomes are densely arranged along the oral slit or scattered in the cytoplasm (Fig. 3C, D, L, M). There are about seven contractile vacuoles arranged along the posterior half of the ventral margin (Fig. 3A, B, H). Usually two spherical macronuclear nodules, about 15 lm in diam. are located centrally in the cell; only one in 24 individuals possess a single macronuclear nodule (Fig. 3M). The micronucleus is not detected. The movement is by slow gliding on the substrate. The ciliary pattern is shown in Fig. 3E, F. There are 13–21 (usually 14) densely ciliated right somatic kineties; anterior intermediate kineties on the right side are shortened anteriorly to form a distinct suture (Fig. 3F, arrowhead). On the left side, basal bodies are mostly loosely arranged and form about five to eight somatic kineties (Fig. 3E). The dorsal brush kinety comprises dikinetids in its anterior 1/4 and monokinetids in the rest (Fig. 3E, K). The first two perioral kineties (PK1 and 2) delimit the oral slit and are composed of closely spaced dikinetids in the anterior 1/3 and of monokinetids posteriorly (Fig. 3E). Amphileptus wilberti sp. n. (Table 1; Fig. 4) In vivo, it is about 180–210 9 50–60 lm in size. The body is oval or pyriform, with inconspicuous neck-like region and broadly rounded posterior end (Fig. 4A, E–G). The cell is bi-laterally compressed about 3:5. The cilia are about 8–10 lm long and densely arranged on the right side, whereas on the left side they are difficult to be detected in vivo. The pellicle is smooth and thin with many sparsely spaced, dot-like cortical granules. The cytoplasm is slightly greyish, with numerous greasily shining globules (1–6 lm in diam.) and several food vacuoles (3–6 lm in diam.), which render the main part of the body more or less opaque (Fig. 4I). The extrusomes (Fig. 4B) are bar-shaped, slightly curved, about 10–12 lm long, sparsely arranged along the anterior part of the oral slit and scattered in the cytoplasm (Fig. 4A, D, I). Usually three contractile vacuoles are distributed along the posterior part of the ventral margin; each vacuole is about



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Figure 2 Amphileptus dragescoi sp. n. in vivo (A–C, F–J) and after protargol impregnation (D, E, K, L). A. Left view of a representative individual, arrow points to contractile vacuole. B. Extrusomes. C. Shape variants. D, E. Left (D) and right (E) view of the same specimen, arrowheads mark the suture. F. Right view. G. Left view, arrowhead shows contractile vacuole. H. Anterior body portion, arrow points to apical extrusomes. I. Portion of left view to show cortical granules. J. Left view, arrows indicate macronuclear nodules. K. Left view showing arrangement of macronuclear nodules and extrusomes. L. Anterior portion of left view, arrow points to apical extrusomes, arrowhead refers to PK1 = doublearrowhead shows dorsal brush; PK1, 2 = perioral kinety 1, 2; DB = dorsal brush. Scale bars = 60 lm.

10 lm in diam. during diastole (Fig. 4A). There are two macronuclear nodules located in the cell centre, about 23 9 17 lm in size. The movement is by gliding slowly on the substrate. The ciliary pattern is illustrated in Fig. 4C, D. On the right side, there are about 15–19 right somatic kineties with basal bodies closely spaced; intermediate kineties are shortened to form a distinct anterior suture (Fig. 4C). The left side is sparsely ciliated with about seven or eight somatic kineties including one perioral kinety and the dorsal brush kinety. The dorsal brush kinety is composed of closely spaced basal body pairs in the anterior 1/3 and sparsely spaced monokinetids posteriorly (Fig. 4D). There are two perioral kineties (PK1, 2): PK1, at left of the oral slit, comprises regularly spaced dikinetids in the

anterior 1/3 and densely spaced monokinetids in the rest; PK2, at right of the oral slit, is composed of close-set dikinetids in anterior half and monokinetids in posterior half (Fig. 4D). SSU rDNA sequences and phylogenetic analyses The complete SSU rRNA gene sequence for A. dragescoi sp. n. (GenBank accession number KF871282) is 1,591 nucleotides in length and its GC content is 42.13%. Alignment of the sequences shows that the primary structure of the SSU rRNA gene of the new sequence is similar to that of other pleurostomatid ciliates. It is revealed that A. dragescoi differs from other Amphileptus spp. in 55–62 nucleotides.


