The morphology of three Loxophyllum species (Ciliophora, Pleurostomatida) from southern China, L. lembum sp. n., L. vesiculosum sp. n. and L. perihoplophorum Buddenbrock, 1920, with notes on the molecular phylogeny of Loxophyllum.

Published by the International Society of Protistologists

The Journal of

Eukaryotic Microbiology

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

The Morphology of Three Loxophyllum Species (Ciliophora, Pleurostomatida) from Southern China, L. lembum sp. n., L. vesiculosum sp. n. and L. perihoplophorum Buddenbrock, 1920, with Notes on the Molecular Phylogeny of Loxophyllum Lei Wua, Ruimin Chena, Zhenzhen Yia, Jiqiu Lia, Alan Warrenb & Xiaofeng Lina a Laboratory of Protozoology, Key Laboratory of Ecology and Environment Science in Guangdong Higher Education, South China Normal University, Guangzhou, 510631, China b Department of Life Sciences, The Natural History Museum, Cromwell Road, London, SW7 5BD, UK

Keywords Brackish water ciliates; Litostomatea; mangrove wetland; new species; SSU rDNA. Correspondence X. Lin, Laboratory of Protozoology, College of Life Science, South China Normal University, Guangzhou 510631, China Telephone/FAX number: +86 20 85210644; e-mail: [email protected] Received: 25 June 2013; revised 12 September 2013; accepted September 30, 2013. doi:10.1111/jeu.12089

ABSTRACT Two new pleurostomatid ciliates, Loxophyllum lembum sp. n., L. vesiculosum sp. n., and the poorly known L. perihoplophorum Buddenbrock, 1920, isolated from brackish waters in coastal regions of southern China, are described following observations of live cells and protargol-impregnated specimens. Loxophyllum lembum sp. n. is distinguished by a combination of characters including two macronuclear nodules, 6–9 contractile vacuoles along the ventral margin, 11–14 right and 6–8 left kineties and the presence of cortical granules. Loxophyllum vesiculosum sp. n. differs from its congeners mainly by the unique distribution of contractile vacuoles, several of which lie along the dorsal margin and one on the ventral margin, and 15–21 right and 6–8 left kineties. Loxophyllum perihoplophorum is characterized by its large cell size (350– 450 lm long in vivo), 3–5 contractile vacuoles along the dorsal margin in the posterior region of the body, and 19–23 right and 7–9 left kineties. An improved diagnosis of L. perihoplophorum is provided. The SSU rDNA sequence of L. perihoplophorum is reported for the first time and its molecular phylogeny is analyzed. Maximum likelihood and Bayesian inference analyses of SSU rDNA sequence data recover the monophyly both of the order Pleurostomatida and of the genus Loxophyllum.

PLEUROSTOMATIDS are characterized by a slit-like oral apparatus and a distinctly flattened cell. They are common inhabitants of marine, brackish and freshwater habitats worldwide (Dragesco 1960; Foissner and Leipe 1995; Kahl 1935; Lin and Song 2004; Lin et al. 2005a,b, 2007, 2009; Song and Wilbert 1989; Song et al. 2004). During recent investigations on the biodiversity of ciliated protozoa in mangrove wetlands and other coastal water habitats of southern China, more than 200 species have been found, about 20% of which are pleurostomatids (Chen et al. 2011; Pan et al. 2010, 2013; Wu et al. 2013). The species diversity of pleurostomatid ciliates in this region is therefore much higher than anticipated compared with previous studies elsewhere (Dragesco 1965; Kahl 1931; Song and Wilbert 1989).

There are 12 genera in the order Pleurostomatida, among which Loxophyllum Dujardin, 1841 is one of the largest (Lynn 2008). This genus is characterized by the right somatic kineties terminating anteriorly along perioral kineties 2 and 3, and the presence of numerous extrusomes along the dorsal margin (Foissner and Leipe 1995; Foissner et al. 1995; Lin et al. 2008). To date, over 60 morphospecies of Loxophyllum have been reported, although only about one-third of these have been investigated in detail with respect to their morphology and ciliary pattern (Dragesco 1954, 1960, 1965; Foissner 1984; Foissner and Leipe 1995; Foissner et al. 1995; Kahl 1931, 1933; Lin et al. 2005c, 2008; Pan et al. 2010, 2013; Petz et al. 1995; Song 1993; Song and Wilbert 1989; Wu et al. 2013). Furthermore, gene sequence data are available for

