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.

Published by the International Society of Protistologists

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

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. Hongbo Pana, Juxiang Hub,c, Jiamei Jianga, Liqing Wanga & Xiaozhong Hud a College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China b Key Laboratory of Ecological Impacts of Hydraulic-Projects and Restoration of Aquatic Ecosystem of Ministry of Water Resources, Wuhan 430079, China c Institute of Hydroecology, Ministry of Water Resources and Chinese Academy of Sciences, Wuhan 430079, China d Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China

Keywords Infraciliature; Pleuronema marinum; psammon ciliates; scuticociliate; SSU rRNA gene. Correspondence X. Hu, Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China Telephone number/FAX number: +86-53282031610; e-mail: [email protected] Received: 26 July 2015; revised 22 September 2015; accepted October 5, 2015. doi:10.1111/jeu.12277

ABSTRACT The morphology and phylogeny of Pleuronema binucleatum n. sp., P. parawiackowskii n. sp., and P. marinum Dujardin 1841, collected from Hangzhou Bay estuary, China, were investigated using standard methods. Pleuronema binucleatum n. sp. can be identified by possessing about 90–120 9 35–50 lm cell size in vivo, reniform body outline, two macronuclei, six to eight preoral kineties, 32–41 somatic kineties, and posterior end of the anterior fragment of membranelle 2 (M2a) hook-like. Pleuronema parawiackowskii n. sp. is characterized by the combination of the following characters: body size about 60–90 9 20–40 lm in vivo, elliptical in outline, four to eight preoral kineties, 20–29 somatic kineties, posterior portion of the M2a slightly curved but nonhooked, and single macronucleus sausage-like. After comparison with other populations of P. marinum, it is suggested that many misidentifications exist in previous studies. And an improved diagnosis of P. marinum was supplied: cell about 95–180 lm long, elliptical in outline; 2–4 preoral kineties and 53–70 somatic kineties; both membranelle 1 and membranelle 3 three-rowed; posterior end of the M2a straight; single contractile vacuole characteristically positioned near mid-body. The small subunit rRNA genes of three forms were sequenced. Phylogenetic analyses indicate that the monophyly of the genus Pleuronema is still not supported.

PLEURONEMA, characterized, interalia, by a conspicuous, sail-like paroral membrane, is a well-known genus of scuticociliates. It is ubiquitous and plays an important role in the microbial food web of the interstitial habitats (Bulit et al. 2003; Cleven and Koenigs 2007; Kalinowska 2008; Madoni 2006). To date, over 20 species have been reported. In previous studies, some morphological characters were proved to be critical and could be relied on for species identification, viz., the structure of membranelle 2 and 3, the position of the contractile vacuole, and the numbers of somatic kineties and preoral kineties (Drais 1986; Pan et al. 2010). Ten gesco and Dragesco-Kerne new species were added to the genus just in the past decade, which might suggest that the species diversity of

Pleuronema is underestimated (Pan et al. 2015a,b; Wang et al. 2008a,b, 2009); besides that, recent molecular phylogenetic work pointed out that some well-known species are complex and need to be re-studied (Gao et al. 2013, 2014). Diversity of marine ciliates in China was investigated mainly in the Yellow and Bohai Seas in the last 20 yr (Luo et al. 2014; Qu et al. 2015; Shao et al. 2014; Song et al. 2009; Yan et al. 2014, 2015). Recently, a faunistic survey of ciliates was conducted in Yangtze Estuary and Hangzhou Bay, where the diversity of ciliates is never known before. Three Pleuronema species were isolated in the region. Two of them were found to be new and another represented the well-known P. marinum. In addition, the

© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2016, 63, 287–298

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Three Pleuronema spp. from Hangzhou Bay, China

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The three species were all collected from the Hangzhou Bay estuary during the ebb tide. Pleuronema marinum was collected on 20 April, 2012 from an intertidal area of Dayangshan Island (30°350 33.8″N, 122°040 59.1″E), with the water temperature 19.5 °C and the salinity 6.8&. Pleuronema binucleatum n. sp. and P. parawiackowskii n. sp. were both sampled from a sand beach of Gouqi Island (30°430 13.5″N, 122°470 33.5″E). The former collected on 25 September, 2014, with the water temperature 14.3 °C, the salinity 13.4&, pH 8.46 and the dissolved oxygen 5.12 mg/L and the latter was collected on 24 April 2015, with the water temperature 16.3 °C, the salinity 20.2&, pH 8.32 and the dissolved oxygen 5.11 mg/L. Isolated specimens were maintained in the laboratory for about 1 wk as raw cultures in Petri dishes for live observation and silver staining. Live cells were observed at 100–1,000X magnifications with bright field and differential interference contrast microscopy (Olympus BX53, Tokyo, Japan). To reveal the infraciliature, the protargol method was employed (Wilbert 1975). Measurements were performed at 200–1,000X magnification, and drawings of stained specimens were conducted with the help of a camera lucida. Terminology and systematics are according to Wang et al. (2008a) and Lynn (2008), respectively.

