Morphology and Phylogeny of Three Trachelocercid Ciliates, with Description of a New Species, Trachelocerca Orientalis Spec

Journal of Eukaryotic Microbiology ISSN 1066-5234

1 REVIEW ARTICLE 2 3 Morphology and Phylogeny of Three Trachelocercid Ciliates, 4 5 with Description of a New Species, Trachelocerca orientalis 6 spec. nov. (Ciliophora, Karyorelictea) 7 8 9 10 Ying Yana, Feng Gaoa, Yuan Xub, Khaled A. S. Al-Rasheidc & Weibo Songa 11 12 a Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao, 266003, China 13 b State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, 200062, China 14 c Zoology Department, King Saud University, Riyadh, 11451, Saudi Arabia 15 16 17 18 19 Keywords ABSTRACT 20 Marine ciliates; SSU rRNA gene; 21 trachelocercids. Three trachelocercid ciliates, Trachelocerca orientalis spec. nov., Prototrache- 22 locerca fasciolata (Sauerbrey, 1928) Foissner, 1996 and Tracheloraphis huangi 23 Correspondence Xu et al., 2011, isolated from marine coastal habitats at Qingdao, China, were 24 F. Gao, Laboratory of Protozoology, Institute taxonomically studied using observation in vivo and silver staining methods. 25 of Evolution & Marine Biodiversity, Ocean The new species T. orientalis spec. nov. can be recognized by the combination 26 University of China, Qingdao 266003, China of its size (600–1,200 lm in vivo), 15–21 somatic kineties and about 13 groups 27 Telephone number: +86 532 8203 1676; of macronuclear nodules forming a strand and the colorless globular cortical 28 FAX number: +86 532 8203 1676; granules. Together with the sequence data of the small subunit ribosomal 29 e-mail: [email protected] RNA (SSU rRNA) gene, the information of a new isolate of P. fasciolata and 30 three populations of T. huangi is also documented based on the present work. 31 Y. Xu, State Key Laboratory of Estuarine According to the molecular data, the phylogeny of three species is estimated 32 and Coastal Research, East China Normal and the analyses show that they are all found within the trachelocercid assem- 33 University, Shanghai 200062, China blage though T. huangi does not cluster with its congeners but with Tracheloc- 34 Telephone number: +86 21 5434 5473; erca species. Nonetheless, the monophyly of Trachelocerca is not rejected by 35 FAX number: +86 21 5434 5473; the approximately unbiased test (p = 0.345 > 0.05), while that of Trachelora- Dispatch: 5.8.14No. of pages: 10 CE: PE: Manikandan 36 e-mail: [email protected] phis is not confirmed (p = 0.0002 < 0.05). 37 Received: 29 March 2014; revised 2 July 38 2014; accepted July 4, 2014. 39 40 doi:10.1111/jeu.12154 41 42 43 44 THE karyorelictean family Trachelocercidae Kent 1881 In last few years, studies on the fauna of trachelocercids 45 were found commonly all over the world, which play sig- have been performed in China seas (Gao et al. 2010; Xu 46 nificant ecological roles in marine habitats (Al-Rasheid et al. 2011b, 2012, 2013a,b; Yan et al. 2013). In this study, 1 47 1996, 1997, 1998, 2001; Carey 1992; Dragesco and Drage- we investigated three trachelocercids isolated from coastal 48 sco-Kerneis 1986; Foissner 1996). Since the first species area at Qingdao, China, with regard to their live morphol- 49 was described over 200 yr ago, definition and classifica- ogy, ciliature, and SSU rRNA gene based phylogenetic, as 50 tion of members within the family were extremely con- a new contribution, the results are documented here. 51 fused due to the highly similar morphology until more 52 reliable characters (somatic and oral ciliary patterns) were J E U 12154-4527 MATERIALS AND METHODS 53 applied to distinguish them (Dragesco 1960; Foissner and Journal Code Manuscript No. 54 Dragesco 1996a; Kahl 1933). Of more than 70 species Sampling and morphological investigations 55 reported within Trachelocercidae, there is detailed informa- 56 tion for only nine (Alekperov et al. 2007; Andreoli et al. Trachelocerca orientalis spec. nov. was isolated on 8 April 57 2009; Carey 1992; Foissner 1996, 1997a,b; Foissner and 2012 from the intertidal zone in Qingdao, China (36°050N, 58 Al-Rasheid 1999; Foissner and Dragesco 1996a,b; Mazei 120°270E), where the water temperature was 15 °C and 59 et al. 2009; Song et al. 2009; Wilbert 1986). salinity was 14& (Fig. 1C, S1).

