Morphology, ontogeny, and molecular phylogeny of two novel bakuellid-like hypotrichs (Ciliophora: Hypotrichia), with establishment of two new genera

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European Journal of Protistology 49 (2013) 78–92

Morphology, ontogeny, and molecular phylogeny of two novel bakuellid-like hypotrichs (Ciliophora: Hypotrichia), with establishment of two new genera Jiamei Jianga,b , Jie Huangb , Liqiong Lic , Chen Shaod , Khaled A.S. Al-Rasheide , Saleh A. Al-Farraje , Zigui Chenb,∗ a

College of Fishery and Life Science, Shanghai Ocean University, Shanghai 201306, China Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China c School of Ocean, Yantai Academe of China Agricultural University, Yantai 264670, China d The Key Laboratory of Biomedical Information Engineering, Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China e Zoology Department, King Saud University, Riyadh 11451, Saudi Arabia b

Received 8 January 2012; received in revised form 11 May 2012; accepted 15 May 2012 Available online 21 July 2012

Abstract The morphology, ontogeny and molecular phylogeny of Apobakuella fusca gen. n., sp. n. and Parabistichella variabilis gen. n., sp. n., from south China were investigated. Apobakuella fusca, brown colored, demonstrates bakuellid-like infraciliature, and a similar ontogenesis as the genus Bakuella. It is argued, however, that this species represents a novel genus, Apobakuella, which is characterized by two or more marginal rows on the right, several buccal and parabuccal cirri, and lack of frontoterminal and caudal cirri. Phylogenetic analysis based on SSU rRNA gene sequences supports the close relationship of Apobakuella with Neobakuella and Diaxonella within the core Urostylida. By contrast, Parabistichella variabilis has a dominant frontoventral row, few midventral pairs, a long midventral row, and one marginal row on each side. Its morphogenesis exhibits: (1) partial reorganization of the parental adoral membranelles; (2) over six frontoventral-transverse cirri anlagen; (3) intrakinetal development of the midventral row; and (4) very likely, formation of the frontoventral row from the midventral row anlage. Both the morphological characteristics and the SSU rRNA gene sequences suggest that it is incertae sedis among the basal hypotrichs. Further investigation of key taxa with additional molecular markers is required to reveal a better understanding on the phylogeny of Parabistichella. © 2012 Elsevier GmbH. All rights reserved. Keywords: Apobakuella; Bakuellid; Hypotrichs; Morphogenesis; SSU rRNA; Parabistichella

Introduction The ciliate subclass Hypotrichia Stein, 1859, has consistently been a focus of ciliatological research and now contains

∗ Corresponding

author. Tel.: +86 532 8203 2673. E-mail address: [email protected] (Z. Chen).

0932-4739/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ejop.2012.05.003

over 1000 nominal species which exhibit extremely diverse morphological/morphogenetical features (e.g. Berger 1999, 2001, 2006, 2008, 2011; Chen et al. 2010b; Foissner et al. 2004, 2010; Hu et al. 2010; Huang et al. 2010; Kamra and Kumar 2010; Li et al. 2010b; Schmidt et al. 2007; Vd’aˇcn´y et al. 2010). The family Bakuellidae Jankowski, 1979 differs from other Urostylida members (e.g., Holostichidae or Urostylidae)

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in having three frontal cirri and a midventral complex composed of anterior midventral pairs and posterior midventral row(s) (Berger 2006; Jankowski 1979). The family contains at least eight genera, i.e., Australothrix, Bakuella, Birojimia, Holostichides, Metaurostylopsis, Neobakuella, Parabirojimia and Paragastrostyla (Berger 2006; Li et al. 2011). Several brief reviews of this family have been made in the past decades (Chen et al. 2010a; Eigner 1994; Eigner and Foissner 1992; Franco et al. 1996; Song et al. 2011), including a recent update in Lynn’s (2008) classification scheme wherein Bakuellidae is a synonym of Urostylidae. Bakuellids are comparatively rare, and molecular data are available for only a few taxa (Li et al. 2011; Song et al. 2011; Yi et al. 2008a,b). In particular, the phylogenetic incongruence between the morphological and molecular data of some of its genera (e.g., Metaurostylopsis and Parabirojimia) challenges our understanding of the system and classification of the family Bakuellidae (Song et al. 2011; Yi et al. 2008a). In this work, we describe two novel bakuellid-like hypotrichs with highly differentiated frontal cirri and midventral complex. We argue that they represent two new genera (Apobakuella gen. n. and Parabistichella gen. n., respectively) and that they are possibly able to be distinguished from each other at family level. Their morphology, ontogeny, and molecular phylogeny are investigated.