armatalis and Uronema elegans were selected as outgroup species. Sequences were aligned using the GUIDANCE algorithm (Penn et al. 2010) with the default parameters in GUIDANCE web server (http://guidance.tau.ac.il/ver2/). Ambiguous columns in the alignment were removed based on confidence scores calculated by GUIDANCE. The final alignment, including 1,728 sites and 48 taxa, was used to construct phylogenetic trees. Maximum-likelihood (ML) analysis was carried out using RAxML-HPC2 on XSEDE v7.3.2 (Stamatakis 2006; Stamatakis et al. 2008) on CIPRES Science Gateway (Miller et al. 2010) using GTR + G + I models as selected by Modeltest v.3.4 (Posada and Crandall 1998). Support for the best ML tree came from 1,000 bootstrap replicates. The Bayesian inference (BI) analysis was performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003) on CIPRES Science Gateway with the same model as selected by AIC criterion in MrModeltest v.2.0 (Nylander 2004). The BI analysis was run with two sets of four chains for 1,000,000 generations and a sampling frequency of 100 generations. The first 25% of sampled trees were discarded as burn-in prior to constructing. The monophyly of the genus Pleuronema was compared to competing phylogenetic hypotheses using the approximately unbiased (AU) test (Shimodaira 2002). A constrained ML tree enforcing the monophyly of Pleuronema was generated based on SSU rDNA alignment. Internal relationships within the constrained group and among the remaining taxa were unspecified. The site-wise likelihoods for the resulting constrained topology and the nonconstrained ML topology were calculated using PAUP (Swofford 2003) and were then analyzed in CONSEL (Shimodaira and Hasegawa 2001) to obtain p-values.

DNA amplification and sequencing

RESULTS AND DISCUSSIONS

Genomic DNA was extracted from cells using the DNeasy Blood & Tissue kit (Qiagen Inc., Valencia, CA) following the manufacturer’s instructions, with the modification that one-tenth of the volume suggested for each reagent solution was used (Gao and Katz 2014). A fragment of approximately 1,700 bp comprising part of SSU rDNA was amplified through nested PCR using universal forward and reverse primers (Medlin et al. 1988). Cycling parameters of PCR was: 5 min initial denaturation (94 °C), 30 cycles of 1 min at 94 °C, 1 min at 58 °C, and 2 min at 72 °C, with a final extension of 7 min at 72 °C. The fragment was cloned into the pMDTM18-T vector (Takara Biotechnology, Dalian Co., Ltd., Dalian, China) and transformed into the competent Escherichia coli strain. Both strands of clones were sequenced on an ABI-PRISM 3730 automatic sequencer (Applied Biosystems, Foster City, CA).

Pleuronema binucleatum n. sp.

phylogeny of these three species was discussed based on the analyses of the small subunit rRNA (SSU rRNA) gene sequences. MATERIALS AND METHODS Sample collection and identification

Phylogenetic analyses The SSU rDNA sequences of 45 other ciliates obtained from the NCBI GenBank database were used in addition to the newly characterized sequences of three pleuronematids (accession numbers refer to Fig. 6). Philasterides

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Description. Cells in vivo about 90–120 9 35–50 lm, body reniform in outline with anterior end broadly rounded and posterior end somewhat tapered (Table 1 and Fig. 1A, 2A–E). Dorsoventrally flattened about 3:2 with ventral side concave and dorsal side convex. Buccal field occupying about 65% of body length and 44% of body width. Cytoplasm grayish, containing numerous granules (mostly 4 lm across) at anterior 25% of body length, which renders this part dark and opaque (Fig. 2A, C, D). Usually, two spherical macronuclei centrally located in anterior part (Fig. 1B, 2F); of 67 individuals, however, three possessing single larger, elliptical macronucleus and five having another two smaller macronuclei. Single contractile vacuole, ca. 15–20 lm across, subterminally positioned near dorsal side of cell (Fig. 2C). Cilia of both paroral membrane and membranelles about 20 lm long. Somatic cilia usually about 10 lm long; 16–22 caudal cilia 25 lm long, always projecting radially from the cell (Fig. 2G). Movement moderately fast, rotating around main body axis, and sometimes motionless for short periods.



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Figure 1 Pleuronema binucleatum n. sp. from life (A) and after protargol impregnation (B–E). A. Ventral view of a representative individual. B. Ventral view to show two macronuclei and oral apparatus. C. Details of oral apparatus. D, E. Ventral and dorsal view to show the infraciliature. M1–3 = membranelle 1–3; M2a = anterior part of membranelle 2; M2b = posterior part of membranelle 2; PM = paroral membrane. Bars: 50 lm.

