IJSEM Papers in Press. Published September 21, 2014 as doi:10.1099/ijs.0.066381-0 Fan et al., Page 1 1
International Journal of Systematic and Evolutionary Microbiology
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Running title: Y. Fan et al. – A new ciliate, Bistichella cystiformans spec. nov.
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Contents category: New Taxa - Eukaryotic Micro-organisms GenBank accession number of new sequence: KJ509196
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Morphology, ontogenetic features and SSU rRNA gene-based phylogeny of a new soil ciliate, Bistichella cystiformans spec. nov. (Protista, Ciliophora, Stichotrichia)
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Yangbo Fan1, Xiaozhong Hu1, Feng Gao1, Saleh A. Al-Farraj2 and Khaled A.S. Al-Rasheid2
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1
Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, College of Fisheries, Ocean University of China, Qingdao 266003, China 2 Zoology Department, King Saud University, Riyadh 11451, Saudi Arabia
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Correspondence Xiaozhong Hu, Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China Telephone number: +86 532 8203 1610; FAX number: +86 532 8203 1610; e-mail: [email protected]
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The morphology, ontogeny and SSU rRNA gene-based phylogeny of Bistichella cystiformans spec. nov., isolated from the slightly saline soil of a mangrove wetland in Zhanjiang, southern China, were investigated. The new species is characterized by having five to eight buccal cirri arranged in a row, three to five transverse cirri, four macronuclear nodules aligned, 17–32 and 20–34 cirri in frontoventral rows V and VI respectively, both extending to the transverse cirri. The main ontogenetic features of the new species are as follows: (1) parental adoral zone of membranelles is completely inherited by the proter; (2) frontoventral and transverse cirri are formed in six-anlagen mode; (3) basically frontal-ventral-transverse cirral anlagen II – V generate one transverse cirrus each at their posterior ends while anlage VI provides no transverse cirrus; (4) both marginal rows and dorsal kineties develop intrakinetally, no dorsal kinety fragment is formed; and (5) the macronuclear nodules fuse into a single mass at the middle stage. Phylogenetic analyses based on the SSU rRNA gene show that the new species groups with the clade containing B. variabilis, Parabistichella variabilis, Uroleptoides magnigranulosus and two species of Orthoamphisiella. Given the present state of knowledge, it is still too early to come to a final conclusion regarding the familial classification of Bistichella and further investigation of key taxa with additional molecular markers is required.
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INRODUCTION
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Stichotrichian ciliates have proven to be one of the most important components of the microbial food web in almost all aquatic and terrestrial ecosystems worldwide, because they consume a significant portion of the bacterial productivity, enhancing nutrient cycles and energy flows to organisms at higher trophic levels (Foissner, 1987, 1998, 1999; Song et al., 2009).
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Berger (2008) established the genus Bistichella as a taxon of unknown position in the Hypotricha (= Stichotrichia Small & Lynn, 1985; Lynn, 2008), with four species transferred from the genus Pseudouroleptus and one from Amphisiella. Based on its diagnostic features, e.g. the presence of two short frontal and two long frontoventral rows, Bistichella should be classified into the order Stichotrichida Fauré-Fremiet, 1961. Although the small subunit ribosomal RNA (SSU rRNA) gene sequence for Bistichella variabilis has recently become available (He & Xu, 2011), there remains a lack of morphogenetic data from which to determine the origin of the two long frontoventral rows. Given the present state of knowledge, therefore, it is premature to assign Bistichella to any known family in the Stichotrichida.
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During a recent survey of ciliates in mangrove wetland near Zhanjiang, southern China, a novel ciliate was isolated from slightly saline soil. The isolate was successfully maintained in raw cultures, giving the opportunity to study its morphology, morphogenesis and phylogeny based on SSU rRNA gene sequence data. We here demonstrate that the population is a new member of the genus Bistichella, and discuss the familial classification of the genus.
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MATERIALS AND METHODS
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Sampling and cultivation. Bistichella cystiformans spec. nov. was isolated from a soil sample collected from Gaoqiao Mangrove National Nature Reserve in Zhanjiang (21°31 30 N; 110°16 58 E), southern China. The wet soil sample was collected from the upper 0–5 cm on April 8, 2012 (Fig. 1a). After being air-dried, the sample was stored in a plastic bag prior to being studied on April 18, 2012. The samples were wetted using the non-flooded Petri dish method (Foissner et al., 2002). Briefly, this method involves placing 50–500 g litter and mineral soil in a Petri dish (13–18 cm wide, 2–3 cm high) and saturating, but not flooding it, with distilled water (Fig. 1b). The species appeared several days after rewetting the sample in the Petri dishes with distilled water at a temperature of about 27 °C and salinity of 5‰. Cells were maintained in the laboratory for about two weeks as raw cultures with smaller colpodids and scuticociliates as well bacteria as food sources. ′
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In order to investigate the cysts, about 20 specimens isolated from the raw culture were added 3
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to a large drop of filtered in situ water in a concave slide, which was kept in a wet chamber (Foissner & Stoeck, 2011). All specimens had encysted within 12 h.
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Morphological and morphogenetic studies. Living cells and cysts were picked out and observed using bright field and differential interference contrast microscopy at 100–1000 ×. Protargol impregnation (Wilbert, 1975) was used to reveal the infraciliature and nuclear apparatus. Measurements of stained specimens were performed with an ocular micrometre. Drawings were made with the help of a camera lucida. To illustrate the changes occurring during the morphogenetic process, parental cirri are depicted in contour lines, whereas new ones are shaded black. Terminology is according to Berger (2008).
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DNA extraction, PCR amplification, and sequencing. Genomic DNA was extracted from cells using the DNeasy Tissue kit (Qiagen, CA), following the manufacturer's instructions. The PCR amplification of the SSU rRNA gene was performed according to Huang et al. (2014) using the primers 82F (5’ - GAA ACT GCG AAT GGC TC - 3’) and 18S-R (5’ - TGA TCC TTC TGC AGG TTC ACC TAC - 3’) (Medlin et al., 1988). Purified PCR product of the appropriate size was directly sequenced in both directions on an ABI 3700 sequencer.
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Phylogenetic analyses. The SSU rRNA gene sequence of the new species, together with the sequences of 60 representative taxa downloaded from the GenBank database, were used in the present phylogenetic analyses. Two choreotrichs and two oligotrichs were selected as the outgroup species. Accession numbers are shown in Fig. 7, except for 13 urostylid species: Pseudokeronopsis flava (DQ227798), Uroleptopsis citrina (FJ870094), Nothoholosticha fasciola (FJ377548), Heterokeronopsis pulchra (JQ083600), Metaurostylopsis salina (EU220229), Monocoronella carnea (FJ775726), Urostyla grandis (EF535731), Anteholosticha manca (DQ503578), Diaxonella pseudorubra (GU942564), Apokeronopsis wrighti (EU417963), Thigmokeronopsis stoecki (EU220226), Hemicycliostyla sphagni (FJ361758), and Pseudourostyla cristata (DQ019318). Sequences were aligned using MUSCLE v3.7 (Penn et al., 2010) with default parameters. The resulting alignment was manually edited using the program BioEdit 7.0.5.2 (Hall, 1999). Ambiguously aligned regions and gaps were excluded prior to phylogenetic analysis, resulting in a matrix of 1,727 characters (available from the authors upon request). Maximum likelihood (ML) analysis was conducted using RAxML-HPC2 v7.3.2 on XSEDE (Stamatakis et al., 2008) on CIPRES Science Gateway (Miller et al., 2010), using the GTR + G model as the optimal choice. Support for the best ML tree came from 1,000 bootstrap replicates with the GTR + CAT model. Bayesian inference (BI) analysis was performed with MrBayes v3.2.2 on XSEDE (Ronquist & Huelsenbeck, 2003) on the CIPRES Science Gateway, using the GTR + I (= 0.5278) + G (= 0.4686) model as selected by MrModeltest v.2.0 (Nylander, 2004). Markov chain Monte Carlo (MCMC) simulations were run with two sets of four chains for 2,000,000 generations with a sampling frequency of 100 and a burn-in of 5,000 trees (25%). All 4
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remaining trees were used to calculate posterior probabilities using a 50% majority rule consensus. TREEVIEW v1.6.6 (Page, 1996) and MEGA 4.0 (Tamura et al., 2007) were used to visualize the tree topologies. The systematic classification follows Lynn (2008).
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RESULTS
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Bistichella cystiformans spec. nov.
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Diagnosis
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Size about 120–200 µm × 40–80 µm; body elongate elliptical with both ends broadly rounded. 33–45 adoral membranelles, five to eight buccal cirri arranged in a row. Four to six cirri in frontal row III; on average, seven cirri in frontal row IV extending usually to the same length as the adoral zone; 17–32 and 20–34 cirri in frontoventral rows V and VI respectively, both extending to the transverse cirri. Three to five transverse cirri, 20–35 left and 25–47 right marginal cirri; three bipolar dorsal kineties. Four macronuclear nodules and three to six micronuclei.
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Type locality
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Slightly saline soil from Gaoqiao Mangrove National Nature Reserve in Zhanjiang (21°31 30 N; 110°16 58 E), southern China. ′
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Deposition of type specimens
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One protargol-impregnated slide with the holotype specimen (Fig. 2e, f) (registration number: FYB2012040801) and a paratype slide (registration number: FYB2012040801-1) have been deposited in the Laboratory of Protozoology, OUC, Qingdao, China.
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Etymology
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cys.ti.for’mans. N.L. fem. n. cystis (from Gr. fem. n. kustis), the bladder and in biology, a cyst; L. part. adj. formans forming; N.L. part. adj. cystiformans cyst-forming. This species-group name indicates that this species can form cyst in response to food starvation. Gene sequence The SSU rRNA gene sequence has been submitted to GenBank database with the accession number KJ509196. Morphological description (Table 1 and Figs 2a–f, 3a–h and 6a–c) 5
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Size 120–200 µm × 40–80 µm in vivo. Body elongate elliptical in outline, with the anterior and posterior end broadly rounded, dorsoventrally flattened about 3:1, highly flexible but not contractile (Figs 2a–c and 3a–b). Adoral zone approximately 2/5 of body length. Pellicle soft, without cortical granules underneath. Cytoplasm colourless, usually with many lipid droplets (approx. 3 µm across) and food vacuoles containing small colpodids, scuticociliates and bacteria, which were observed in both living and protargol-impregnated specimens. Contractile vacuole about 25 µm across, slightly above mid-body near left body margin (Fig. 2a), pulsating at intervals of about 30 s. Four macronuclear nodules, spherical to ellipsoid, conspicuous in vivo, arranged slightly left of cell. Three to six spherical micronuclei, attached or near to the macronuclear nodules. Slowly crawling on substrates or rotating around main body axis when swimming. Body folding or twisting when crawling over debris.