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–10 1 Morphology and Phylogeny of Three Trachelocercid Ciliates Yan et al.

1 et al. 2008, 2010; Edgar 2004) using the default parame- 2 ters. The resulting alignment was manually edited using 3 BioEdit 7.0.5.2 (Hall 1999). The final alignment, including 4 1,706 sites and 42 taxa, was used to construct phyloge- 5 COLOR netic trees (The alignment file is available upon request). 6 The program MrModeltest v.2.0 (Nylander 2004) selected 7 the GTR + I + G model as the best model with AIC crite- 8 rion, which was then used for Bayesian inference (BI). 9 The BI analysis was performed using MrBayes v3.1.2 10 (Ronquist and Huelsenbeck 2003) with the chain length of 11 5,000,000 generations and a sampling frequency of 100 12 generations. The first 25% of sampled trees were dis- 13 carded as burn-in. Maximum-likelihood (ML) analysis was 14 carried out online using RAxML-HPC2 on XSEDE (8.0.24) 15 on the CIPRES Science Gateway (Stamatakis 2006; Sta- 3 16 matakis et al. 2008) with the GTRGAMMA model as this 4 Figure 1 Photograph of the sample site, the intertidal zone of Dia- 17 was the best fitting model available in RAxML. Searches osuyuan sandy beach in Qingdao, China. A. Map of China. B, C. Por- 18 for the best tree were conducted starting from 50 random tion of the map of China, showing location of Qingdao. 19 trees. Support for the best ML tree came from 1,000 20 bootstrap replicates (Zhang et al. 2012). 21 Prototrachelocerca fasciolata was collected on 18 April The statistical possibility of the alternative phylogenetic 22 2012 from a sandy beach near Qingdao (35°550N, hypotheses was evaluated using approximately unbiased 23 120°120E) (Fig. 1C, S2), where the water temperature was (AU) tests (Shimodaira 2002). Constrained ML trees com- 24 28°°C and salinity was 13.5&. pelling the monophyly of Trachelocerca and Tracheloraphis 25 Tracheloraphis huangi was collected on 13 June 2012 were generated using the same toolkit as the uncon- 26 from a sandy beach in north Qingdao (36°140N, 120°400E) strained ML trees. The resulting constrained topologies 27 (Fig. 1C, S3), where the water temperature was 27°°C were then compared to the nonconstrained ML topologies 28 and salinity was 25&. using the AU test option implemented in CONSEL v0.1 29 The sampling method was mainly as described in Xu (Shimodaira and Hasegawa 2001). 30 et al. (2013b). Living cells were studied using bright field 31 and differential interference contrast microscopy RESULTS 32 (100–1,000X magnifications) (Chen et al. 2013). The ciliary 33 pattern was revealed by the protargol impregnation Trachelocerca orientalis spec. nov. (Table 1; Fig. 2) 34 method (Wilbert 1975) using the following fixative: 10 ml 35 saturated, aqueous mercuric chloride and 3 ml Bouin’s Fully extended cells are about 600–1,200 9 20–30 lmin 36 solution, mixed just before use (Xu et al. 2011a). Counts vivo, but mostly about 800–1,000 lm in length; flexible 37 and measurements of stained specimens were performed and contractile (Fig. 2A–C, K–N). Posterior end is rounded 38 at a magnification of 1,000X. Drawings were made with while neck is distinctly separated from trunk with a trian- 39 the help of a camera lucida. Terminology is mainly accord- gular head (Fig. 2A, K–N). Cortical granules are globular, 40 ing to Foissner (1996). about 1 lm in diam., and colorless at high magnification 41 (Fig. 2H). The distribution of cortical granules is in stripes 42 between ciliary rows and densely spaced in glabrous zone Phylogenetic analyses 43 (Fig. 2H, P). Cytoplasm is grayish and opaque, and packed 44 Genomic DNA was extracted from cells using DNeasy with ellipsoid and globular granules which are about 1 lm 45 2 Tissue kit (Qiagen, CA) following the manufacturer’s in diam. (Fig. 2G, Q). Seven to 22-nuclear groups, each 46 instructions. PCR amplification of the SSU rRNA gene was consisting of two to four macronuclei, form a distinct 47 performed using the primers Euk A (50-AAC CTG GTT GAT strand in trunk (Fig. 2C, D, G, J, Q, U). Locomotion is by 48 CCT GCC AGT-30) or 82F (50-GAA ACT GCG AAT GGC gliding along bottom of Petri dish. 49 TC-30) and Euk-B (50-TGA TCC TTC TGC AGG TTC ACC The cell surface is ciliated except for the glabrous 50 TAC-30) (Elwood et al. 1985; Medlin et al. 1988). Cloning stripe, which is narrow, occupies approximately the width 51 and sequencing were performed according to Gao et al. of two somatic kineties, and extends the whole body 52 (2013). length in the midline of left side (Fig. 2I, R). The entire in- 53 Other than the newly characterized SSU rRNA gene fraciliature consists of dikinetids. There are ca. 15–21 54 sequences, the sequences used in the present analyses somatic kineties, of which cilia are about 10 lm long and 55 were obtained from the NCBI GenBank database. Six het- arranged in longitudinal rows (Fig. 2I, J, T). The glabrous 56 erotrichs were selected as the outgroup species. stripe is bordered by the bristle kinety of which the kinet- 57 Sequence alignment was carried out on the web server ids are more widely spaced and irregularly arranged than 58 Phylogeny.fr (http://www.phylogeny.fr/version2_cgi/index. those of the somatic ciliary rows (Fig. 2E, I, T). Anterior 59 cgi) with the alignment algorithm MUSCLE (Dereeper and posterior secant systems form on the left side of gla-