About 32–41 somatic kineties, each composed of dikinetids in anterior 2/3 and monokinetids in the rest, extending over entire length of cell to form a small, bald apical plate at anterior end of cell (Fig. 1D, E). Somatic kinety 1 (SK1) comprised of about 60 kinetosomes. Six to eight preoral kineties to left of buccal field (Fig. 2J), and one or two postoral kineties behind buccal field (Fig. 2K, L). Oral apparatus stable within population: membranelle 1 (M1) consisting of one short and two long rows of kinetosomes (Fig. 1C, 2H, I); membranelle 2 bipartite, anterior one (M2a) linear, two-rowed in anterior and posterior portions and single-rowed in the middle, and its posterior end hook-like (Fig. 1C, 2I); posterior one (M2b) V-shaped, with two rows of kinetosomes arranged in zig-zag pattern (Fig. 1C, 2J, L); membranelle 3 (M3) three-rowed (Fig. 1C, D, 2J, L); paroral membrane (PM) occupying about 60% of body length (Fig. 1C). SSU rRNA gene sequence. The SSU rRNA gene sequence of P. binucleatum n. sp. has been deposited in the GenBank database with the accession number, length and G + C content as follows: KT033424, 1,762 bp, 42.96%. Comments on P. binucleatum n. sp. Regarding the structure of oral apparatus, body size and shape, and the number of somatic kineties, seven species should be compared, namely, Pleuronema coronatum Kent, 1881, P. borrori Dragesco 1968; P. balli Small and Lynn, 1985, P. salmastra Dragesco, 1986, P. arctica Agatha, 1993, P. lynni Fernandez-Leborans and Novillo 1994; and P. glaciale Corliss and Snyder 1986. Pleuronema binucleatum n. sp. resembles P. coronatum most, especially in terms of the number of somatic kin-

eties. However, the former can be separated by having two macronuclei (vs. one in P. coronatum), more kinetosomes in SK1 (about 60 vs. 21–30), and more preoral kineties (6–8 vs. 3–6; Song 2000). Furthermore, its SSU rRNA gene sequence is different from that of P. coronatum by 54–58 bp (Gao et al. 2013). Wang et al. (2008a) described two populations of P. coronatum, some individuals of which had two macronuclei. Meanwhile, we rechecked the slides, and found that their May population comprises specimens with the same numbers of the preoral kineties and the somatic kineties as the new species. Therefore, it is likely that the May population is a complex including P. binucleatum n. sp. Concerning recent molecular work pointed out that P. coronatum is a complex (Gao et al. 2013), it is necessary to compare P. borrori and P. balli with the new species separately, although they were treated as junior synonym of P. coronatum by Wang et al. (2008a). Different from P. borrori, P. binucleatum has more preoral kineties (6–8 vs. 2–6), fewer somatic kineties (32–41 vs. 41–46), and mostly two macronuclei (vs. one). Compared with P. balli, the new species can be identified by possessing fewer somatic kineties (32–41 vs. 39–45) and two macronuclei (vs. one; Dragesco 1968). Pleuronema glaciale and P. salmastra, can be distinguished from the new species by the number of somatic kineties (44–58 in P. glaciale, 43–63 in P. salmastra vs. 32–41; Corliss and Snyder 1986; Dragesco and Dragescois 1986). And the structure of M2a of P. glaciale is Kerne different from that of P. binucleatum (completely tworowed vs. only anteriorly and posteriorly two-rowed), and the length of M2a is shorter compared to M1 in the former (Corliss and Snyder 1986).


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Figure 2 Pleuronema binucleatum n. sp. from life (A–G) and after protargol impregnation (H–L). A. Ventral view of a typical individual. B–E. Shape variants among different individuals, arrow (C) points to the contractile vacuole. F. Details of the cytoplasm. G. Posterior portion, arrows mark caudal cilia. H. Anterior of the buccal field. I. Anterior portion of the oral apparatus. J. Posterior portion of the oral apparatus, arrows point to preoral kineties. K, L. Posterior portion of the two different cells, arrows mark postoral kineties. M1, 3 = membranelle 1, 3; M2a = anterior part of membranelle 2; M2b = posterior part of membranelle 2; Ma = macronucleus. Bars: 50 lm (A–E); 20 lm (F–K); 15 lm (L).

Pleuronema arctica is similar to P. binucleatum n. sp. concerning the number of preoral kineties. However, it is characterized by possessing 39–61 somatic kineties (vs. 32–41 in P. binucleatum n. sp.) and M2a nearly wholly two-rowed (vs. only anteriorly and posteriorly two-rowed; Agatha et al. 1993). Pleuronema lynni differs from P. binucleatum n. sp. by the structure of M2a (entirely two-rowed vs. the anterior and the posterior portions two-rowed while the middle portion single-rowed) and M2b (not V-shaped vs. Vshaped; Fernandez-Leborans and Novillo 1994).