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Cells easily encyst when food is scarce. Cysts usually globular and about 40 µm in diameter; cortex thin and indistinct, cortical granules absent (Fig. 3h). Cyst contents dominated by the nuclear apparatus and cyst plasm with many globular inclusions 2–3 µm in diameter.
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Infraciliature as shown in Figs 2d–f, 6a–c. Adoral zone composed of 33–45 membranelles, with its distal portion located at the right anterior end of the body. Paroral and endoral membrane almost equal in length and parallel to each other, the former slightly longer than the latter. Cirral pattern and number of cirri usually variable, except for the three enlarged frontal cirri, with the rightmost one immediately behind the distal end of the adoral zone of membranelles. Five to eight buccal cirri arranged in a longitudinal row close to the paroral membrane. No frontoterminal cirri present. Four to six cirri in frontal row III, located at same level as the row of buccal cirri. Frontal row IV composed of seven cirri on average, extending usually to the proximal end of the adoral zone; in eight out of 25 individuals this row comprised 16 cirri, extending nearly to mid-body. Frontoventral row V composed of 17–32 cirri, starting from anterior quarter of the body and extending slightly obliquely to the leftmost transverse cirrus. Frontoventral row VI composed of 20–34 cirri, starting near the anterior end of the right marginal row and terminating close to the rightmost transverse cirrus. Usually, four transverse cirri, arranged in an oblique pseudorow, protruding beyond the posterior end of the body. In three out of 25 individuals, an additional frontoventral row and five transverse cirri present. One left and one right marginal row confluent posteriorly, composed of 20–35 and 25–47 cirri, respectively. Three bipolar dorsal kineties with about 24 dikinetids in each row, slightly shortened anteriorly and posteriorly; dorsal cilia approx. 3 µm long in vivo. A short row with about four to six dikinetids present on the anterior-left of the leftmost dorsal kinety in four out of 25 individuals. Caudal cirri absent.
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Morphogenesis during binary fission (Figs 4a–j, 5a–d and 6d–j)
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Stomatogenesis and frontal-ventral-transverse cirral anlagen. Several critical morphogenetic stages were observed. Fig. 4a shows a relatively early divider demonstrating that new adoral membranelles begin to organize from anterior to posterior in the oral primordium of the opisthe and that six frontal-ventral-transverse cirral anlagen are recognizable; the parental undulating membranes are dedifferentiated to form the proter’s anlage I; while in the opisthe, anlage I is detached from the oral primordium (Figs 6d, f).
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Later, the new adoral membranelles continue to develop posteriorly in the opisthe; the anterior part of anlage I develops into the leftmost frontal cirrus in both dividers (Figs 4c, d and 6e, g); with the rest of it beginning to split longitudinally, giving rise to the paroral and endoral membranes.
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Gradually, all cirral anlagen develop into new cirri. The anterior-most cirri from anlagen II and III, together with the cirrus from anlage I, become three enlarged frontal cirri; the rear cirri derived from anlage II (except the rearmost one) migrate towards the newly formed paroral membrane and become a row of buccal cirri; anlagen II to V (sometimes III to V) provide the rearmost cirri to form the transverse cirri, while anlage VI does not form transverse cirrus at all; the remaining cirri from anlagen III to VI form the two frontal and two frontoventral rows (Figs 4f, g, i, j, 5a, b and 6i, h). Rarely, an additional anlage is present between the normal anlagen V and VI in the opisthe (Figs 4c, d), which may contribute one transverse cirrus and an extra ventral row (Fig. 5c).
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In a late divider, the anterior part of the new adoral zone curves to the right in the opisthe, while the parental adoral zone of membranelles remains completely intact throughout the morphogenetic process (Figs 5c, d and 6j).
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Anlagen of marginal rows and dorsal kineties. The formation of the marginal rows and dorsal kineties proceeds intrakinetally in a conventional manner for hypotrichs. The anlagen of the marginal rows are formed by the dedifferentiation of some cirri within each parental marginal row (Fig. 4a). These anlagen then stretch longitudinally and gradually replace the parental structures completely (Figs 4c, f, i, 5a, c and 6d, e, i). The dorsal kineties anlagen are also formed within the old rows in both dividers such that, with the proliferation of basal bodies, they elongate and supplant the parental structures (Figs 4b, e, h, j and 5b, d).
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Nuclear apparatus. The macronuclear nodules fuse into a single mass in middle dividers and then divide to form four nodules for filial cells. The divisional process of the micronuclei was unclear, although they were recognizable in some dividers (Figs 4b, e, h, j, 5b, d and 6f, g, j).
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SSU rRNA gene sequence and phylogenetic analyses (Fig. 7)
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The SSU rRNA gene sequence of Bistichella cystiformans spec. nov. was deposited in the GenBank database with the accession number KJ509196. The length and GC content of the SSU rRNA gene are 1,694 bp and 45.81%, respectively.
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We included a broad selection of 61 species in the present phylogenetic analyses, containing almost all available SSU rRNA gene sequences from both the orders Stichotrichida and Sporadotrichida. The topologies of the ML and BI trees were basically congruent and, therefore, a single topology is presented based on the ML tree with support values from both algorithms indicated on branches (Fig. 7). In the phylogenetic trees, Bistichella cystiformans spec. nov. clusters in the clade containing B. variabilis, Parabistichella variabilis, Uroleptoides magnigranulosus and two Orthoamphisiella species (47% ML, 0.87 BI). Sequence comparisons indicate a similar result, with the sequence similarities between B. cystiformans spec. nov. and the species in the Bistichella–Parabistichella–Uroleptoides–Orthoamphisiella clade ranging from 99.2% to 99.4%.
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DISCUSSION
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Morphological comparison with congeners (Table 2)
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Berger (2008) established the genus Bistichella with five species assembled in it, namely B. buitkampi (Foissner, 1982) Berger, 2008 (the type of the genus, basionym: Paraurostyla buitkampi Foissner, 1982), B. namibiensis (Foissner, Agatha & Berger, 2002) Berger, 2008 (basionym: Amphisiella namibiensis Foissner, Agatha & Berger, 2002), B. procera (Berger & Foissner, 1987) Berger, 2008 (basionym: Pseudouroleptus procerus Berger & Foissner, 1987), B. terrestris (Hemberger, 1985) Berger, 2008 (basionym: Pseudouroleptus terrestris Hemberger, 1985) and B. humicola (Gellért, 1956) Berger, 2008 (basionym: Uroleptus humicola Gellért, 1956). Simultaneously, Foissner et al. (2008) transferred two of them, i.e., Pseudouroleptus procerus and Pseudouroleptus terrestris into the newly erected genus Metauroleptus. He & Xu (2011) described a new species, Bistichella variabilis, and noted that the assignment of Foissner et al. (2008) tends to make the genus Bistichella monophyletic, containing only members without caudal cirri. Considering that the presence or absence of caudal cirri is widely used to separate hypotrich genera (e.g. the oxytrichid genus Tachysoma Stokes, 1887 and the urostyloid genus Caudiholosticha Berger, 2003) (Berger, 1999, 2006), the pisciform shape with a narrowed posterior end and the presence of caudal cirri in Pseudouroleptus procerus and P. terrestris, make these more suitable to be members of the genus Metauroleptus (Berger & Foissner, 1987; Hemberger, 1985; Berger, 2008). We therefore agree with the exclusion of these two species from Bistichella, and accordingly compare the new species B. cystiformans with its four congeners. 8
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In terms of the infraciliature, Bistichella cystiformans spec. nov. is closely related to B. variabilis. The former, however, can be recognized by the position of the rightmost frontal cirrus (immediately behind the distal end of the adoral zone of membranelles vs. positioned anteriorly at the same level as the other two frontal cirri) and the two rightmost frontoventral rows (V extending posteriorly to the leftmost transverse cirrus vs. distinctly away from transverse cirri; VI commencing near the anterior end of the right marginal row and terminating close to the rightmost transverse cirrus vs. mostly short and located in the posterior quarter of the body). Moreover, the new species differs in having fewer adoral membranelles (33–45 vs. 46–63), buccal cirri (5–8 vs. 7–12), and fewer left (20–35 vs. 37–63) and right (25–47 vs. 44–78) marginal cirri (He & Xu, 2011).
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Bistichella buitkampi resembles B. cystiformans spec. nov. in body size and shape. However, the former can be separated from the latter in having fewer buccal cirri (2–4 vs. 5–8), fewer cirri in frontal rows III (2–3 vs. 4–6) and IV (3–7 vs. 5–18), a shorter frontoventral row VI (far away from transverse cirri vs. close to transverse cirri), an absence of transverse cirri (vs. presence) and in the arrangement of the macronuclear nodules (in two separated pairs vs. four, in a row) (Foissner, 1982).
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Bistichella namibiensis is very similar to B. cystiformans spec. nov. in the arrangement of the two frontoventral rows, but differs from the latter in having two macronuclear nodules (vs. four), fewer buccal cirri (2–5 vs. 5–8), fewer cirri in frontal row III (approx. 1 vs. 4–6) and IV (approx. 3 vs. 5–18), as well as in having more cirri in frontoventral row VI (38–50 vs. 20–34) and more left (44–58 vs. 20–35) and right (42–61 vs. 25–47) marginal cirri (Foissner et al., 2002).
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Bistichella humicola was classified as a taxon incertae sedis in Bistichella by Berger (2008). It is described mainly from fixed and opal blue-stained specimens and, as a result, the validity of the cirral pattern cannot be reliably ascertained. Nevertheless, it can be easily distinguished from B. cystiformans spec. nov. in having only one buccal cirrus (vs. 5–8), absence of transverse cirri (vs. 3–5) and presence of many (approx. 32) macronuclear nodules (vs. four) (Gellért, 1956).
