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ScienceDirect European Journal of Protistology 51 (2015) 1–14
Morphology, morphogenesis and molecular phylogeny of a soil ciliate, Pseudouroleptus caudatus caudatus Hemberger, 1985 (Ciliophora, Hypotricha), from Lhalu Wetland, Tibet Lingyun Chena,b,1 , Xiaolu Zhaoa,1 , Honggang Maa , Alan Warrenc , Chen Shaob,∗ , Jie Huangd,∗ a
Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China The Key Laboratory of Biomedical Information Engineering Ministry of Education, Department of Biology and Engineering, 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 d Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China b
Received 16 December 2013; received in revised form 1 August 2014; accepted 1 September 2014 Available online 6 September 2014
Abstract Pseudouroleptus caudatus caudatus Hemberger, 1985, a soil ciliate isolated from Tibet, was studied in vivo and after protargol impregnation. The Tibetan population is mainly characterized by: elongate body with narrowly rounded anterior end and tapered posterior end; length of buccal area relative to body length ca. 20–25%; cortical granules colourless, round, densely distributed throughout sub-pellicular layer of cell; one parabuccal cirrus; post-peristomial cirrus lacking in 75% of specimens analyzed; left and right ventral rows commence at same level; four dorsal kineties; 3–6 inconspicuous caudal cirri; two macronuclear nodules; 2–7 micronuclei; contractile vacuole located at about 33% of body length near left margin. Morphogenesis is characterized by: (1) parental adoral zone of membranelles retained completely; (2) anterior segments of streaks VI and IV and the whole of streak V form the anterior, middle, posterior segments of the mixed row, respectively; (3) right ventral row originates de novo in both daughter cells; (4) marginal rows develop intrakinetally; (5) dorsal kinety anlage 3 develops de novo in the proter and intrakinetally in the opisthe; and (6) the two macronuclear nodules fuse into a single mass which then divides. Molecular phylogenies corroborate the morphological identification and support the close relationship between Pseudouroleptus and Strongylidium. © 2014 Elsevier GmbH. All rights reserved.
Keywords: Hypotricha; Morphogenesis; Phylogeny; Pseudouroleptus caudatus caudatus; Taxonomy
Introduction
∗ Corresponding
authors. E-mail addresses: [email protected] (C. Shao), [email protected] (J. Huang). 1 Co-first authors. http://dx.doi.org/10.1016/j.ejop.2014.09.001 0932-4739/© 2014 Elsevier GmbH. All rights reserved.
The Hypotricha Stein, 1859 is a speciose and morphologically diverse group that inhabits a wide range of biotopes (Berger 2008; Chen et al. 2013a,b; Fan et al. 2014; Foissner 2012; Jiang et al. 2013; Kahl 1932; Küppers and Claps 2013; Li et al. 2010a,b, 2013; Liu et al. 2010; Lv et al. 2013; Paiva
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et al. 2012; Pan et al. 2012, 2013; Park et al. 2013; Song et al. 2009; Weisse et al. 2013). The genus Pseudouroleptus was established by Hemberger (1985) with P. caudatus as the type species by original designation. Subsequently, P. caudatus was split into two subspecies, P. caudatus caudatus and P. caudatus namibiensis, mainly according to the distinct difference in the number of transverse cirri. The former subspecies has a widespread geographic distribution, occurring in both terrestrial and limnetic habitats, whereas the latter has so far only been reported from the type location (Foissner et al. 2002). Until recently there were five species of Pseudouroleptus that lacked dorsal kinety fragmentation. However, Berger (2008) assigned four of these to Bistichella leaving only the type species, P. caudatus, in the genus. The ciliature of Pseudouroleptus is generally similar to that of Strongylidium, with each possessing buccal, frontal, parabuccal and post-peristomial cirri, two marginal rows and two long ventral cirral rows. However, during morphogenesis the right ventral cirral row forms de novo in Pseudouroleptus versus intrakinetally in Strongylidium (Chen et al. 2013b). To date, morphogenesis has been described in detail for only one of the two subspecies of P. caudatus, namely P. caudatus caudatus (Hemberger 1985; Berger 2008). Likewise, only one small subunit (SSU) rRNA gene sequence of P. caudatus is available in GenBank, that of a Brazilian population that was not identified to subspecies level (Paiva et al. 2009). Therefore, in order to increase knowledge and understanding of the systematics and biodiversity of the genus Pseudouroleptus, morphological, morphogenetic and molecular studies from a wider range of isolates are needed. In the spring of 2011, the subspecies Pseudouroleptus caudatus caudatus Hemberger, 1985 was isolated from soil samples collected from a wetland in Tibet, China. We here describe its morphology, morphogenesis, and molecular phylogeny based on SSU rRNA gene sequence data.
Material and Methods Sampling and cultivation Soil samples were collected on 1st May 2011 from the Lhalu Wetland in the northwest of Lhasa, Tibet (29◦ 40 45 N, 91◦ 05 48 E) (Fig. 1A–D). This wetland is at an altitude of 3645 m above sea level where the annual average temperature is 7.6 ◦ C and the annual average precipitation is 439 mm. Water enters the wetland from two heavy-sedimentcarrying rivers, the River Duodi and River Niangre, and also in the form of industrial effluent from the Najin Power Station. The dominant hydrophyte in the wetland is the common reed Phragmites australis. The sample comprised 500 g of soil from the top 10 cm layer. Ciliates were stimulated to excyst and emerge from the soil
sample by employing the non-flooded Petri dish method (Foissner 1987; Foissner et al. 2002). Ciliates were isolated and cultured at room temperature (20 ◦ C) in Petri dishes containing distilled water with squeezed rice grains to enrich the availability of bacterial food (Chen et al. 2013c).
Morphology and morphogenesis Cells were examined both in vivo using bright field and differential interference contrast microscopy, and following protargol staining in order to reveal the infraciliature (Wilbert 1975). Counts and measurements of morphological characters in protargol-stained specimens were performed at a magnification of ×1000. Drawings were made with the help of a camera lucida (Shao et al. 2013a,b). Two voucher slides with protargol-stained specimens have been deposited, one in the laboratory of Protozoology, Ocean University of China (registration number: CLY2011050103) and the other in the Natural History Museum, London (registration number: NHMUK 2013.12.15.1). Terminology mainly follows Hemberger (1985) and Küppers and Claps (2013). Systematic follows Lynn (2008). Numbering of the cirri follows Berger (1999, 2008).
DNA extraction, PCR amplification and sequencing Genomic DNA was extracted from cells washed with autoclaved freshwater using DNeasy Tissue Kit (Qiagen, CA) according to Chen et al. (2013d). PCR amplification of SSU rDNA was performed using the universal primers EukA and EukB (Medlin et al. 1988; Yi and Song 2011). Highfidelity Taq polymerase (Takara Ex Taq; Takara Biomedicals) was used to minimize the possibility of amplification errors. The amplification cycles were as follows: 5 min at 94 ◦ C, followed by 30 cycles of 94 ◦ C for 30 s, 56 ◦ C for 1 min, 72 ◦ C for 1 min 50 s; the final extension was 7 min at 72 ◦ C (Huang et al. 2010). The PCR product was purified using SanPrep DNA Gel Extraction Kit (Sangon Bio. Co., Shanghai, China) and then inserted into the pMDTM 19-T vector (Takara Biotechnology, Dalian Co., Ltd.). Sequencing was performed bidirectionally on an ABI 3700 sequencer (Invitrogen sequencing facility, Shanghai, China) using primers M1347 (5 -CGCCAGGGTTTTCCCAGTCACGAC-3 ), M13-48 (5 -AGCGGATAACAATTTCACACAGGA-3 ) and three internal primers 900F (5 -CGATCAGATACCGTCCTAGT3 ), 900R (5 -ACTAGGACGGTATCTGATCG-3 ) and Pro B (5 -GGTTAAAAAGCTCGTAGT-3 ).
Phylogenetic analyses Phylogenetic analyses were carried out on the SSU rRNA gene sequence of the Tibetan population of
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Fig. 1. ((A)–(D)). The sample site and surrounding area. (A) The location of the Lhalu Wetland, arrow indicates the Potala Palace; inset shows the location of Tibet. ((B)–(D)) Photographs showing the locality where the samples containing Pseudouroleptus caudatus caudatus were collected. (B) Showing the Potala Palace.
Pseudouroleptus caudatus caudatus and of 53 other sequences downloaded from GenBank (see Fig. 7 for accession numbers except those of the following 13 hypotrichs: Hemicycliostyla sphagni FJ361758, Pseudourostyla cristata FJ598608, Anteholosticha sp. HM568416, Heterokeronopsis pulchra JQ083600, Nothoholosticha fasciola FJ377548, Pseudokeronopsis carnae AY881633, Uroleptopsis citrina FJ870094, Monocoronella carnea FJ775726, Metaurostylopsis salina EU220229, Apokeronopsis wrighti EU417963, Thigmokeronopsis stoecki EU220226, Diaxonella trimarginata DQ190950 and Urostyla grandis AF508781). Sequences were aligned using Clustal W implemented in Bioedit 7.0.9 with default parameters (Hall 1999). Regions that could not be aligned unambiguously were removed and ends were trimmed manually, resulting in a matrix of 1731 characters.