Material and Methods

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illustrate the changes during the morphogenetic process, parental cirri are depicted by contour whereas new ones are shaded black. General terminology is mainly according to Lynn (2008). For an explanation of group-specific terms, see Berger (2006, 2008). The classification and nomenclature of hypotrichs follow Berger (1999, 2006, 2008).

DNA extraction, PCR amplification, and sequencing Genomic DNA extraction, PCR amplification, and sequencing of the SSU rRNA gene were performed according to Jiang and Song (2010). The universal eukaryotic primers Euk A and Euk B (Medlin et al. 1988) were used to amplify the SSU rRNA gene. Cycling parameters were as follows: a pre-run of 5 min at 94 ◦ C; then 35 cycles of 30 s at 94 ◦ C, 1 min at 60 ◦ C, and 2 min at 72 ◦ C; and, finally, one cycle of 7 min at 72 ◦ C. Sequencing in both directions was carried out on an ABI 3700 sequencer. Multiple clones (≥2) from the same PCR products were sequenced to verify the target gene. The SSU rRNA gene sequences were submitted to the NCBI/GenBank database, with accession numbers of JN008942 for Apobakuella fusca sp. n. (1766 bp) and JN008943 for Parabistichella variabilis sp. n. (1774 bp).

Sampling and cultivation Samples of Apobakuella fusca gen. n., sp. n. were collected from the surface of intertidal gravel in the Futian mangrove forest, Shenzhen, South China, on April 1, 2009, at a water temperature of 21 ◦ C, pH 7.5 and salinity 14‰. Samples of Parabistichella variabilis gen. n., sp. n., meanwhile, were collected from the intertidal zone at the estuary of the Pearl River, Seagull Village, Guangzhou, South China, on April 14, 2009, at a water temperature of 20 ◦ C and salinity 0.3‰ (freshwater). The surface sediments were transferred to Petri dishes with their original water and maintained as a raw culture in the laboratory for several days at room temperature. Rice grains were added to enrich the bacteria. About four days later, it was observed that the novel species, together with some euplotids and cyrtophorids, had become predominant.

Morphological and morphogenetic studies Specimens were examined in vivo using bright field and Nomarski differential interference contrast microscopy. Protargol silver impregnation (Wilbert 1975) was applied to reveal the infraciliature and an ocular micrometer was used to measure the stained specimens. Drawings were made with the aid of a drawing device (Li et al. 2010a). To

Phylogenetic analyses In order to assess the phylogenetic positions of Apobakuella fusca gen. n., sp. n. and Parabistichella variabilis gen. n., sp. n., the SSU rRNA gene sequences were aligned using MUSCLE v3.7 (Edgar 2004), and trimmed using Bioedit v7.0.5.2 so that both ends were 1725 bp in length (Hall 1999). Preliminary Maximum Likelihood (ML) analyses were performed on CIPRES Portal V 2.0 (http://www.phylo.org) and showed similar topologies for different selections of taxa. We therefore selected a set of 52 species representing key taxa of hypotrichs for the phylogenetic analysis, with two choreotrichs and two oligotrichs as an outgroup. ML bootstrapping analyses were carried out using RAxML with the setting as described in Stamatakis (2006). Bayesian inference (BI) was performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003) with the GTR+I+G model as selected by AIC in MrModeltest v.2.0 (Nylander 2004). Markov chain Monte Carlo (MCMC) simulations were run with two sets of four chains using the default settings: i.e. a chain length of 1,000,000 generations, with trees sampled every 100 generations. The first 25% of sampled trees were considered burn-in ones and were discarded prior to constructing a 50% majority rule consensus tree.

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Results Apobakuella gen. n. Diagnosis: Bakuellids with one left and two or more right marginal rows; frontal cirri clearly differentiated but no frontoterminal ones; one buccal and several parabuccal rows or fragments; midventral complex typically present with one or more midventral rows; transverse cirri present; three dorsal kineties; caudal cirri lacking. Type species: Apobakuella fusca sp. n. Etymology: Composite of the Greek prefix apo- (derived from) and the generic name Bakuella; feminine gender. Species assignable: The type species only.