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists 2 Journal of Eukaryotic Microbiology 2014, 0, 1–10 Yan et al. Morphology and Phylogeny of Three Trachelocercid Ciliates

1 Table 1. Morphometric data (lm) for Trachelocerca orientalis spec. brous stripe, which extends the whole length of the 2 nov. (first line), Prototrachelocerca fasciolata (second line) and Trachel- body, is about half of the body width (Fig. 3K). Six to 18 3 oraphis huangi (third line) macronuclear nodules form a strand in the middle of the 4 trunk (Fig. 3C, L, W) while micronuclei are hardly to Characters Min Max Mean CV n 5 detect. Locomotion is by gliding over sand grains and 6 Body length (lm) 114 296 215.2 25.7 20 organic debris. 7 231 809 490.2 38.7 17 The entire infraciliature consists of dikinetids bearing 8 197 356 277.8 14.0 20 8–10 lm long cilia. Twenty-two to 38 somatic kineties 9 Body width (lm) 34 154 108.1 35.4 20 cover the body surface. The anterior and posterior 10 65 167 117.4 24.8 18 secant system forms on the left side of the glabrous 11 63 141 101.6 21.5 20 stripe, which is bordered by the bristle kinety (Fig. 3E). 12 SK in head, number 10 14 11.4 9.4 19 The glabrous stripe, which extends the whole body 13 11 24 18.3 16.9 19 length in the midline of the left side, is as wide as 14 14 21 17.8 12.5 20 about half of the body width (Fig. 3K). The oral infracilia- 15 SK in trunk, number 15 21 17.1 8.7 18 ture comprises two rows of circumoral kineties, which 16 22 38 32.4 12.9 20 are interrupted by the inserted brosse kineties (Fig. 3D, 23 37 28.7 12.8 20 17 E, T, U). 18 Anterior end to NG, distance 29 107 64.0 32.6 20 19 76 218 157.4 22.6 13 13 207 135.1 42.6 13 5 20 Tracheloraphis huangi Xu et al. 2011a, 2011b (Table 1; NG, number 7 22 12.9 28.6 20 21 Fig. 4) 6 18 11.9 30.2 11 22 11 1 020This species was recently described by Xu et al. (2011a). 23 Brosse rows, number –– – – –Here, we add some additional data on the new population 24 22 2 011here. 25 2 3 2.8 16.8 20 Cells in vivo are about 500–800 9 20–30 lm, highly 26 Width of GS (lm) 20 41 26.8 22.4 18 flexible and contractile (Fig. 4A–C, J, K, M). Head and 27 38 62 47.3 18.2 13 neck are distinguishable from trunk (Fig. 4A, J). Posterior 28 21 38 27.9 21.0 19 region is relatively narrow and cuneiform (Fig. 4A–C, J, K, 29 Length of a NG (lm) 9 12 10.8 17.1 16 M). Cells are grayish at low magnification in bright-field 30 7 20 13.0 33.8 13 microscopy (Fig. 4J, K, M). Cortical granules are ca. 0.8– 31 23 32 27.9 11.3 15 1.5 lm in diam., colorless, scattered between ciliary rows 32 Width of a NG (lm) 9 13 10.5 15.9 16 and densely packed in glabrous stripe (Fig. 4D, O, R). 