© 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 61, 115–125

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only about 10 species (Pan et al. 2010, 2013; Wu et al. 2013). Consequently, much work needs to be carried out, redescribing poorly known species, characterizing unknown species and populating gene sequence databases, to gain a better understanding of the taxonomy and systematics of the genus. In the present study, three Loxophyllum species, collected from inshore waters, a shrimp-culturing pond and a mangrove wetland, respectively, near Zhanjiang, southern China, were investigated in terms of their morphology and ciliary pattern. In addition, the SSU rDNA gene of L. perihoplophorum was sequenced and its systematic position was analyzed. MATERIALS AND METHODS Loxophyllum lembum sp. n. was isolated on 15 December 2009 from inshore waters of the South China Sea near Zhanjiang (21°11′03″N, 110°24′38″E), southern China. Loxophyllum vesiculosum sp. n. was collected on the same day from a shrimp-culturing pond on Techeng Island (21°09′12″N, 110°25′20″E) in Zhanjiang. Loxophyllum perihoplophorum was isolated on 7 April 2012 from a mangrove wetland on Techeng Island (21°09′10″N, 110°26′18″ E) in Zhanjiang. All the samples were collected using 20lm mesh plankton nets. Cells of each species were maintained in the laboratory at about 25 °C in Petri dishes with raw water from the collection site and some rice grains to enrich the growth of bacteria as a food source for the ciliates. Observations of living cells were carried out using bright field and differential interference contrast (DIC) microscopy. The number, size, location, and pulsation interval of contractile vacuoles were recorded based on live observations. The protargol impregnation method according to Wilbert (1975) was used to reveal the ciliary pattern. Living cells were examined at 100–1,000X magnifications; measurements of stained specimens were performed at a magnification of 1,000X. Drawings of impregnated specimens were made with the help of a camera lucida at a magnification of 1,000X. Classification and terminology are according to Lynn (2008). Genomic DNA extraction, gene amplification, and gene sequencing of L. perihoplophorum were conducted according to the method described by Wu et al. (2013). It was not possible to obtain SSU rDNA sequences of L. lembum or L. vesiculosum due to technical problems. Apart from the sequence of L. perihoplophorum, all SSU rDNA gene sequences used in the phylogenetic analyses were obtained from the NCBI GenBank database (for GenBank accession numbers, see Fig. 7). These included all available SSU rDNA gene sequences of the order Pleurostomatida, 10 taxa of Trichostomatia with GenBank accession numbers as follows: AF298821, AB534184, AB530165, AF298820, AM158448, AM158457, U57763, AM158473, AM158467, FM201781, and 9 taxa of Haptoria with GenBank accession numbers as follows: DQ411860, DQ411862, DQ777746, EU242508, HM581673, JF263444, JF263447, L26447, U57771. Nyctotheroides deslierresae

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and N. parvus (class Armophorea, order Clevelandellida) were selected as the outgroup taxa. Sequences were aligned with CLUSTAL W implemented in Bioedit v.7.0 (Hall 1999) and edited manually by removing both the primers and the highly variable regions. The final alignment of 1,595 characters and 39 taxa was used to construct phylogenetic trees. The maximum likelihood (ML) analysis was performed online using RaxM-HPC2 v7.2.8 on the CIPRES Portal V 1.15 (http://www.phylo.org) with parameter settings as reported by Stamatakis et al. (2008). The reliability of internal branches was estimated by using a nonparametric bootstrap method with 1,000 replicates. A Bayesian inference (BI) analysis was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the Markov chain Monte Carlo algorithm under the GTR + G + I evolutionary model indicated by MrModeltest v.2 (Nylander 2004). The program was run for 1,000,000 generations with a sample frequency of 100 generations. The first 2,500 generations were discarded as burn-in. RESULTS Loxophyllum lembum sp. n. (Table 1; Fig. 1, 2) Cells in vivo are 125–235 lm (usually about 150 lm) long 9 45–70 lm wide. The body shape is highly variable, but usually narrow boat-shaped with a beak-like anterior end, a bluntly pointed posterior end and a neck region is about 25–30% of the cell length (Fig. 1A, B, 2A–E). The body is laterally compressed about 2–3:1 and often has a well-defined hyaline fringe (Fig. 1A, B, 2A). The two macronuclear nodules are ovoid in shape, each about 20– 35 lm in length, and are usually readily detectable in vivo under DIC as two large transparent areas in the central region of the cell (Fig. 1F, 2G). One micronucleus is positioned between the two macronuclear nodules (Fig. 2G). There are 6–9 contractile vacuoles, the largest of which is 8–10 lm in diam. and subterminally positioned, the others are 5–7 lm in diam. and distributed along the ventral margin in the posterior half of the cell (Fig. 1A, B, 2B). The extrusomes are slender, bar-shaped, about 7–9 lm long (Fig. 1G, 2H); some are sparsely and uniformly arranged along the hyaline edge of the ventral margin, whereas others are clustered together to form 7–10 warts along the dorsal margin (Fig. 1A, B, 2B). The pellicle is thin and with small (< 0.5 lm across), densely spaced, colorless, dot-like cortical granules between the ciliary rows on both sides of the cell (Fig. 1E, 2I). The right side is flat and densely ciliated with cilia about 4–5 lm long (Fig. 1A); the left side is slightly vaulted in the central body region and sparsely ciliated with cilia that are difficult to detect in life; the bristles of the dorsal brush are about 3 lm long and detectable in vivo at high magnifications (Fig. 2F). The cytoplasm is colorless to grayish, often with numerous tiny, shining globules 2–5 lm across that render the main part of the body is more or less opaque, especially at low magnifications (Fig. 1B, 2A, H). Locomotion is by gliding moderately fast


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Table 1. Morphological characteristics of Loxophyllum lembum sp. n. (first line), L. vesiculosum sp. n. (second line) and L. perihoplophorum Buddenbrock, 1920 (third line) Characters