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Figure 3 Amphileptus marinus from life (A–C, G–J, L) and after protargol impregnation (D–F, K, M). A. Right view of a representative individual. B. Shape variants. C. Extrusomes. D. To show distribution of extrusomes. E, F. Left and right view of the same specimen, arrowheads indicate the suture. G–I. Left view, arrowheads refer to contractile vacuoles. J. Portion of left view of body, showing cortical granules. K. Anterior portion of left view of body, arrow and arrowhead mark dorsal brush and PK1 respectively. L. Portion of cytoplasm, arrowheads refer to extrusomes. M. Right view of an abnormal individual with a single macronuclear nodule. PK1, 2 = perioral kinety 1, 2; DB = dorsal brush. Scale bars = 100 lm (A, E–I, M); 4 lm (C).

As shown in the phylogenetic tree based on the SSU rRNA gene (Fig. 5), all the species of the order Pleurostomatida form a monophyletic group. The family Amphileptidae is divided into two groups: A. dragescoi groups with Pseudoamphileptus macrostoma and Amphileptus procerus, and form a sister clade to other two Amphileptus spp.; while the second group includes only one species, Epiphyllum shenzhenense. DISCUSSION Comments on A. dragescoi sp. n At present, it is agreed that the number and position of the contractile vacuoles and the possession of an apical group of extrusomes are reliable and important features

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for species discrimination within the genus Amphileptus (Chen et al. 2011; Foissner 1984; Lin et al. 2005a, 2007; Song 1991). To date, only three congeners have the apical group of extrusomes and a single contractile vacuole: Amphileptus affinis Song and Wilbert 1989; Amphileptus yuianus Lin et al. 2005; and Amphileptus fusidens (Kahl, 1926) Song and Wilbert 1989. Amphileptus dragescoi is very similar to A. affinis in the number of somatic kineties and body shape. But it can be distinguished from A. affinis by the position of the contractile vacuole (terminally vs. ventrally), the shape of the extrusomes (clavate vs. bar-shaped) and the marine habitat (vs. freshwater; Song and Wilbert 1989). Like A. dragescoi, A. yuianus is also a marine species. However, it differs from the new form by the larger cell size (150–200 9 25–50 lm vs. 90–140 9 10–15 lm) and



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Figure. 4 Amphileptus wilberti sp. n. from life (A, D–H) and after protargol impregnation (B, C). A. Right view of a representative individual. B. Extrusomes. C, D. Right (C) and left (D) view of the same specimen, arrowheads indicate the suture. E–H. Shape variants. I. Portion of cytoplasm, arrows refer to extrusomes. PK1, 2 = perioral kinety 1, 2; DB = dorsal brush. Scale bars = 90 lm (A); 70 lm (B, C).

the higher number of the right somatic kineties (18–22 vs. 12–15; Lin et al. 2005a). Compared with A. fusidens, A. dragescoi can be identified by the longer body (90–140 lm vs. 40–55 lm) and the position of the contractile vacuole (terminally positioned vs. ventrally; Song and Wilbert 1989). Comments on A. marinus (Kahl 1931) Song et al. 2004 Amphileptus marinus was originally reported by Kahl (1931) under the name Hemiophrys marina. In terms of

cell size, shape, contractile vacuoles, nuclear apparatus, especially the distribution of extrusomes, our population corresponds well with the original description, and thus both are conspecific. Borror (1963) reported another population from USA, and noted that the extrusomes were exactly arranged along the oral slit. But in his description, contractile vacuoles are scattered in the posterior of the body and a posterior suture is present. Song et al. (2004) thought that the US population was possibly misinterpreted, and we agree with them.