33 7 15 11.0 29.8 13 Cytoplasm is colorless and packed with colorless ellipsoi- 34 18 27 23.7 11.0 15 dal granules which are 3–4 lm long and globular granules 35 Ma nodules in a NG, number 2 5 3.3 32.9 20 which are 1–3 lm in diam. Locomotion is by gliding over 36 6 18 11.9 30.2 11 sand grains and organic debris. –– – – – 37 The infraciliature consists entirely of dikinetids 38 GS = glabrous stripe, Ma = macronuclear, NG = nuclear group, (Fig. 4H, I). Cilia are about 10 lm long. There are 23–37 39 SK = somatic kineties. somatic kineties in the trunk, with 14–21 extending to 40 the head region (Fig. 4F–I, L, P, S). The anterior and 41 posterior secant system forms on the left side of the 42 brous stripe where some somatic kineties abut to the glabrous stripe where some kineties abut to the bristle 43 bristle kinety (Fig. 2E, I). Oral infraciliature consists of a kinety (Fig. 4I, L). The glabrous stripe is narrow, usually 44 simple, continuous circumoral kinety, without brosse about the width of two or three ciliary rows (Fig. 4I). 45 (Fig. 2E, F, I, J, O, S). Oral infraciliature comprises a circumoral kinety, which is 46 interrupted by the inserted brosse kineties (Fig. 4F, G, L, 47 N, P). 48 Prototrachelocerca fasciolata (Sauerbrey 1928) 49 Foissner 1996 (Table 1; Fig. 3) Molecular phylogeny based on SSU rRNA gene 50 This species was redescribed in detail by Foissner (1996). sequences (Fig. 5) 51 Therefore, only a brief description of the Qingdao popula- 52 tion is provided here. The newly characterized SSU rRNA gene sequences of 53 Cells in vivo are about 1,000–2,000 9 30–60 lm, flexi- the five isolates have been deposited in the GenBank 54 ble and contractile (Fig. 3A, B, M–P). Body is flattened database with the length (bp), GC content and GenBank 55 and dark brownish due to cortical granules, with an accession numbers as follows: The new species, T. orien- 56 indistinct neck and wedge-shaped tail (Fig. 3A, M–P). talis spec. nov. – 1,558, 46.66%, KJ609036; P. fasciolata 57 Cortical granules are rounded, ca. 0.5 lm in diam., yel- – 1,570, 47.96%, KJ609035; T. huangi pop1 – 1,552, 58 lowish-brown, located between ciliary rows and narrowly 47.62%, KJ609039; T. huangi pop2 – 1,636, 47.25%, 59 spaced in the glabrous stripe (Fig. 3F, Q, S). The gla- KJ609038; T. huangi pop3 – 1,467, 48.44%, KJ609037.

