Available online at www.sciencedirect.com
ScienceDirect European Journal of Protistology 50 (2014) 524–537
Morphology, morphogenesis and molecular phylogeny of a new marine ciliate, Trichototaxis marina n. sp. (Ciliophora, Urostylida) Xiaoteng Lua , Feng Gaoa , Chen Shaob , Xiaozhong Hua,∗ , Alan Warrenc a
Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China The Key Laboratory of Biomedical Information Engineering, Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China c Department of Life Sciences, Natural History Museum, London SW7 5BD, UK b
Received 16 May 2014; received in revised form 15 August 2014; accepted 15 August 2014 Available online 23 August 2014
Abstract The live morphology, infraciliature and morphogenesis of a new urostylid ciliate, Trichototaxis marina n. sp., collected from coastal water in Qingdao, China, were studied based on the observations of live and silver stained specimens. The new species is characterised as follows: body very flexible and contractile, slight to brick-reddish in colour due to irregularly-shaped, brick-red pigments; ca. 70 adoral membranelles; about 17 frontal cirri arranged in a bicorona; average 67 midventral pairs, the right base of each pair being conspicuously larger than the left base; five to seven transverse cirri; constantly two frontoterminal, one buccal and two pretransverse ventral cirri; two or three left marginal rows; right and innermost left marginal rows with 56–92 and 66–106 cirri, respectively; six bipolar dorsal kineties; more than 100 macronuclear nodules. The characteristic morphogenetic feature in T. marina is the development of the left marginal rows, that is, only one left marginal row is newly built the other one or two being retained from the parental cell. Phylogenetic analyses based on small subunit ribosomal gene sequence data reveal a close relationship of T. marina with members of family Pseudokeronopsidae. © 2014 Elsevier GmbH. All rights reserved.
Keywords: Infraciliature; Marine ciliates; Ontogenesis; SSU rDNA; Trichototaxis marina n. sp
Introduction Hypotrichs with their highly diverse cirral and morphogenetic patterns play a major role in understanding the systematics and evolutionary relationships among ciliates (Berger, 2006; Foissner and Stoeck, 2008). Furthermore, recent studies have revealed a large, previously undescribed diversity of hypotrich ciliates (e.g. Berger, 2006; Chen et al.,
∗ Corresponding author. Tel.: +86 532 8203 1610; fax: +86 532 8203 1610.
E-mail address: [email protected] (X. Hu). http://dx.doi.org/10.1016/j.ejop.2014.08.002 0932-4739/© 2014 Elsevier GmbH. All rights reserved.
2013a,b,c,d,e,f; Fan et al., 2014; Foissner, 2012; Huang et al. 2012; Jiang et al., 2013a,b,c; Li et al., 2013; Lv et al., 2013; Paiva et al., 2012; Pan et al., 2013; Shao et al., 2013a,b, 2014; Singh and Kamra, 2013; Singh et al., 2013; Song et al., 2009; Weisse et al., 2013). The hypotrich genus Trichototaxis was established by Stokes (1891) with T. stagnatilis as the type species by original designation, its main distinguishing character being the presence of multiple left marginal rows (Berger, 2006; Stokes, 1891). Four other species has since been described, namely T. multinucleata Burkovsky, 1970, T. rubentis Sarmiento and Guerra, 1960, T. villaensis Sarmiento and
X. Lu et al. / European Journal of Protistology 50 (2014) 524–537
Guerra, 1960 and Trichototaxis songi Chen et al., 2007. However T. multinucleata, T. rubentis and T. villaensis still remain species indeterminata (Berger, 2006). Plückebaum et al. (1997) briefly described a red-to-purple coloured freshwater hypotrich which they considered might be a new species of Trichototaxis, and suggested the name T. rubra. However, it lacks a bicoronal arrangement of frontal cirri, so cannot be assigned into Trichototaxis. Considering its high resemblance to Diaxonella pseudorubra (Kaltenbach, 1960), Berger (2006) synonymised it with the latter. In October 2013, an unknown urostylid ciliate was isolated from coastal waters of the Yellow Sea near the Olympic Centre in Qingdao (Tsingtao), northern China. Observations of its morphology both in vivo and following protargol staining demonstrate that it represents a novel species within the genus Trichototaxis. In this paper we describe its morphology and morphogenesis during division and regeneration. The small subunit ribosomal DNA (SSU rDNA) of the new isolate was sequenced and analysed in order to assess its phylogenetic position.