Min

Max

Mean

SD

CV

n

Body length

125 135 215 45 45 45 11 15 19 6 6 7 2 2 2 20 25 28 15 18 12 8 5 7 50 45 38

230 255 360 65 100 80 14 21 23 8 8 9 2 2 2 35 60 48 25 40 20 11 13 9 165 110 90

157.4 184.6 259.8 54.1 69.3 55.4 13.0 18.4 20.7 6.8 6.7 8.3 2.0 2.0 2.0 27.3 35.5 38.6 21.4 26.9 16.5 8.8 9.1 7.5 99.8 70.3 56.6

26.77 32.84 32.19 6.98 13.37 9.79 1.00 1.68 1.03 0.55 0.75 0.66 0 0 0 4.28 6.86 5.69 3.69 4.59 2.46 1.17 1.90 0.69 33.17 14.36 13.57

17.0 17.8 12.4 12.9 19.3 17.7 7.7 9.1 5.0 8.1 11.2 7.9 0 0 0 15.7 19.3 14.7 17.2 17.1 14.9 13.3 2.9 9.2 33.2 20.4 24.0

21 28 20 21 28 20 21 31 20 21 19 20 21 28 20 20 27 20 20 27 20 20 23 20 19 23 20

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

Length of nematodesmata

All measurements in lm. Data based on protargol-impregnated specimens. CV = coefficient of variation in%; Max = maximum; Mean = arithmetic mean; Min = minimum; n = sample size; SD = standard deviation. a Perioral kineties 2, 3 included. b Perioral kinety 1 and dorsal brush kinety included.

on the substrate, or by swimming with a slow clockwise rotation about the longitudinal axis, with the neck region constantly swaying from side to side. The ciliary pattern is shown in Fig. 1C, D and 2J, K. There are 11–14 right kineties, including kineties 2 and 3; the right somatic kineties are densely ciliated and shortened anteriorly along perioral kinety 3 (Fig. 1C, 2J). There are 6–8 left kineties, including perioral kinety 1 and the dorsal brush kinety; the left somatic kineties terminate anteriorly along perioral kinety 1 (Fig. 1D, 2K). The dorsal brush extends to about 1/3 of the body length as regularly spaced dikinetids and continues posteriorly as a row of closely spaced monokinetids (Fig. 1D, 2K). There are three perioral kineties around the oral slit. Perioral kinety 1, which lies to the left of the cytostome, has widely spaced basal body pairs in the anterior half and continues posteriorly as a row of monokinetids (Fig. 1D). Perioral kineties 2 and 3 lie to the right of the cytostome; perioral kinety 2 is composed of closely spaced dikinetids in the anterior half and monokinetids in the posterior half;

Figure 1 Morphology and ciliary pattern of Loxophyllum lembum sp. n. drawn from life (A, B, E, G) and after protargol impregnation (C, D, F). A, B. Right (A) and left (B) side view of typical individuals. C, D. Ciliary pattern of right (C) and left (D) side. E. Cortical granules. F. Macronuclear nodules. G. Extrusomes. DB = dorsal brush; PK1-3 = perioral kinety 1, 2 and 3. Scale bars, 80 lm in (A–D); 10 lm in (F, G).

perioral kinety 3 is composed entirely of monokinetids (Fig. 1C, D). The nematodesmata are highly developed and up to 165 lm in length (Fig. 1D). Loxophyllum vesiculosum sp. n. (Table 1; Fig. 3, 4) Cells in vivo are 200–420 9 55–85 lm in size. The body shape is variable from slender belt-like to broad leaf-shaped, with a beak-like anterior end and a bluntly pointed posterior end (Fig. 3A, 4A–E). The cell margin has a well-defined hyaline fringe (Fig. 4B–D). The body is laterally compressed about 3–4:1 when fully extended, but when the cell contracts, the main body region is hunched on the left side. There are two ovoid to ellipsoidal macronuclear nodules, each about 30–45 9 25–35 lm in size, usually detectable in vivo as two large transparent areas in the equatorial region of the cell (Fig. 4A, E). The single micronucleus is ovoid in shape, about 4–6 lm in length, and is situated between the two macronuclear nodules. There are 5–7 contractile vacuoles, each about 5–12 lm in diam. and located in the posterior half of the body; most are positioned along the dorsal margin although the largest one is usually terminally located and another, which pulses infrequently, is located near the ventral margin (Fig. 3A, 4A, E). The extrusomes are slender bar-shaped, straight to slightly curved, 7–11 lm long (Fig. 3D, 4H), and are evenly distributed along the entire ventral margin, clustered to form 9–13 warts along the dorsal margin and or scattered in cytoplasm (Fig. 3A, 4D). The pellicle is thin and with numerous small (< 0.5 lm across), sparsely distributed, colorless, dot-like cortical granules (Fig. 3B, 4F, G). There are 4–6 longitudinal ridges that are conspicuous at low magnifications on the left side (Fig. 4D). The right side is densely ciliated with cilia about 6–8 lm long; the cilia on the left side are sparsely distributed and difficult to detect in life. The cytoplasm is slightly grayish, often with numerous greasily shining globules