Five species of the subclasses Oligotrichia and Choreotrichia were chosen as the outgroup taxa. The nucleotide substitution model GTR + I + G (Shape = 0.4685, Pinvar = 0.5003) was selected under AIC criteria using MrModeltest 2.2 (Nylander 2004). This model was used to perform Bayesian Inference (BI) analysis with MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003). Markov chain Monte Carlo (MCMC) simulations were run with two sets of four chains for 1000,000 generations with a sampling frequency of 100. The first 25% were discarded as burn-in prior to constructing 50% majority rule consensus trees. The maximum likelihood (ML) tree was constructed with the PhyML V2.4.4 program (Guindon and Gascuel 2003) using the model GTR + I + G (Shape = 0.4724, Pinvar = 0.5018) selected in Modeltest (Posada and Crandall 1998). The reliability of internal branches was evaluated by posterior probabilities (BI) and 1000 non-parametric bootstrap
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Fig. 2. ((A)–(D)). Morphology of Pseudouroleptus caudatus caudatus from life ((A), (B)) and after protargol staining ((C), (D)). (A) Ventral view of a representative individual. (B) Detail of cell (dorsal view), to show the distribution of cortical granules (possibly mitochondria). ((C), (D)) Ventral and dorsal view of the same individual, showing infraciliature and nuclear apparatus, arrow points to the post-peristomial cirrus and arrowhead indicates the buccal cirrus. AZM, adoral zone of membranelles; CC, caudal cirri; CV, contractile vacuole; FC, frontal cirri; III/2, cirrus III/2; LMR, left marginal row; LVR, left ventral row; Ma, macronuclear nodules; Mi, micronuclei; RMR, right marginal row; RVR, right ventral row; 1–4, dorsal kineties. Bars, 150 m.
pseudoreplicates (ML). MEGA 5 (Kumar et al. 2008) was used to visualize tree topologies.
Morphological description of Tibet population (Figs 2A–D, 3A–I; Table 1)
Results
This subspecies has been redescribed by Foissner et al. (2002) and Küppers and Claps (2013), hence, here we only include data that are specific to the Tibetan population.
Class Spirotrichea Bütschli, 1889 Order Stichotrichida Fauré-Fremiet, 1961 Family Spirofilidae von Gelei, 1929 Genus Pseudouroleptus Hemberger, 1985 Pseudouroleptus caudatus caudatus Hemberger, 1985
Morphology Body 230–320 × 45–65 m in vivo, flexible and slightly contractile, outline more or less fusiform, narrowly rounded anteriorly and tapered posteriorly to form a short tail that is
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Fig. 3. ((A)–(I)). Photomicrographs of Pseudouroleptus caudatus caudatus from life ((A)–(E)) and after protargol staining ((F)–(I)). (A) Ventral view of a representative specimen. (B) Showing a bending specimen. (C) Detail of cell surface in dorsal view, to show the distribution of cortical granules, possibly mitochondria (arrows). (D) Nuclear apparatus. (E) Posterior body region, to show the food vacuoles (arrows). (F) Ventral view of anterior portion of cell, to show, inter alia, the buccal cirrus (arrowhead) and the post-peristomial cirrus (arrow). (G) Dorsal view of anterior portion of cell, to show the macronuclear nodules and micronuclei. (H) Ventral view of the midbody region. (I) Posterior region of cell showing the caudal cirri (arrowheads). CV, contractile vacuole; FC, frontal cirrus; III/2, cirrus III/2; LMR, left marginal row; LVR, left ventral row; Ma, macronuclear nodules; Mi, micronuclei; RMR, right marginal row; RVR, right ventral row. Bar, 150 m.
more flexible and contractile than the rest of the cell; length to width ratio about 5:1 in vivo on average, 2.5:1 in protargolstained cells; right margin sigmoidal, curving to right in posterior region, left margin varying from almost straight to sigmoidal (Figs 2A, C 3A, B). Cytoplasm colourless to greyish, usually with numerous lipid droplets ca. 3–5 m in diameter, and food vacuoles 5–10 m in diameter containing
algae that render cell dark when observed at low magnification (Fig. 3A, E). Contractile vacuole about 20 m across, located at about 33% of body length near left cell margin (Figs 2A, 3A). Cortical granules (possibly mitochondria) colourless, round, about 1.5 m in diameter, and densely distributed in the sub-pellicular layer throughout the entire cell (Figs 2B, 3C). Invariably two macronuclear nodules in anterior half
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Table 1. Morphometric data for the Tibet population of Pseudouroleptus caudatus caudatus. Charactera
Min
Max
Mean
SD
Body (length) Body (width) Adoral zone of membranelles (length) Adoral membranelles (number) Buccal cirri (number) Frontal cirri (number) Frontoventral cirri (number) Postperistomial ventral cirri (number) Left ventral cirri (number) Right ventral cirri (number) Left marginal cirri (number) Right marginal cirri (number) Caudal cirri (number) Caudal cirri in DK1 (number) Caudal cirri in DK2 (number) Dorsal kineties (number) Dikinetids in DK1 (number) Dikinetids in DK2 (number) Dikinetids in DK3 (number) Dikinetids in DK4 (number) Macronuclear nodules (number) Length of macronuclear nodules Width of macronuclear nodules Micronuclei (number) Length of micronuclei Width of micronuclei
230 88 70 54 1 3 1 0 60 51 56 57 3 1 1 4 28 28 25 22 2 21 9 2 5 3
325 138 90 67 1 3 1 1 77 65 72 76 6 4 2 4 44 43 47 34 2 45 24 7 10 5
276.3 109.3 77.8 59.1 1.0 3.0 1.0 0.3 69.7 59.6 63.2 67.6 4.9 3.07 1.67 4.0 34.2 34.0 32.8 28.0 2.0 28.8 13.5 3.4 7.5 4.1
27.36 13.75 4.79 3.39 0 0 0 0.44 5.02 3.83 4.59 5.49 0.85 1.10 0.49 0 4.01 4.40 5.45 3.71 0 5.04 3.69 1.28 1.04 0.53
CV
n
9.9 12.6 6.2 5.7 0 0 0 146.7 7.2 6.4 7.3 8.1 17.3 35.9 29.3 0 11.7 12.9 16.6 13.3 0 17.5 27.3 37.6 13.9 12.9
20 20 20 20 20 20 20 20 20 20 20 20 20 15 15 20 20 20 20 20 25 28 28 14 47 47
a All data are based on protargol-impregnated specimens. Measurements in m. Abbreviations: CV, coefficient of variation in %; DK1–4, dorsal kineties 1–4; Max, maximum; Mean, arithmetic mean; Min, minimum; n, number of cells measured; SD, standard deviation.
of body, anterior one in cell midline, posterior one slightly left of midline; nodules ellipsoidal, length: width ratio about 2:1, with small to moderately large nucleoli. Two-to-seven micronuclei (Figs 2D, 3D, F). Locomotion usually by moderately rapid swimming while rotating clockwise about main body axis, or occasionally by gliding on substrate. Adoral zone occupies nearly 20–25% of body length in vivo (about 28% in protargol-stained cells), curving across anterior cell margin and extending conspicuously down right side of body, i.e., DE-value 0.37 in the holotype specimen (Fig. 3F; for explanation of DE-value, see Berger 2006: 18). Adoral zone comprises 59 membranelles on average, bases of largest membranelles about 15 m wide, cilia up to 20 m long (Fig. 2C). Paroral and endoral approximately equal in length, distinctly curved, intersecting optically at posterior quarter. Paroral consists of dikinetids whereas endoral is composed of monokinetids. Three clearly differentiated frontal cirri; one conspicuous parabuccal cirrus (III/2) located below rightmost frontal cirrus: cilia of frontal cirri and parabuccal cirrus about 15 m long (Figs 2C, 3F). Single buccal cirrus located about 10% down body length, right of paroral. In five out of 20 specimens investigated one post-peristomial cirrus was present, located behind proximal end of adoral zone (Figs 2C, 3F). Two long, oblique, sigmoid ventral cirral rows, beginning approximately near right frontal cirrus
and terminating at posterior end of cell, comprising 60–77 (left) and 51–65 (right) cirri, respectively (Figs 2C, 3H). Left and right marginal rows composed of 56–72 and 57–76 cirri, respectively, cilia of which are 10–15 m long, terminating at posterior end of cell (Figs 2A, C, 3F, G). After protargol staining, some fibres were observed associated with cirri of ventral rows (Fig. 3F). Transverse cirri absent. Four dorsal kineties extending almost entire length of cell; dorsal cilia 5 m long; no dorsomarginal kineties formed (Figs 2D, 3G). Three to six caudal cirri, densely packed and located at posterior body margin (Figs 2D, 3I).