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Figure 5 The maximum likelihood (ML) tree inferred from small-subunit rDNA sequences showing the position of Amphileptus among the order Pleurostomatida. Nodal support for branches in the ML and BI trees are marked in order. “–” indicates full support in analyses. Clades with a different topology in the BI tree are shown by n. The scale bar corresponds to five substitutions per 100 nucleotide positions. Species newly sequenced in this work is in bold. Asterisk points to the species newly sequenced by Prof. Weibo Song’s Lab (not published until now).

Table 2. List of Amphileptus-like species with two macronuclear nodules and several only ventrally positioned contractile vacuoles

Species Amphileptus Amphileptus Amphileptus Amphileptus et al. 2011

marinus songi sp. n. wilberti sp. n. salignus Chen

Amphileptus eigneri Lin et al. 2007 Amphileptus gui Lin et al. 2005a,b Amphileptus ensiformis Song and Wilbert 1989 Amphileptus rotundus (Kahl 1931) Amphileptus muscicola (Kahl 1931) Amphileptus pectinatus (Kahl, 1926) Amphileptus meleagris (Ehrenberg, 1835)

Body length (lm)

No. of LSK/RSK

150–300 200–450 180–210 180–360

5–8/13–21 10–12/20–27 7–8/15–19 4/24–29

100–200 150–300 100–200

Shape and distribution of extrusomes

Biotope

Sources

Marine Marine Brackish water Brackish water

Present work Song et al. (2004) Present work Chen et al. (2011)

6–9/14–18 7–11/37–50 5–6/18–22

Fusiform; oral area Bar-shaped; ventral margin Bar-shaped; anterior of oral area Type I, long bar-shaped, oral area; Type II, short bar-shaped, under pellicle Bar-shaped; anterior of oral area Bar-shaped; apical Bar-shaped; apical

Marine Marine Freshwater

160–200 ca. 130 ca. 200

–/15–16 –/ca. 8 –/ca. 10

Bar-shaped; dorsal side Bar-shaped; oral area Bar-shaped; oral area

Freshwater Freshwater Freshwater

Lin et al. (2007) Lin et al. (2005a) Song and Wilbert (1989) Kahl (1931) Kahl (1931) Kahl (1931)

200–300

–

Needle-like; oral area

Freshwater

Kahl (1931)

LSK/RSK = left somatic kineties/right somatic kineties.

Song et al. (2004) described a form from China under the name A. marinus (Kahl 1931). However, the cell size of their isolate was larger (200–450 lm vs. 150–300 lm in length, p < 0.05) and the extrusomes were arranged along the entire ventral margin (vs. only along the oral slit in both original and our populations). Because the distribution pat-

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tern of extrusomes is species-specific for pleurostomatids (Foissner 1984; Foissner et al. 1995; Lin et al. 2007, 2008), the form reported by Song et al. (2004) should be a misidentification. It very likely represents a new species of Amphileptus and thus Amphileptus songi sp. n. (basionym A. marinus sensu Song et al. 2004) is proposed here.