Morphology of Apobakuella fusca sp. n. (Figs 1A–I, 2A–L; Table 1) Diagnosis: Brown-yellowish Apobakuella; size about 190 ␮m × 60 ␮m in vivo; two types of cortical granules: smaller ones colorless, larger ones green or yellow; about 45 adoral membranelles, 3–9 buccal cirri, 5–9 parabuccal cirri arranged in two or three rows, 7–13 transverse cirri; midventral complex composed of 14–27 pairs of cirri extending to posterior half of body, and 1–4 midventral rows in posterior portion; numerous macronuclear nodules; brackish water habitat. Type specimens: The slide containing the protargolstained holotype specimen (Figs 1H, I, 2K) is deposited

in the Laboratory of Protozoology, OUC, China (No. JJM2009040101). A paratype slide is deposited in the Natural History Museum, London, UK (accession number: NHMUK2011:10:27:2). Etymology: The Latin adjective fusca (brown) refers to the color of the live specimen. Type locality: The Futian mangrove forest in Shenzhen, South China (22◦ 31 N, 114◦ 00 E). Description: Body flexible but not contractile; size about 150–210 ␮m × 50–65 ␮m in vivo; cell brown-yellowish, elongate and elliptical in outline with both ends rounded and left margin more convex than right one; dorsoventrally flattened with the ratio about 2.5:1 (body width to thickness) (Figs 1A, B, 2A–C). Buccal cavity prominent, about one third of body length (Fig. 1A). Pellicle thin, two types of cortical granules on ventral and dorsal side, recognizable only at 400× or higher magnifications (Figs 1C, F, G, 2D–F, J): larger ones moss-green or olivecolored, about 1 ␮m across, arranged along cirral rows, but also grouped and distributed in rows in other areas of dorsal and ventral side (Figs 1C, 2D–F, J); smaller ones pale or greyish, about 0.2 ␮m across, densely arranged around cirri (Figs 1C, 2E) and sparsely arranged within rows of larger ones (Figs 1C, 2D). Cytoplasm colorless, usually packed with many small globules (about 2–5 ␮m across) and large food vacuoles containing flagellates, scuticociliates and bacteria (Figs 1A, B, 2J). Contractile vacuole about 20 ␮m across, positioned usually near posterior end of adoral zone of membranelles (Figs 1A, B, 2C), contracting at intervals of about 5–10 min. Only one individual observed with a different

Table 1. Morphometric data on Apobakuella fusca gen. n., sp. n. Character

Min

Max

Mean

SD

CV

n

Body length (␮m) Body width (␮m) Adoral zone of membranelles length (␮m) Number of adoral membranelles Number of frontal cirri Number of buccal cirri Number of parabuccal cirral rows Number of parabuccal cirri from anlage III Number of midventral pairs Number of midventral rows Number of cirri in midventral row 1a Number of cirri in midventral row na Number of cirri in left marginal row Number of cirri in right marginal row 1b Number of cirri in right marginal row 2b Number of transverse cirri Number of dorsal kineties Number of dikinetids in middle dorsal kinety

175 62 80 37 3 3 2 3 14 1 3 5 45 47 37 7 3 38

265 175 130 52 3 9 3 4 27 4 21 23 60 70 65 13 3 47

215.5 125.8 96.9 46.8 3.0 7.3 2.7 3.7 20.5 2.3 8.3 14.3 51.0 55.2 49.9 9.2 3.0 41.4

28.1 27.9 10.4 3.8 0 1.2 0.4 0.5 2.8 0.9 4.9 4.4 3.4 5.8 7.2 1.7 0 3.0

13.1 22.2 10.8 8.1 0 16.6 16.3 13.5 13.9 37.1 58.8 31.1 6.6 10.4 14.4 18.6 0 7.2

25 25 25 25 21 25 27 27 22 25 25 25 25 25 25 25 19 12

Measurements are based on protargol-impregnated specimens. Abbreviations: CV, coefficient of variation in %; max, maximum; mean, arithmetic mean; min, minimum; n, number of cells measured; SD, standard deviation. a Midventral row 1 is the leftmost row, midventral row n is the rightmost one. If n = 1 (5 out of 25 cells checked), then midventral row 1 and row n means the same row. b For designation of groups, see Fig. 1H.