Morphogenesis (Figs 4A–F, 5A–F, 6A–I) Oral apparatus and frontal-ventral-transverse cirral anlagen Opisthe. In the first stage of morphogenesis a long field of basal bodies develops left of the mid-region of the left ventral row (Figs 4A, 6A). At the same time, a streak is formed in the left ventral row, at about the level of the anterior end of the oral primordium (OP; Fig. 4A). Based on subsequent stages, we deduce that this streak is the frontal-ventral-transverse cirral anlage (FVTA) V (Fig. 4B). During the next stage the oral primordium continues to grow by further proliferation
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Fig. 4. A–F. Divisional morphogenesis in Pseudouroleptus caudatus caudatus from early to late stages (after protargol staining). (A) Ventral view of an early divider, note the oral primordium is an elongated field, arrow and double-arrowhead point to the dedifferentiated buccal cirrus and the appearance of the oral primordium of the proter, respectively. Arrowhead indicates the frontal–ventral–transverse cirral anlage formed within the old ventral left row. ((B), (C)) Ventral (B) and dorsal (C) view of the same specimen in the early stage of morphogenesis. In B, white arrows point to the parental right ventral row. In C, arrow shows the apokinetal formation of the dorsal kineties anlage 3 in the proter, arrowheads mark the intrakinetal development of the other dorsal kineties anlagen. ((D), (E)) Ventral (D) and dorsal (E) view of a middle divider, arrows mark the differentiation of frontal–ventral–transverse cirral anlagen in both proter and opisthe, double-arrowheads and arrowheads indicate the anlagen for left and right ventral rows, respectively. (F) Ventral view of a later divider, arrows point to the left frontal cirri formed from the undulating membranes anlagen, arrowheads mark the buccal cirri, double-arrowheads show the post-peristomial cirri, white arrowheads indicate the median segment of the left ventral cirral row and white arrows indicate the anterior segment of the left ventral cirral row. I–VI, frontal–ventral–transverse cirral anlagen; LMA, anlagen of left marginal row; Ma, macronuclear nodules; Mi, micronuclei; OP, oral primordium; RMA, anlagen of right marginal row; 1–3, dorsal kineties. Bars, 150 m.
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Fig. 5. ((A)–(F)). Late dividers of Pseudouroleptus caudatus caudatus, after protargol staining. ((A)–(D)) Ventral ((A), (C)) and dorsal ((B), (D)) views. In A and C, arrows point to the left frontal cirri formed from the undulating membranes anlagen, arrowheads mark the buccal cirri, double-arrowheads show the post-peristomial cirri, white arrowheads and white arrows mark the median and anterior segments of the left ventral cirral row, respectively. In B and D, arrows point to the segmentation of dorsal kineties anlagen 3. Note the caudal cirri are formed from the posterior ends of the dorsal kinety anlagen (arrowheads in D). ((E), (F)) Ventral (E) and dorsal (F) views of a very late divider, showing that all cirri have migrated to their final positions. Arrowheads in E indicate the buccal cirri, double-arrowheads in E point to the post-peristomial cirri, and arrowheads in F mark the caudal cirri. LMA, anlagen of left marginal row; LMR, left marginal row; LVR, left ventral row; Ma, macronuclear nodules; Mi, micronuclei; RMA, anlagen of right marginal row; RMR, right marginal row; RVR, right ventral row; 1–4, dorsal kineties. Bars, 150 m ((A)–(D)) and 85 m ((E), (F)).
of basal bodies, the new membranelles differentiate in a posteriad direction and the distal end of the developing adoral zone bends slightly rightwards (Figs 4B, D, 6B). At the same time, FVTA V continues to develop, FVTA I-IV are formed between the OP and FVTA V, and FVTA VI originates to the right of FVTA V (Figs 4B, D, 6B). Later, the undulating membranes anlage begins to split forming the paroral and endoral membranes and also gives rise to the left frontal cirrus. By this time the differentiation of the FVTA is almost finished: FVTA I differentiates into the left frontal cirrus; FVTA II generates the middle frontal cirrus and the buccal cirrus; FVTA
III contributes the right frontal cirrus, cirrus III/2 and another smaller cirrus which will be resorbed during the following stages; FVTA IV develops the median segment of the left ventral cirral row and the post-peristomial cirrus; FVTA V differentiates into the posterior segment of the left ventral row; and FVTA VI generates the right ventral row and the anterior segment of the left ventral cirral row (Figs 4F, 5A, C, 6E). In the last stages, the paroral and endoral are completely separated, the frontoventral ciliature is completely differentiated and the migration process is almost finished (Fig. 5E). The median segment of the left ventral cirral row migrates
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Fig. 6. ((A)–(I)). Photomicrographs of Pseudouroleptus caudatus caudatus during divisional morphogenesis (after protargol staining). (A) Ventral view of an early divider, showing the appearance of the oral primordium of the opisthe, white arrow points to the appearance of the oral primordium on the surface of the cell. (B) Ventral view of a later divider, arrows mark the frontal–ventral–transverse cirral anlagen in both proter and opisthe, double-arrowheads show the anlagen for the left ventral rows formed intrakinetally, arrowheads mark the anlagen for the right ventral rows, and white arrows point to the parental right ventral rows. (C) Dorsal view of anterior region of cell, showing the dorsal kineties anlagen (arrows). (D) Ventral view of the frontal area, white arrows point to the parental right ventral rows. (E) Ventral view of a middle divider, arrows point to the left frontal cirri in both proter and opisthe, arrowheads mark the buccal cirri, double-arrowheads show the post-peristomial cirri, white arrowheads and white arrows mark the median and anterior segments of the left ventral cirral row, respectively. (F) Dorsal view, to show the segmentation of the anlage of dorsal kinety 3 (arrowheads). (G) Ventral view of the proter in late morphogenetic stage, double-arrowhead indicates the post-peristomial cirrus. (H) Dorsal view of the opisthe, to indicate the newly formed caudal cirri (arrowheads) and the left marginal row (arrow). (I) Dorsal view of the posterior portion of the proter, arrowheads point to the caudal cirri developing from the ends of dorsal kinety anlagen 1 and 2. I–VI, frontal-ventral-transverse cirral anlagen; III/2, cirrus III/2; LMA, left marginal row anlage of proter; Ma, macronuclear nodules; Mi, micronuclei; OP, oral primordium; RMA, right marginal row anlage of proter; 1–3, dorsal kineties. Bars, 30 m ((C), (D), (G)) and 100 m ((B), (E)).
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Fig. 7. Maximum likelihood (ML) phylogenetic tree based on the small subunit rRNA (SSU rRNA) gene sequences. Numbers at the nodes represent the bootstrap values of ML analyses and posterior probability of BI analyses. Fully supported (100%/1.00) branches are marked with solid circles. Asterisk (*) represents support values less than 50% and the disagreement between BI and the reference ML tree. The scale bar corresponds to two substitutions per 100 nucleotide positions. The newly sequenced species in the present study is shown in bold and the red box shows the close relationship between P. caudatus caudatus and Pseudouroleptus caudatus.
ahead of the left ventral row, and the anterior segment of the left ventral cirral row migrates in front of the median segment of the left ventral cirral row, collectively forming a mixed (left ventral) row (Fig. 5E).
Proter. Some key stages during the ontogenetic process were not observed, so the origin of the FVTA is not clear. Morphogenesis begins with the differentiation of the buccal cirrus (Fig. 4A) and the appearance of the oral primordium on the surface of the cell (Fig. 4A). As in most other hypotrichs, the parental adoral zone is retained completely intact by the proter and no new membranelles are formed, so changes of the oral structure are confined to the paroral and endoral. In the next stage, the undulating membranes anlage is formed, probably from dedifferentiation of the parental paroral and endoral, and FVTA V and VI appear (Figs 4B, D, 6B, D). In the following stages, the development of the undulating membranes anlage and FVTA is the same as that in the opisthe (Fig. 6G).
During the late stage of the divisional process, the ventral cirral pattern of interphase cells can be entirely characterized on the basis of cirral migration: (i) cirri I/1, II/3 and III/3 correspond the three frontal cirri, and cirri II/2, III/2 and IV/2 correspond the buccal cirrus, the parabuccal cirrus, and the post-peristomial cirrus, respectively (Figs 4F, 5A, C, E, 6E, G); (ii) the anterior segments of streaks VI and IV and the whole of streak V form the anterior, middle, and posterior segment, respectively, of the left ventral row which is thus a mixed row (Figs 4F, 5A, C, 6E, G); (iii) the posterior segment of streak VI develops into the right ventral cirral row (Figs 4F, 5A, C, 6E, G).
Marginal cirri. The new marginal rows originate in the usual way for hypotrichs, i.e. in middle dividers one anlage develops each for the proter and the opisthe within each parental row (Figs 4B, D, F, 5A, C, 6B, E). These anlagen then
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stretch mainly posteriad and gradually replace the parental rows (Fig. 5E).