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Morphogenetic pattern in Bistichella
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Prior to this investigation, no morphogenetic data were available for any species of Bistichella. Our study of Bistichella cystiformans spec. nov. attempts to remedy this defect, however, we were unable to fully determine the earliest stages of morphogenesis in our specimens and, therefore, the origin of the oral primordium and cirral anlagen is not available. Accordingly, 9
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morphogenesis in B. cystiformans can be summarized as follows: (1) in the proter, the parental adoral zone of membranelles is completely retained, while in the opisthe, the oral primordium is formed apokinetally in the posterior half of the cell; (2) the frontoventral and transverse cirri are formed by the conventional six-anlagen mode; (3) the cirral streaks II – V (or III – V) generate one transverse cirrus each at their posterior ends while streak VI provides no transverse cirrus; (4) the marginal rows occur within the old structures in a secondary mode; (5) the dorsal kineties develop intrakinetally and no fragments are formed; (6) all macronuclear nodules fuse into a single mass and then divide twice.
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One of the most remarkable morphogenetic features in Bistichella cystiformans spec. nov. is that the rightmost frontal-ventral-transverse cirral anlage (= VI) does not form a transverse cirrus at its rear end, which, according to current knowledge, is unique in stichotrichs with six frontal-ventral-transverse cirral anlagen. Without exception in these species, the FVT-anlage VI contributes one transverse cirrus (e.g. Amphisiella, Gonostomum, Fragmocirrus, Trachelostyla, Oxytricha, Notohymena) (Berger, 1999, 2008, 2011; Chen et al., 2013a, b, c; Lv et al., 2013; Shao et al., 2013). Whether or not the absence of a special group of cirri is driven by environmental changes cannot be determined at present. Considering, however, that some taxa (e.g. Apoholosticha and Heterokeronopsis) found in mangroves always exhibit a lack of specific cirral groups, the possibility that the observed difference is due to the effect of variable mangrove environments upon the phenotype cannot be excluded (Fan et al., 2014; Pan et al., 2013).
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Familial classification of Bistichella
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Bistichella was established with species previously allocated into Pseudouroleptus and Amphisiella (Berger, 2008). Unlike the oxytrichid genus Pseudouroleptus, Bistichella lacks dorsomarginal kineties and dorsal kinety fragmentation, strongly indicating that it does not belong to the dorsomarginalian part of the Hypotricha, and should not be included in the family Oxytrichidae (Berger, 1999). Likewise, Bistichella lacks the amphisiellid cirral row, a critical character of the name-bearing type genus Amphisiella, and therefore it can also not be included in the family Amphisiellidae (Berger, 2008; Chen et al., 2013b). This position was also taken by Berger (2008) in his review of the amphisiellids, in which he classified Bistichella as a taxon of unknown position in the Hypotricha. The separation of Bistichella from Pseudouroleptus and Amphisiella is also supported by molecular trees in which the former is quite distant from the latter two (He & Xu, 2011).
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Present and previous phylogenetic studies based on SSU rRNA gene sequences show that Orthamphisiella clusters into a clade including Bistichella with relatively low bootstrap values (He & Xu, 2011). The molecular data of Orthamphisiella were only available for O. breviseries and an unidentified species and the assignment of both species into the 10
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Orthamphisiella is uncertain based on morphological data (Berger, 2008). Considering that the SSU rRNA gene sequence of the type species O. stramenticola is unavailable, the systematic position of Orthoamphisiella remains uncertain. Moreover, Orthoamphisiella differs distinctly from Bistichella in having no or only one long frontoventral row (vs. two), and in that the frontoventral row of the proter is composed of parental cirri in the anterior portion and newly formed cirri in the posterior portion (vs. only newly formed cirri), so both genera cannot have the same familial assignment from a morphological and morphogenetic perspective (Eigner & Foissner, 1991). Eigner (1997) established the family Orthoamphisiellidae to include Orthoamphisiella and some other genera, based primarily on the formation of within-anlagen in the two rightmost ventral cirral rows. Actually, except for the type species Orthoamphisiella stramenticola, none of the taxa originally included corresponded with the familial classification (Berger, 2008). Thus, this family is redundant and not widely accepted (Berger, 2008; Lynn, 2008).
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Bistichella has some ontogenetic traits in common with kahliellid genera, such as Kahliella Corliss, 1960, the type genus of the family Kahliellidae, e.g. the cirral anlagen are formed within the parental rows and dorsal kinety fragmentation is lacking (Berger, 2011). Kahliellids are also characterized by their tendency to preserve parental marginal rows and/or dorsal kineties in a variable number of divisions and in presenting dorsomarginal kineties. Bistichella, however, bears no parental marginal rows and/or dorsal kineties in divisions, and lacks dorsomarginal kineties, and therefore it cannot be included in the family Kahliellidae.
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Metauroleptus Foissner et al., 2008 is very similar to Bistichella in having three frontal cirri, two long and several short frontoventral rows, a row of buccal cirri, one left and one right marginal row (Foissner et al., 2008). In addition, the frontoventral-transverse cirral anlagen appear as the conventional six streaks, and the anlagen of marginal rows and dorsal kineties occur intrakinetally in both genera. Molecular data were unavailable, however, and therefore the systematic position of Metauroleptus remains unknown at present.
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The current study confirms that Bistichella has a close relationship with Parabistichella Jiang et al., 2013. This is in accordance with the morphological data, since both genera resemble each other very well in having three frontal cirri, long frontoventral rows, one left and one right marginal row, and no caudal cirri (Jiang et al., 2013). It is still too early, however, to provide a definite familial assignment for these two genera, since the classification is based on only a limited number of taxa and is, in any case, far from settled given the low bootstrap values in the phylogenetic trees.
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ACKNOWLEDGEMENTS
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This work was supported by the Natural Science Foundation of China (Project numbers: 41176119, 31030059 and 31111120437) and by King Saud University, Deanship of Scientific Research, International Research Group Program (Project number: IRG14-22). Many thanks are due to Ms. Wenping Chen (SCNU) for sample collection and to Ms. An Liu (OUC) for gene sequencing. Our thanks are also given to Prof. Xiaofeng Lin (SCNU) for ensuring institutional support for this research.
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REFERENCES
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Berger, H. (1999). Monograph of the Oxytrichidae (Ciliophora, Hypotrichia) (Monographiae Biologicae, vol. 78). Dordrecht: Springer. Berger, H. (2006). Monograph of the Urostyloidae (Ciliophora, Hypotricha) (Monographiae Biologicae, vol. 85). Dordrecht: Springer. Berger, H. (2008). Monograph of the Amphisiellidae and Trachelostylidae (Ciliophora, Hypotricha). (Monographiae Biologicae, vol. 88). Dordrecht: Springer. Berger, H. (2011). Monograph of the Gonostomatidae and Kahliellidae (Ciliophora, Hypotricha) (Monographiae Biologicae, vol. 90). Dordrecht: Springer. Berger, H. & Foissner, W. (1987). Morphology and biometry of some soil hypotrichs (Protozoa: Ciliophora). Zool Jb Syst 114, 193–239. Chen, X., Yan, Y., Hu, X., Zhu, M., Ma, H. & Warren, A. (2013a). Morphology and morphogenesis of a soil ciliate, Rigidohymena candens (Kahl, 1932) Berger, 2011 (Ciliophora, Hypotricha, Oxytrichidae), with notes on its molecular phylogeny based on SSU rDNA sequence data. Int J Syst Evol Microbiol 63, 1912–1921. Chen, X., Shao, C., Lin, X., Clamp, J. C. & Song, W. (2013b). Morphology and molecular phylogeny of two new brackish-water species of Amphisiella (Ciliophora, Hypotrichia), with notes on morphogenesis. Eur J Protistol 49, 453–466. Chen, X., Shao, C., Liu, X., Huang, J. & Al-Rasheid, K. A. S. (2013c). Morphology and phylogenies of two hypotrichous brackish-water ciliates from China, Neourostylopsis orientalis n. sp. and Protogastrostyla sterkii (Wallengren, 1900) n. comb., with establishment of a new genus Neourostylopsis n. gen. (Protista, Ciliophora, Hypotrichia). Int J Syst Evol Microbiol 63, 1197–1209. Eigner, P. (1997). Evolution of morphogenetic processes in the Orthoamphisiellidae n. fam., Oxytrichidae, and Parakahliellidae n. fam., and their depiction using a computer method (Ciliophora, Hypotrichida). J Eukaryot Microbiol 44, 553–573. Eigner, P. & Foissner, W. (1991). Orthoamphisiella stramenticola nov. gen., nov. spec., a new hypotrichous ciliate (Ciliophora: Hypotrichida) occurring in walnut leaf litter. Acta Protozool 30, 129–133. Fan, Y., Chen, X., Hu, X., Shao, C., Al-Rasheid, K. A. S., Al-Farraj, S. A. & Lin, X. (2014). Morphology and morphogenesis of Apoholosticha sinica n. g., n. sp. (Ciliophora, 12