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Fig. 1. A–I. Apobakuella fusca gen. n., sp. n. in life (A–D, F, G) and after protargol impregnation (E, H, I). A. Ventral view of a representative individual. B. Ventral view to show another general body shape, arrow shows the contractile vacuole. C. Ventral view to show the arrangement of two types of cortical granules (arrows and arrowheads). D. Variation of the position of the contractile vacuole (arrow) in one individual. E. Detailed ventral view of the anterior portion. F, G. Ventral (F) and dorsal (G) view to show the distribution of cortical granules. H, I. Ventral (H) and dorsal (I) view of the holotype to show the infraciliature; cirri in gray are absent in the majority, arrows show the parabuccal cirri, arrowhead indicates the short outer midventral row. AZM, adoral zone of membranelles; BC, buccal cirri; DK, dorsal kineties; E, endoral; FC, frontal cirri; LMR, left marginal row; Ma, macronuclear nodules; MP, midventral pairs; MVR1, 2, n, midventral row 1, 2, n; PBC, parabuccal cirri; P, paroral; RMR1, 2, right marginal row 1, 2; TC, transverse cirri. Scale bars: 60 ␮m (A, B); 40 ␮m (E); 80 ␮m (H).

contractile vacuole position. At first this was located in the usual position but, later, it appeared in the posterior region of the body (Figs 1D, 2G), and we postulate, therefore, that this variance was in this case a result of the pressure of the cover slip. Numerous (over 200) macronuclear nodules, oval to elongated in shape, scattered throughout cytoplasm, thus difficult to observe in vivo (Fig. 1I). Micronucleus not observed. Locomotion with no specialties, crawling moderately quickly on substrate. Adoral zone of membranelles about one-third of body length, comprising 37–52 membranelles, with cilia up to 18 ␮m in vivo. Paroral and endoral almost equal in length and intersecting spatially in their posterior region (Fig. 1E). Most somatic cirri relatively fine with cilia about 15 ␮m long in vivo. Constantly three frontal cirri (Fig. 1E, H), 3–9 buccal cirri arranged in longitudinal row close to paroral (Fig. 1E), 5–9 parabuccal cirri arranged in two or three rows to

right of buccal cirri (Figs 1E, H, 2K). No frontoterminal cirri present. Midventral complex composed of 14–27 pairs of cirri arranged in typical zig-zag pattern, extending to about 50% or more of body length, together with 1–4 short to long midventral rows each comprising 3–23 cirri (Figs 1H, 2L). Usually right midventral rows composed of more cirri than left ones, although rarely (in three out of 25 specimens checked) right one relatively short (Fig. 1H). Transverse cirri row near end of inmost midventral row, slightly curved, composed of 7–13 narrowly spaced and slightly enlarged cirri (Fig. 2I). Invariably one left and two right marginal rows. Right marginal row 2 comprising 37–65 cirri, beginning dorsolaterally, extending to ventral side and then curving leftwards posteriorly to terminate near midline of cell; right marginal row 1 comprising 47–70 cirri, starting at level of rightmost frontal cirrus and similarly terminating near midline. Left marginal row composed of 45–60 cirri, commencing to left of posterior

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Fig. 2. A–V. Microphotographs of Apobakuella fusca gen. n., sp. n. in life (A–G, J) and after protargol impregnation (H, I, K, L, show specimens in interphase stages; M–V show specimens during morphogenesis). A–C, G. Ventral view of different specimens, arrow indicates the contractile vacuole. D–F, J. Dorsal (D, F, J) and ventral (E) view to show the arrangement of the cortical granules; arrows show the smaller ones, arrowheads indicate the bigger ones. H. Dorsal view, showing a dorsal kinety. I. Ventral view of the posterior part, arrow marks the transverse cirri. K. Ventral view of the anterior portion, arrows show the parabuccal cirri. L. Ventral view of the posterior portion, arrowheads show the midventral pairs. M. Ventral view of an early divider, arrowheads indicate the small patches which later fuse to the oral primordium in the opisthe. N, O. Ventral view of the same specimens to show the oral primordium in the proter (N) and the opisthe (O). P, Q. Ventral view of a later divider showing the development of the oral primordium in the proter (P) and the opisthe (Q). R. Dorsal view, to show the anlagen of dorsal kineties (arrowhead) and left marginal row (arrow). S. Ventral view of the proter, arrow shows the leftmost frontal cirrus. T, U. Ventral view of the proter (T) and opisthe (U) of the same individual, arrowheads point to the buccal cirri, arrows indicate the parabuccal cirri. V. The fusing macronuclear nodules of a divider at a middle stage. Ma, macronuclear nodules; MVR1, 2, midventral row 1, 2. Scale bars: 70 ␮m.