Dorsal ciliature. The development of the dorsal kineties anlagen (DKA) commences in early dividers (Figs 4C, 6C). DKA1 and 2 develop intrakinetally in both opisthe and proter (Fig. 4C). DKA3 emerges as a row of basal bodies that develops de novo to the right of parental dorsal kinety 3 in the proter, but intrakinetally within the parental structure in the opisthe (Fig. 4C). The DKA then stretch mainly posteriad and gradually replace the parental rows (Fig. 4E). In late dividers, DKA3 fragments in the posterior region forming two anlagen. Thus, four rows of dorsal kineties are formed in each daughter cell (Figs 5B, D, 6F). During the morphogenetic process, three or four caudal cirri are formed at the posterior end of DKA1, and one or two are formed at the end of DKA2 (Figs 5D, F, 6H, I).
Nuclear apparatus. The division of the nuclear apparatus proceeds in the usual way for hypotrichs (Berger 2008). The two macronuclear nodules fuse into a single mass which subsequently makes successive amitotic divisions to produce the species-specific number of nodules (two in the case of Pseudouroleptus caudatus caudatus) in each filial product (Figs 4E, F, 5B, D, 6F–H). The micronuclei divide mitotically (Figs 5D, F, 6G–I).
SSU rRNA gene sequence and phylogenetic analyses (Fig. 7) The SSU rRNA gene sequence of Pseudouroleptus caudatus caudatus is deposited in GenBank with the accession number KF591597. The length and G + C content of the new sequence are 1773 bp and 45.23%, respectively. Phylogenetic trees constructed using BI and ML methods show basically congruent topologies (Fig. 7). The Tibetan population of P. caudatus caudatus clusters with the Brazilian population of P. caudatus with high support (82% ML, 1.00 BI), which together form a fully supported clade with two Strongylidium species (100% ML, 1.00 BI). The difference in branch lengths of the Tibetan and the Brazilian populations of P. caudatus suggests that the latter might be another subspecies, namely P. caudatus namibiensis. More molecular data of these taxa are needed to test this hypothesis.
Discussion Identification of the Tibetan population of P. caudatus caudatus Pseudouroleptus caudatus was first isolated from freshwater samples collected in Peru (Hemberger 1985; Berger 2008). Its original description, however, was based only on silverstained specimens. Subsequently, populations were reported
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from samples collected in Spain (see Berger 2008) and Argentina (Küppers and Claps 2013). Furthermore, Foissner et al. (2002) described two African populations as separate subspecies, namely P. caudatus caudatus and P. caudatus namibiensis. The main diagnostic feature separating these two subspecies is the length of the right ventral row (labelled as transverse cirri in Foissner et al. 2002): in the former it extends anteriorly to near the level of the buccal cirrus whereas in the latter it terminates anteriorly atin the midbody region. Based on this criterion, all three previously described populations (Peru, Spain, Argentina) of P. caudatus are the subspecies P. caudatus caudatus. For comparative purposes, details of the main morphological features of these populations are given in Table 2.
Special morphogenetic characters of Pseudouroleptus and related genera Based on the Tibetan population, the most characteristic events during morphogenesis in Pseudouroleptus caudatus caudatus can be summarized as follows: (i) the parental adoral zone of membranelles is retained completely; (ii) the left ventral row is formed from three parts, i.e. the anterior segments of streaks VI and IV, and the entire streak V form the anterior, middle, and posterior segments, respectively; (iii) the right ventral row originates de novo in both daughter cells; (iv) the marginal rows develop intrakinetally; (v) dorsal kineties anlagen 1 and 2 develop intrakinetally in both opisthe and proter, but dorsal kinety anlage 3 forms differently in each daughter, i.e. de novo in the proter and intrakinetally in the opisthe; and (vi) the nuclear apparatus divides in the usual way for hypotrichs. Thus, the Tibetan population has a basically similar process of morphogenesis to that of the Peruvian population (Hemberger 1985; Berger 1999). Morphologically, Hemiamphisiella Foissner, 1988 and Strongylidium Sterki, 1878 are the most similar genera to Pseudouroleptus, and share the same arrangement of the left ventral cirral row. Our phylogenetic analyses based on SSU rRNA gene sequence data support the close relationship of Pseudouroleptus and Strongylidium. Molecular data for Hemiamphisiella are not yet available. The pattern of replication of the dorsal kineties, however, differs significantly among these genera with the third dorsal kinety forming de novo without fragmentation in Strongylidium and Hemiamphisiella (vs. third kinety fragments in Pseudouroleptus), and the fourth dorsal kinety does not fragment in Strongylidium and Hemiamphisiella (vs. the fourth kinety in Pseudouroleptus is the result of fragmentation of kinety 3) (Berger 2008). The pattern of formation of the right ventral row also differs among these genera, developing de novo to the right of the parental structure in Pseudouroleptus and Hemiamphisiella versus intrakinetally in Strongylidium (Paiva and Silva-Neto 2007; Chen et al. 2013b).
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Table 2. Comparison of various populations of Pseudouroleptus caudatus caudatus and P. caudatus namibiensis.
1
2
3
4
5
6
Body, length in vivo (m) Body, width in vivo (m) Length:width ratio in vivo Body (length) (protargol) (m) Body, (width) (protargol) (m) Length:width ratio (protargol) Cortical granules Position of CV
– – – 250 50 4:1a Brownishb – – ca. 25% – 33–50 1 3 1 1 42 55 45 55 – 4 – – – – – 2 – – 2–3 – – Soil & freshwater Hemberger (1982)
180–280 40–60 5:1 150–255 39–55 2.5:1 Colourless 50% of body length, near left cell margin Absent 21–37% 42–75 40–63 1 3 1 1 40–70 35–60 35–53 43–70 – 4 – – – – – 2 20–30 7–11 2–4 7–10 5–6 Soil Foissner et al. (2002)
180–330 40–60 – 167–295 40–55 4.7:1 Colourless –
Collecting canals Ratio of AZM in vivo AZM (length) (m) AZM (number) Buccal cirri (number) Frontal cirri (number) Parabuccal cirri (number) Post-peristomial ventral cirri (number) Left ventral cirri (number) Right ventral cirri (number) Left marginal cirri (number) Right marginal cirri (number) Caudal cirri (number) Dorsal kineties (number) Dikinetids in DK1 (number) Dikinetids in DK2 (number) Dikinetids in DK3 (number) Dikinetids in DK4 (number) Dikinetids in DK5 (number) Macronuclear nodules (number) Macronuclear nodules (length) (m) Macronuclear nodules (width) (m) Micronuclei (number) Micronuclei (length) (m) Micronuclei (width) (m) Habitat Data source
150–270 40–70 5:1a 160–280 45–90 4:1a Colourless 50% of body length, near left cell margin – ca. 33% 55–85 40–60 1 3 1–2 0–2 40–63 47–68 39–62 48–68 3–4 4–5 30–55c 30–55c 30–55c 30–55c – 2 35–50 18–25 2–5 8–12 6–10 Sediment Olmo-Rísquez (1998)
161–252 56–105 – 196–308 49–112 – Colourless 33% of body length, near left cell margin Present 30% 63–91 43–63 1 3 1 1–2 42–63 42–56 40–70 50–75 4–5 4 – – – – – 2 27–43 8–17 2–5 5–11 4–6 Soil Küppers and Claps (2013)
230–320 45–65 5:1 230–325 88–138 4.3:1 Colourless 33% of body length, near left cell margin Absent 20–25% 70–90 54–67 1 3 1 0–1 60–77 51–65 56–72 57–76 3–6 4 28–44 28–43 25–47 22–34 – 2 21–45 9–24 2–7 5–10 3–5 Soil Original
– – 46–71 33–51 1 3 1 1–3 50–67 20–31 44–61 47–70 1–4 4 – – – – – 2 18–27 6–12 1–4 5–7 4–5 Soil & freshwater Foissner et al. (2002)
Population: 1 – Peru population; 2 – Spain population; 3 – Africa population (Pseudouroleptus caudatus caudatus); 4 – Africa population (P. caudatus namibiensis); 5 – Argentina population; 6 – Tibet population. a Data from drawings. Abbreviations: AZM – adoral zone of membranelles; CV – contractile vacuole; DK1–4 – dorsal kineties. b According to Foissner (2002), a feature likely caused by the dense cortical granulation. c According to Olmo-Rísquez (1998), measurement of dorsal kineties 1–4 is likely inaccurate.
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Character
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Acknowledgements This work was supported by the National Natural Science Foundation of China (project numbers: 31372148 to C. Shao and H. Ma, 31172041 to C. Shao and Z. Yi) and the China Scholarship Council, which funded an extended visit by the principal author to North Carolina Central University, USA. We are grateful to the associate editor Helmut Berger for helpful critical suggestions and comments on the manuscript.