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–10 3 Morphology and Phylogeny of Three Trachelocercid Ciliates Yan et al.

1 2 3 4 5 COLOR 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Figure 2 Trachelocerca orientalis spec. nov. in vivo (A–D, G, H, K–N, P, Q) and after protargol impregnation (E, F, I, J, O, R–U). O, S from the 47 holotype. A. Typical individual; B. contracted individual; C. shape variants; D. nuclear groups; E, F. left (E) and right (F) side of anterior body region, 48 showing bristle kinety (arrowhead) and continuous (uninterrupted) circumoral kinety consisting of one row of dikinetids; G. mid-region of cell mark- 49 ing a nuclear group; H. distribution of cortical granules (arrowheads) between ciliary rows; I, J. ciliary pattern of left (I) and right (J) side, showing 50 glabrous stripe occupying the width of about two ciliary rows, anterior secant system (arrows in I) and bristle kinety (arrowheads in I); K, L, N. 51 extended gliding individual; M. contracted individual; O, S. (from the holotype) anterior body region showing circumoral kinety; P. distribution of 52 cortical granules (arrows); Q. nuclear groups (arrowhead); R. somatic kineties; T. ciliary pattern of mid-body, to show glabrous stripe and bristle 53 kinety; U. overview showing ciliary pattern and nuclear groups. BK = bristle kinety, CK = circumoral kinety, GS = glabrous stripe, NG = nuclear 54 groups, SK = somatic kineties. Bars: 300 lm (A, K, N), 200 lm (B), 150 lm (L, M), 100 lm (I, J, U), 20 lm (E, F). 55 56 57 The SSU rDNA-based trees were constructed as shown is presented with support values from both algorithms 58 in Fig. 5. The topologies of the ML and BI trees were indicated on branches. Except for the family Wilbertomor- 59 basically accordant; therefore, a single topology of ML tree phidae that is represented by only one species, all the four

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists 4 Journal of Eukaryotic Microbiology 2014, 0, 1–10 Yan et al. Morphology and Phylogeny of Three Trachelocercid Ciliates

1 2 3 4 5 6 COLOR 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Figure 3 Prototrachelocerca fasciolata from life (A–C, F, G–J, M–S) and after protargol impregnation (D, E, K, L, T–V). A. Typical individual; B. 45 shape variants; C. macronuclei; D, E. left (E) and right (D) side of anterior body region, showing bristle kinety (arrowhead in E) and compound cir- 46 cumoral kineties interrupted by two rows of brosse; F. distribution of cortical granules between ciliary rows and in glabrous stripe (arrowheads); 47 G. mid-region of cell marking macronuclei; H–J. general view (from Foissner 1996); K, L. Ciliary pattern of left (K) and right (L) side, showing gla- 48 brous stripe occupying the width of more than 10 ciliary rows; M, N, P. three extended individuals; O. contracted individual; Q, S. distribution of 49 cortical granules (arrowheads); R. macronuclei; T, U. anterior body region indicating brosse and circumoral kineties; V. ciliature in mid-region of 50 body to show somatic kineties and bristle kinety (arrowheads). B = brosse, CK = circumoral kinety, GS = glabrous stripe, Ma = macronuclei, 51 SK = somatic kineties. Bars: 600 lm (H, J), 500 lm (A, B), 300 lm(M–P), 250 lm (K, L), 100 lm (I), 15 lm (D, E). 52 53 54 families within Karyorelictea (Geleiidae, Loxodidae, Trache- Thus, all ﬁve populations of three forms, that is, T. orien- 55 locercidae, and Kentrophoridae) are monophyletic with talis spec. nov., P. fasciolata, and T. huangi are falling in the 56 high support values (97% ML, 0.97 BI; 97% ML, 1.00 BI; clade of Trachelocercidae as expected. However, three iso- 57 94% ML, 0.98 BI; and 100% ML, 1.00 BI). These lates of T. huangi do not cluster with its congener, Trachel- 58 results are in concordance with previous studies (Xu et al. oraphis sp. L31520, the only known one available from 59 2013a,b). Genbank, but groups in the clade of Trachelocerca species

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–10 5 Morphology and Phylogeny of Three Trachelocercid Ciliates Yan et al.