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Comments on A. songi sp. n Except for the morphological differences discussed above, A. songi can also be distinguished from the present population of A. marinus by the number of ciliary rows (10–12 left and 20–27 right somatic kineties vs. 5–8 left and 13–21 right). Regarding the cell size, the presence of two macronuclear nodules and only ventrally positioned contractile vacuoles, there are seven other Amphileptus species similar to A. songi (Table 2), namely, A. salignus Chen et al. 2011; A. eigneri Lin et al. 2007; A. gui Lin et al. 2005; A. ensiformis Song and Wilbert 1989; A. rotundus (Kahl 1931) Carey 1991, A. pectinatus (Kahl, 1926), A. meleagris (Ehrenberg, 1835). Of these, A. pectinatus resembles A. songi most. However, it can be distinguished by the habitat (freshwater vs. marine) and fewer right kineties (ca. 10 vs. 20–27; Kahl 1931). Amphileptus eigneri and A. gui are marine species as well. But both can be distinguished from A. songi by the arrangement of extrusomes and the number of ciliary rows (Table 2). Although the ciliary pattern of the freshwater species, Amphileptus rotundus and A. meleagris, is not known yet, the former can be identified from A. songi by the distribution of extrusomes (arranged in dorsal side of cytoplasm vs. along the ventral margin of the cell) and the latter can be separated by having a pointed caudal end (vs. rounded) and longer extrusomes (17 lm vs. ca. 12 lm; Kahl 1931). Amphileptus ensiformis can be easily distinguished from A. songi by the presence of a distinct apical group of extrusomes (Song and Wilbert 1989). A. salignus, meanwhile, has two types of extrusomes (vs. one type in A. marinus) and fewer left somatic kineties (4 vs. 10–12; Chen et al. 2011). Comments on A. wilberti sp. n Besides A. marinus and A. songi discussed above, there are still seven species, which should be compared with A. wilberti sp. n. in terms of having two macronuclear nodules, ventrally positioned contractile vacuoles and extrusomes arranged along the oral slit (Table 2). In general morphology, A. wilberti sp. n. is nearly the same as A. eigneri. It can be distinguished, however, by the presence of cortical granules (vs. absence), oval or pyriform body shape (vs. elongate elliptical body shape) and fewer contractile vacuoles (ca. 3 vs. 5–8; Lin et al. 2007). Amphileptus muscicola, A. pectinatus and A. marinus are similar to A. wilberti concerning cell size and contractile vacuoles. The first two species can be identified by fewer right somatic kineties and the distribution of extrusomes (in the anterior of oral slit vs. in the entire oral slit; Kahl 1931), while the last can be separated by its elongate body shape (vs. oval or pyriform body shape). Compared with A. songi and A. meleagris, A. wilberti is smaller (180–210 lm long vs. 200–450 lm, p < 0.05, and

200–300 lm long respectively; Kahl 1931; Song et al. 2004). In addition, A. wilberti has fewer somatic kineties than A. songi (15–19 right, 7 or 8 left vs. 20–27 right, 10–12 left). Amphileptus salignus is also a brackish species; however, it differs from A. wilberti in having two types of extrusomes (vs. only one type) and more right somatic kineties (24–29 vs. 15–19; Chen et al. 2011). Is Amphileptus monophyletic or not? Due to limited sampling, it seems that Amphileptus was considered monophyletic in most previous studies (Pan et al. 2013; Vd’acny and Foissner 2013; Vd’acny et al. 2011; Wu et al. 2013; Zhang et al. 2010, 2012) except for the Bayesian tree of Gao et al. (2010) and phylogenetic trees of Vd’acny et al. (2014), where it was paraphyletic. After expanding sampling and adding two new sequences of Amphileptus, the present analyses suggest that the genus Amphileptus is possible paraphyletic. The results of the AU test (Table 3), however, indicated that the monophyly of Amphileptidae or Amphileptus was not statistically significantly rejected (p = 0.219/0.200 > 0.05). So, considering the molecular results above and the specialized morphological characteristics, more molecular information is still needed to resolve the problem as to whether the family Amphileptidae or the genus Amphileptus is monophyletic or not. TAXONOMIC SUMMARY Order Pleurostomatida Schewiakoff, 1896 €tschli, 1889 Family Amphileptidae Bu Genus Amphileptus Ehrenberg, 1830 Amphileptus dragescoi sp. n Diagnosis. Medium-sized marine Amphileptus about 90– 140 9 10–15 lm in vivo; body elongated or lanceolate shaped with colourless cortical granules underneath the pellicle; 12–15 right and consistently five left kineties; extrusomes clavate, forming an apical group; two macronuclear nodules; single contractile vacuole terminally positioned. Type locality. Coastal waters of Donghai Island (22°550 18″N, 110°310 00″E), Guangdong, China.