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ScienceDirect European Journal of Protistology 51 (2015) 1–14
Morphology, morphogenesis and molecular phylogeny of a soil ciliate, Pseudouroleptus caudatus caudatus Hemberger, 1985 (Ciliophora, Hypotricha), from Lhalu Wetland, Tibet Lingyun Chena,b,1 , Xiaolu Zhaoa,1 , Honggang Maa , Alan Warrenc , Chen Shaob,∗ , Jie Huangd,∗ a
Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China The Key Laboratory of Biomedical Information Engineering Ministry of Education, Department of Biology and Engineering, 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 d Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China b
Received 16 December 2013; received in revised form 1 August 2014; accepted 1 September 2014 Available online 6 September 2014
Abstract Pseudouroleptus caudatus caudatus Hemberger, 1985, a soil ciliate isolated from Tibet, was studied in vivo and after protargol impregnation. The Tibetan population is mainly characterized by: elongate body with narrowly rounded anterior end and tapered posterior end; length of buccal area relative to body length ca. 20–25%; cortical granules colourless, round, densely distributed throughout sub-pellicular layer of cell; one parabuccal cirrus; post-peristomial cirrus lacking in 75% of specimens analyzed; left and right ventral rows commence at same level; four dorsal kineties; 3–6 inconspicuous caudal cirri; two macronuclear nodules; 2–7 micronuclei; contractile vacuole located at about 33% of body length near left margin. Morphogenesis is characterized by: (1) parental adoral zone of membranelles retained completely; (2) anterior segments of streaks VI and IV and the whole of streak V form the anterior, middle, posterior segments of the mixed row, respectively; (3) right ventral row originates de novo in both daughter cells; (4) marginal rows develop intrakinetally; (5) dorsal kinety anlage 3 develops de novo in the proter and intrakinetally in the opisthe; and (6) the two macronuclear nodules fuse into a single mass which then divides. Molecular phylogenies corroborate the morphological identification and support the close relationship between Pseudouroleptus and Strongylidium. © 2014 Elsevier GmbH. All rights reserved.
Keywords: Hypotricha; Morphogenesis; Phylogeny; Pseudouroleptus caudatus caudatus; Taxonomy
Introduction
∗ Corresponding
authors. E-mail addresses: [email protected] (C. Shao), [email protected] (J. Huang). 1 Co-first authors. http://dx.doi.org/10.1016/j.ejop.2014.09.001 0932-4739/© 2014 Elsevier GmbH. All rights reserved.
The Hypotricha Stein, 1859 is a speciose and morphologically diverse group that inhabits a wide range of biotopes (Berger 2008; Chen et al. 2013a,b; Fan et al. 2014; Foissner 2012; Jiang et al. 2013; Kahl 1932; Küppers and Claps 2013; Li et al. 2010a,b, 2013; Liu et al. 2010; Lv et al. 2013; Paiva
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et al. 2012; Pan et al. 2012, 2013; Park et al. 2013; Song et al. 2009; Weisse et al. 2013). The genus Pseudouroleptus was established by Hemberger (1985) with P. caudatus as the type species by original designation. Subsequently, P. caudatus was split into two subspecies, P. caudatus caudatus and P. caudatus namibiensis, mainly according to the distinct difference in the number of transverse cirri. The former subspecies has a widespread geographic distribution, occurring in both terrestrial and limnetic habitats, whereas the latter has so far only been reported from the type location (Foissner et al. 2002). Until recently there were five species of Pseudouroleptus that lacked dorsal kinety fragmentation. However, Berger (2008) assigned four of these to Bistichella leaving only the type species, P. caudatus, in the genus. The ciliature of Pseudouroleptus is generally similar to that of Strongylidium, with each possessing buccal, frontal, parabuccal and post-peristomial cirri, two marginal rows and two long ventral cirral rows. However, during morphogenesis the right ventral cirral row forms de novo in Pseudouroleptus versus intrakinetally in Strongylidium (Chen et al. 2013b). To date, morphogenesis has been described in detail for only one of the two subspecies of P. caudatus, namely P. caudatus caudatus (Hemberger 1985; Berger 2008). Likewise, only one small subunit (SSU) rRNA gene sequence of P. caudatus is available in GenBank, that of a Brazilian population that was not identified to subspecies level (Paiva et al. 2009). Therefore, in order to increase knowledge and understanding of the systematics and biodiversity of the genus Pseudouroleptus, morphological, morphogenetic and molecular studies from a wider range of isolates are needed. In the spring of 2011, the subspecies Pseudouroleptus caudatus caudatus Hemberger, 1985 was isolated from soil samples collected from a wetland in Tibet, China. We here describe its morphology, morphogenesis, and molecular phylogeny based on SSU rRNA gene sequence data.
Material and Methods Sampling and cultivation Soil samples were collected on 1st May 2011 from the Lhalu Wetland in the northwest of Lhasa, Tibet (29◦ 40 45 N, 91◦ 05 48 E) (Fig. 1A–D). This wetland is at an altitude of 3645 m above sea level where the annual average temperature is 7.6 ◦ C and the annual average precipitation is 439 mm. Water enters the wetland from two heavy-sedimentcarrying rivers, the River Duodi and River Niangre, and also in the form of industrial effluent from the Najin Power Station. The dominant hydrophyte in the wetland is the common reed Phragmites australis. The sample comprised 500 g of soil from the top 10 cm layer. Ciliates were stimulated to excyst and emerge from the soil
sample by employing the non-flooded Petri dish method (Foissner 1987; Foissner et al. 2002). Ciliates were isolated and cultured at room temperature (20 ◦ C) in Petri dishes containing distilled water with squeezed rice grains to enrich the availability of bacterial food (Chen et al. 2013c).
Morphology and morphogenesis Cells were examined both in vivo using bright field and differential interference contrast microscopy, and following protargol staining in order to reveal the infraciliature (Wilbert 1975). Counts and measurements of morphological characters in protargol-stained specimens were performed at a magnification of ×1000. Drawings were made with the help of a camera lucida (Shao et al. 2013a,b). Two voucher slides with protargol-stained specimens have been deposited, one in the laboratory of Protozoology, Ocean University of China (registration number: CLY2011050103) and the other in the Natural History Museum, London (registration number: NHMUK 2013.12.15.1). Terminology mainly follows Hemberger (1985) and Küppers and Claps (2013). Systematic follows Lynn (2008). Numbering of the cirri follows Berger (1999, 2008).
DNA extraction, PCR amplification and sequencing Genomic DNA was extracted from cells washed with autoclaved freshwater using DNeasy Tissue Kit (Qiagen, CA) according to Chen et al. (2013d). PCR amplification of SSU rDNA was performed using the universal primers EukA and EukB (Medlin et al. 1988; Yi and Song 2011). Highfidelity Taq polymerase (Takara Ex Taq; Takara Biomedicals) was used to minimize the possibility of amplification errors. The amplification cycles were as follows: 5 min at 94 ◦ C, followed by 30 cycles of 94 ◦ C for 30 s, 56 ◦ C for 1 min, 72 ◦ C for 1 min 50 s; the final extension was 7 min at 72 ◦ C (Huang et al. 2010). The PCR product was purified using SanPrep DNA Gel Extraction Kit (Sangon Bio. Co., Shanghai, China) and then inserted into the pMDTM 19-T vector (Takara Biotechnology, Dalian Co., Ltd.). Sequencing was performed bidirectionally on an ABI 3700 sequencer (Invitrogen sequencing facility, Shanghai, China) using primers M1347 (5 -CGCCAGGGTTTTCCCAGTCACGAC-3 ), M13-48 (5 -AGCGGATAACAATTTCACACAGGA-3 ) and three internal primers 900F (5 -CGATCAGATACCGTCCTAGT3 ), 900R (5 -ACTAGGACGGTATCTGATCG-3 ) and Pro B (5 -GGTTAAAAAGCTCGTAGT-3 ).
Phylogenetic analyses Phylogenetic analyses were carried out on the SSU rRNA gene sequence of the Tibetan population of
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Fig. 1. ((A)–(D)). The sample site and surrounding area. (A) The location of the Lhalu Wetland, arrow indicates the Potala Palace; inset shows the location of Tibet. ((B)–(D)) Photographs showing the locality where the samples containing Pseudouroleptus caudatus caudatus were collected. (B) Showing the Potala Palace.
Pseudouroleptus caudatus caudatus and of 53 other sequences downloaded from GenBank (see Fig. 7 for accession numbers except those of the following 13 hypotrichs: Hemicycliostyla sphagni FJ361758, Pseudourostyla cristata FJ598608, Anteholosticha sp. HM568416, Heterokeronopsis pulchra JQ083600, Nothoholosticha fasciola FJ377548, Pseudokeronopsis carnae AY881633, Uroleptopsis citrina FJ870094, Monocoronella carnea FJ775726, Metaurostylopsis salina EU220229, Apokeronopsis wrighti EU417963, Thigmokeronopsis stoecki EU220226, Diaxonella trimarginata DQ190950 and Urostyla grandis AF508781). Sequences were aligned using Clustal W implemented in Bioedit 7.0.9 with default parameters (Hall 1999). Regions that could not be aligned unambiguously were removed and ends were trimmed manually, resulting in a matrix of 1731 characters.