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Pleuronema marinum Dujardin 1841 Description. Cells in vivo about 95–120 9 30–50 lm, elongate elliptical in outline with both ends broadly rounded (Table 1 and Fig. 3A, E, 4A, B). Right and left margins somewhat parallel under low magnification (Fig. 4B). Dorsoventrally flattened about 4:3 with ventral side flat and dorsal side convex (Fig. 3B, 4C). Buccal field occupying about 60% of body length and 38% of body width. Cytoplasm filled with numerous ellipsoidal granules (mostly 6– 8 9 3–5 lm) in anterior 60–75% of body, which renders



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this part black under low magnification (Fig. 3A, B, 4A, B, E). Usually 4–7 macronuclei gathered in center of anterior part; however, in some individuals only one large, globular, or irregularly shaped macronucleus present (Fig. 3D, 4G). Single contractile vacuole characteristically positioned in middle portion of body near dorsal side (Fig. 4E, F). Cilia of both paroral membrane and membranelles about 20 lm long. Somatic cilia about 12 lm long; about 13 caudal cilia 25 lm long, were always projecting radially from cell (Fig. 4I). Movement moderately fast, rotating around the main body axis, and sometimes motionless for short periods. About 53–70 nearly bipolar somatic kineties form a small, bald apical plate at anterior end of cell and a suture on posterior ventral side (Fig. 3F). Somatic kineties composed of dikinetids in anterior 2/3 of the body and monokinetids in the rest. Two to four preoral kineties (Fig. 3F). Details of the oral apparatus shown in Fig. 3C, 4D, H, K: M1 consisting of one very short and two long rows of kinetosomes (Fig. 3C, 4D); M2a, whose anterior and posterior parts are two-rowed while the middle part is singlerowed, almost straight with posterior end somewhat curved to the right; M2b V-shaped with kinetosomes arranged in zig-zag pattern (Fig. 4K); M3 three-rowed; PM about 60% of body length.

SSU rRNA gene sequence. The SSU rRNA gene sequence of Pleuronema marinum has been deposited in the GenBank database with the accession number, length and G + C content as follows: KF206428, 1,678 bp, 42.61%. Comments on Pleuronema marinum. In original description, Dujardin (1841) only described the elongate shape of this species and supplied a superficial illustration. Subsequently, Kahl (1931, 1933) described it in detail from live cells, and he emphasized that the position of its contractile vacuole was in the middle of the dorsal side, which was considered as a diagnostic feature of this species by him. Our population corresponds with previous descriptions very well in the body shape and size as well as the position of contractile vacuole, and thus they must be conspecific although our form has a more variable number of macronuclei (1–7 vs. 1). Two American populations of P. marinum were described by Noland (1937) and Borror (1963). Different from Kahl’s and our populations, however, these two isolates have a posteriorly located contractile vacuole and fewer somatic kineties (50, 40–55 vs. 53–70 in the present form). Therefore, they are likely misidentifications. Dragesco (1968) identified a French isolate of P. marinum, the infraciliature of which is similar to ours. How-

Figure 3 Pleuronema marinum from life (A, B, E) and after protargol impregnation (C, D, F, G). A. Ventral view of a representative individual, arrowheads point to caudal cilia. B. Lateral view. C. Details of the oral apparatus. D. Macronuclei from different individuals. E. Shape variants. F, G. Ventral and dorsal view to show infraciliature, arrowhead indicates the contractile vacuole pore. M1, 3 = membranelle 1, 3; M2a = anterior part of membranelle 2; M2b = posterior part of membranelle 2; PM = paroral membrane. Bars: 40 lm.


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Figure 4 Pleuroneam marinum from life (A–C, E, F, I) and after protargol impregnation (D, G, H, J, K). A, B. Ventral view. C. Lateral view, arrowhead marks the macronucleus. D. Anterior portion of the buccal field. E, F. Dorsal view, arrowheads indicate the contractile vacuole. G. Variants of macronuclei. H. Middle part of the oral apparatus. I. Posterior end of the cell, arrowheads point to caudal cilia. J. To show the contractile vacuole pore (arrowhead). K. Posterior portion of the oral apparatus. M1, 3 = membranelle 1, 3; M2a = anterior part of membranelle 2; M2b = posterior part of membranelle 2; PM = paroral membrane. Bars = 40 lm (A–C, E, F); 15 lm (D, H–K).