portion of adoral zone and ending obviously away from right marginal rows (Fig. 1H). Three bipolar dorsal kineties with cilia about 4–5 ␮m long in vivo. Dikinetids arranged more or less evenly in each row (Figs 1I, 2H). About 38–47 dikinetids in middle one. Caudal cirri lacking.

Morphogenesis of Apobakuella fusca sp. n. (Figs 2M–V, 3A–J) Stomatogenesis: Division commences with the formation of long and narrow oral primordia near the left midventral cirri in the opisthe (Figs 2M, 3A) and along the parental

endoral in the proter (Fig. 3A). Later, the opisthe’s undulating membrane anlage develops nearby, possibly from the oral primordium (Figs 2O, 3B, C). Simultaneously, the proter’s undulating membrane anlage forms from the oral primordium to its anterior right while the parental endoral is still intact (Fig. 3C). In the remaining stages, the development of oral primordia and undulating membrane anlagen follows the normal pattern as in other hypotrichs, and the new oral apparatuses are formed (Fig. 3D, F, I). Development of frontal, midventral, and transverse cirri: In the intermediate stage, several streaks are formed to the right of the oral primordia which develop into the frontoventral-transverse cirri anlagen (Fig. 3C). Meanwhile, the parental buccal cirri dedifferentiate from anterior to

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Fig. 3. A–J. Morphogenesis of Apobakuella fusca gen. n., sp. n. at early to later stages after protargol impregnation. A. Ventral view of an early divider, arrow and arrowheads show the oral primordium for the opisthe and the proter, respectively. B. A slightly later divider, arrow points to the oral primordium for the proter. C. A divider in the following stage, double-arrowhead shows the undulating membrane anlage, arrowheads demonstrate the old buccal cirri and the anterior portion of the paroral in dedifferentiation, arrow points to the oral primordium in the opisthe. D, E. Ventral and dorsal view of the same specimen. Arrows point to the marginal cirri anlagen. F, G. Ventral (F) and dorsal (G) view of the same specimen at a later stage, arrows show the new inner marginal rows. H–J. Ventral (H, I) and dorsal (J) view of the same specimen at a middle stage, (H) is a detailed view of the anterior portion of (I), arrowhead in (H) points to the additional cirrus in absorption; arrowheads in (I) show the new midventral rows, double-arrowheads show the parabuccal cirri, arrows point to the new inner marginal rows. Scale bars: 80 ␮m (A, also applies to B, C; F, also applies to G, I, J).

posterior (Figs 2P, 3C), and possibly contribute to the formation of the proter’s frontoventral-transverse cirri anlagen (Fig. 3C). With further kinetosome proliferation, about 20 oblique streaks are differentiated for each daughter cell (Fig. 3D). Subsequently, a single cirrus develops from the anterior end of anlage I and becomes the leftmost frontal cirrus (Fig. 2S). Commencing in the anterior frontoventral-transverse cirri streaks and then proceeding posteriad, cirri form at the left ends of the anlagen in the proter and opisthe (Fig. 3F).

In the next stage, the frontal, midventral, and transverse cirri are almost formed. The following description refers to the specimen shown in Fig. 3H–J. In the proter, anlagen II and III produce the frontal cirri (two slightly enlarged ones), the buccal cirri (from anlage II), and the parabuccal cirri (from anlage III). Anlagen IV and V form a midventral pair and two parabuccal cirri each; anlagen VI–XVI produce a midventral pair each; anlagen XVII–XXII form a midventral pair and a transverse cirrus each, as well as additional cirri (Fig. 3H) which are possibly resorbed in the later stages. The