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Figure 2 Photomicrographs of Loxophyllum lembum sp. n. from life (A–I, DIC) and after protargol impregnation (J, K). A. Left side view of a typical individual, arrows show the hyaline margin. B. Right side view, arrows point to the contractile vacuoles, arrowheads mark the warts. C–E. Contorted specimens. F. The anterior part of left side, to show the dorsal brush (arrows) and perioral kinety 1 (arrowheads). G. Nuclear pattern showing the two macronuclear nodules and a single micronucleus (arrow) in between. H. Detail of the middle part of the cell, to show the extrusomes (arrowhead) and shining globules (arrow). I. Cortical granules (arrows). J. Right side view, to show the perioral kinety 1 (arrowhead) and perioral kinety 2 (arrow). K. Left side view, to show the dorsal brush kinety (arrow), the perioral kinety 1 (arrowhead) and the nematodesmata. DIC = differential interference contrast microscopy. Ma = macronuclear nodules. Scale bars, 80 lm in (A–E); 20 lm in (F–I); 50 lm in (J, K).

(2–6 lm across) that render the main body region is more or less opaque, even at low magnifications (Fig. 4B–D). There are numerous food vacuoles, each about 4–15 lm in diam., often containing algae. Locomotion is by slowly gliding on the substrate usually with the body continually stretching and contracting, rarely swimming. The ciliary pattern is shown in Fig. 3C, E and 4I–L. The right side of the body has 15–21 kineties, including perior-

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al kineties 2 and 3; the right somatic kineties terminate anteriorly along perioral kinety 3 (Fig. 3E). The left side has 6–8 kineties including perioral kinety 1 and the dorsal brush kinety; the left somatic kineties terminate anteriorly along perioral kinety 1 (Fig. 3C). The dorsal brush kinety extends nearly to the mid-body as densely spaced dikinetids and continues posteriorly as a row of monokinetids that become increasingly widely spaced (Fig. 3C, 4J).


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Figure 3 Morphology and ciliary pattern of Loxophyllum vesiculosum sp. n. drawn from life (A, B, D) and after protargol impregnation (C, E). A. Right side view of a typical individual. B. Cortical granules. C, E. The ciliary pattern of left (C) and right (D) side. D. Extrusomes. DB = dorsal brush; PK1–3 = perioral kinety 1, 2, and 3. Scale bars, 150 lm in (A, C, E); 10 lm in (D).

There are three perioral kineties around the oral slit. Perioral kinety 1, to the left of the cytostome, comprises dikinetids in the anterior half and continues to the posterior as a row of monokinetids (Fig. 3C, 4J). Perioral kinety 2, to the right of the cytostome, consists of closely spaced dikinetids in the anterior 3/5 and monokinetids in the posterior 2/5 (Fig. 3E, 4I). Perioral kinety 3 is composed entirely of monokinetids (Fig. 3E). The well-developed nematodesmata originate from the kinetosomes of the right perioral kineties and extend posteriorly to about 1/3 of the cell length (Fig. 3C, 4K, L). Loxophyllum perihoplohorum Buddenbrock, 1920 (Table 1; Fig. 5, 6) Since the original report by Buddenbrock (1920), L. perihoplophorum has never been redescribed. Consequently, its ciliary pattern and other aspects of its morphology, e.g. the cortical granules, remained unknown. Here, a detailed redescription based on the Chinese population is presented. The cell size is highly variable, about 200–500 lm 9 55– 150 lm in vivo, usually 350–450 lm long. The body is flexible and variable in shape, from slender to broad leafshaped. The cell fringe is thin, hyaline and well-defined, about 3–4 lm in width (Fig. 5A, 6A, B). The body is laterally compressed about 3–5:1 with the right side flat and the left side slightly to distinctly hunched due to the presence of numerous food vacuoles (Fig. 6A, B). There are two ellipsoidal macronuclear nodules, each about 25–35 lm 9 15–20 lm in size, and usually detectable in vivo as two transparent areas in the mid-body region (Fig. 5A, 6C). The single micronucleus is ellipsoidal, about 3 lm across, and located between the two macronuclear nodules. There are 3–5 contractile vacuoles, each about 5–10 lm in diam.; most are distributed along the dorsal margin in the posterior region of the cell, apart from the largest one which is located subterminally (Fig. 5A, 6B). The extrusomes are