Material and Methods
525
For the phylogenetic analyses, the SSU rDNA sequences of 52 other urostyloid and oxytrichid ciliates obtained from the NCBI GenBank database were used in addition to the newly characterised sequence of Trichototaxis marina n. sp (for accession numbers see Fig. 7). Four euplotids were selected as the outgroup species. Sequences were aligned using MUSCLE v3.7 (Edgar, 2004) with the default parameters on the web server (http://www.phylogeny.fr/version2 cgi) (Dereeper et al., 2008, 2010). The resulting alignment was manually edited using BioEdit v7.0.5.2 (Hall, 1999). The final alignment, including 1838 sites and 57 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 with default parameters. Support for the best ML tree came from 1000 bootstrap replicates. The Bayesian inference (BI) analysis was performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) with the GTR + I + G 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 50% majority rule consensus trees.
Sampling and cultivation Samples were collected on 10th October 2013 from a coastal water in Qingdao (Tsingtao, 36◦ 08 N; 120◦ 59 E), China. Glass slides were used as artificial substrates to collect the ciliates. Briefly, the slides were immersed at a depth of 1 m for about 7–10 d to allow colonisation by ciliates to take place. After this time the slides were retrieved by transferring into Petri dishes along with seawater from the sampling site. Individual Trichototaxis cells were isolated in the laboratory using micropipettes. Cultures were established at room temperature (20 ◦ C) using boiled seawater with diatoms (Phaeodactylum tricornutum) as the food source.
Morphology and morphogenesis Live observations were carried out using bright field and differential interference contrast microscopy. The protargol staining method was used to reveal the infraciliature (Wilbert, 1975). Drawings were made with the help of a camera lucida at a magnification of 1250× To illustrate the changes during morphogenesis, parental cirri are depicted by contour whereas new ones are shaded black. Terminology and systematics follow Berger (2006) and Lynn (2008) respectively.
Results Trichototaxis marina n. sp. Diagnosis Marine Trichototaxis with elongate elliptical outline, size 250–300 × 40–60 m in vivo and brick-reddish cell colour; pigment granules, spherical or roughly elliptical-shaped, brick-reddish of variable size (1–10 m), densely distributed under pellicle; one contractile vacuole positioned to left of buccal vertex; about 70 adoral membranelles; 13–19 frontal cirri in a bicorona; constantly two frontoterminal, one buccal and two pretransverse ventral cirri; 48–87 midventral pairs extending to level of transverse cirri, right base of each pair being conspicuously larger than left base; 5–7 transverse cirri; one right marginal row with 56–92 cirri; two or three left marginal rows; innermost (first) of which bears 66–106 narrowly spaced cirri; six bipolar dorsal kineties; more than 100 macronuclear nodules. Type locality Coastal waters of the Yellow Sea near the Olympic Centre in Qingdao (Tsingtao, 36◦ 08 N; 120◦ 59 E), China.
Phylogenetic analyses Genomic DNA was extracted from cells using DNeasy Tissue kit (Qiagen, CA) following the manufacturer’s instructions. PCR amplification and sequencing of the SSU rDNA were performed according to Gao et al. (2014).