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midventral rows, meanwhile, and the rightmost transverse cirri, originate from the last three anlagen (XXIII–XXV). Essentially the same process is observed in the opisthe, although the number of streaks is different in this case (Figs 2U, 3I). No later stages were observed, however, considering that the frontoterminal cirri are absent in non-dividing stages, the anteriormost cirri of the rightmost anlage most probably do not migrate anteriorly. Development of marginal rows and dorsal kineties: The formation of the marginal rows and dorsal kineties proceeds in a conventional manner with two primordia being formed within each row/kinety (Figs 2R, 3D–J). Division of nuclear apparatus: Apobakuella fusca has many macronuclear nodules scattered throughout the cell. Prior to division, the nodules fuse gradually (Fig. 3E). However, due to the limited stages of division that were observed, it is unclear whether the nodules fuse to a single mass or whether they make a complex before subsequent divisions (Figs 2V, 3G, J). Two micronuclei, however, divide in a conventional manner during the middle stage (Fig. 3J).

Parabistichella gen. n. Diagnosis: Hypotrich with continuous adoral zone of membranelles. Three frontal cirri clearly differentiated; buccal cirrus(i) present; few midventral pairs followed by one midventral row; no frontoterminal cirri; frontoventral row present; one marginal row on each side; transverse cirri present; three dorsal kineties; caudal cirri absent. Type species: Parabistichella variabilis sp. n. Etymology: Composite of Greek para- (beside, from, against) and the generic name Bistichella; feminine gender. Species assignable: The type species only.

Morphology of Parabistichella variabilis sp. n. (Figs 4A–K, 5A–K; Table 2) Diagnosis: Size about 130–240 ␮m × 65–85 ␮m; cells elongated oval in vivo. Colorless cortical granules about 3 ␮m across, arranged along infraciliature and in buccal area. Single contractile vacuole positioned near left margin at about 35% of body length. 43–51 adoral membranelles and one buccal cirrus; midventral complex extending to near transverse cirri, composed of three or four midventral pairs and a long midventral row comprising 15–36 cirri; long frontoventral row made of 48–76 cirri; 5–8 transverse cirri; 43–67 cirri in left and 40–56 cirri in right marginal row respectively. Occasionally some extra left marginal and midventral row(s) significantly shortened or fragmented. Two macronuclear nodules and two micronuclei. Freshwater habitat. Type specimens: The slide containing the protargolstained holotype specimen (Fig. 4D, E) is deposited in the Laboratory of Protozoology, OUC, China (No. JJM2009041403). A paratype slide is deposited in the Natural History Museum, London, UK (accession number: NHMUK 2011:10:27:2). Etymology: “variabilis” means variable, referring to the high variability of the infraciliature. Type locality: Estuary of the Pearl River in Seagull Village, Guangzhou, South China (22◦ 52 N, 113◦ 33 E). Description: Size about 130–240 ␮m × 65–85 ␮m in vivo; cell slightly to dark grey colored due to many inclusions; outline elongated oval with both ends widely rounded and margins slightly convex (Figs 4A, 5A–C). In well-fed cells, anterior body portion wider than that in normal ones (Fig. 5D, E). Body flexible and bending distinctly when crawling on substrate, about 2:1 flattened dorsoventrally (Fig. 5F).

Table 2. Morphometric data on Parabistichella variabilis gen. n., sp. n. Character

Min

Max

Mean

SD

CV

n

Body length (␮m) Body width (␮m) Adoral zone of membranelles length (␮m) Number of adoral membranelles Number of buccal cirri Number of frontal cirri Number of midventral pairs Number of cirri in midventral rowa Number of cirri in frontoventral row Number of cirri in left marginal row Number of cirri in right marginal row Number of transverse cirri Distance from the rear end of the cell to the rearmost transverse cirrus (␮m) Number of dorsal kineties Number of macronuclear nodules

167 84 64 43 1 3 3 15 48 43 40 5 11 3 2

268 192 96 51 1 3 4 36 76 67 56 8 28 3 3

203.3 117.5 80.2 47.0 1.0 3.0 3.1 30.7 60.9 53.3 47.0 6.8 19.7 3.0 2.1

26.8 21.9 6.9 2.2 0 0 0.2 4.9 8.4 5.8 4.6 0.7 5.3 0 0.3

13.2 18.7 8.6 4.8 0 0 7.3 16.1 13.8 10.9 9.9 10.6 26.9 0 14.7

20 20 20 20 20 20 20 20 20 20 20 20 22 19 20

Measurements are based on protargol-impregnated specimens. Abbreviations: CV, coefficient of variation in %; max, maximum; mean, arithmetic mean; min, minimum; n, number of cells measured; SD, standard deviation. a The data are from the specimens possessing one midventral row, those with irregular fragmented midventral or frontoventral rows, as shown in Fig. 4F–K, are not included.