bar-shaped, 5–7 lm long, some are evenly distributed along the entire ventral margin (Fig. 5C, 6D), others are grouped together to form about 12–15 warts along the dorsal margin (Fig. 5A, 6A, B). The pellicle is thin with a large number of tiny (~0.3 lm across), colorless, dot-like cortical granules densely packed between the ciliary rows on both sides of the body (Fig. 5B, 6E). The right side is densely ciliated with cilia ~8 lm long that are easily detected in vivo at high magnifications (Fig. 6F); the cilia on the left side are sparsely distributed and difficult to detect in life; the bristles of the dorsal brush are about 3 lm long and detectable in vivo at high magnifications (Fig. 6E). The cytoplasm is slightly grayish, often with many small (2–5 lm across), greasily shining globules that render the main body region grayish and opaque, especially at low magnifications (Fig. 6A, B). Locomotion is by gliding on the substrate with the body gently extending and contracting, or swimming while rotating clockwise about the longitudinal body axis. The ciliary pattern is shown in Fig. 5F, G and 6G, H. There are 19–23 kineties on the right side, including perioral kineties 2 and 3; the right somatic kineties are densely arranged and terminate anteriorly along perioral kinety 3 (Fig. 5F). The left side is sparsely ciliated and has 7–9 kineties, including perioral kinety 1 and dorsal brush kinety; the left somatic kineties terminate anteriorly along perioral kinety 1. The dorsal brush kinety consists of dikinetids in the anterior half and monokinetids in the posterior half (Fig. 5G, 6G). There are three perioral kineties around the oral slit. Perioral kinety 1, to the left of the oral slit, consists of comparatively widely spaced dikinetids in the anterior 2/5 and monokinetids in the posterior 3/5 (Fig. 5G, 6G). Perioral kineties 2 and 3 lie to the right of the oral slit (Fig. 5F); perioral kinety 2 is composed of dikinetids in the anterior 2/5 and monokinetids in the posterior 3/5; perioral kinety 3 is formed entirely of monokinetids (Fig. 5F, 6H). The nematodesmata was not observed. SSU rDNA gene sequences and phylogenetic analyses The SSU rDNA gene sequence of Loxophyllum perihoplohorum is deposited in the GenBank database with length, GC content, and accession number as follows: 1,638 bp, 42.12%, KC493570. The topologies of trees constructed using each of the two algorithms were almost identical, therefore only the ML tree is shown (Fig. 7). In both analyses, the members of the order Pleurostomatida formed a monophyletic group nested within the class Litostomatea with high support (99% ML; 1.00 BI). All the species of Loxophyllum formed a monophyletic group with high support (98% ML, 1.00 BI). Loxophyllum perihoplohorum clustered with L. rugosum with moderate support (71% ML, 0.92 BI). DISCUSSION Comments on Loxophyllum lembum sp. n. Loxophyllum lembum is partly characterized by the distribution of its contractile vacuoles, with one located


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Figure 4 Photomicrographs of Loxophyllum vesiculosum sp. n. from life (A–H, DIC) and after protargol impregnation (I–L). A. Right side view of a typical individual, arrows mark the contractile vacuoles, and white arrowheads show the macronuclear nodules. B–D. Different cell shapes in life, arrowheads mark the dorsal extrusome warts, white arrows show the furrows on left side, black arrows mark the hyaline margin. E. Left side view of a typical individual, arrows show the contractile vacuoles, white arrowheads mark the macronuclear nodules. F, G. Cortical granules (arrows). H. Extrusomes (arrows). I. Ciliary pattern of right side, arrow shows perioral kinety 2. J. Ciliary pattern of left side, arrow shows perioral kinety 1, arrowheads denote the dorsal brush kinety. K. Mid-region of right side of cell, arrows mark the well-developed nematodesmata. L. Right view of a typical individual, arrow shows the nematodesmata. DIC = differential interference contrast microscopy; Ma = macronuclear nodule. Scale bars, 150 lm in (A–E); 20 lm in (F–H, K); 100 lm in (I, J, L).

subterminally and the others distributed along the ventral margin. Based on this feature, four Loxophyllum spp. resemble L. lembum, namely L. chinense Pan et al., 2013; L. salinum Wu et al., 2013; L. grande (Entz, 1879) Kahl, 1931; and L. multinucleatum Kahl, 1931 (Table 2; Kahl 1931; Pan et al. 2013; Wu et al. 2013). Although Loxophyllum chinense is very similar to the new species in having two macronuclear nodules, a similar cell size and the same number of left kineties, L. lem-

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bum can be clearly separated from L. chinense by the presence (vs. absence) of cortical granules and having fewer (11–14 vs. 13–18) right kineties and more (7–9 vs. 3–5) contractile vacuoles along the ventral margin (Pan et al. 2013). Loxophyllum lembum also resembles the brackish water species L. salinum in terms of its general morphology. However, the former can be separated from the latter in the number and arrangement of the contractile vacuoles


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Moreover, L. vesiculosum differs: from L. australe by having more (5–7 vs. 2) contractile vacuoles and more right (15–21 vs. 10–12) and left (6–8 vs. 3 or 4) kineties; from L. magnum by having fewer (5–7 vs. 10) contractile vacuoles and more (2 vs. 1) macronuclear nodules; from L. lionotiforme var. plurivacuolatum by having more (5–7 vs. 4) contractile vacuoles and a larger body size (200–420 lm vs. 200 lm in length in vivo); and from S. utriculariae by its larger body size (200–420 lm vs. 140–200 lm in length in vivo) and by having more right (15–21 vs. 4–7) and left (6–8 vs. 3 or 4) kineties (Table 3; Foissner and O’Donoghue 1990; Foissner and Leipe 1995; Vuxanovici 1959). Comments on Loxophyllum perihoplophorum Buddenbrock, 1920 Figure 5 Morphology and ciliary pattern of Loxophyllum perihoplophorum drawn from life (A–E) and after protargol impregnation (F, G). A. Right side view of a typical individual. B. Cortical granules. C. Extrusomes. D, E. Loxophyllum perihoplophorum from Kahl (1931) (D) and Buddenbrock (1920) (E), respectively. F, G. Ciliary pattern of right (F) and left (G) side. DB = dorsal brush; PK1–3 = perioral kinety 1–3. Scale bars, 150 lm in (A, D–G); 10 lm in (C).