Table 3. Approximately unbiased (AU) test results Topology constraints Unconstrained Monophyly of the genus Amphileptus Monophyly of the family Amphileptidae

Ln likelihood

AU value (p)

5,357.79123 5,363.11998

0.819 0.200

5,362.89267

0.219

p < 0.05 refute monophyly; p > 0.05 do not refute the possibility of monophyly.


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Type material. A protargol slide with the holotype specimen is deposited in the Laboratory of Protozoology, OUC, China, with registration number PHB-09121507-1. Another slide with several paratype specimens is deposited in the same place with the registration number: PHB-091215072. Relevant specimens are marked by black ink circles on the cover slip. Dedication. We dedicate this species to Prof. Dr. Jean Dragesco, the great French protozoologist, in recognition of his significant contribution to taxonomic study of ciliates. Amphileptus marinus (Kahl 1931) Song et al. 2004 Litonotus (Hemiophrys) marina Kahl 1931, Die Tierwelt Deutschlands, 21:188, fig. 30, 25 (original description). Litonotus (Hemiophrys) marina – Borror 1963, Arch. Protistenkd., 106:484, fig. 35 and 36. non Amphileptus marinus (Kahl 1931) Song et al. 2004 – Song, Wilbert, & Hu, 2004, Eur. J. Protistol., 40:2, fig. 1 and 2 (combining authors, misidentification). Improved diagnosis. Large marine Amphileptus, usually 150–300 9 30–80 lm in vivo; elongated or slender lanceolate in outline, with blunt rounded posterior end; 13–21 right and five to eight left somatic kineties; colourless cortical granules densely distributed underneath pellicle; fusiform-shaped extrusomes arranged along oral slit; usually two macronuclear nodules; several contractile vacuoles ventrally positioned. Type locality. Sea water moat in Sylt (Kahl 1931). Type material. Not available. Voucher material. One voucher slide with protargolimpregnated specimens was deposited in Laboratory of Protozoology, OUC, China, with registration number PHB09121503-1. Amphileptus songi sp. n Amphileptus marinus (Kahl 1931) Song et al. 2004 – Song, Wilbert, & Hu, 2004, Eur. J. Protistol., 40:2, fig. 1 and 2. Diagnosis. Large marine Amphileptus 200–450 lm long in vivo with a slender lanceolate body; two macronuclear nodules; 10–12 left and 20–27 right somatic kineties; several contractile vacuoles positioned ventrally in the posterior half of the cell; extrusomes bar-shaped, densely arranged along the entire ventral margin. Type locality. An open-water scallop-farm off Wendong, Shandong Province (the Yellow Sea). The water temperature was about 24 °C, salinity ca. 33&. Type material. According to Song et al. (2004), a protargol slide with the holotype specimen was deposited in the Natural History Museum, London, UK (Registration no. 2003:4:24:1). Amphileptus wilberti sp. n Diagnosis. Brackish Amphileptus about 180–210 9 50– 60 lm in vivo in size; body oval or pyriform with posterior end broadly rounded; colourless cortical granules sparsely