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Fig. 4. A–L. Parabistichella variabilis gen. n., sp. n. in life (A–C) and after protargol impregnation (D–L). A. Ventral view of a typical individual. B, C. Cortical granules along the cirri (B) and around the dorsal cilia (C). D, E. Ventral (D) and dorsal (E) view of the holotype to show the infraciliature, double-arrowhead points to the smaller transverse cirrus in several cells. F–K. Ventral view of some other specimens to show the variety in the infraciliature, arrows show the fragmental ventral and frontoventral rows, arrowheads point to the additional left marginal cirri, double-arrowheads indicate the smaller transverse cirri. L. Ventral view of a cell in early reorganization. DK1–3, dorsal kinety 1–3; FVR, frontoventral row; FVRA, frontoventral row anlage; LMR, left marginal row; MP, midventral pairs; MVR, midventral row; MVRA, midventral row anlage; RMR, right marginal row; TC, transverse cirri. Scale bars: 70 ␮m.

Buccal cavity prominent and about one third of body length (Fig. 4A). Cortical granules colorless, about 3 ␮m across, arranged along ciliature and in buccal field, where they are abundant (Figs 4B, C, 5G, I). Cytoplasm colorless, usually filled with some large, globular to clod-shaped inclusions and several food vacuoles containing Aspidisca, Chlamydonella, scuticociliates, and medium hypotrichs (Fig. 5A–D). Contractile vacuole about 25 ␮m across, slightly ahead of middle of body, near lefthand cell margin (Figs 4A, 5A, C). Two macronuclear nodules, long ellipsoidal, conspicuous in vivo, arranged slightly left of midline in middle third of cell (Figs 4A, 5H). Micronucleus not observed. Movement moderately rapid on substrate. Adoral zone of membranelles about one third of body length, comprising 43–51 membranelles. Paroral and endoral almost equal in length and optically intersecting in their

middle region (Fig. 4D). Consistently three enlarged frontal cirri; rightmost one exactly behind distal end of adoral zone of membranelles. Single buccal cirrus positioned close to middle-right of paroral (Figs 4D, 5K). No frontoterminal cirri present. Midventral complex composed of three to four midventral pairs in a typical zig-zag pattern together with a long midventral row. Midventral pairs extending to about rear fourth of adoral zone (Fig. 5K), while midventral row consisted of 15–36 cirri, terminating close to transverse cirri. Mostly one long frontoventral row composed of 48–76 cirri starting at level of rightmost frontal cirrus and terminating near level of transverse cirri. Five to eight transverse cirri arranged densely, near end of midventral row. Bases of transverse cirri noticeably larger than that of other cirri. However, in six specimens out of 29 measured, bases of some transverse cirri were significantly shorter, only 33–66% of length

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Fig. 5. A–K. Microphotographs of Parabistichella variabilis gen. n., sp. n. from life (A–I) and after protargol impregnation (J, K). A–E. Ventral view of different individuals, arrows show the contractile vacuole. F. Lateral view. G. Ventral view, to show the cortical granules along the cirri (arrows). H. Macronuclear nodules (arrows). I. Dorsal view, to show the dorsal cilia (arrowheads) and the cortical granules around (arrows). J. Ventral view of the posterior portion, arrow shows the additional left marginal cirri. K. Ventral view, midventral pairs are circled; arrow shows cirrus III/2, arrowhead points to the buccal cirrus. FVR, frontoventral row; Ma, macronuclear nodules; MVR, midventral row; RMR, right marginal row; TC, transverse cirri. Scale bars: 90 ␮m (B), 130 ␮m (E).

of other cirri (Fig. 4D, F, H). One left and one right marginal row, composed of 43–67 and 40–56 cirri, respectively. Three bipolar dorsal kineties with cilia about 2–3 ␮m long in vivo. Caudal cirri absent. It is noteworthy that the patterns of midventral and frontoventral rows are rather variable in these specimens. In seven out of 29 individuals, additional short midventral rows are present, while in two out of 29 specimens, the long frontoventral row is replaced by several irregular short rows (Fig. 4F–K). Also, additional left marginal cirri are present linearly or otherwise in seven cells out of the 29 observed (Figs 4F, I, K, 5J). These data are not included in Table 2.