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Hypotrichia), with consideration of its systematic position among urostylids. Eur J Protistol 50, 78–88. Foissner, W. (1982). Ökologie und Taxonomie der Hypotrichida (Protozoa: Ciliophora) einiger österreichischer Böden. Eur J Protistol 34, 195–235. Foissner, W. (1987). Soil protozoa : fundamental problems, ecological significance, adaptations in ciliates and testaceans, bioindicators, and guide to the literature. Progr Protistol 2, 69–212. Foissner, W. (1998). An updated compilation of world soil ciliates (Protozoa, Ciliophora), with ecological notes, new records, and descriptions of new species. Arch Protistenk 126, 19–143. Foissner, W. (1999). Soil protozoa as bioindicators: pros and cons, methods, diversity representative examples. Agric Ecosyst Environ 74, 95–112. Foissner, W., Agatha, S. & Berger, H. (2002). Soil ciliates (Protozoa, Ciliophora) from Namibia (Southwest Africa), with emphasis on two contrasting environments, the Etosha region and the Namib Desert. Part I: text and line drawings. Part II: photographs. Denisia 5, 1–1459. Foissner, W., Quintela-Alonso, P. & Al-Rasheid, K. A. S. (2008). Soil ciliates from Saudi Arabia, including descriptions of two new genera and six new species. Acta Protozool 47, 317–352. Foissner, W. & Stoeck, T. (2011). Cotterillia bromelicola nov. gen., nov. spec., a gonostomatid ciliate (Ciliophora, Hypotricha) from tank bromeliads (Bromeliaceae) with de novo originating dorsal kineties. Eur J Protistol 47, 29–50. Gellért, J. (1956). Ciliaten des sic hunter dem Moosrasen auf Felsen gebildeten Humus. Acta Biol Hung 6, 337–359. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98. He, Y. & Xu, K. (2011). Morphology and small subunit rDNA phylogeny of a new soil ciliate, Bistichella variabilis n. sp. (Ciliophora, Stichotrichia). J Eukaryot Microbiol 58, 332–338. Hemberger, H. (1985). Neue Gattungen und Arten hypotricher Ciliaten. Arch Protistenk 130, 397–417. Huang, J., Chen, Z., Song, W. & Berger, H. (2014). Three-gene based phylogeny of the Urostyloidea (Protista, Ciliophora, Hypotricha), with notes on classification of some core taxa. Mol Phylogenet Evol 70, 337–347. Jiang, J., Huang, J., Li, L., Shao, C., Al-Rasheid, K. A. S., Al-Farraj, S. A. & Chen, Z. (2013). Morphology, ontogeny, and molecular phylogeny of two novel bakuellid-like hypotrichs (Ciliophora: Hypotrichia), with establishment of two new genera. Eur J Protistol 49, 78–92. Lv, Z., Chen, L., Chen, L., Shao, C., Miao, M. & Warren, A. (2013). Morphogenesis and molecular phylogeny of a new freshwater ciliate, Notohymena apoaustralis n. sp. 13
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(Ciliophora, Oxytrichidae). J Eukaryot Microbiol 60, 455–466. Lynn, D. H. (2008). The Ciliated Protozoa: Characterization, Classification, and Guide to the Literature, 3rd ed. Dordrecht: Springer. Medlin, L., Elwood, H. J., Stickel, S. & Sogin, M. L. (1988). The characterization of enzymatically amplified eukaryotes 16S-like ribosomal RNA coding regions. Gene 71, 491–500. Miller, M. A., Pfeiffer, W. & Schwartz, T. (2010). "Creating the CIPRES Science Gateway for inference of large phylogenetic trees" in Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans LA. p. 1–8. Nylander, J. A. A. (2004). MrModeltest 2.2. Program distributed by the author. Uppsala: Evolutionary Biology Centre, Uppsala University. Page, R. D. M. (1996). TREEVIEW: an application to view phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358. Pan, Y., Li, J., Hu, X., Warren, A. & Song, W. (2013). Ontogeny and phylogeny of a new urostylid ciliate, Heterokeronopsis pulchra g. n., sp. n. (Protozoa, Ciliophora). Eur J Protistol 49, 298–311. Penn, O., Privman, E., Ashkenazy, H., Landan, G., Graur, D. & Pupko, T. (2010). GUIDANCE: a web server for assessing alignment confidence scores. Nucleic Acids Res 38(Web Server issue), W23–W28. Ronquist, F. & Huelsenbeck, J. P. (2003). MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Shao, C., Pan, X., Jiang, J., Ma, H., Al-Rasheid, K. A. S., Warren, A. & Lin, X. (2013). A redescription of the oxytrichid Tetmemena pustulata (Müller, 1786) Eigner, 1999 and notes on morphogenesis in the marine urostylid Metaurostylopsis salina Lei et al., 2005 (Ciliophora, Hypotrichia). Eur J Protistol 49, 272–282. Song, W., Warren, A. & Hu, X. (2009). Free-living Ciliates in the Bohai and Yellow Seas, China. Beijing: Science Press (in both Chinese and English). Stamatakis, A., Hoover, P. & Rougemont, J. (2008). A rapid bootstrap algorithm for the RAxML Web-Servers. Syst Biol 75, 758–771. Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA 4: Molecular evolutionary genetics analysis (MEGA) software Ver. 4.0. Mol Biol Evol 24, 1596–1599. Wilbert, N. (1975). Eine verbesserte Technik der Protargolimprägnation für Ciliaten. Mikrokosmos 64, 171–179.
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1 2
FIGURE LEGENDS
3
Fig. 1. (a) Photograph of the sample site, Gaoqiao Mangrove National Nature Reserve in Zhanjiang, southern China. (b) Portion of a map of southern China, showing the location of Zhanjiang. (c) Photograph showing the non-flooded Petri dish.
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Fig. 2. Bistichella cystiformans spec. nov. in vivo (a–c) and after protargol impregnation (d–f). (a) Ventral view of a representative individual. (b) Left lateral view. (c) Swimming movement. (d) Detailed ventral view of the anterior portion, the frontal cirri and buccal cirri row are shaded, while the broken lines connect cirri generated in the same anlage. (e, f) Ventral (e) and dorsal (f) view of the holotype to show the infraciliature and nuclear apparatus. AZM, adoral zone of membranelles; BC, buccal cirri; CV, contractile vacuole; DK1–3, dorsal kineties; EM, endoral membrane; FC, frontal cirri; LMR, left marginal row; Ma, macronuclear nodules; Mi, micronuclei; PM, paroral membrane; RMR, right marginal row; TC, transverse cirri; III, IV, frontal rows; V, VI, frontoventral rows. Bars, 70 µm.
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Fig. 3. Photomicrographs of Bistichella cystiformans spec. nov. in vivo. (a, b) Ventral views of different, freely motile individuals. (c) Ventral view of a squeezed specimen to show the grooves along the marginal cirral row and frontoventral rows (arrowheads). (d) Ventral view of the anterior portion, arrowheads mark the tooth-like structures inserted between membranelles. (e) Ventral view of buccal field, showing the endoral membrane (arrowhead) and paroral membrane (arrow). (f) Cytoplasm with many food vacuoles and four macronuclear nodules (arrowheads). (g) Ventral view of the posterior portion to show the frontoventral rows V, VI, and transverse cirri. (h) Resting cysts. FV, food vacuoles; TC, transverse cirri; V, VI, frontoventral rows V and VI. Bars, 50 µm (a–c), 70 µm (h).
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Fig. 4. Morphogenesis of Bistichella cystiformans spec. nov. after protargol impregnation. (a, b) Ventral (a) and dorsal (b) view of an early-middle divider, to show the formation of the frontal-ventral-transverse cirral anlagen and how the anterior end of the undulating membranes anlage contributes the leftmost frontal cirri (arrowhead), arrow points to the formation of the left marginal anlage within the old cirral row. (c–e) Ventral (c, d) and dorsal (e) views of a mid-stage divider, showing formation of the leftmost frontal cirri (arrowheads) and the frontoventral cirri anlagen beginning to differentiate into cirri (d). Note the macronuclear nodules fuse into a single mass. (f–h) Ventral (f, g) and dorsal (h) views of a divider in the middle stage, arrowheads denote the separation of the undulating membranes. Note the macronuclear mass is about to divide. (i, j) Ventral (i) and dorsal (j) view of a late divider, showing the almost complete segmentation of the frontoventral cirri anlagen, arrows mark the new transverse cirri. DK, dorsal kineties; DKA, dorsal kineties anlagen; LMA, left marginal anlage; LMR, left marginal row; Ma, macronuclear nodules; Mi, micronuclei; OP, opisthe’s oral primordium; RMA, right marginal anlage; RMR, right marginal row. Bars, 70 15
1
µm.
2 3 4 5 6 7 8
Fig. 5. Late stages of morphogenesis of Bistichella cystiformans spec. nov. after protargol impregnation. (a, b) Ventral (a) and dorsal (b) view of a late divider, note the dividing macronuclear nodules. (c, d) Ventral (c) and dorsal (d) view of a very late divider, to show the cirri almost in their final positions, arrowheads showing the transverse cirri migrating to their final positions. DK, dorsal kineties; LMR, left marginal row; Ma, macronuclear nodules; Mi, micronuclei; RMR, right marginal row. Bars, 70 µm.
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Fig. 6. Photomicrographs of Bistichella cystiformans spec. nov. in interphase stages (a–c) and during binary division (d–j) after protargol impregnation. (a) Ventral view of a representative cell with four transverse cirri. (b) Ventral view of another specimen to show the elongated frontal row IV (arrowhead). (c) Ventral view of the anterior portion. (d, f) Ventral view of an early divider, to show the frontoventral cirri anlagen of the opisthe. (e, g) Ventral view of a mid-stage divider, arrowhead marks the undulating membranes anlage contributing the leftmost frontal cirrus. (h, i) Ventral view of a divider in the middle stage, arrowheads mark the three new frontal cirri of the proter. (j) Ventral view of a very late divider. Note the dividing macronuclear nodules. BC, buccal cirri; EM, endoral membrane; FC, frontal cirri; PM, paroral membrane. Bars, 70 µm.
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Fig. 7. Maximum likelihood (ML) tree based on SSU rRNA gene sequences, showing the position of Bistichella cystiformans spec. nov. (bold, arrow). Bootstrap values above 50 for ML and posterior probability values above 0.5 for Bayesian inference (BI) are given near nodes. Fully supported (100/1.00) branches are marked with solid circles. All branches are drawn to scale. The scale bar corresponds to two substitutions per 100 nucleotide positions.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Table 1. Morphometric characterization of Bistichella cystiformans spec. nov. Character a Min Max Body, length 86 146 Body, width 26 86 AZM, length 39 82 Adoral membranelles, number 33 45 Distance between body anterior 7 13 end and distal end of AZM Frontal cirri, number 3 3 Buccal cirri, number 5 8 Cirri in frontal row III, number 4 6 Cirri in frontal row IV, number 5 18 Distance betweenbody anterior end and 15 30 anterior end of frontoventral row V Cirri in frontoventral row V, number 17 42 Cirri in frontoventral row VI, number 20 34 Transverse cirri, number 3 5 Distance between body posterior end and 6 25 the rearmost transverse cirrus Cirri in left marginal row, number 20 35 Cirri in right marginal row, number 25 47 Dorsal kineties, number 3 4 Dikinetids in dorsal kinety 1, number 20 32 Dikinetids in dorsal kinety 2, number 18 30 Dikinetids in dorsal kinety 3, number 20 29 Macronuclear nodules, number 4 4 Anteriormost macronuclear nodule, length 17 28 Anteriormost macronuclear nodule, width 13 24 Micronuclei, number 3 6 Micronuclei, length 2 4
Mean SD 106.3 16.43 41.3 15.08 47.5 9.37 37.5 2.66 10.0 1.65
CV 15.5 36.5 19.7 7.1 16.5
n 25 25 25 25 15
3.0 6.5 4.5 10.7 19.8
0 0.77 0.65 3.76 4.71
0 11.9 14.6 35.2 23.8
25 25 25 25 15
24.3 27.1 4.0 14.1
4.84 3.29 0.50 5.57
19.9 12.2 12.5 39.4
25 25 25 15
27.8 33.2 3.2 24.0 24.6 24.9 4.0 21.9 17.6 4.1 2.9
3.53 4.99 0.41 3.42 3.09 2.39 0 3.37 3.09 0.83 0.52
12.7 15.0 12.8 14.3 12.6 9.6 0 15.4 17.6 20.2 18.0
25 25 20 15 15 15 25 15 15 25 15
a
All data are based on protargol-impregnated specimens. Measurements in µm. Abbreviations: AZM, adoral zone of membranelles; CV, coefficient of variation in %; Max, maximum; Mean, arithmetic mean; Min, minimum; n, sample size; SD, standard deviation.