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Table 1. Morphometric data of Pleuronema binucleatum n. sp. (first line), P. marinum (second line) and P. parawiackowskii n. sp. (third line) Characters

Min

Max

Mean

Median

Body length

91 89 64 39 29 30 62 60 57 16 10 15 32 53 23 6 2 6 0 0 0 3 3 3 1 1 1

137 143 96 75 56 55 95 95 78 33 22 26 41 70 29 8 4 8 2 0 0 3 3 3 11 7 1

116.4 113.6 79.0 56.1 42.1 40.7 80.2 72.4 67.8 24.6 15.9 21.4 36.1 59.0 25.0 6.6 3.5 7.3 1.0 0 0 3.0 3.0 3.0 2.2 4.9 1.0

115 116 80 55 41 41 80 70 69 25 16 22 36 58 25 7 3.5 7 1 0 0 3 3 3 2 5 1

Body width

Length of buccal field Width of buccal field No. of SK

No. of PK

No. of PoK

No. of kinetosome rows in M3 No. of Ma

SD

CV

n

10.00 14.23 9.64 8.20 5.72 7.19 6.16 8.41 3.79 2.91 2.31 2.83 2.46 4.27 1.33 0.74 0.60 0.65 0.59 0 0 0 0 0 1.16 2.15 0

8.6 12.5 12.2 14.6 13.6 17.7 7.7 11.6 10.0 11.8 14.5 13.2 6.8 7.2 5.3 11.2 17.1 8.9 59.0 0 0 0 0 0 52.7 43.9 0

67 23 19 67 23 19 65 23 19 65 22 19 67 23 19 67 22 19 67 23 19 36 23 19 67 23 12

All data are based on protargol-impregnated specimens. Measurements in lm. CV = coefficient of variation in %; M3 = membranelle 3; Ma = macronucleus; Max = maximum; Mean = arithmetic mean; Min = minimum; n = number of specimens investigated; No. = number; PK = preoral kineties; PoK = postoral kineties; SD = standard deviation of the mean; SK = somatic kineties.

ever, the contractile vacuole of his population was subterminally positioned, which was different from Kahl’s form (e.g. positioned in the middle of the body). Regarding the position of the contractile vacuole is very stable in scuticociliates (de Castro et al. 2014; Fan et al. 2011, 2014; Foissner et al. 1994, 2002, 2014), his population is also misidentified. Thereafter, Pleuronema marinum are reported on several occasions (Agamaliev 1971; Aladro-Lubel 1984; Alekperov and Asadullayeva 1999; Burkovsky 1970). However, the identifications of all the forms in those studies were based on the descriptions of Borror (1963) or Dragesco (1968), and none of them has a mid-body positioned contractile vacuole (Table 2). Therefore, they cannot be P. marinum. To date, eight congeners have a straight posterior end of M2a as in P. marinum. However, none of them has a mid-body positioned contractile vacuole (vs. present in P. marinum). With respect to the general morphology, four species should be compared with P. marinum, namely P. arenicola Dragesco 1960; P. oculata Dragesco 1960; P. sinica Wang et al. 2009; and P. tardum Czapik and Jordan 1977. Pleuronema arenicola can be clearly distinguished by M3 two-rowed (vs. three-rowed; Dragesco 1960). Pleuronema oculata differs from P. marinum by the shape of M2b (straight vs. V-shaped; Dragesco 1960). With respect to the body shape, Pleuronema sinica is very similar to P. marinum. However, it can be separated from the latter by possessing fewer somatic kineties (40– 49 vs. 53–70), and a different appearance of M2b (the right branch is twice length of the left branch vs. the right branch is one and a half length of the left branch; Wang et al. 2009). In addition, P. sinica differs from P. marinum by 47 bp in the SSU rRNA gene sequences. Our population of Pleuronema marinum resembles P. tardum in possessing several macronuclei. But, it can be identified by more somatic kineties (53–70 vs. 40–50),

Table 2. Comparison of different forms under the name Pleuronema marinum Source

Length (lm)

Shape

Position of CV

No. of SK

No. of PK

No. of Ma

Present work Dujardin (1841) Kahl (1931) Kahl (1933) Noland (1937)a Borror (1963)a Dragesco (1968)a Aladro-Lubel (1984)a Alekperov and Asadullayeva (1999)a Burkovsky (1970)a Agamaliev (1971)a

95–120 100 120–180 100–180 51–126 74–170 95–141 136.5–155.5 60–75 90–130 80

Elongated Elongated Slim kidney shaped

Middle of dorsal side – Middle Middle Subterminal Subterminal Subterminal Terminal – Terminal –

53–70 – – – ca. 50 40–55b 58–66 – 35–38 32–36 35–38

2–4 – – – – 4 1–2 – – 2 2

1 to several – 1 1 – 1 1 1 1 – 7–10

Oval Oval – Elongated Oval Elongated Elongated

CV = contractile vacuole; Ma = macronucleus; PK = preoral kineties; SK = somatic kineties. –, data not available. a Misidentification. b Data from the illustration.