along the ventral margin (7–9 in posterior region vs. 3 or 4 in mid-body region) and in having more (6–8 vs. 4–6) left kineties (Wu et al. 2013). Loxophyllum grande differs from L. lembum by its significantly larger body size (400 lm vs. 125–230 lm in length), and by having more (16 vs. 11–14) right kineties and fewer (4 or 5 vs. 6–8) left kineties (Kahl 1931). Loxophyllum multinucleatum can be separated from L. lembum by having more (10 vs. 6–8) left kineties, more (several vs. 2) macronuclear nodules, fewer (ca. 3 vs. 7–9) contractile vacuoles and by the absence (vs. presence) of warts along the dorsal margin (Kahl 1931). Comments on Loxophyllum vesiculosum sp. n. The most conspicuous characteristic of Loxophyllum vesiculosum is the arrangement of its contractile vacuoles, all of which are located in the posterior half of the body with several located along the dorsal margin and one positioned near the ventral margin. In addition, the number of somatic kineties, the number of macronuclear nodules, and the biotope can also clearly distinguish L. vesiculosum from its most similar congeners, i.e., L. compressum Dragesco, 1965; L. australe Foissner and O’Donoghue, 1990; L. magnum Vuxanovici, 1959; L. lionotiforme var. plurivacuolatum Vuxanovici, 1959; and L. contractile var. polyvacuolatum Vuxanovici, 1959; and also Siroloxophyllum utriculariae (Penard, 1922) Foissner and Leipe, 1995. Like Loxophyllum vesiculosum, L. compressum has several contractile vacuoles distributed along both the dorsal and ventral margins. However, the former can be separated from the latter by having fewer (6–8 vs. 10) left kineties and fewer (2 vs. 6–8) macronuclear nodules (Dragesco 1965). Loxophyllum vesiculosum differs from the other five similar species by its brackish (vs. freshwater) habitat.

The Chinese population described in the present study was identified as L. perihoplophorum based on the following characters: (1) the presence of several contractile vacuoles along the dorsal margin in the posterior half of the cell; (2) the general body shape and appearance in vivo, e.g. extrusomes grouped in warts along the dorsal margin and evenly distributed along the ventral margin; (3) the body size in vivo, i.e., 350–450 lm long vs. ca. 400 lm long in Buddenbrock’s population; (4) the presence of two macronuclei; and (5) the number of right kineties, i.e., 19– 23 vs. about 20 in Buddenbrock’s (1920) population. The only significant difference between the original description and the Chinese population of L. perihoplophorum is the habitat, the former having been isolated from a marine biotope and the latter from a brackish water biotope. It should be noted, however, that although the Chinese population was collected from waters with a salinity of 21.8&, it was able to grow in the laboratory with salinities up to 30&, suggesting that this species can tolerate a range of salinity. We therefore concluded that, based on the available data, the Chinese population and the population of L. perihoplophorum described by Buddenbrock (1920) are conspecific. In terms of the body size and the number and location of the contractile vacuoles, L. perihoplophorum is very similar to L. psammophilum. Although the ciliary pattern of L. psammophilum is unknown, the former can be distinguished from the latter by having fewer (2 vs. > 10) macronuclear nodules and by the absence (vs. presence in L. perihoplophorum) of warts (Dragesco 1954). Comments on the phylogenetic analysis All pleurostomatid species included in the present analysis formed a well-supported monophyletic group (99% ML; 1.00 BI) nested within the class Litostomatea, which is consistent with previous studies based on SSU €der-Kypke et al. 2006; rDNA gene sequence data (Stru Vd’acny et al. 2010, 2011; Zhang et al. 2012). Loxophyllum perihoplophorum and L. rugosum clustered together on the SSU rDNA gene tree in a clade with moderate support (71% ML; 0.92 BI). These two taxa differ from each other in 42 nucleotides and have a sequence


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Figure 6 Photomicrographs of Loxophyllum perihoplophorum from life (A–F, DIC) and after protargol impregnation (G, H). A. Right side view of a typical individual, arrows show the dorsal extrusome warts. B. Left side view, arrowheads point to the contractile vacuoles, arrow marks the hyaline margin. C. Detail of cell showing a macronuclear nodule. D. Detail of the middle part of the cell, to show the extrusomes (arrows). E. Anterior part of the left side, to show the dorsal brush (arrowheads) and cortical granules (arrows). F. Right side of cell, to show the cilia (arrows). G. Left side view, to show the dorsal brush kinety (arrowheads) and perioral kinety 1 (arrows). H. Right side view, arrows mark perioral kinety 2. DIC = differential interference contrast microscopy; Ma = macronuclear nodules. Scale bars, 40 lm in (A, B); 20 lm in (G, H).

similarity of 97.44%, confirming their validity as separate species. TAXONOMIC SUMMARY Order Pleurostomatida Schewiakoff, 1896 Family Litonotidae Kent, 1882 Genus Loxophyllum Dujardin, 1841