17
1
Table 2. Morphological comparison of Bistichella cystiformans spec. nov. with congeners. Character
B. cystiformans spec. nov.
B. variabilis
B. buitkampi
B. namibiensis
B. humicola
Body size in vivo in µm
120–200 × 40–80
170–270 × 50–90
135–180 × 50–70
140–300 × 40–100
120 a
Body shape
Elongate ellipsoid
Elongate ellipsoid
Dumbbell-shaped to sigmoidal
Elongate ellipsoid
Elongate ellipsoid
33–45
46–63
34–40
39–48
31
5–8
7–12
2–4
2–5
1
2–3
1
b
9
3
b
Possibly lacking
Adoral membranelles, number Buccal cirri, number Cirri in frontal row III, number Cirri in frontal row IV, number
4–6
6–12
5–18
7–18
3–7
Cirri in frontoventral row V, number
17–42
35–62
25–37
33–46
24
Arrangement of frontoventral row V
Extending to the leftmost transverse cirrus
Extending posteriorly to about 80% of body length
Extending posteriorly to about 75% of body length
Extending obliquely to transverse cirri
Extending posteriorly to about 64% of body length
Cirri in frontoventral row VI, number
20–34
7–38
20–30
38–50
44
Arrangement of frontoventral row VI
Commencing behind distal end of adoral zone and extending to the rightmost transverse cirrus
Mostly short and located in the posterior quarter of body and extending to the rightmost transverse cirrus
Commencing behind distal end of adoral zone and extending posteriorly to about 75% of body length
Commencing behind distal end of adoral zone and extending obliquely to transverse cirri
Commencing behind distal end of adoral zone and extending posteriorly to rear cell end
3–5
5–7
6–15
4–6
Absent
Cirri in left marginal row, number
20–35
37–63
35–52
44–58
35
Cirri in right marginal row, number
25–47
44–78
39–49
42–61
41
3–4
3–4
3
3
Data unavailable
4
4
4
2
32
3–6
2–7
2
2–6
Data unavailable
Present study
He & Xu (2011)
Foissner (1982)
Foissner et al. (2002)
Gellért (1956) Berger (2008)
Transverse cirri, number
Dorsal kineties, number Macronuclear nodules, number Micronuclei, number Data source
2
a
3
b
Method (in vivo or after fixation) not indicated. Data from the holotype specimen in Foissner et al. (2002).
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International Journal of Systematic and Evolutionary Microbiology
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Running title: Y. Fan et al. – A new ciliate, Bistichella cystiformans spec. nov.
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Contents category: New Taxa - Eukaryotic Micro-organisms GenBank accession number of new sequence: KJ509196
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Morphology, ontogenetic features and SSU rRNA gene-based phylogeny of a new soil ciliate, Bistichella cystiformans spec. nov. (Protista, Ciliophora, Stichotrichia)
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Yangbo Fan1, Xiaozhong Hu1, Feng Gao1, Saleh A. Al-Farraj2 and Khaled A.S. Al-Rasheid2
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1
Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, College of Fisheries, Ocean University of China, Qingdao 266003, China 2 Zoology Department, King Saud University, Riyadh 11451, Saudi Arabia
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Correspondence Xiaozhong Hu, Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China Telephone number: +86 532 8203 1610; FAX number: +86 532 8203 1610; e-mail: [email protected]
Fan et al., Page 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
The morphology, ontogeny and SSU rRNA gene-based phylogeny of Bistichella cystiformans spec. nov., isolated from the slightly saline soil of a mangrove wetland in Zhanjiang, southern China, were investigated. The new species is characterized by having five to eight buccal cirri arranged in a row, three to five transverse cirri, four macronuclear nodules aligned, 17–32 and 20–34 cirri in frontoventral rows V and VI respectively, both extending to the transverse cirri. The main ontogenetic features of the new species are as follows: (1) parental adoral zone of membranelles is completely inherited by the proter; (2) frontoventral and transverse cirri are formed in six-anlagen mode; (3) basically frontal-ventral-transverse cirral anlagen II – V generate one transverse cirrus each at their posterior ends while anlage VI provides no transverse cirrus; (4) both marginal rows and dorsal kineties develop intrakinetally, no dorsal kinety fragment is formed; and (5) the macronuclear nodules fuse into a single mass at the middle stage. Phylogenetic analyses based on the SSU rRNA gene show that the new species groups with the clade containing B. variabilis, Parabistichella variabilis, Uroleptoides magnigranulosus and two species of Orthoamphisiella. Given the present state of knowledge, it is still too early to come to a final conclusion regarding the familial classification of Bistichella and further investigation of key taxa with additional molecular markers is required.
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INRODUCTION
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Stichotrichian ciliates have proven to be one of the most important components of the microbial food web in almost all aquatic and terrestrial ecosystems worldwide, because they consume a significant portion of the bacterial productivity, enhancing nutrient cycles and energy flows to organisms at higher trophic levels (Foissner, 1987, 1998, 1999; Song et al., 2009).
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Berger (2008) established the genus Bistichella as a taxon of unknown position in the Hypotricha (= Stichotrichia Small & Lynn, 1985; Lynn, 2008), with four species transferred from the genus Pseudouroleptus and one from Amphisiella. Based on its diagnostic features, e.g. the presence of two short frontal and two long frontoventral rows, Bistichella should be classified into the order Stichotrichida Fauré-Fremiet, 1961. Although the small subunit ribosomal RNA (SSU rRNA) gene sequence for Bistichella variabilis has recently become available (He & Xu, 2011), there remains a lack of morphogenetic data from which to determine the origin of the two long frontoventral rows. Given the present state of knowledge, therefore, it is premature to assign Bistichella to any known family in the Stichotrichida.
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During a recent survey of ciliates in mangrove wetland near Zhanjiang, southern China, a novel ciliate was isolated from slightly saline soil. The isolate was successfully maintained in raw cultures, giving the opportunity to study its morphology, morphogenesis and phylogeny based on SSU rRNA gene sequence data. We here demonstrate that the population is a new member of the genus Bistichella, and discuss the familial classification of the genus.
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MATERIALS AND METHODS
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Sampling and cultivation. Bistichella cystiformans spec. nov. was isolated from a soil sample collected from Gaoqiao Mangrove National Nature Reserve in Zhanjiang (21°31 30 N; 110°16 58 E), southern China. The wet soil sample was collected from the upper 0–5 cm on April 8, 2012 (Fig. 1a). After being air-dried, the sample was stored in a plastic bag prior to being studied on April 18, 2012. The samples were wetted using the non-flooded Petri dish method (Foissner et al., 2002). Briefly, this method involves placing 50–500 g litter and mineral soil in a Petri dish (13–18 cm wide, 2–3 cm high) and saturating, but not flooding it, with distilled water (Fig. 1b). The species appeared several days after rewetting the sample in the Petri dishes with distilled water at a temperature of about 27 °C and salinity of 5‰. Cells were maintained in the laboratory for about two weeks as raw cultures with smaller colpodids and scuticociliates as well bacteria as food sources. ′
′
′′
′′
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In order to investigate the cysts, about 20 specimens isolated from the raw culture were added 3
Fan et al., Page 4 1 2
to a large drop of filtered in situ water in a concave slide, which was kept in a wet chamber (Foissner & Stoeck, 2011). All specimens had encysted within 12 h.
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Morphological and morphogenetic studies. Living cells and cysts were picked out and observed using bright field and differential interference contrast microscopy at 100–1000 ×. Protargol impregnation (Wilbert, 1975) was used to reveal the infraciliature and nuclear apparatus. Measurements of stained specimens were performed with an ocular micrometre. Drawings were made with the help of a camera lucida. To illustrate the changes occurring during the morphogenetic process, parental cirri are depicted in contour lines, whereas new ones are shaded black. Terminology is according to Berger (2008).
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DNA extraction, PCR amplification, and sequencing. Genomic DNA was extracted from cells using the DNeasy Tissue kit (Qiagen, CA), following the manufacturer's instructions. The PCR amplification of the SSU rRNA gene was performed according to Huang et al. (2014) using the primers 82F (5’ - GAA ACT GCG AAT GGC TC - 3’) and 18S-R (5’ - TGA TCC TTC TGC AGG TTC ACC TAC - 3’) (Medlin et al., 1988). Purified PCR product of the appropriate size was directly sequenced in both directions on an ABI 3700 sequencer.
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Phylogenetic analyses. The SSU rRNA gene sequence of the new species, together with the sequences of 60 representative taxa downloaded from the GenBank database, were used in the present phylogenetic analyses. Two choreotrichs and two oligotrichs were selected as the outgroup species. Accession numbers are shown in Fig. 7, except for 13 urostylid species: Pseudokeronopsis flava (DQ227798), Uroleptopsis citrina (FJ870094), Nothoholosticha fasciola (FJ377548), Heterokeronopsis pulchra (JQ083600), Metaurostylopsis salina (EU220229), Monocoronella carnea (FJ775726), Urostyla grandis (EF535731), Anteholosticha manca (DQ503578), Diaxonella pseudorubra (GU942564), Apokeronopsis wrighti (EU417963), Thigmokeronopsis stoecki (EU220226), Hemicycliostyla sphagni (FJ361758), and Pseudourostyla cristata (DQ019318). Sequences were aligned using MUSCLE v3.7 (Penn et al., 2010) with default parameters. The resulting alignment was manually edited using the program BioEdit 7.0.5.2 (Hall, 1999). Ambiguously aligned regions and gaps were excluded prior to phylogenetic analysis, resulting in a matrix of 1,727 characters (available from the authors upon request). Maximum likelihood (ML) analysis was conducted using RAxML-HPC2 v7.3.2 on XSEDE (Stamatakis et al., 2008) on CIPRES Science Gateway (Miller et al., 2010), using the GTR + G model as the optimal choice. Support for the best ML tree came from 1,000 bootstrap replicates with the GTR + CAT model. Bayesian inference (BI) analysis was performed with MrBayes v3.2.2 on XSEDE (Ronquist & Huelsenbeck, 2003) on the CIPRES Science Gateway, using the GTR + I (= 0.5278) + G (= 0.4686) model as selected by MrModeltest v.2.0 (Nylander, 2004). Markov chain Monte Carlo (MCMC) simulations were run with two sets of four chains for 2,000,000 generations with a sampling frequency of 100 and a burn-in of 5,000 trees (25%). All 4
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remaining trees were used to calculate posterior probabilities using a 50% majority rule consensus. TREEVIEW v1.6.6 (Page, 1996) and MEGA 4.0 (Tamura et al., 2007) were used to visualize the tree topologies. The systematic classification follows Lynn (2008).