Five species of the subclasses Oligotrichia and Choreotrichia were chosen as the outgroup taxa. The nucleotide substitution model GTR + I + G (Shape = 0.4685, Pinvar = 0.5003) was selected under AIC criteria using MrModeltest 2.2 (Nylander 2004). This model was used to perform Bayesian Inference (BI) analysis with MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003). Markov chain Monte Carlo (MCMC) simulations were run with two sets of four chains for 1000,000 generations with a sampling frequency of 100. The first 25% were discarded as burn-in prior to constructing 50% majority rule consensus trees. The maximum likelihood (ML) tree was constructed with the PhyML V2.4.4 program (Guindon and Gascuel 2003) using the model GTR + I + G (Shape = 0.4724, Pinvar = 0.5018) selected in Modeltest (Posada and Crandall 1998). The reliability of internal branches was evaluated by posterior probabilities (BI) and 1000 non-parametric bootstrap
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Fig. 2. ((A)–(D)). Morphology of Pseudouroleptus caudatus caudatus from life ((A), (B)) and after protargol staining ((C), (D)). (A) Ventral view of a representative individual. (B) Detail of cell (dorsal view), to show the distribution of cortical granules (possibly mitochondria). ((C), (D)) Ventral and dorsal view of the same individual, showing infraciliature and nuclear apparatus, arrow points to the post-peristomial cirrus and arrowhead indicates the buccal cirrus. AZM, adoral zone of membranelles; CC, caudal cirri; CV, contractile vacuole; FC, frontal cirri; III/2, cirrus III/2; LMR, left marginal row; LVR, left ventral row; Ma, macronuclear nodules; Mi, micronuclei; RMR, right marginal row; RVR, right ventral row; 1–4, dorsal kineties. Bars, 150 m.
pseudoreplicates (ML). MEGA 5 (Kumar et al. 2008) was used to visualize tree topologies.
Morphological description of Tibet population (Figs 2A–D, 3A–I; Table 1)
Results
This subspecies has been redescribed by Foissner et al. (2002) and Küppers and Claps (2013), hence, here we only include data that are specific to the Tibetan population.
Class Spirotrichea Bütschli, 1889 Order Stichotrichida Fauré-Fremiet, 1961 Family Spirofilidae von Gelei, 1929 Genus Pseudouroleptus Hemberger, 1985 Pseudouroleptus caudatus caudatus Hemberger, 1985
Morphology Body 230–320 × 45–65 m in vivo, flexible and slightly contractile, outline more or less fusiform, narrowly rounded anteriorly and tapered posteriorly to form a short tail that is
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Fig. 3. ((A)–(I)). Photomicrographs of Pseudouroleptus caudatus caudatus from life ((A)–(E)) and after protargol staining ((F)–(I)). (A) Ventral view of a representative specimen. (B) Showing a bending specimen. (C) Detail of cell surface in dorsal view, to show the distribution of cortical granules, possibly mitochondria (arrows). (D) Nuclear apparatus. (E) Posterior body region, to show the food vacuoles (arrows). (F) Ventral view of anterior portion of cell, to show, inter alia, the buccal cirrus (arrowhead) and the post-peristomial cirrus (arrow). (G) Dorsal view of anterior portion of cell, to show the macronuclear nodules and micronuclei. (H) Ventral view of the midbody region. (I) Posterior region of cell showing the caudal cirri (arrowheads). CV, contractile vacuole; FC, frontal cirrus; III/2, cirrus III/2; LMR, left marginal row; LVR, left ventral row; Ma, macronuclear nodules; Mi, micronuclei; RMR, right marginal row; RVR, right ventral row. Bar, 150 m.
more flexible and contractile than the rest of the cell; length to width ratio about 5:1 in vivo on average, 2.5:1 in protargolstained cells; right margin sigmoidal, curving to right in posterior region, left margin varying from almost straight to sigmoidal (Figs 2A, C 3A, B). Cytoplasm colourless to greyish, usually with numerous lipid droplets ca. 3–5 m in diameter, and food vacuoles 5–10 m in diameter containing
algae that render cell dark when observed at low magnification (Fig. 3A, E). Contractile vacuole about 20 m across, located at about 33% of body length near left cell margin (Figs 2A, 3A). Cortical granules (possibly mitochondria) colourless, round, about 1.5 m in diameter, and densely distributed in the sub-pellicular layer throughout the entire cell (Figs 2B, 3C). Invariably two macronuclear nodules in anterior half
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Table 1. Morphometric data for the Tibet population of Pseudouroleptus caudatus caudatus. Charactera
Min
Max
Mean
SD
Body (length) Body (width) Adoral zone of membranelles (length) Adoral membranelles (number) Buccal cirri (number) Frontal cirri (number) Frontoventral cirri (number) Postperistomial ventral cirri (number) Left ventral cirri (number) Right ventral cirri (number) Left marginal cirri (number) Right marginal cirri (number) Caudal cirri (number) Caudal cirri in DK1 (number) Caudal cirri in DK2 (number) Dorsal kineties (number) Dikinetids in DK1 (number) Dikinetids in DK2 (number) Dikinetids in DK3 (number) Dikinetids in DK4 (number) Macronuclear nodules (number) Length of macronuclear nodules Width of macronuclear nodules Micronuclei (number) Length of micronuclei Width of micronuclei
230 88 70 54 1 3 1 0 60 51 56 57 3 1 1 4 28 28 25 22 2 21 9 2 5 3
325 138 90 67 1 3 1 1 77 65 72 76 6 4 2 4 44 43 47 34 2 45 24 7 10 5
276.3 109.3 77.8 59.1 1.0 3.0 1.0 0.3 69.7 59.6 63.2 67.6 4.9 3.07 1.67 4.0 34.2 34.0 32.8 28.0 2.0 28.8 13.5 3.4 7.5 4.1
27.36 13.75 4.79 3.39 0 0 0 0.44 5.02 3.83 4.59 5.49 0.85 1.10 0.49 0 4.01 4.40 5.45 3.71 0 5.04 3.69 1.28 1.04 0.53
CV
n
9.9 12.6 6.2 5.7 0 0 0 146.7 7.2 6.4 7.3 8.1 17.3 35.9 29.3 0 11.7 12.9 16.6 13.3 0 17.5 27.3 37.6 13.9 12.9
20 20 20 20 20 20 20 20 20 20 20 20 20 15 15 20 20 20 20 20 25 28 28 14 47 47
a All data are based on protargol-impregnated specimens. Measurements in m. Abbreviations: CV, coefficient of variation in %; DK1–4, dorsal kineties 1–4; Max, maximum; Mean, arithmetic mean; Min, minimum; n, number of cells measured; SD, standard deviation.
of body, anterior one in cell midline, posterior one slightly left of midline; nodules ellipsoidal, length: width ratio about 2:1, with small to moderately large nucleoli. Two-to-seven micronuclei (Figs 2D, 3D, F). Locomotion usually by moderately rapid swimming while rotating clockwise about main body axis, or occasionally by gliding on substrate. Adoral zone occupies nearly 20–25% of body length in vivo (about 28% in protargol-stained cells), curving across anterior cell margin and extending conspicuously down right side of body, i.e., DE-value 0.37 in the holotype specimen (Fig. 3F; for explanation of DE-value, see Berger 2006: 18). Adoral zone comprises 59 membranelles on average, bases of largest membranelles about 15 m wide, cilia up to 20 m long (Fig. 2C). Paroral and endoral approximately equal in length, distinctly curved, intersecting optically at posterior quarter. Paroral consists of dikinetids whereas endoral is composed of monokinetids. Three clearly differentiated frontal cirri; one conspicuous parabuccal cirrus (III/2) located below rightmost frontal cirrus: cilia of frontal cirri and parabuccal cirrus about 15 m long (Figs 2C, 3F). Single buccal cirrus located about 10% down body length, right of paroral. In five out of 20 specimens investigated one post-peristomial cirrus was present, located behind proximal end of adoral zone (Figs 2C, 3F). Two long, oblique, sigmoid ventral cirral rows, beginning approximately near right frontal cirrus
and terminating at posterior end of cell, comprising 60–77 (left) and 51–65 (right) cirri, respectively (Figs 2C, 3H). Left and right marginal rows composed of 56–72 and 57–76 cirri, respectively, cilia of which are 10–15 m long, terminating at posterior end of cell (Figs 2A, C, 3F, G). After protargol staining, some fibres were observed associated with cirri of ventral rows (Fig. 3F). Transverse cirri absent. Four dorsal kineties extending almost entire length of cell; dorsal cilia 5 m long; no dorsomarginal kineties formed (Figs 2D, 3G). Three to six caudal cirri, densely packed and located at posterior body margin (Figs 2D, 3I).