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Loxophyllum lembum sp. n. Diagnosis: Medium-sized Loxophyllum about 125– 235 9 45–70 lm in vivo; two macronuclear nodules; one micronucleus; 11–14 right and 6–8 left kineties; based on live observations, 6–9 contractile vacuoles distributed along ventral margin; extrusomes long, bar-shaped, evenly spaced along ventral margin and clustered together to


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Figure 7 Maximum likelihood tree based on 39 SSU rDNA sequences of litostomatean and armophorean ciliates. Numbers at nodes indicate bootstrap values of maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BI). The arrow depicts the newly sequenced species. GenBank accession numbers are given after names of species. The scale bar corresponds to five substitutions per 100 nucleotide positions.

form 7–10 warts along dorsal margin; brackish water habitat. Type locality and ecological features: Isolated on 15 December 2009 from inshore waters at a dock near Zhanjiang, China (21°11′03″N, 110°24′38″E). Water temperature 26.0 °C, salinity 27.2&, pH 7.8. Type slides: A protargol slide with the holotype specimen circled in black ink is deposited in the Laboratory of Protozoology, South China Normal University (SCNU), China, with registration number CR-20091215-04. One paratype slide with protargol-impregnated specimens is deposited in the Natural History Museum, London, UK, with registration number NHMUK 2012. 10.2.1. Etymology: The Latin adjective lembus, -a, -um ([m, f, n]; cymbiform) refers to the boat-like cell shape.

Loxophyllum vesiculosum sp. n. Diagnosis: Medium to large Loxophyllum 200–420 9 55– 85 lm in vivo; cell shape variable from extremely slender belt-like to broad leaf-shaped; two macronuclear nodules; 15–21 right and 6–8 left kineties; based on live observations, 5–7 contractile vacuoles in posterior part of body, most located along dorsal margin, one near ventral margin; extrusomes slender, bar-shaped, some scattered in cytoplasm, others evenly distributed along ventral margin or clustered together to form 9–13 warts on dorsal margin; brackish water habitat. Type locality and ecological features: Isolated on 15 December 2009 from a shrimp-culturing pond in Techeng Island (21°09′12″N, 110°25′20″E) in Zhanjiang, China. Water temperature 23.8 °C, salinity 24.8&, pH 7.4.


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Table 2. Comparison of Loxophyllum lembum sp. n. with morphologically similar species

Species L. L. L. L. L.

lembum sp. n. chinense salinum grande multinucleatum

Body length in vivo (lm)

Number of Ma

RSKa/LSKb

No. and position of CVs

Warts

Cortical granules

Biotope

Data source

125–235 120–200 90–180 400 140–160

2 2 2 2 Several

11–14a, 6–8b 13–18a, 6–8b 12–15a, 4–6b 16a, 4–5b –, 10b

7–9, ventral margin 3–5, ventral margin 3–4, ventral margin 7*, ventral margin 3*, ventral margin

Present Present Present – Absent

Present Absent Present – –

Brackish Marine Brackish Marine Brackish

Present work Pan et al. (2013) Wu et al. (2013) Kahl (1931) Kahl (1931)

CVs = contractile vacuoles; Ma = macronucleus; RSK/LSK = right somatic kineties/left somatic kineties; – = data not available; * = data from illustration. a Perioral kineties 2, 3 included. b Perioral kinety 1 and dorsal brush kinety included.

Table 3. Comparison of Loxophyllum vesiculosum sp. n. with morphologically similar species whose contractile vacuoles are located along both the ventral and dorsal margins Species

Body length

RSKa/LSKb a

b

No. of Ma

No. of CVs

Habitat

Data source

L. vesiculosum sp. n. L. compressum L. australe

200–420 280 120–180

15–21 /6–8 14–16a/10b 10–12a/3 or 4b

2 6–8 4

5–7 ca. 4 or 5* 2

Brackish Marine Freshwater

L. magnum L. lionoliforme var. plurivacuolatum L. contractile var. polyvacuolatum Siroloxophyllum utriculariae

460 205

–, – –, –

1 2

10 4

Freshwater Freshwater

Present work Dragesco (1965) Foissner and O’Donoghue (1990) Vuxanovici (1959) Vuxanovici (1959)

60

–, –

2

7

Freshwater

Vuxanovici (1959)

140–200

4–7a/3 or 4b

2

2

Freshwater

Foissner and Leipe (1995)

RSK/LSK = right somatic kineties/left somatic kineties; CVs = contractile vacuoles; D = dorsal margin; V = ventral margin; – = data not available; * = data from illustration. a Perioral kineties 2, 3 included. b Perioral kinety 1 and dorsal brush kinety included.

Type slides: A protargol slide with the holotype specimen circled in black ink is deposited in the Laboratory of Protozoology, SCNU, China, with registration number CR2009-1215-02. One paratype slide with protargol-impregnated specimens is deposited in the Natural History Museum, London, UK, with registration number NHMUK 2012.10.2.2. Etymology: The Latin adjective vesiculosus, -a, -um ([m, f, n]; blister) refers to the presence of several contractile vacuoles on both sides in the posterior region of the cell. Loxophyllum perihoplohorum Buddenbrock, 1920 Improved diagnosis (based on the original and the Chinese populations): Medium to large Loxophyllum, 200–500 9 55–150 lm in vivo; cell shape highly variable; two macronuclear nodules; 19–23 right and 7–9 left kineties; based on live observations, several contractile vacuoles are located dorsally in posterior 1/3 of cell; extrusomes bar-shaped, evenly distributed along entire ventral margin and clustered together forming 12–15 warts on dorsal margin; marine or brackish water habitat.