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RESULTS
7 8
Bistichella cystiformans spec. nov.
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Diagnosis
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Size about 120–200 µm × 40–80 µm; body elongate elliptical with both ends broadly rounded. 33–45 adoral membranelles, five to eight buccal cirri arranged in a row. Four to six cirri in frontal row III; on average, seven cirri in frontal row IV extending usually to the same length as the adoral zone; 17–32 and 20–34 cirri in frontoventral rows V and VI respectively, both extending to the transverse cirri. Three to five transverse cirri, 20–35 left and 25–47 right marginal cirri; three bipolar dorsal kineties. Four macronuclear nodules and three to six micronuclei.
19 20
Type locality
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Slightly saline soil from Gaoqiao Mangrove National Nature Reserve in Zhanjiang (21°31 30 N; 110°16 58 E), southern China. ′
′′
′
′′
24 25
Deposition of type specimens
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One protargol-impregnated slide with the holotype specimen (Fig. 2e, f) (registration number: FYB2012040801) and a paratype slide (registration number: FYB2012040801-1) have been deposited in the Laboratory of Protozoology, OUC, Qingdao, China.
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Etymology
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cys.ti.for’mans. N.L. fem. n. cystis (from Gr. fem. n. kustis), the bladder and in biology, a cyst; L. part. adj. formans forming; N.L. part. adj. cystiformans cyst-forming. This species-group name indicates that this species can form cyst in response to food starvation. Gene sequence The SSU rRNA gene sequence has been submitted to GenBank database with the accession number KJ509196. Morphological description (Table 1 and Figs 2a–f, 3a–h and 6a–c) 5
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Size 120–200 µm × 40–80 µm in vivo. Body elongate elliptical in outline, with the anterior and posterior end broadly rounded, dorsoventrally flattened about 3:1, highly flexible but not contractile (Figs 2a–c and 3a–b). Adoral zone approximately 2/5 of body length. Pellicle soft, without cortical granules underneath. Cytoplasm colourless, usually with many lipid droplets (approx. 3 µm across) and food vacuoles containing small colpodids, scuticociliates and bacteria, which were observed in both living and protargol-impregnated specimens. Contractile vacuole about 25 µm across, slightly above mid-body near left body margin (Fig. 2a), pulsating at intervals of about 30 s. Four macronuclear nodules, spherical to ellipsoid, conspicuous in vivo, arranged slightly left of cell. Three to six spherical micronuclei, attached or near to the macronuclear nodules. Slowly crawling on substrates or rotating around main body axis when swimming. Body folding or twisting when crawling over debris.
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Cells easily encyst when food is scarce. Cysts usually globular and about 40 µm in diameter; cortex thin and indistinct, cortical granules absent (Fig. 3h). Cyst contents dominated by the nuclear apparatus and cyst plasm with many globular inclusions 2–3 µm in diameter.
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Infraciliature as shown in Figs 2d–f, 6a–c. Adoral zone composed of 33–45 membranelles, with its distal portion located at the right anterior end of the body. Paroral and endoral membrane almost equal in length and parallel to each other, the former slightly longer than the latter. Cirral pattern and number of cirri usually variable, except for the three enlarged frontal cirri, with the rightmost one immediately behind the distal end of the adoral zone of membranelles. Five to eight buccal cirri arranged in a longitudinal row close to the paroral membrane. No frontoterminal cirri present. Four to six cirri in frontal row III, located at same level as the row of buccal cirri. Frontal row IV composed of seven cirri on average, extending usually to the proximal end of the adoral zone; in eight out of 25 individuals this row comprised 16 cirri, extending nearly to mid-body. Frontoventral row V composed of 17–32 cirri, starting from anterior quarter of the body and extending slightly obliquely to the leftmost transverse cirrus. Frontoventral row VI composed of 20–34 cirri, starting near the anterior end of the right marginal row and terminating close to the rightmost transverse cirrus. Usually, four transverse cirri, arranged in an oblique pseudorow, protruding beyond the posterior end of the body. In three out of 25 individuals, an additional frontoventral row and five transverse cirri present. One left and one right marginal row confluent posteriorly, composed of 20–35 and 25–47 cirri, respectively. Three bipolar dorsal kineties with about 24 dikinetids in each row, slightly shortened anteriorly and posteriorly; dorsal cilia approx. 3 µm long in vivo. A short row with about four to six dikinetids present on the anterior-left of the leftmost dorsal kinety in four out of 25 individuals. Caudal cirri absent.
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Morphogenesis during binary fission (Figs 4a–j, 5a–d and 6d–j)
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Stomatogenesis and frontal-ventral-transverse cirral anlagen. Several critical morphogenetic stages were observed. Fig. 4a shows a relatively early divider demonstrating that new adoral membranelles begin to organize from anterior to posterior in the oral primordium of the opisthe and that six frontal-ventral-transverse cirral anlagen are recognizable; the parental undulating membranes are dedifferentiated to form the proter’s anlage I; while in the opisthe, anlage I is detached from the oral primordium (Figs 6d, f).
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Later, the new adoral membranelles continue to develop posteriorly in the opisthe; the anterior part of anlage I develops into the leftmost frontal cirrus in both dividers (Figs 4c, d and 6e, g); with the rest of it beginning to split longitudinally, giving rise to the paroral and endoral membranes.
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Gradually, all cirral anlagen develop into new cirri. The anterior-most cirri from anlagen II and III, together with the cirrus from anlage I, become three enlarged frontal cirri; the rear cirri derived from anlage II (except the rearmost one) migrate towards the newly formed paroral membrane and become a row of buccal cirri; anlagen II to V (sometimes III to V) provide the rearmost cirri to form the transverse cirri, while anlage VI does not form transverse cirrus at all; the remaining cirri from anlagen III to VI form the two frontal and two frontoventral rows (Figs 4f, g, i, j, 5a, b and 6i, h). Rarely, an additional anlage is present between the normal anlagen V and VI in the opisthe (Figs 4c, d), which may contribute one transverse cirrus and an extra ventral row (Fig. 5c).
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In a late divider, the anterior part of the new adoral zone curves to the right in the opisthe, while the parental adoral zone of membranelles remains completely intact throughout the morphogenetic process (Figs 5c, d and 6j).
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Anlagen of marginal rows and dorsal kineties. The formation of the marginal rows and dorsal kineties proceeds intrakinetally in a conventional manner for hypotrichs. The anlagen of the marginal rows are formed by the dedifferentiation of some cirri within each parental marginal row (Fig. 4a). These anlagen then stretch longitudinally and gradually replace the parental structures completely (Figs 4c, f, i, 5a, c and 6d, e, i). The dorsal kineties anlagen are also formed within the old rows in both dividers such that, with the proliferation of basal bodies, they elongate and supplant the parental structures (Figs 4b, e, h, j and 5b, d).
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Nuclear apparatus. The macronuclear nodules fuse into a single mass in middle dividers and then divide to form four nodules for filial cells. The divisional process of the micronuclei was unclear, although they were recognizable in some dividers (Figs 4b, e, h, j, 5b, d and 6f, g, j).
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SSU rRNA gene sequence and phylogenetic analyses (Fig. 7)
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The SSU rRNA gene sequence of Bistichella cystiformans spec. nov. was deposited in the GenBank database with the accession number KJ509196. The length and GC content of the SSU rRNA gene are 1,694 bp and 45.81%, respectively.
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We included a broad selection of 61 species in the present phylogenetic analyses, containing almost all available SSU rRNA gene sequences from both the orders Stichotrichida and Sporadotrichida. The topologies of the ML and BI trees were basically congruent and, therefore, a single topology is presented based on the ML tree with support values from both algorithms indicated on branches (Fig. 7). In the phylogenetic trees, Bistichella cystiformans spec. nov. clusters in the clade containing B. variabilis, Parabistichella variabilis, Uroleptoides magnigranulosus and two Orthoamphisiella species (47% ML, 0.87 BI). Sequence comparisons indicate a similar result, with the sequence similarities between B. cystiformans spec. nov. and the species in the Bistichella–Parabistichella–Uroleptoides–Orthoamphisiella clade ranging from 99.2% to 99.4%.
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DISCUSSION
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Morphological comparison with congeners (Table 2)
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Berger (2008) established the genus Bistichella with five species assembled in it, namely B. buitkampi (Foissner, 1982) Berger, 2008 (the type of the genus, basionym: Paraurostyla buitkampi Foissner, 1982), B. namibiensis (Foissner, Agatha & Berger, 2002) Berger, 2008 (basionym: Amphisiella namibiensis Foissner, Agatha & Berger, 2002), B. procera (Berger & Foissner, 1987) Berger, 2008 (basionym: Pseudouroleptus procerus Berger & Foissner, 1987), B. terrestris (Hemberger, 1985) Berger, 2008 (basionym: Pseudouroleptus terrestris Hemberger, 1985) and B. humicola (Gellért, 1956) Berger, 2008 (basionym: Uroleptus humicola Gellért, 1956). Simultaneously, Foissner et al. (2008) transferred two of them, i.e., Pseudouroleptus procerus and Pseudouroleptus terrestris into the newly erected genus Metauroleptus. He & Xu (2011) described a new species, Bistichella variabilis, and noted that the assignment of Foissner et al. (2008) tends to make the genus Bistichella monophyletic, containing only members without caudal cirri. Considering that the presence or absence of caudal cirri is widely used to separate hypotrich genera (e.g. the oxytrichid genus Tachysoma Stokes, 1887 and the urostyloid genus Caudiholosticha Berger, 2003) (Berger, 1999, 2006), the pisciform shape with a narrowed posterior end and the presence of caudal cirri in Pseudouroleptus procerus and P. terrestris, make these more suitable to be members of the genus Metauroleptus (Berger & Foissner, 1987; Hemberger, 1985; Berger, 2008). We therefore agree with the exclusion of these two species from Bistichella, and accordingly compare the new species B. cystiformans with its four congeners. 8
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In terms of the infraciliature, Bistichella cystiformans spec. nov. is closely related to B. variabilis. The former, however, can be recognized by the position of the rightmost frontal cirrus (immediately behind the distal end of the adoral zone of membranelles vs. positioned anteriorly at the same level as the other two frontal cirri) and the two rightmost frontoventral rows (V extending posteriorly to the leftmost transverse cirrus vs. distinctly away from transverse cirri; VI commencing near the anterior end of the right marginal row and terminating close to the rightmost transverse cirrus vs. mostly short and located in the posterior quarter of the body). Moreover, the new species differs in having fewer adoral membranelles (33–45 vs. 46–63), buccal cirri (5–8 vs. 7–12), and fewer left (20–35 vs. 37–63) and right (25–47 vs. 44–78) marginal cirri (He & Xu, 2011).