Deposition of type slides One protargol-stained slide with the holotype specimen circled in ink (Fig. 1G and H) is deposited in the Laboratory of Protozoology, Ocean University of China (OUC, registration number: LuX2013101001-18). One paratype slide is
526
X. Lu et al. / European Journal of Protistology 50 (2014) 524–537
Fig. 1. (A–H) Morphology and infraciliature of Trichototaxis marina n. sp. from life (A and B) and after protargol staining (C–H). (A) Ventral view of a typical individual. (B) Different body shapes. (C) Ventral view to show the frontoventral ciliature. Note the infraciliary fibres of midventral pairs and marginal rows (arrows); arrowhead indicates the frontoterminal cirri. The region highlighted in yellow depicts the bicorona. (D and E) Basal bodies and infraciliary fibres of marginal cirri and midventral pairs. (F) Infraciliary fibres of transverse cirri. (G and H) Ventral and dorsal view of the infraciliature of the holotype specimen, arrow marks the buccal cirrus. AZM, adoral zone of membranelles; BiC, bicorona of frontal cirri; E, endoral membrane; FT, frontoterminal cirri; LMR, left marginal row; Ma, macronuclear nodules; MP, midventral pairs; P, paroral membrane; PTVC, pretransverse ventral cirri; RMR, right marginal row; TC, transverse cirri. 1–6, dorsal kineties. Scale bars = 50 m (A and C); 100 m (B, G and H); 30 m (F).
deposited in the Natural History Museum, London, UK (registration number NHMUK 2014.5.16.1). Etymology The species-group name marina (Latin adjective; living in the sea) refers to the habitat where the species was discovered.
Morphology of Trichototaxis marina n. sp. (Figs 1A–H, 2A–Q) Table 1. Body 250–300 × 40–60 m in vivo; ratio of length to width about 4–8:1, usually 5:1; dorsoventrally flattened ca. 2:1 (Figs 1A, 2A). Body conspicuously brick-reddish in colour at low magnification, elongate-elliptical or sigmoid in outline, widest at anterior third. Pellicle very flexible and highly contractile (Figs 1B, 2B–G). Cortical granules not observed, but spherical or roughly elliptical-shaped, brickreddish pigment granules of variable size (1–10 m across) densely distributed under pellicle, which render cells red
(Fig. 2K). Endoplasm usually packed with numerous small (
ScienceDirect European Journal of Protistology 50 (2014) 524–537
Morphology, morphogenesis and molecular phylogeny of a new marine ciliate, Trichototaxis marina n. sp. (Ciliophora, Urostylida) Xiaoteng Lua , Feng Gaoa , Chen Shaob , Xiaozhong Hua,∗ , Alan Warrenc a
Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China The Key Laboratory of Biomedical Information Engineering, Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China c Department of Life Sciences, Natural History Museum, London SW7 5BD, UK b
Received 16 May 2014; received in revised form 15 August 2014; accepted 15 August 2014 Available online 23 August 2014
Abstract The live morphology, infraciliature and morphogenesis of a new urostylid ciliate, Trichototaxis marina n. sp., collected from coastal water in Qingdao, China, were studied based on the observations of live and silver stained specimens. The new species is characterised as follows: body very flexible and contractile, slight to brick-reddish in colour due to irregularly-shaped, brick-red pigments; ca. 70 adoral membranelles; about 17 frontal cirri arranged in a bicorona; average 67 midventral pairs, the right base of each pair being conspicuously larger than the left base; five to seven transverse cirri; constantly two frontoterminal, one buccal and two pretransverse ventral cirri; two or three left marginal rows; right and innermost left marginal rows with 56–92 and 66–106 cirri, respectively; six bipolar dorsal kineties; more than 100 macronuclear nodules. The characteristic morphogenetic feature in T. marina is the development of the left marginal rows, that is, only one left marginal row is newly built the other one or two being retained from the parental cell. Phylogenetic analyses based on small subunit ribosomal gene sequence data reveal a close relationship of T. marina with members of family Pseudokeronopsidae. © 2014 Elsevier GmbH. All rights reserved.