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distributed beneath the pellicle; 15–19 right and seven or eight left kineties; extrusomes bar-shaped, attached to the anterior portion of oral slit; usually three contractile vacuoles arranged along posterior portion of ventral margin; two macronuclear nodules. Type locality. A mangrove wetland in Techeng Island (21°080 52″N, 110°260 20″E), Guangdong, China. Type material. A protargol slide with the holotype specimen is deposited in the Laboratory of Protozoology, OUC, China, with registration number PHB-10040704-1. Another slide with several paratype specimens is deposited in the same place with the registration number: PHB-100407042. Relevant specimens are marked by black ink circles on the cover slip. Dedication. We dedicate this species to our respected colleague, the eminent ciliatologist, Prof. Norbert Wilbert, University of Bonn, Germany, in recognition of his significant contributions to the study of ciliates. ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (projects nos 31201703, 31071898, 31222050), Innovation Program of Shanghai Municipal Education Commission (no. 13YZ095), Shanghai Universities First-class Disciplines Project of Fisheries, and International Research Group Program (no. IRG14-22), Deanship of Scientific Research, King Saud University. Many thanks are due to Prof. Dr. Weibo Song for his helpful suggestions, and Dr. Jie Huang for her gene sequencing work. We also appreciate two anonymous reviewers for their constructive comments. LITERATURE CITED Borror, A. C. 1963. Morphology and ecology of the benthic ciliate protozoa of Alligator Harbour, Florida. Arch. Protistenkd., 106:465–534. Canella, M. F. 1960. Contributo ad una revisione dei generi Amphileptus, Hemiophrys e Litonotus (Ciliata, Holotricha, Gymnostomata). Ann. Univ. Ferrara (N. S. Sect. III), 2:47–95. Carey, P. G. 1991. Marine Interstitial Ciliates: An Illustrated Key. Chapman and Hall, London. Chen, R., Lin, X. & Warren, A. 2011. A new pleurostomatid ciliate, Amphileptus salignus n. sp. (Protozoa, Ciliophora), from mangrove wetlands in southern China. Zootaxa, 3048:62–68. s me sopsammiques littoraux, syste maDragesco, J. 1960. Cilie cologie. Trav. Stat. Biol. Roscoff, 12:1–356. tique, morphologie, e is, A. 1986. Cilie s libres de l’AfriDragesco, J. & Dragesco-Kerne tude que intertropicale. Introduction a la connaissance et a l’e s. Faune Tropicale, 26:1–559. des Cilie € Foissner, W. 1984. Taxonomie und Okologie einiger Ciliaten (Protozoa, Ciliophora) des Saprobiensystems. I: Genera Litonotus, Amphileptus, Opisthodon. Hydrobiologia, 119:193–208. Foissner, W., Berger, H., Blatterer, H. & Kohmann, F. 1995. Taxo€kologische Revision der Ciliaten des Saprobiennomische und o systems – Band IV: Gymnostomatea, Loxodes, Suctoria. € r WasserwirtsInformationsberichte des Bayer. Landesamtes fu chaft, 1(95):1–540. Gao, F., Katz, L. A. & Song, W. 2013. Multigene-based analyses on evolutionary phylogeny of two controversial ciliate orders:



Pan et al.