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Figure 5 Pleuronema parawiackowskii n. sp. from life (A, C, G–I, K) and after protargol impregnation (B, D–F, J, L–O). A. Ventral view of a typical individual. B. Macronuclei from different individuals. C. Ventral view of a slender individual. D, E. Ventral and dorsal view to show infraciliature. F. Details of the oral apparatus, arrow shows posterior end of M2a. G, H, K. Ventral view of different individuals, arrowheads point to caudal cilia, arrows mark the paroral membrane. I. Lateral view. J. Anterior portion of the oral apparatus. L. Posterior part, arrowheads indicate preoral kineties. M, N. Macronucleus. O. Part of the oral apparatus, arrow points to posterior end of M2a. M1, 3 = membranelle 1, 3; M2a = anterior part of membranelle 2; M2b = posterior part of membranelle 2; PM = paroral membrane. Bars = 30 lm (A, C–E, J–I, K); 20 lm (J, L, O).

structure of M1 (three-rowed vs. two-rowed), and the appearance of M2b (the right branch is triple the length of the left one vs. the right branch is one and a half length of the left branch; Czapik and Jordan 1977).

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Pleuronema parawiackowskii n. sp. Description. Cell size ca. 65–90 9 25–40 lm in vivo, and body elliptical in outline with both ends blunt rounded



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(Table 1 and Fig. 5A, C, G, H, K). Dorsoventrally compressed about 2:1 with ventral side flat and dorsal side convex (Fig. 5I). Buccal field very large, occupying ca. 85% of body length and about half of body width. Cytoplasm slightly grayish and containing many greasily shining globules in the anterior portion (Fig. 5A, G, K). Extrusomes 4 lm long, densely arranged beneath the pellicle. Single macronucleus sausage-shaped (Fig. 5B, M, N). One contractile vacuole subterminally positioned near dorsal side (Fig. 5A, C). Cilia of both paroral membrane membranelles ca. 40 lm long, forming a conspicuous sail-like structure (Fig. 5G, H, I). Somatic cilia ca. 12 lm long. About 15 caudal cilia, 50 lm long, extending radially from cell. When swimming, cells rotating around the main body axis, and sometimes motionless for short periods. About 23–29 somatic kineties, each composed of dikinetids in anterior three quarters of body length and monokinetids in posterior portion (Fig. 5D, E). Usually, 6–8 preoral kineties (Fig. 5L). Oral apparatus typical of genus (Fig. 5D, F, J, O): M1 about 1/3 of M2a in length, composed of three kineties, outmost of which is extremely short and composed of no more than eight kinetosomes (Fig. 5F, J); M2a composed of two-rowed kinetosomes except for one short middle section, which is single-rowed, and its posterior part bending to the inside of the buccal field but not to form a hook-shaped structure (Fig. 5D, F, J, O); M2b V-shaped; M3 consisting of three-rowed kinetosomes; PM occupying about 80% of the cell length. SSU rRNA gene sequence. The SSU rRNA gene sequence of Pleuronema parawiackowskii n. sp. has been deposited in the GenBank database with the accession number, length and G + C content as follows: KT033423, 1,762 bp, 42.17%. Comments on Pleuronema parawiackowskii n. sp. In terms of general morphology, Pleuronema parawiackowskii n. sp. should be compared with at least four species, P. wiackowskii Wang et al., 2008, P. arenicola Dragesco 1960; P. marinum Dujardin 1841; and P. oculata Dragesco 1960. Pleuronema parawiackowskii n. sp. resembles P. wiackowskii most in the shapes of the body and the macronucleus (Wang et al. 2008b). However, it can be distinguished from the latter by smaller size in vivo (65– 90 lm long vs. 95–120 lm long), fewer somatic kineties (23–29 vs. 27–35) and more preoral kineties (6–8 vs. 3–5). In addition, the posterior portion of M2a in P. parawiackowskii n. sp. bends to the inside of the buccal field (vs. straight). Therefore, we conclude that P. parawiackowskii n. sp. should be a distinct form. SSU rRNA gene sequence of Pleuronema wiackowskii in the Genbank database is only different from the new species by 5 bp. According to its definition and our personal communication, the sequence is from the population described by Zhao et al. (2011), instead of the original one. However, it is nearly the same as P. parawiackowskii n. sp. in all morphological details, especially the numbers of somatic kineties and preoral kineties and the detailed structure of M2a. Therefore, P. wiackowskii sensu Zhao