Morphogenesis (Figs 4A–F, 5A–F, 6A–I) Oral apparatus and frontal-ventral-transverse cirral anlagen Opisthe. In the first stage of morphogenesis a long field of basal bodies develops left of the mid-region of the left ventral row (Figs 4A, 6A). At the same time, a streak is formed in the left ventral row, at about the level of the anterior end of the oral primordium (OP; Fig. 4A). Based on subsequent stages, we deduce that this streak is the frontal-ventral-transverse cirral anlage (FVTA) V (Fig. 4B). During the next stage the oral primordium continues to grow by further proliferation
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Fig. 4. A–F. Divisional morphogenesis in Pseudouroleptus caudatus caudatus from early to late stages (after protargol staining). (A) Ventral view of an early divider, note the oral primordium is an elongated field, arrow and double-arrowhead point to the dedifferentiated buccal cirrus and the appearance of the oral primordium of the proter, respectively. Arrowhead indicates the frontal–ventral–transverse cirral anlage formed within the old ventral left row. ((B), (C)) Ventral (B) and dorsal (C) view of the same specimen in the early stage of morphogenesis. In B, white arrows point to the parental right ventral row. In C, arrow shows the apokinetal formation of the dorsal kineties anlage 3 in the proter, arrowheads mark the intrakinetal development of the other dorsal kineties anlagen. ((D), (E)) Ventral (D) and dorsal (E) view of a middle divider, arrows mark the differentiation of frontal–ventral–transverse cirral anlagen in both proter and opisthe, double-arrowheads and arrowheads indicate the anlagen for left and right ventral rows, respectively. (F) Ventral view of a later divider, arrows point to the left frontal cirri formed from the undulating membranes anlagen, arrowheads mark the buccal cirri, double-arrowheads show the post-peristomial cirri, white arrowheads indicate the median segment of the left ventral cirral row and white arrows indicate the anterior segment of the left ventral cirral row. I–VI, frontal–ventral–transverse cirral anlagen; LMA, anlagen of left marginal row; Ma, macronuclear nodules; Mi, micronuclei; OP, oral primordium; RMA, anlagen of right marginal row; 1–3, dorsal kineties. Bars, 150 m.
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Fig. 5. ((A)–(F)). Late dividers of Pseudouroleptus caudatus caudatus, after protargol staining. ((A)–(D)) Ventral ((A), (C)) and dorsal ((B), (D)) views. In A and C, arrows point to the left frontal cirri formed from the undulating membranes anlagen, arrowheads mark the buccal cirri, double-arrowheads show the post-peristomial cirri, white arrowheads and white arrows mark the median and anterior segments of the left ventral cirral row, respectively. In B and D, arrows point to the segmentation of dorsal kineties anlagen 3. Note the caudal cirri are formed from the posterior ends of the dorsal kinety anlagen (arrowheads in D). ((E), (F)) Ventral (E) and dorsal (F) views of a very late divider, showing that all cirri have migrated to their final positions. Arrowheads in E indicate the buccal cirri, double-arrowheads in E point to the post-peristomial cirri, and arrowheads in F mark the caudal cirri. LMA, anlagen of left marginal row; LMR, left marginal row; LVR, left ventral row; Ma, macronuclear nodules; Mi, micronuclei; RMA, anlagen of right marginal row; RMR, right marginal row; RVR, right ventral row; 1–4, dorsal kineties. Bars, 150 m ((A)–(D)) and 85 m ((E), (F)).
of basal bodies, the new membranelles differentiate in a posteriad direction and the distal end of the developing adoral zone bends slightly rightwards (Figs 4B, D, 6B). At the same time, FVTA V continues to develop, FVTA I-IV are formed between the OP and FVTA V, and FVTA VI originates to the right of FVTA V (Figs 4B, D, 6B). Later, the undulating membranes anlage begins to split forming the paroral and endoral membranes and also gives rise to the left frontal cirrus. By this time the differentiation of the FVTA is almost finished: FVTA I differentiates into the left frontal cirrus; FVTA II generates the middle frontal cirrus and the buccal cirrus; FVTA
III contributes the right frontal cirrus, cirrus III/2 and another smaller cirrus which will be resorbed during the following stages; FVTA IV develops the median segment of the left ventral cirral row and the post-peristomial cirrus; FVTA V differentiates into the posterior segment of the left ventral row; and FVTA VI generates the right ventral row and the anterior segment of the left ventral cirral row (Figs 4F, 5A, C, 6E). In the last stages, the paroral and endoral are completely separated, the frontoventral ciliature is completely differentiated and the migration process is almost finished (Fig. 5E). The median segment of the left ventral cirral row migrates
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Fig. 6. ((A)–(I)). Photomicrographs of Pseudouroleptus caudatus caudatus during divisional morphogenesis (after protargol staining). (A) Ventral view of an early divider, showing the appearance of the oral primordium of the opisthe, white arrow points to the appearance of the oral primordium on the surface of the cell. (B) Ventral view of a later divider, arrows mark the frontal–ventral–transverse cirral anlagen in both proter and opisthe, double-arrowheads show the anlagen for the left ventral rows formed intrakinetally, arrowheads mark the anlagen for the right ventral rows, and white arrows point to the parental right ventral rows. (C) Dorsal view of anterior region of cell, showing the dorsal kineties anlagen (arrows). (D) Ventral view of the frontal area, white arrows point to the parental right ventral rows. (E) Ventral view of a middle divider, arrows point to the left frontal cirri in both proter and opisthe, arrowheads mark the buccal cirri, double-arrowheads show the post-peristomial cirri, white arrowheads and white arrows mark the median and anterior segments of the left ventral cirral row, respectively. (F) Dorsal view, to show the segmentation of the anlage of dorsal kinety 3 (arrowheads). (G) Ventral view of the proter in late morphogenetic stage, double-arrowhead indicates the post-peristomial cirrus. (H) Dorsal view of the opisthe, to indicate the newly formed caudal cirri (arrowheads) and the left marginal row (arrow). (I) Dorsal view of the posterior portion of the proter, arrowheads point to the caudal cirri developing from the ends of dorsal kinety anlagen 1 and 2. I–VI, frontal-ventral-transverse cirral anlagen; III/2, cirrus III/2; LMA, left marginal row anlage of proter; Ma, macronuclear nodules; Mi, micronuclei; OP, oral primordium; RMA, right marginal row anlage of proter; 1–3, dorsal kineties. Bars, 30 m ((C), (D), (G)) and 100 m ((B), (E)).
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Fig. 7. Maximum likelihood (ML) phylogenetic tree based on the small subunit rRNA (SSU rRNA) gene sequences. Numbers at the nodes represent the bootstrap values of ML analyses and posterior probability of BI analyses. Fully supported (100%/1.00) branches are marked with solid circles. Asterisk (*) represents support values less than 50% and the disagreement between BI and the reference ML tree. The scale bar corresponds to two substitutions per 100 nucleotide positions. The newly sequenced species in the present study is shown in bold and the red box shows the close relationship between P. caudatus caudatus and Pseudouroleptus caudatus.
ahead of the left ventral row, and the anterior segment of the left ventral cirral row migrates in front of the median segment of the left ventral cirral row, collectively forming a mixed (left ventral) row (Fig. 5E).
Proter. Some key stages during the ontogenetic process were not observed, so the origin of the FVTA is not clear. Morphogenesis begins with the differentiation of the buccal cirrus (Fig. 4A) and the appearance of the oral primordium on the surface of the cell (Fig. 4A). As in most other hypotrichs, the parental adoral zone is retained completely intact by the proter and no new membranelles are formed, so changes of the oral structure are confined to the paroral and endoral. In the next stage, the undulating membranes anlage is formed, probably from dedifferentiation of the parental paroral and endoral, and FVTA V and VI appear (Figs 4B, D, 6B, D). In the following stages, the development of the undulating membranes anlage and FVTA is the same as that in the opisthe (Fig. 6G).
During the late stage of the divisional process, the ventral cirral pattern of interphase cells can be entirely characterized on the basis of cirral migration: (i) cirri I/1, II/3 and III/3 correspond the three frontal cirri, and cirri II/2, III/2 and IV/2 correspond the buccal cirrus, the parabuccal cirrus, and the post-peristomial cirrus, respectively (Figs 4F, 5A, C, E, 6E, G); (ii) the anterior segments of streaks VI and IV and the whole of streak V form the anterior, middle, and posterior segment, respectively, of the left ventral row which is thus a mixed row (Figs 4F, 5A, C, 6E, G); (iii) the posterior segment of streak VI develops into the right ventral cirral row (Figs 4F, 5A, C, 6E, G).