Keywords: Infraciliature; Marine ciliates; Ontogenesis; SSU rDNA; Trichototaxis marina n. sp
Introduction Hypotrichs with their highly diverse cirral and morphogenetic patterns play a major role in understanding the systematics and evolutionary relationships among ciliates (Berger, 2006; Foissner and Stoeck, 2008). Furthermore, recent studies have revealed a large, previously undescribed diversity of hypotrich ciliates (e.g. Berger, 2006; Chen et al.,
∗ Corresponding author. Tel.: +86 532 8203 1610; fax: +86 532 8203 1610.
E-mail address: [email protected] (X. Hu). http://dx.doi.org/10.1016/j.ejop.2014.08.002 0932-4739/© 2014 Elsevier GmbH. All rights reserved.
2013a,b,c,d,e,f; Fan et al., 2014; Foissner, 2012; Huang et al. 2012; Jiang et al., 2013a,b,c; Li et al., 2013; Lv et al., 2013; Paiva et al., 2012; Pan et al., 2013; Shao et al., 2013a,b, 2014; Singh and Kamra, 2013; Singh et al., 2013; Song et al., 2009; Weisse et al., 2013). The hypotrich genus Trichototaxis was established by Stokes (1891) with T. stagnatilis as the type species by original designation, its main distinguishing character being the presence of multiple left marginal rows (Berger, 2006; Stokes, 1891). Four other species has since been described, namely T. multinucleata Burkovsky, 1970, T. rubentis Sarmiento and Guerra, 1960, T. villaensis Sarmiento and
X. Lu et al. / European Journal of Protistology 50 (2014) 524–537
Guerra, 1960 and Trichototaxis songi Chen et al., 2007. However T. multinucleata, T. rubentis and T. villaensis still remain species indeterminata (Berger, 2006). Plückebaum et al. (1997) briefly described a red-to-purple coloured freshwater hypotrich which they considered might be a new species of Trichototaxis, and suggested the name T. rubra. However, it lacks a bicoronal arrangement of frontal cirri, so cannot be assigned into Trichototaxis. Considering its high resemblance to Diaxonella pseudorubra (Kaltenbach, 1960), Berger (2006) synonymised it with the latter. In October 2013, an unknown urostylid ciliate was isolated from coastal waters of the Yellow Sea near the Olympic Centre in Qingdao (Tsingtao), northern China. Observations of its morphology both in vivo and following protargol staining demonstrate that it represents a novel species within the genus Trichototaxis. In this paper we describe its morphology and morphogenesis during division and regeneration. The small subunit ribosomal DNA (SSU rDNA) of the new isolate was sequenced and analysed in order to assess its phylogenetic position.
Material and Methods
525
For the phylogenetic analyses, the SSU rDNA sequences of 52 other urostyloid and oxytrichid ciliates obtained from the NCBI GenBank database were used in addition to the newly characterised sequence of Trichototaxis marina n. sp (for accession numbers see Fig. 7). Four euplotids were selected as the outgroup species. Sequences were aligned using MUSCLE v3.7 (Edgar, 2004) with the default parameters on the web server (http://www.phylogeny.fr/version2 cgi) (Dereeper et al., 2008, 2010). The resulting alignment was manually edited using BioEdit v7.0.5.2 (Hall, 1999). The final alignment, including 1838 sites and 57 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 with default parameters. Support for the best ML tree came from 1000 bootstrap replicates. The Bayesian inference (BI) analysis was performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) with the GTR + I + G 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 50% majority rule consensus trees.
Sampling and cultivation Samples were collected on 10th October 2013 from a coastal water in Qingdao (Tsingtao, 36◦ 08 N; 120◦ 59 E), China. Glass slides were used as artificial substrates to collect the ciliates. Briefly, the slides were immersed at a depth of 1 m for about 7–10 d to allow colonisation by ciliates to take place. After this time the slides were retrieved by transferring into Petri dishes along with seawater from the sampling site. Individual Trichototaxis cells were isolated in the laboratory using micropipettes. Cultures were established at room temperature (20 ◦ C) using boiled seawater with diatoms (Phaeodactylum tricornutum) as the food source.