Pleuronematida and Loxocephalida (Protista, Ciliophora, Oligohymenophorea). Mol. Phylogenet. Evol., 68:55–63. € der-Kypke, M. C., Al-Rasheid, K. A. S., Lin, X. & Gao, S., Stru Song, W. 2010. Molecular phylogeny of three ambiguous ciliate genera: Kentrophoros, Trachelolophos and Trachelotractus (Alveolata, Ciliophora). Zool. Scr., 39:305–313. Gao, S., Song, W., Ma, H., Yi, Z., Clamp, J. C., Al-Rasheid, K. A. S., Al-Khedhairy, A. A. & Lin, X. 2008. Phylogeny of six genera of the subclass Haptoria (Ciliophora, Litostomatea) inferred from sequences of small subunit rRNA genes. J. Eukaryot. Microbiol., 55:562–566. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser., 41:95–98. Jiang, J., Huang, J., Li, L., Shao, C., Al-Farraj, S. A. & Chen, Z. 2013. Morphology, ontogeny, and molecular phylogeny of two novel bakuellid-like hypotrichs (Ciliophora: Hypotricha), with establishment of two new genera. Eur. J. Protistol., 49:78–92. Kahl, A. 1931. Urtiere oder Protozoa I: Wimpertiere oder Ciliata (Infusoria) 2. Holotricha außer den im 1. Teil behandelten Prostomata. Tierwelt Dtl., 21:181–389. Lin, X., Song, W. & Warren, A. 2005a. Two new marine pleurostomatid ciliates from China, Amphileptus gui nov spec. and Amphileptus yuianus nov spec. (Ciliophora, Pleurostomatida). Eur. J. Protistol., 41:163–173. Lin, X., Song, W. & Warren, A. 2005b. Taxonomic studies on three marine pleurostomatid ciliates: Kentrophyllum verrucosum (Stokes, 1893) Petz, Song et Wilbert, 1995, Epiphyllum -Fre miet, 1908) gen. n., comb. n. and Amphisoliforme (Faure leptus sikorai sp. n., with the establishment of a new genus Epiphyllum (Ciliophora: Pleurostomatida). Acta Protozool., 44:129–145. Lin, X., Song, W. & Li, J. 2007. Amphileptus aeschtae nov. spec. and Amphileptus eigneri nov. spec. (Ciliophora, Pleurostomatida), two new marine pleurostomatid ciliates from China. Eur. J. Protistol., 43:77–86. Lin, X., AL-Rasheid, K. A. S., AL-Quraishy, S. A., AL-Farraj, S. A. & Song, W. 2008. Identification of three highly confused marine Loxophyllum (Ciliophora: Pleurostomatida) with a key to seven congeners from the China Sea. J. Eukaryot. Microbiol., 55:331– 342. Lynn, D. 2008. The Ciliated Protozoa, Characterization, Classification, and Guide to the Literature. Springer, Dordrecht. p. 605. Medlin, L., Elwood, H. J., Stickel, S. & Sogin, M. L. 1988. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene, 71:491–499. Miller, M. A., Pfeiffer, W. & Schwartz, T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), New Orleans, LA. p. 1–8. Nylander, J. A. A. 2004. MrModeltest v2. Evolutionary Biology Centre, Uppsala University, Uppsala. Pan, H., Gao, F., Lin, X., Warren, A. & Song, W. 2013. Three new Loxophyllum species (Ciliophora: Pleurostomatida) from China with a brief review of the marine and brackish Loxophyllum species. J. Eukaryot. Microbiol., 60:44–56. Pan, H., Gao, F., Li, J., Lin, X., Al-Farraj, S. A. & Al-Rasheid, K. A. S. 2010. Morpholgy and phylogeny of two new pleurostomatid ciliates, Epiphyllum shenzhenense n. sp. and Loxophyllum spirellum n. sp. (Protozoa, Ciliophora) from a mangrove wetland, South China. J. Eukaryot. Microbiol., 57:421–428. Penn, O., Privman, E., Ashkenazy, H., Landan, G., Graur, D. & Pupko, T. 2010a. GUIDANCE: a web server for assessing align-