et al. 2011 must be conspecific with P. parawiackowskii n. sp. Although the description of Pleuronema arenicola is not sufficient, P. parawiackowskii n. sp. can be separated from it by M3 three rowed (vs. two-rowed) and possessing more preoral kineties (6–8 vs. 3; Dragesco 1960). Pleuronema parawiackowskii n. sp. can be easily distinguished from P. marinum by possessing more preoral kineties (6–8 vs. 2–4), fewer somatic kineties (23–29 vs. 53– 70), the shape of macronucleus (sausage-shaped vs. spherical), and the position of contractile vacuole (subterminal vs. central; Kahl 1931, 1933). Compared with Pleuronema oculata, the new species differs by possessing fewer somatic kineties (23–29 vs. 50), the shape of M2b (V-shaped vs. straight) and the shape of macronucleus (sausage-shaped vs. spherical; Dragesco 1960). Phylogenetic analyses Phylogenetic trees inferred from the SSU rRNA gene sequences using BI and ML analyses generate similar topologies, and therefore a single topology (ML tree) is presented here with support values from both algorithms (Fig. 6). The phylogenetic relationships of pleuronematids has recently been examined using SSU rDNA, ITS1-5.8SITS2, and LSU rDNA gene sequences (Gao et al. 2014; Pan et al. 2015a,b). The present analyses reveal a similar relationship among families within the order Pleuronematida, for example, the paraphyly of the order Pleuronematida, and the monophyly of the families Pleuronematidae, Histiobalantiidae, Eurystomateliidae, and Ctedoctematidae. With the addition of three new sequence data, Pleuronema is still not monophyletic in ML tree with a rather low support value (25%), with Schizocalyptra spp. nested within it; and it clustered into two clades which formed a soft polytomy with the genus Schizocalyptra in the BI tree. Nevertheless, the hypothesis that the genus Pleuronema is monophyletic is not rejected by the AU test (Table 3, p = 0.795 > 0.05). Three newly sequenced species fall well in the genus Pleuronema. Two populations of P. parawiackowskii n. sp., one of which is misidentified as P. wiackowskii (as discussed above), group together, forming a sister to P. czapikae, and then cluster with P. sp. 1 with full support. Pleuronema binucleatum n. sp. shows a close relationship with P. elegans and P. coronatum isolate 2 (JX310014) (91% ML, 1.00 BI) while P. marinum groups with Pleuronema sp. 2 (FJ848876), branching sister to P. sinica with full support. Table 3. Approximately unbiased (AU) test results Topology constraints

ln likelihood

AU value (p)

Unconstrained Pleuronema monophyletic

13,252.18038 13,253.25358

0.834 0.795

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


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Figure 6 Maximum-likelihood (ML) tree inferred from the small subunit ribosomal RNA (SSU rRNA) gene sequences, showing the positions of Pleuronema binucleatum n. sp., P. marinum, and P. parawiackowskii n. sp. (in bold). Numbers at nodes represent the bootstrap values of maximum likelihood (ML) of 1,000 replicates and the posterior probability of Bayesian analysis (BI). Fully supported (100%/1.00) branches are marked with solid circles. Hyphen (-) refers to the disagreement between ML and BI. The asterisk indicates this population of Pleuronema wiackowskii is designated as P. parawiackowskii n. sp. The scale bar corresponds to five substitutions per 100 nucleotide positions.

TAXONOMIC SUMMARY Subclass Scuticociliatia Small, 1967 -Fremiet in Corliss, 1956 Order Pleuronematida Faure Family Pleuronematidae Kent, 1881 Genus Pleuronema Dujardin, 1841 Pleuronema binucleatum n. sp. Diagnosis. Cells about 90–120 9 35–50 lm in vivo, oval or reniform in outline; 16–22 prolonged caudal cilia; buccal area occupying 65% of body length; 6–8 preoral kineties, and 32–41 somatic kineties; M1 three-rowed; posterior end of the anterior fragment of membranelle 2 (M2a) hook-like; M3 three-rowed; single contractile vac-

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uole subterminally positioned near dorsal side; usually two spherical macronuclei. Type locality. An intertidal sand beach in Gouqi Island (30°430 13.5″N,122°470 33.5″E), China. Type materials. One protargol slide with the holotype specimen (marked by a black ink circle) was deposited in the National History Museum, London, with registration number NHMUK 2015.7.13.1. Another slide with paratype specimens was deposited in the Laboratory of Protozoology, Ocean University of China, with registration number of PHB 2015042406-1. Etymology. The specific epithet “binucleatum” is derived by adding the Latin prefix “bi-” (meaning “two”) to the Latin word “nucleatus” (meaning “having kernel”), in recognition that this species usually possesses two macronuclei.