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Ecological features (Chinese population): Water temperature 22.1 °C, salinity 21.8&, pH 7.6. Voucher material: One voucher slide with protargolimpregnated specimens is deposited in the Laboratory of Protozoology, SCNU, China, with registration number WL2012040702. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (project numbers: 31222050, 31030059) and the Scientific Research Foundation of Graduate School of South China Normal University (project number: 2013KYJJ012). We thank Mr. Xudong Cui and Ms. Wenping Chen (SCNU, China) and Ms. Yangbo Fan (OUC, China) for sample collection and Prof. Dr. Weibo Song (OUC, China) for his helpful suggestions. LITERATURE CITED € ber einige neue oder Buddenbrock, W. 1920. Beobachtungen u wenig bekannte marine Infusorien. Arch. Protistenk., 41:341– 364.


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

Morphology and Phylogeny of Three Pleuronema Species (Ciliophora, Scuticociliatia) from Hangzhou Bay, China, with Description of Two New Species, P. binucleatum n. sp. and P. parawiackowskii n. sp.

Morphology and Molecular Phylogeny of Three Cyrtophorid Ciliates (Protozoa, Ciliophora) from China, Including Two New Species, Chilodonella parauncinata sp. n. and Chlamydonella irregularis sp. n.

Two New Brackish Ciliates, Amphileptus spiculatus sp. n. and A. bellus sp. n. from Mangrove Wetlands in Southern China, with Notes on the Molecular Phylogeny of the Family Amphileptidae (Protozoa, Ciliophora, Pleurostomatida).

Morphology, morphogenesis and molecular phylogeny of a new marine ciliate, Trichototaxis marina n. sp. (Ciliophora, Urostylida).

Morphology, morphogenesis, and molecular phylogeny of Sterkiella tetracirrata n. sp. (Ciliophora, Oxytrichidae), from the Silent Valley National Park, India.

Morphology, morphogenesis, and molecular phylogeny of Paraparentocirrus sibillinensis n. gen., n. sp., a "Stylonychine Oxytrichidae" (Ciliophora, Hypotrichida) without transverse cirri.

Morphology and phylogeny of Bryophryoides ocellatus n. g., n. sp. (Ciliophora, Colpodea) from in situ soil percolates of Idaho, U.S.A.

Morphology and molecular phylogeny of three marine Condylostoma species from China, including two new ones (Ciliophora, Heterotrichea).

Morphology and molecular phylogeny of Apoterritricha lutea n. g., n. sp. (Ciliophora, Spirotrichea, Hypotrichia): a putative missing link connecting Cyrtohymena and Afrokeronopsis.

Morphology, morphogenesis and molecular phylogeny of a novel soil ciliate, Pseudouroleptus plestiensis n. sp. (Ciliophora, Oxytrichidae), from the uplands of Colfiorito, Italy.

New Species of the Fern Genus Lindsaea (Lindsaeaceae) from New Guinea with Notes on the Phylogeny of L. sect. Synaphlebium.

Synthesis and conformational study of sequential polypeptides, (L-ala-L-val-gly)n and (L-val-L-ala-gly)n.

Morphology and molecular analysis of Paratylenchus nanjingensis n. sp. (Nematoda: Paratylenchinae) from the rhizosphere soil of Pinus massoniana in China.

Standard Thermodynamic Functions of Tripeptides N-Formyl-l-methionyl-l-leucyl-l-phenylalaninol and N-Formyl-l-methionyl-l-leucyl-l-phenylalanine Methyl Ester.

Podocoryna loyola, n. sp. (Hydrozoa, Hydractiniidae): a probably introduced species on artificial substrate from southern Brazil.

Effects of L-beta-N-methylamino-L-alanine (L-BMAA) on the cortical cholinergic and glutamatergic systems of the rat.

Morphology and morphometrics of Heterodorus qinghaiensis n. sp. (Dorylaimida, Nordiidae) from soil samples in China.

Description of Myxobolus balatonicus n. sp. (Myxozoa: Myxobolidae) from the common carp Cyprinus carpio L. in Lake Balaton.

Morphology and Molecular Phylogeny of Pseudocyrtohymena koreana n. g., n. sp. and Antarctic Neokeronopsis asiatica Foissner et al., 2010 (Ciliophora, Sporadotrichida), with a Brief Discussion of the Cyrtohymena Undulating Membranes Pattern.

Synthesis, crystal structure, and molecular conformation of N-Ac-dehydro-Phe-L-Val-L-Val-OCH3.

Synthesis, crystal structure, and molecular conformation of N-Boc-L-Phe-dehydro-Leu-L-Val-OCH3.

Enzymatic synthesis and characterization of N(5)-(carboxymethyl)-L-ornithine and N (6)-(carboxymethyl)-L-lysine.

Bozasella gracilis n. sp. (Ciliophora, Entodiniomorphida) from Asian elephant and phylogenetic analysis of entodiniomorphids and vestibuliferids.