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5 COLOR 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 – – 47 Figure 4 Tracheloraphis huangi from life (A E) and after protargol impregnation (F I). A. Typical individual; B. contracted individual; C. shape vari- 48 ants; D. transverse section of cell marking bristle kinety and cortical granules; E. nuclear group; F, G. left (F) and right (G) side of anterior body 49 region, indicating bristle kinety (arrowhead), brosse and interrupted circumoral kinety; H, I. ciliary pattern of left (I) and right (H) side, showing glabrous stripe occupying the width of about three ciliary rows, and anterior (arrow in I) and posterior secant system (arrowheads in I); J, K. 50 extended gliding individual; L, P. ciliary pattern in anterior body end of left (L) and right (P) side, noting brosse, circumoral kinety, bristle kineties 51 (arrowhead in L) and anterior secant system (arrow in L); M. contracted individual; N. anterior body region showing circumoral kinety; O, R. distri- 52 bution of cortical granules between ciliary rows (O) and in glabrous stripe (R) (arrowheads); Q. macronuclei indicating (protein?) crystals (arrow- 53 head); S. Overview showing ciliary pattern and the single nuclear group; T. macronuclei forming a single nuclear group. B = brosse, BK = bristle 54 kinety, C = crystal, CK = circumoral kinety, CG = cortical granules, GS = glabrous stripe, Ma = macronuclei, NG = nuclear group, SK = somatic 55 kineties. Bars: 300 lm (A, J, K), 200 lm (C, H, I), 150 lm (M, S), 100 lm (B), 30 lm (F, G). 56 57 with high support values (99% ML, 1.00 BI). Therefore, Trachelocerca could not be rejected by the AU test 58 both Trachelocerca and Tracheloraphis are not monophy- (p = 0.345 > 0.05), while the hypothesis that Trachelora- 59 letic. However, the AU test shows that the monophyly of phis is monophyletic is rejected (p = 0.0002 < 0.05).

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists 6 Journal of Eukaryotic Microbiology 2014, 0, 1–10 Yan et al. Morphology and Phylogeny of Three Trachelocercid Ciliates

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5 COLOR 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Figure 5 Maximum-likelihood (ML) tree inferred from SSU rRNA gene sequences showing the positions of Tracheloraphis huangi, Trachelocerca 37 orientalis spec. nov., and Prototrachelocerca fasciolata (in bold). Numbers at nodes represent the bootstrap values of maximum likelihood out of 38 1,000 replicates and the posterior probability of Bayesian analysis. Fully supported (100%/1.00) branches are marked with solid circles. All 39 branches are drawn to the scale bar, which corresponds to five substitutions per 100 nucleotide positions. 40 – – 41 somatic kineties (22 25 vs. 15 21) (Dragesco 1960; Rai- DISCUSSION – 42 kov 1962; Table 2; Fig. 6A E). Moreover, Dragesco (1960) reported that T. kahli had “the mouth funnel 43 Comments on T. orientalis spec. nov 44 asymmetric (showing a central deep notch)” (Fig. 6D), 45 Since the generic classification of most species in the which indicates that it may have a brosse in the oral 46 family Trachelocercidae remain questionable, the compari- area, thus, this is another possible feature to separate 47 son between T. orientalis spec. nov. and its most closely our new species from T. kahli. 48 related morphospecies should not be limited to the genus Although the detailed information of infraciliature 49 Trachelocerca (Foissner 1996, 1997b; Foissner and Drage- remains unclear, Tracheloraphis grassei Dragesco 1960 50 sco 1996a,b; Xu et al. 2011a). In terms of the body size, can be separated from our new species by the tail shape 51 the number of somatic kineties and the number of ma- (pointed tail vs. narrowly rounded tail) and the number of – 52 cronuclei, there are five nominal congeners which resem- somatic kineties (ca. 14 vs. 15 21) (Dragesco 1960; 53 ble T. orientalis spec. nov. (Table 2). Table 2; Fig. 6M, N). 54 Tracheloraphis kahli Raikov 1962 (synonyms: Trache- Tracheloraphis margaritatus Dragesco and Dragesco-Ker- 55 locerca phoenicopterus Cohn 1866; Tracheloraphis phoe- neis 1986 differs from the new species in having broader 56 nicopterus (Cohn 1866) Dragesco 1960) is the most glabrous stripe (the width of ca. 10 somatic kineties vs. of – 57 closely related species to the new form, which can be ca. 2 somatic kineties) and more somatic kineties (26 30 – 58 separated from the new species by the shape of its tail vs. 15 21) (Dragesco and Dragesco-Kerneis 1986; Table 2; – 59 (beak-shaped tail vs. rounded tail) and the number of Fig. 6F G).