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Bistichella buitkampi resembles B. cystiformans spec. nov. in body size and shape. However, the former can be separated from the latter in having fewer buccal cirri (2–4 vs. 5–8), fewer cirri in frontal rows III (2–3 vs. 4–6) and IV (3–7 vs. 5–18), a shorter frontoventral row VI (far away from transverse cirri vs. close to transverse cirri), an absence of transverse cirri (vs. presence) and in the arrangement of the macronuclear nodules (in two separated pairs vs. four, in a row) (Foissner, 1982).
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Bistichella namibiensis is very similar to B. cystiformans spec. nov. in the arrangement of the two frontoventral rows, but differs from the latter in having two macronuclear nodules (vs. four), fewer buccal cirri (2–5 vs. 5–8), fewer cirri in frontal row III (approx. 1 vs. 4–6) and IV (approx. 3 vs. 5–18), as well as in having more cirri in frontoventral row VI (38–50 vs. 20–34) and more left (44–58 vs. 20–35) and right (42–61 vs. 25–47) marginal cirri (Foissner et al., 2002).
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Bistichella humicola was classified as a taxon incertae sedis in Bistichella by Berger (2008). It is described mainly from fixed and opal blue-stained specimens and, as a result, the validity of the cirral pattern cannot be reliably ascertained. Nevertheless, it can be easily distinguished from B. cystiformans spec. nov. in having only one buccal cirrus (vs. 5–8), absence of transverse cirri (vs. 3–5) and presence of many (approx. 32) macronuclear nodules (vs. four) (Gellért, 1956).
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Morphogenetic pattern in Bistichella
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Prior to this investigation, no morphogenetic data were available for any species of Bistichella. Our study of Bistichella cystiformans spec. nov. attempts to remedy this defect, however, we were unable to fully determine the earliest stages of morphogenesis in our specimens and, therefore, the origin of the oral primordium and cirral anlagen is not available. Accordingly, 9
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morphogenesis in B. cystiformans can be summarized as follows: (1) in the proter, the parental adoral zone of membranelles is completely retained, while in the opisthe, the oral primordium is formed apokinetally in the posterior half of the cell; (2) the frontoventral and transverse cirri are formed by the conventional six-anlagen mode; (3) the cirral streaks II – V (or III – V) generate one transverse cirrus each at their posterior ends while streak VI provides no transverse cirrus; (4) the marginal rows occur within the old structures in a secondary mode; (5) the dorsal kineties develop intrakinetally and no fragments are formed; (6) all macronuclear nodules fuse into a single mass and then divide twice.
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One of the most remarkable morphogenetic features in Bistichella cystiformans spec. nov. is that the rightmost frontal-ventral-transverse cirral anlage (= VI) does not form a transverse cirrus at its rear end, which, according to current knowledge, is unique in stichotrichs with six frontal-ventral-transverse cirral anlagen. Without exception in these species, the FVT-anlage VI contributes one transverse cirrus (e.g. Amphisiella, Gonostomum, Fragmocirrus, Trachelostyla, Oxytricha, Notohymena) (Berger, 1999, 2008, 2011; Chen et al., 2013a, b, c; Lv et al., 2013; Shao et al., 2013). Whether or not the absence of a special group of cirri is driven by environmental changes cannot be determined at present. Considering, however, that some taxa (e.g. Apoholosticha and Heterokeronopsis) found in mangroves always exhibit a lack of specific cirral groups, the possibility that the observed difference is due to the effect of variable mangrove environments upon the phenotype cannot be excluded (Fan et al., 2014; Pan et al., 2013).
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Familial classification of Bistichella
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Bistichella was established with species previously allocated into Pseudouroleptus and Amphisiella (Berger, 2008). Unlike the oxytrichid genus Pseudouroleptus, Bistichella lacks dorsomarginal kineties and dorsal kinety fragmentation, strongly indicating that it does not belong to the dorsomarginalian part of the Hypotricha, and should not be included in the family Oxytrichidae (Berger, 1999). Likewise, Bistichella lacks the amphisiellid cirral row, a critical character of the name-bearing type genus Amphisiella, and therefore it can also not be included in the family Amphisiellidae (Berger, 2008; Chen et al., 2013b). This position was also taken by Berger (2008) in his review of the amphisiellids, in which he classified Bistichella as a taxon of unknown position in the Hypotricha. The separation of Bistichella from Pseudouroleptus and Amphisiella is also supported by molecular trees in which the former is quite distant from the latter two (He & Xu, 2011).
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Present and previous phylogenetic studies based on SSU rRNA gene sequences show that Orthamphisiella clusters into a clade including Bistichella with relatively low bootstrap values (He & Xu, 2011). The molecular data of Orthamphisiella were only available for O. breviseries and an unidentified species and the assignment of both species into the 10
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Orthamphisiella is uncertain based on morphological data (Berger, 2008). Considering that the SSU rRNA gene sequence of the type species O. stramenticola is unavailable, the systematic position of Orthoamphisiella remains uncertain. Moreover, Orthoamphisiella differs distinctly from Bistichella in having no or only one long frontoventral row (vs. two), and in that the frontoventral row of the proter is composed of parental cirri in the anterior portion and newly formed cirri in the posterior portion (vs. only newly formed cirri), so both genera cannot have the same familial assignment from a morphological and morphogenetic perspective (Eigner & Foissner, 1991). Eigner (1997) established the family Orthoamphisiellidae to include Orthoamphisiella and some other genera, based primarily on the formation of within-anlagen in the two rightmost ventral cirral rows. Actually, except for the type species Orthoamphisiella stramenticola, none of the taxa originally included corresponded with the familial classification (Berger, 2008). Thus, this family is redundant and not widely accepted (Berger, 2008; Lynn, 2008).
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Bistichella has some ontogenetic traits in common with kahliellid genera, such as Kahliella Corliss, 1960, the type genus of the family Kahliellidae, e.g. the cirral anlagen are formed within the parental rows and dorsal kinety fragmentation is lacking (Berger, 2011). Kahliellids are also characterized by their tendency to preserve parental marginal rows and/or dorsal kineties in a variable number of divisions and in presenting dorsomarginal kineties. Bistichella, however, bears no parental marginal rows and/or dorsal kineties in divisions, and lacks dorsomarginal kineties, and therefore it cannot be included in the family Kahliellidae.
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Metauroleptus Foissner et al., 2008 is very similar to Bistichella in having three frontal cirri, two long and several short frontoventral rows, a row of buccal cirri, one left and one right marginal row (Foissner et al., 2008). In addition, the frontoventral-transverse cirral anlagen appear as the conventional six streaks, and the anlagen of marginal rows and dorsal kineties occur intrakinetally in both genera. Molecular data were unavailable, however, and therefore the systematic position of Metauroleptus remains unknown at present.
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The current study confirms that Bistichella has a close relationship with Parabistichella Jiang et al., 2013. This is in accordance with the morphological data, since both genera resemble each other very well in having three frontal cirri, long frontoventral rows, one left and one right marginal row, and no caudal cirri (Jiang et al., 2013). It is still too early, however, to provide a definite familial assignment for these two genera, since the classification is based on only a limited number of taxa and is, in any case, far from settled given the low bootstrap values in the phylogenetic trees.
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ACKNOWLEDGEMENTS
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This work was supported by the Natural Science Foundation of China (Project numbers: 41176119, 31030059 and 31111120437) and by King Saud University, Deanship of Scientific Research, International Research Group Program (Project number: IRG14-22). Many thanks are due to Ms. Wenping Chen (SCNU) for sample collection and to Ms. An Liu (OUC) for gene sequencing. Our thanks are also given to Prof. Xiaofeng Lin (SCNU) for ensuring institutional support for this research.
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REFERENCES
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(Ciliophora, Oxytrichidae). J Eukaryot Microbiol 60, 455–466. Lynn, D. H. (2008). The Ciliated Protozoa: Characterization, Classification, and Guide to the Literature, 3rd ed. Dordrecht: Springer. Medlin, L., Elwood, H. J., Stickel, S. & Sogin, M. L. (1988). The characterization of enzymatically amplified eukaryotes 16S-like ribosomal RNA coding regions. Gene 71, 491–500. Miller, M. A., Pfeiffer, W. & Schwartz, T. (2010). "Creating the CIPRES Science Gateway for inference of large phylogenetic trees" in Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans LA. p. 1–8. Nylander, J. A. A. (2004). MrModeltest 2.2. Program distributed by the author. Uppsala: Evolutionary Biology Centre, Uppsala University. Page, R. D. M. (1996). TREEVIEW: an application to view phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358. Pan, Y., Li, J., Hu, X., Warren, A. & Song, W. (2013). Ontogeny and phylogeny of a new urostylid ciliate, Heterokeronopsis pulchra g. n., sp. n. (Protozoa, Ciliophora). Eur J Protistol 49, 298–311. Penn, O., Privman, E., Ashkenazy, H., Landan, G., Graur, D. & Pupko, T. (2010). GUIDANCE: a web server for assessing alignment confidence scores. Nucleic Acids Res 38(Web Server issue), W23–W28. Ronquist, F. & Huelsenbeck, J. P. (2003). MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Shao, C., Pan, X., Jiang, J., Ma, H., Al-Rasheid, K. A. S., Warren, A. & Lin, X. (2013). A redescription of the oxytrichid Tetmemena pustulata (Müller, 1786) Eigner, 1999 and notes on morphogenesis in the marine urostylid Metaurostylopsis salina Lei et al., 2005 (Ciliophora, Hypotrichia). Eur J Protistol 49, 272–282. Song, W., Warren, A. & Hu, X. (2009). Free-living Ciliates in the Bohai and Yellow Seas, China. Beijing: Science Press (in both Chinese and English). Stamatakis, A., Hoover, P. & Rougemont, J. (2008). A rapid bootstrap algorithm for the RAxML Web-Servers. Syst Biol 75, 758–771. Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA 4: Molecular evolutionary genetics analysis (MEGA) software Ver. 4.0. Mol Biol Evol 24, 1596–1599. Wilbert, N. (1975). Eine verbesserte Technik der Protargolimprägnation für Ciliaten. Mikrokosmos 64, 171–179.