Marginal cirri. The new marginal rows originate in the usual way for hypotrichs, i.e. in middle dividers one anlage develops each for the proter and the opisthe within each parental row (Figs 4B, D, F, 5A, C, 6B, E). These anlagen then
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stretch mainly posteriad and gradually replace the parental rows (Fig. 5E).
Dorsal ciliature. The development of the dorsal kineties anlagen (DKA) commences in early dividers (Figs 4C, 6C). DKA1 and 2 develop intrakinetally in both opisthe and proter (Fig. 4C). DKA3 emerges as a row of basal bodies that develops de novo to the right of parental dorsal kinety 3 in the proter, but intrakinetally within the parental structure in the opisthe (Fig. 4C). The DKA then stretch mainly posteriad and gradually replace the parental rows (Fig. 4E). In late dividers, DKA3 fragments in the posterior region forming two anlagen. Thus, four rows of dorsal kineties are formed in each daughter cell (Figs 5B, D, 6F). During the morphogenetic process, three or four caudal cirri are formed at the posterior end of DKA1, and one or two are formed at the end of DKA2 (Figs 5D, F, 6H, I).
Nuclear apparatus. The division of the nuclear apparatus proceeds in the usual way for hypotrichs (Berger 2008). The two macronuclear nodules fuse into a single mass which subsequently makes successive amitotic divisions to produce the species-specific number of nodules (two in the case of Pseudouroleptus caudatus caudatus) in each filial product (Figs 4E, F, 5B, D, 6F–H). The micronuclei divide mitotically (Figs 5D, F, 6G–I).
SSU rRNA gene sequence and phylogenetic analyses (Fig. 7) The SSU rRNA gene sequence of Pseudouroleptus caudatus caudatus is deposited in GenBank with the accession number KF591597. The length and G + C content of the new sequence are 1773 bp and 45.23%, respectively. Phylogenetic trees constructed using BI and ML methods show basically congruent topologies (Fig. 7). The Tibetan population of P. caudatus caudatus clusters with the Brazilian population of P. caudatus with high support (82% ML, 1.00 BI), which together form a fully supported clade with two Strongylidium species (100% ML, 1.00 BI). The difference in branch lengths of the Tibetan and the Brazilian populations of P. caudatus suggests that the latter might be another subspecies, namely P. caudatus namibiensis. More molecular data of these taxa are needed to test this hypothesis.
Discussion Identification of the Tibetan population of P. caudatus caudatus Pseudouroleptus caudatus was first isolated from freshwater samples collected in Peru (Hemberger 1985; Berger 2008). Its original description, however, was based only on silverstained specimens. Subsequently, populations were reported
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from samples collected in Spain (see Berger 2008) and Argentina (Küppers and Claps 2013). Furthermore, Foissner et al. (2002) described two African populations as separate subspecies, namely P. caudatus caudatus and P. caudatus namibiensis. The main diagnostic feature separating these two subspecies is the length of the right ventral row (labelled as transverse cirri in Foissner et al. 2002): in the former it extends anteriorly to near the level of the buccal cirrus whereas in the latter it terminates anteriorly atin the midbody region. Based on this criterion, all three previously described populations (Peru, Spain, Argentina) of P. caudatus are the subspecies P. caudatus caudatus. For comparative purposes, details of the main morphological features of these populations are given in Table 2.
Special morphogenetic characters of Pseudouroleptus and related genera Based on the Tibetan population, the most characteristic events during morphogenesis in Pseudouroleptus caudatus caudatus can be summarized as follows: (i) the parental adoral zone of membranelles is retained completely; (ii) the left ventral row is formed from three parts, i.e. the anterior segments of streaks VI and IV, and the entire streak V form the anterior, middle, and posterior segments, respectively; (iii) the right ventral row originates de novo in both daughter cells; (iv) the marginal rows develop intrakinetally; (v) dorsal kineties anlagen 1 and 2 develop intrakinetally in both opisthe and proter, but dorsal kinety anlage 3 forms differently in each daughter, i.e. de novo in the proter and intrakinetally in the opisthe; and (vi) the nuclear apparatus divides in the usual way for hypotrichs. Thus, the Tibetan population has a basically similar process of morphogenesis to that of the Peruvian population (Hemberger 1985; Berger 1999). Morphologically, Hemiamphisiella Foissner, 1988 and Strongylidium Sterki, 1878 are the most similar genera to Pseudouroleptus, and share the same arrangement of the left ventral cirral row. Our phylogenetic analyses based on SSU rRNA gene sequence data support the close relationship of Pseudouroleptus and Strongylidium. Molecular data for Hemiamphisiella are not yet available. The pattern of replication of the dorsal kineties, however, differs significantly among these genera with the third dorsal kinety forming de novo without fragmentation in Strongylidium and Hemiamphisiella (vs. third kinety fragments in Pseudouroleptus), and the fourth dorsal kinety does not fragment in Strongylidium and Hemiamphisiella (vs. the fourth kinety in Pseudouroleptus is the result of fragmentation of kinety 3) (Berger 2008). The pattern of formation of the right ventral row also differs among these genera, developing de novo to the right of the parental structure in Pseudouroleptus and Hemiamphisiella versus intrakinetally in Strongylidium (Paiva and Silva-Neto 2007; Chen et al. 2013b).
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Table 2. Comparison of various populations of Pseudouroleptus caudatus caudatus and P. caudatus namibiensis.
1
2
3
4
5
6
Body, length in vivo (m) Body, width in vivo (m) Length:width ratio in vivo Body (length) (protargol) (m) Body, (width) (protargol) (m) Length:width ratio (protargol) Cortical granules Position of CV
– – – 250 50 4:1a Brownishb – – ca. 25% – 33–50 1 3 1 1 42 55 45 55 – 4 – – – – – 2 – – 2–3 – – Soil & freshwater Hemberger (1982)
180–280 40–60 5:1 150–255 39–55 2.5:1 Colourless 50% of body length, near left cell margin Absent 21–37% 42–75 40–63 1 3 1 1 40–70 35–60 35–53 43–70 – 4 – – – – – 2 20–30 7–11 2–4 7–10 5–6 Soil Foissner et al. (2002)
180–330 40–60 – 167–295 40–55 4.7:1 Colourless –
Collecting canals Ratio of AZM in vivo AZM (length) (m) AZM (number) Buccal cirri (number) Frontal cirri (number) Parabuccal cirri (number) Post-peristomial ventral cirri (number) Left ventral cirri (number) Right ventral cirri (number) Left marginal cirri (number) Right marginal cirri (number) Caudal cirri (number) Dorsal kineties (number) Dikinetids in DK1 (number) Dikinetids in DK2 (number) Dikinetids in DK3 (number) Dikinetids in DK4 (number) Dikinetids in DK5 (number) Macronuclear nodules (number) Macronuclear nodules (length) (m) Macronuclear nodules (width) (m) Micronuclei (number) Micronuclei (length) (m) Micronuclei (width) (m) Habitat Data source
150–270 40–70 5:1a 160–280 45–90 4:1a Colourless 50% of body length, near left cell margin – ca. 33% 55–85 40–60 1 3 1–2 0–2 40–63 47–68 39–62 48–68 3–4 4–5 30–55c 30–55c 30–55c 30–55c – 2 35–50 18–25 2–5 8–12 6–10 Sediment Olmo-Rísquez (1998)
161–252 56–105 – 196–308 49–112 – Colourless 33% of body length, near left cell margin Present 30% 63–91 43–63 1 3 1 1–2 42–63 42–56 40–70 50–75 4–5 4 – – – – – 2 27–43 8–17 2–5 5–11 4–6 Soil Küppers and Claps (2013)
230–320 45–65 5:1 230–325 88–138 4.3:1 Colourless 33% of body length, near left cell margin Absent 20–25% 70–90 54–67 1 3 1 0–1 60–77 51–65 56–72 57–76 3–6 4 28–44 28–43 25–47 22–34 – 2 21–45 9–24 2–7 5–10 3–5 Soil Original
– – 46–71 33–51 1 3 1 1–3 50–67 20–31 44–61 47–70 1–4 4 – – – – – 2 18–27 6–12 1–4 5–7 4–5 Soil & freshwater Foissner et al. (2002)
Population: 1 – Peru population; 2 – Spain population; 3 – Africa population (Pseudouroleptus caudatus caudatus); 4 – Africa population (P. caudatus namibiensis); 5 – Argentina population; 6 – Tibet population. a Data from drawings. Abbreviations: AZM – adoral zone of membranelles; CV – contractile vacuole; DK1–4 – dorsal kineties. b According to Foissner (2002), a feature likely caused by the dense cortical granulation. c According to Olmo-Rísquez (1998), measurement of dorsal kineties 1–4 is likely inaccurate.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (project numbers: 31372148 to C. Shao and H. Ma, 31172041 to C. Shao and Z. Yi) and the China Scholarship Council, which funded an extended visit by the principal author to North Carolina Central University, USA. We are grateful to the associate editor Helmut Berger for helpful critical suggestions and comments on the manuscript.
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