Morphology and morphogenesis Live observations were carried out using bright field and differential interference contrast microscopy. The protargol staining method was used to reveal the infraciliature (Wilbert, 1975). Drawings were made with the help of a camera lucida at a magnification of 1250× To illustrate the changes during morphogenesis, parental cirri are depicted by contour whereas new ones are shaded black. Terminology and systematics follow Berger (2006) and Lynn (2008) respectively.
Results Trichototaxis marina n. sp. Diagnosis Marine Trichototaxis with elongate elliptical outline, size 250–300 × 40–60 m in vivo and brick-reddish cell colour; pigment granules, spherical or roughly elliptical-shaped, brick-reddish of variable size (1–10 m), densely distributed under pellicle; one contractile vacuole positioned to left of buccal vertex; about 70 adoral membranelles; 13–19 frontal cirri in a bicorona; constantly two frontoterminal, one buccal and two pretransverse ventral cirri; 48–87 midventral pairs extending to level of transverse cirri, right base of each pair being conspicuously larger than left base; 5–7 transverse cirri; one right marginal row with 56–92 cirri; two or three left marginal rows; innermost (first) of which bears 66–106 narrowly spaced cirri; six bipolar dorsal kineties; more than 100 macronuclear nodules. Type locality Coastal waters of the Yellow Sea near the Olympic Centre in Qingdao (Tsingtao, 36◦ 08 N; 120◦ 59 E), China.
Phylogenetic analyses Genomic DNA was extracted from cells using DNeasy Tissue kit (Qiagen, CA) following the manufacturer’s instructions. PCR amplification and sequencing of the SSU rDNA were performed according to Gao et al. (2014).
Deposition of type slides One protargol-stained slide with the holotype specimen circled in ink (Fig. 1G and H) is deposited in the Laboratory of Protozoology, Ocean University of China (OUC, registration number: LuX2013101001-18). One paratype slide is
526
X. Lu et al. / European Journal of Protistology 50 (2014) 524–537
Fig. 1. (A–H) Morphology and infraciliature of Trichototaxis marina n. sp. from life (A and B) and after protargol staining (C–H). (A) Ventral view of a typical individual. (B) Different body shapes. (C) Ventral view to show the frontoventral ciliature. Note the infraciliary fibres of midventral pairs and marginal rows (arrows); arrowhead indicates the frontoterminal cirri. The region highlighted in yellow depicts the bicorona. (D and E) Basal bodies and infraciliary fibres of marginal cirri and midventral pairs. (F) Infraciliary fibres of transverse cirri. (G and H) Ventral and dorsal view of the infraciliature of the holotype specimen, arrow marks the buccal cirrus. AZM, adoral zone of membranelles; BiC, bicorona of frontal cirri; E, endoral membrane; FT, frontoterminal cirri; LMR, left marginal row; Ma, macronuclear nodules; MP, midventral pairs; P, paroral membrane; PTVC, pretransverse ventral cirri; RMR, right marginal row; TC, transverse cirri. 1–6, dorsal kineties. Scale bars = 50 m (A and C); 100 m (B, G and H); 30 m (F).
deposited in the Natural History Museum, London, UK (registration number NHMUK 2014.5.16.1). Etymology The species-group name marina (Latin adjective; living in the sea) refers to the habitat where the species was discovered.
Morphology of Trichototaxis marina n. sp. (Figs 1A–H, 2A–Q) Table 1. Body 250–300 × 40–60 m in vivo; ratio of length to width about 4–8:1, usually 5:1; dorsoventrally flattened ca. 2:1 (Figs 1A, 2A). Body conspicuously brick-reddish in colour at low magnification, elongate-elliptical or sigmoid in outline, widest at anterior third. Pellicle very flexible and highly contractile (Figs 1B, 2B–G). Cortical granules not observed, but spherical or roughly elliptical-shaped, brickreddish pigment granules of variable size (1–10 m across) densely distributed under pellicle, which render cells red
(Fig. 2K). Endoplasm usually packed with numerous small (