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FIGURE LEGENDS
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Fig. 1. (a) Photograph of the sample site, Gaoqiao Mangrove National Nature Reserve in Zhanjiang, southern China. (b) Portion of a map of southern China, showing the location of Zhanjiang. (c) Photograph showing the non-flooded Petri dish.
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Fig. 2. Bistichella cystiformans spec. nov. in vivo (a–c) and after protargol impregnation (d–f). (a) Ventral view of a representative individual. (b) Left lateral view. (c) Swimming movement. (d) Detailed ventral view of the anterior portion, the frontal cirri and buccal cirri row are shaded, while the broken lines connect cirri generated in the same anlage. (e, f) Ventral (e) and dorsal (f) view of the holotype to show the infraciliature and nuclear apparatus. AZM, adoral zone of membranelles; BC, buccal cirri; CV, contractile vacuole; DK1–3, dorsal kineties; EM, endoral membrane; FC, frontal cirri; LMR, left marginal row; Ma, macronuclear nodules; Mi, micronuclei; PM, paroral membrane; RMR, right marginal row; TC, transverse cirri; III, IV, frontal rows; V, VI, frontoventral rows. Bars, 70 µm.
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Fig. 3. Photomicrographs of Bistichella cystiformans spec. nov. in vivo. (a, b) Ventral views of different, freely motile individuals. (c) Ventral view of a squeezed specimen to show the grooves along the marginal cirral row and frontoventral rows (arrowheads). (d) Ventral view of the anterior portion, arrowheads mark the tooth-like structures inserted between membranelles. (e) Ventral view of buccal field, showing the endoral membrane (arrowhead) and paroral membrane (arrow). (f) Cytoplasm with many food vacuoles and four macronuclear nodules (arrowheads). (g) Ventral view of the posterior portion to show the frontoventral rows V, VI, and transverse cirri. (h) Resting cysts. FV, food vacuoles; TC, transverse cirri; V, VI, frontoventral rows V and VI. Bars, 50 µm (a–c), 70 µm (h).
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Fig. 4. Morphogenesis of Bistichella cystiformans spec. nov. after protargol impregnation. (a, b) Ventral (a) and dorsal (b) view of an early-middle divider, to show the formation of the frontal-ventral-transverse cirral anlagen and how the anterior end of the undulating membranes anlage contributes the leftmost frontal cirri (arrowhead), arrow points to the formation of the left marginal anlage within the old cirral row. (c–e) Ventral (c, d) and dorsal (e) views of a mid-stage divider, showing formation of the leftmost frontal cirri (arrowheads) and the frontoventral cirri anlagen beginning to differentiate into cirri (d). Note the macronuclear nodules fuse into a single mass. (f–h) Ventral (f, g) and dorsal (h) views of a divider in the middle stage, arrowheads denote the separation of the undulating membranes. Note the macronuclear mass is about to divide. (i, j) Ventral (i) and dorsal (j) view of a late divider, showing the almost complete segmentation of the frontoventral cirri anlagen, arrows mark the new transverse cirri. DK, dorsal kineties; DKA, dorsal kineties anlagen; LMA, left marginal anlage; LMR, left marginal row; Ma, macronuclear nodules; Mi, micronuclei; OP, opisthe’s oral primordium; RMA, right marginal anlage; RMR, right marginal row. Bars, 70 15
1
µm.
2 3 4 5 6 7 8
Fig. 5. Late stages of morphogenesis of Bistichella cystiformans spec. nov. after protargol impregnation. (a, b) Ventral (a) and dorsal (b) view of a late divider, note the dividing macronuclear nodules. (c, d) Ventral (c) and dorsal (d) view of a very late divider, to show the cirri almost in their final positions, arrowheads showing the transverse cirri migrating to their final positions. DK, dorsal kineties; LMR, left marginal row; Ma, macronuclear nodules; Mi, micronuclei; RMR, right marginal row. Bars, 70 µm.
9 10 11 12 13 14 15 16 17 18 19
Fig. 6. Photomicrographs of Bistichella cystiformans spec. nov. in interphase stages (a–c) and during binary division (d–j) after protargol impregnation. (a) Ventral view of a representative cell with four transverse cirri. (b) Ventral view of another specimen to show the elongated frontal row IV (arrowhead). (c) Ventral view of the anterior portion. (d, f) Ventral view of an early divider, to show the frontoventral cirri anlagen of the opisthe. (e, g) Ventral view of a mid-stage divider, arrowhead marks the undulating membranes anlage contributing the leftmost frontal cirrus. (h, i) Ventral view of a divider in the middle stage, arrowheads mark the three new frontal cirri of the proter. (j) Ventral view of a very late divider. Note the dividing macronuclear nodules. BC, buccal cirri; EM, endoral membrane; FC, frontal cirri; PM, paroral membrane. Bars, 70 µm.
20 21 22 23 24 25
Fig. 7. Maximum likelihood (ML) tree based on SSU rRNA gene sequences, showing the position of Bistichella cystiformans spec. nov. (bold, arrow). Bootstrap values above 50 for ML and posterior probability values above 0.5 for Bayesian inference (BI) are given near nodes. Fully supported (100/1.00) branches are marked with solid circles. All branches are drawn to scale. The scale bar corresponds to two substitutions per 100 nucleotide positions.
16
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Table 1. Morphometric characterization of Bistichella cystiformans spec. nov. Character a Min Max Body, length 86 146 Body, width 26 86 AZM, length 39 82 Adoral membranelles, number 33 45 Distance between body anterior 7 13 end and distal end of AZM Frontal cirri, number 3 3 Buccal cirri, number 5 8 Cirri in frontal row III, number 4 6 Cirri in frontal row IV, number 5 18 Distance betweenbody anterior end and 15 30 anterior end of frontoventral row V Cirri in frontoventral row V, number 17 42 Cirri in frontoventral row VI, number 20 34 Transverse cirri, number 3 5 Distance between body posterior end and 6 25 the rearmost transverse cirrus Cirri in left marginal row, number 20 35 Cirri in right marginal row, number 25 47 Dorsal kineties, number 3 4 Dikinetids in dorsal kinety 1, number 20 32 Dikinetids in dorsal kinety 2, number 18 30 Dikinetids in dorsal kinety 3, number 20 29 Macronuclear nodules, number 4 4 Anteriormost macronuclear nodule, length 17 28 Anteriormost macronuclear nodule, width 13 24 Micronuclei, number 3 6 Micronuclei, length 2 4
Mean SD 106.3 16.43 41.3 15.08 47.5 9.37 37.5 2.66 10.0 1.65
CV 15.5 36.5 19.7 7.1 16.5
n 25 25 25 25 15
3.0 6.5 4.5 10.7 19.8
0 0.77 0.65 3.76 4.71
0 11.9 14.6 35.2 23.8
25 25 25 25 15
24.3 27.1 4.0 14.1
4.84 3.29 0.50 5.57
19.9 12.2 12.5 39.4
25 25 25 15
27.8 33.2 3.2 24.0 24.6 24.9 4.0 21.9 17.6 4.1 2.9
3.53 4.99 0.41 3.42 3.09 2.39 0 3.37 3.09 0.83 0.52
12.7 15.0 12.8 14.3 12.6 9.6 0 15.4 17.6 20.2 18.0
25 25 20 15 15 15 25 15 15 25 15
a
All data are based on protargol-impregnated specimens. Measurements in µm. Abbreviations: AZM, adoral zone of membranelles; CV, coefficient of variation in %; Max, maximum; Mean, arithmetic mean; Min, minimum; n, sample size; SD, standard deviation.
17
1
Table 2. Morphological comparison of Bistichella cystiformans spec. nov. with congeners. Character
B. cystiformans spec. nov.
B. variabilis
B. buitkampi
B. namibiensis
B. humicola
Body size in vivo in µm
120–200 × 40–80
170–270 × 50–90
135–180 × 50–70
140–300 × 40–100
120 a
Body shape
Elongate ellipsoid
Elongate ellipsoid
Dumbbell-shaped to sigmoidal
Elongate ellipsoid
Elongate ellipsoid
33–45
46–63
34–40
39–48
31
5–8
7–12
2–4
2–5
1
2–3
1
b
9
3
b
Possibly lacking
Adoral membranelles, number Buccal cirri, number Cirri in frontal row III, number Cirri in frontal row IV, number
4–6
6–12
5–18
7–18
3–7
Cirri in frontoventral row V, number
17–42
35–62
25–37
33–46
24
Arrangement of frontoventral row V
Extending to the leftmost transverse cirrus
Extending posteriorly to about 80% of body length
Extending posteriorly to about 75% of body length
Extending obliquely to transverse cirri
Extending posteriorly to about 64% of body length
Cirri in frontoventral row VI, number
20–34
7–38
20–30
38–50
44
Arrangement of frontoventral row VI
Commencing behind distal end of adoral zone and extending to the rightmost transverse cirrus
Mostly short and located in the posterior quarter of body and extending to the rightmost transverse cirrus
Commencing behind distal end of adoral zone and extending posteriorly to about 75% of body length
Commencing behind distal end of adoral zone and extending obliquely to transverse cirri
Commencing behind distal end of adoral zone and extending posteriorly to rear cell end
3–5
5–7
6–15
4–6
Absent
Cirri in left marginal row, number
20–35
37–63
35–52
44–58
35
Cirri in right marginal row, number
25–47
44–78
39–49
42–61
41
3–4
3–4
3
3
Data unavailable
4
4
4
2
32
3–6
2–7
2
2–6
Data unavailable
Present study
He & Xu (2011)
Foissner (1982)
Foissner et al. (2002)
Gellért (1956) Berger (2008)
Transverse cirri, number
Dorsal kineties, number Macronuclear nodules, number Micronuclei, number Data source
2
a
3
b
Method (in vivo or after fixation) not indicated. Data from the holotype specimen in Foissner et al. (2002).
18