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–10 7 Morphology and Phylogeny of Three Trachelocercid Ciliates Yan et al.

1 Table 2. Related species of Trachelocerca orientalis spec. nov 2 3 Species BL (lm) SK, no. NG, no. Data source 4 Trachelocerca orientalis spec. nov. 600–1,200 15–21 7–22 Present study 5 Tracheloraphis kahli 600–1,000 22–25 3–11 Raikov (1962) 6 Tracheloraphis grassei 800–1,000 14 14 Dragesco (1960) 7 Tracheloraphis margaritatus Up to 900 26–30 8–12 Dragesco and Dragesco-Kerneis (1986) 8 Tracheloraphis teissieri 500–900 9–10 18–21 Dragesco (1960) 9 Tracheloraphis poljanskyi 600–900 12 11–48 Raikov (1963) 10 = = = 11 BL body length (in vivo), SK somatic kineties, NG nuclear group. 12 13 width of the glabrous stripe (half of body width in the 14 Qingdao population vs. about one-third of body width in 15 the Roscoff population). As this species is flexible 16 and contractile, we believe this feature is population- 17 dependent and the Qingdao isolate is a population of P. 18 fasciolata. 19 20 21 Comments on T. huangi Xu et al. 2011a, 2011b 22 The original description of T. huangi was reported by Xu 23 et al. (2011a). The new population corresponds well with 24 the former Qingdao population in terms of body size, the 25 width of the glabrous stripe, the number of somatic kin- 26 Figure 6 Morphology of some closely related congeners of Trache- eties and the number of macronuclei. The only minor dif- 27 locerca orientalis spec. nov. A–C. Tracheloraphis kahli (from Raikov ference is that the tail shape of the new population is 28 1962); D, E. Tracheloraphis (Trachelocerca) phoenicopterus (from slightly broader than the former population, which is prob- 29 Dragesco 1960); F, G. Tracheloraphis margaritatus (from Dragesco ably population-dependent. 30 and Dragesco-Kerneis 1986); H, I. Tracheloraphis poljanskyi (from Rai- 31 kov 1963); J–L. Tracheloraphis teissieri (from Dragesco 1960); M, N. Comments on the phylogenetic analyses 32 Tracheloraphis grassei (from Dragesco 1960). Ma = macronuclei, 33 NG = nuclear group. Bars: 300 lm (A, B, E, G, N), 200 lm (H, J, K). Morphologically, the main characters used to distinguish 34 the three genera are the brosse (present/absent) and the 35 circumoral kinety (interrupted/uninterrupted; simple/com- 36 Tracheloraphis teissieri Dragesco 1960 can be distin- plex) (Foissner 1996; Xu et al. 2011b). Trachelocerca spe- 37 guished from the new form by the tail shape (pointed tail cies have an uninterrupted simple circumoral kinety 38 vs. rounded tail) and fewer somatic kineties (9–10 vs. 15– without brosse. The circumoral kineties in Prototracheloc- 39 21) (Dragesco 1960; Table 2; Fig. 6J–K). erca and Tracheloraphis species are all interrupted by 40 Tracheloraphis poljanskyi Raikov 1963 differs from the three rows of brosse kineties. The main difference is that 41 new species in having a broader glabrous stripe (as wide the circumoral kineties are composed of two rows in Pro- 42 as ca. 12 somatic kineties vs. width of ca. two somatic totrachelocerca species, while single-rowed in Trachelora- 43 kineties), a different tail shape (pointed tail vs. round tail) phis species. The fact that all the three isolates of T. 44 and fewer somatic kineties (12 vs. 15–21) (Raikov 1963; huangi collected from different sites or at and different 45 Table 2; Fig. 6H–I). times formed a monophyletic group with full statistical 46 support corroborates their conspecificity. 47 48 Comments on P. fasciolata (Sauerbrey 1928) Foissner 49 1996 TAXONOMIC SUMMARY 50 Due to the brief original description, several synonyms of Order Protostomatida Small and Lynn, 1985 51 P. fasciolata have been reported since it was first Family Trachelocercidae Kent 1881 52 described by Sauerbrey (1928) as Trachelocerca fasciola- Genus Trachelocerca Ehrenberg, 1840 53 ta. The genus Prototrachelocerca was defined by Foiss- 54 ner (1996) following a detailed examination of a Trachelocerca orientalis spec. nov 55 population of P. fasciolata from Roscoff, France. The 56 Qingdao population of P. fasciolata corresponds well with Diagnosis. Extended cells in vivo about 600–1,200 9 20– 57 the Roscoff population (Foissner 1996) in terms of its 30 lm in size; about 15–21 somatic kineties; glabrous 58 body shape, the number of somatic kineties and the stripe about the width of two somatic kineties; 7–22 59 number of macronuclei, while the only difference is the nuclear groups, each consisting of two to four macronu-