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Pleuronema marinum Dujardin 1841

LITERATURE CITED

Remarks. Pleuronema marinum is a large form in the genus. However, after comparison with other populations (see “Results and discussions” section), we find it is still a confused species and a lot of misidentifications exist. Therefore, we supply an improved diagnosis here. Improved diagnosis. Cell about 95–180 lm long in vivo, elongated or elliptical in outline, dorsoventrally flattened about 4:3; ca. 13 prolonged caudal cilia; buccal area occupying 60% of body length; 2–4 preoral kineties and 53–70 somatic kineties; membranelle 1 three-rowed; posterior end of the anterior fragment of membranelle 2 (M2a) straight; membranelle 3 three-rowed; contractile vacuole characteristically mid-body positioned; one or several spherical macronuclei. Deposition of voucher slides. One voucher slide with protargol stained specimens was deposited in the Natural History Museum, London, with registration number NHMUK 2015.7.13.3.

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Pleuronema parawiackowskii n. sp. Syn. Pleuronema wiackowskii sensu Zhao et al. 2011 Diagnosis. Cell about 60–90 9 20–40 lm in vivo with elliptical body shape in outline; buccal area about 85% of body length; 15 prolonged caudal cilia; four to eight preoral kineties, and 20–29 somatic kineties; posterior portion of M2a slightly curved with the end not hooked; M3 threerowed; single contractile vacuole subterminally positioned near dorsal margin; one sausage-shaped macronucleus. Type locality. An intertidal sand beach in Gouqi Island (30°430 13.5″N,122°470 33.5″E), China. Type materials. A protargol slide with the holotype specimen (marked by a black ink circle) was deposited in the Natural History Museum, London, with registration number NHMUK 2015.7.13.2. Another slide with paratype specimens was deposited in the Laboratory of Protozoology, Ocean University of China, with registration numbers of PHB 2014092501-1. Etymology. The specific epithet “parawiackowskii” is derived by adding the Greek prefix “para-” (meaning “similar to, resembling”) to the epithet “wiackowskii”, in recognition that this species resembles P. wiackowskii in morphology. ACKNOWLEDGMENT This study was supported by the National Natural Science Foundation of China (project numbers: 51279112 to J. Hu, 31201703 to H. Pan, 41206123 to J. Jiang, and 41376141 to X. Hu), Innovation Program of Shanghai Municipal Education Commission (No. 13YZ095), the PhD Start-up Fund of Shanghai Ocean University, and the Excellent Young Teachers Support Program of Shanghai. We are grateful to Zhishuai Qu, a PhD student in OUC, for checking the specimens of Pleuronema wiackowskii sensu Zhao et al. 2011. We also thank Mr. Hao Kong, a graduate student in Shanghai Ocean University, for sampling.


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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.

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

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

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.

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

Taxonomy and phylogeny of two species of the genus Deviata (Protista, Ciliophora) from China, with description of a new soil form, Deviata parabacilliformis sp. nov.

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

Morphology and phylogeny of three trachelocercid ciliates, with description of a new species, Trachelocerca orientalis spec. nov. (Ciliophora, Karyorelictea).

Morphology and phylogeny of two species of Loxodes (Ciliophora, Karyorelictea), with description of a new subspecies, Loxodes striatus orientalis subsp. n.

Pythium rishiriense sp. nov. from water and P. alternatum sp. nov. from soil, two new species from Japan.

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

Description of Paracrobeles deserticola sp. n. and Nothacrobeles hebetocaudatus sp. n. (Nematoda: Rhabditida: Cephalobidae) from Iran and the phylogenetic relationships of these two species.

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

Diversity of Phytophthora Species from Declining Mediterranean Maquis Vegetation, including Two New Species, Phytophthora crassamura and P. ornamentata sp. nov.

Lithobius (Monotarsobius) zhangi sp. n., a new species from Eastern China (Chilopoda, Lithobiomorpha, Lithobiidae).

Pseudomacrochenus wusuae sp. n., a new species from Sichuan, China (Coleoptera, Cerambycidae, Lamiinae).

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).

Parasitic copepods from two species of commercial fishes collected off Iraq, with description of Hatschekia shari n. sp.

Kakunataibaiensis sp. n. and a newly recorded species of Dicranotropis (Hemiptera, Fulgoroidea, Delphacidae) from China.

Two new species of Echinoderes (Kinorhyncha, Cyclorhagida), E. romanoi sp. n. and E. joyceae sp. n., from the Gulf of Mexico.

Description of a new tick species, Ixodes collaris n. sp. (Acari: Ixodidae), from bats (Chiroptera: Hipposideridae, Rhinolophidae) in Vietnam.

Morphology and phylogenetic analysis of two oxytrichid soil ciliates from China, Oxytricha paragranulifera n. sp. and Oxytricha granulifera Foissner and Adam, 1983 (Protista, Ciliophora, Hypotrichia).

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

A new genus and species (Cornucollis gen. n. masoalensis sp. n.) of praying mantis from northern Madagascar (Mantodea, Iridopterygidae, Tropidomantinae).