ment confidence scores. Nucleic Acids Res., 38:W23–W28. doi:10.1093/nar/gkq443. Penn, O., Privman, E., Landan, G., Graur, D. & Pupko, T. 2010b. An alignment confidence score capturing robustness to guidetree uncertainty. Mol. Biol. Evol., 27:1759–1767. Posada, D. & Crandall, K. A. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics, 14:817–818. Qinn, G. P. & Keough, M. J. 2002. Experimental Design and Data Analysis for Biologists. Cambridge University Press, Cambridge. p. 537. Ronquist, F. & Huelsenbeck, J. P. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19:1572–1574. Shimodaira, H. 2002. An approximately unbiased test of phylogenetic tree selection. Syst. Biol., 51:492–508. Shinmodaira, H. & Hasegawa, M. 2001. Consel: for assessing the confidence of phylogenetic tree selection. Bioinformatics, 17:1246–1247. Song, W. 1991. A new marine ciliate, Amphileptus litonotiformis nov. sp. (Protozoa Ciliophora). Chin. J. Oceanol. Limnol., 9:300– 305. Song, W. & Wilbert, N. 1989. Taxonomische Untersuchungen an Aufwuchsciliaten (Protozoa, Ciliophora) im Poppelsdorfer Weiher, Bonn. Lauterbornia, 3:2–221. Song, W., Wilbert, N. & Hu, X. 2004. Redescription and neotypification of Amphileptus marinus (Kahl, 1931) nov. comb. (Ciliophora, Pleurostomatida), and reactivation of A. parafusidens Song & Wilbert, 1989. Eur. J. Protistol., 40:1–11. Song, W., Warren, A. & Hu, X. 2009. Free Living Ciliates in the Bohai and Yellow Seas, China. Science Press, Beijing. p. 518. Sonntag, B. & Foissner, W. 2004. Urotricha psenneri n. sp. and Amphileptus piger (Vuxanovici, 1962) n. comb., two planktonic ciliates (Protozoa, Ciliophora) from an oligotrophic lake in Austria. J. Eukaryot. Microbiol., 51:670–677. Stamatakis, A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22:2688–2690. Stamatakis, A., Hoover, P. & Rougemont, J. 2008. A rapid bootstrap algorithm for the RAxML web servers. Syst. Biol., 57:758–771. Swofford, D. L. 2002. PAUP*. Phylogenetic Analysis Using Parsimony (and other methods). Version 4, Sinauer Associates, Sunderland, MA. Vd’acny, P. & Foissner, W. 2013. Synergistic effects of combining morphological and molecular data in resolving the phylogenetic position of Semispathidium (Ciliophora, Haptoria) with description of Semispathidium breviarmatum sp. n. from tropical Africa. Zool. Scr., 42:529–549. Vd’acny, P., Bourland, W. A., Orsi, W., Epstein, S. S. & Foissner, W. 2011. Phylogeny and classification of the Litostomatea (Protista, Ciliophora), with emphasis on free-living taxa and the 18S rRNA gene. Mol. Phylogenet. Evol., 59:510–522. Vd’acny, P., Breiner, H.-W., Yashchenko, V., Dunthorn, M., Stoeck, T. & Foissner, W. 2014. The chaos prevails: molecular phylogeny of the Haptoria (Ciliophora, Litostomatea). Protist, 165:93–111. Wilbert, N. 1975. Eine verbesserte Technik der Protargolimpr€ agna€ r Ciliaten. Mikrokosmos, 64:171–179. tion fu Wu, L., Chen, R., Yi, Z., Li, J., Warren, A. & Lin, X. 2013. Morphology and phylogeny of three new Loxophyllum species (Ciliophora, Pleurostomatida) from mangrove wetlands of southern China. J. Eukaryot. Microbiol., 60:267–281. Wu, L., Chen, R., Yi, Z., Li, J., Warren, A. & Lin, X. 2014. The morphology of three Loxophyllum species (Ciliophora, Pleurostomatida) from southern China, L. lembum sp. n., L. vesiculo-


653


Pan et al.

sum sp. n. and L. perihoplophorum Buddenbrock, 1920, with notes on the molecular phylogeny of Loxophyllum. J. Eukaryot. Microbiol., 61:115–125. Zhang, Q., Simpson, A. & Song, W. 2012. Insights into the phylogeny of systematically controversial haptorian ciliates (Ciliophora, Litostomatea) based on multigene analyses. Proc. R. Soc. Lond. Ser. B Biol. Sci., 279:2625–2635.

654

Zhang, Q., Yi, Z., Song, W., Al-Rasheid, K. A. S. & Warren, A. 2010. The systematic position of Paraspathidium Noland, 1937 (Ciliophora, Litostomatea?) inferred from primary SSU rRNA gene sequences and predicted secondary rRNA structure. Eur. J. Protistol., 46:280–288.


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