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists 8 Journal of Eukaryotic Microbiology 2014, 0, 1–10 Yan et al. Morphology and Phylogeny of Three Trachelocercid Ciliates

1 clei, forming a strand; globular cortical granules colorless, Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Cheve- 2 1 lm in diam. net, F., Dufayard, J. F., Guindon, S., Lefort, V., Lescot, M., 3 Type locality. Intertidal zone of a sandy beach in Qingdao Claverie, J. M. & Gascuel, O. 2008. Phylogeny.fr: robust phylo- 4 (36°050N, 120°270E), China. genetic analysis for the non-specialist. Nucleic Acids Res., – 5 Type material. A protargol-impregnated slide containing 36:465 469. Dragesco, J. 1960. Cilies mesopsammiques littoraux, systema- 6 the holotype specimen marked with an ink circle is depos- tique, morphologie, ecologie. Trav. Stn Biol. Roscoff (N. S.), 7 ited in the Laboratory of Protozoology, OUC, China (slide 12:1–356. 8 no. YY2013040801). Dragesco, J. & Dragesco-Kerneis, A. 1986. Cilies libres de l’Afri- 9 Etymology. The species-group name orientalis (eastern que intertropicale. 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(Prototrachelocerci- 19 ACKNOWLEDGMENTS dae nov. fam.), with a redescription of P. fasciolata (Sauerbrey, 20 1928) nov. comb. and P. caudata (Dragesco & Raikov, 1966) 21 This study was supported by the Natural Science Founda- nov. comb. Eur. J. Protistol., 32:336–355. 22 tion of China (project numbers: 41276139, 31111120437) Foissner, W. 1997a. Updating the trachelocercids (Ciliophora, Kar- 23 and the Research group project no. RGP-VPP-083, King yorelictea). V. Redescription of Kovalevaia sulcata (Kovaleva, 24 Saud University Deanship of Scientific Research. Many 1966) gen. n., comb. n. and Trachelocerca incaudata Kahl, – 25 thanks are to Dr. Jie Huang, Ms. Pu Wang and Mr. Weibo 1933. Acta Protozool., 36:197 219. Foissner, W. 1997b. Updating the trachelocercids (Ciliophora, Kar- 26 Zheng (OUC) for their help in gene sequencing. yorelictea). IV. Transfer of Trachelocerca entzi Kahl, 1927 to the 27 Gymnostomatea as a new genus, Trachelotractus gen. n. 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© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–10 9 Morphology and Phylogeny of Three Trachelocercid Ciliates Yan et al.

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