Morphogenesis of Parabistichella variabilis sp. n. (Fig. 6A–G) A limited number of morphogenetic stages is available for Parabistichella variabilis sp. n. Fig. 6A shows a proter in a middle stage demonstrating that the proximal portion of

the parental adoral zone is renewed and six streaks are recognizable; one anlage (the midventral row anlage) occurs intrakinetally in the frontoventral row, while the frontoventral row anlage is formed on the right, perhaps from the midventral row anlage. In a later divider, new cirri, undulating membranes and adoral membranelles are present in both proter and opisthe (Fig. 6B). The leftmost frontal cirrus forms, as is usual, from the anterior end of anlage I. In the proter, anlage II forms the middle frontal, the buccal cirrus, and the leftmost transverse cirrus; the rightmost frontal cirrus (III/3) and a transverse cirrus are produced by anlage III; while anlagen IV–VII each mainly form another midventral pair and a transverse cirrus. As shown in Fig. 6B, anlagen IV–VII in the opisthe each produce additional 1–4 cirri; however, it is unclear whether these cirri are resorbed later. The newly formed midventral and frontoventral rows appear in both dividers. Each row gives rise to a transverse cirrus at its rear end (Fig. 6B). The marginal row and dorsal kineties anlagen occur within each row or kinety in a conventional manner (Fig. 6B, C).

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Fig. 6. A–G. Morphogenesis of Parabistichella variabilis gen. n., sp. n. after protargol impregnation. A. Ventral view of the proter at an early stage, arrowheads show the cirral streaks, arrow points to the frontoventral row anlage; double-arrowhead shows the new posterior adoral membranelles formed through dedifferentiation. B, C. Ventral (B) and dorsal (C) view of a middle divider, arrowheads show the transverse cirri from ventral and frontoventral row anlagen. D. Ventral view of another middle divider which presents more cirral anlagen (arrows), arrowheads show the transverse cirri formed by the rightmost anlagen. E. To show the fragmentation of the left marginal anlagen (double-arrowhead). F, G. Ventral (F) and dorsal (G) view of the same specimen at a late stage, arrowheads point to the fragmental rows. FVRA, frontoventral row anlage; LMA, left marginal row anlage; MVRA, midventral row anlage; RMA, right marginal row anlage; I–VII, anlage I–VII. Scale bars: 40 ␮m (A), 70 ␮m (B–E), 80 ␮m (F, G).

Macronuclear nodules fuse to a single mass before dividing twice (Fig. 6C). In another middle divider, more frontoventral-transverse cirri anlagen are formed (Fig. 6D). An extra anlage appears next to the newly formed left marginal row in the opisthe (Fig. 6D) or in both dividers (Fig. 6E). Eventually, each divider begins to elongate, the new ciliary structures move further apart as they migrate toward their final positions while the nuclei divide normally (Fig. 6F, G). The midventral row in the proter and the frontoventral row in the opisthe seem to be composed of

some fragmented rows that originate from additional anlagen (Fig. 6D, F).

Physiological regeneration of Parabistichella variabilis sp. n. (Fig. 4L) One early reorganizer shows the morphogenetic process as being similar to that in a proter (Fig. 4L): (1) the posterior portion of the parental adoral zone of the membranelles is renewed; (2) six or more anlagen are formed to the right of

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the buccal area; (3) the frontoventral row anlage develops de novo to the right of the parental one; and (4) the midventral row anlage is present within the frontoventral row.

definition of the family more fully (i.e., molecular sequences of multiple markers for key taxa).

Apobakuella gen. n. Molecular phylogeny of Apobakuella fusca sp. n. and Parabistichella variabilis sp. n. (Fig. 7) The SSU rRNA gene sequences show the closest relationship between Apobakuella fusca and Neobakuella flava with a pairwise similarity of 99.1%. This is consistent with the observation of significant similarities between these two species in their morphology and ontogeny. Apobakuella, Neobakuella, and Diaxonella form a monophyletic group within the core Urostylida clade. Parabistichella variabilis shares the highest SSU rRNA gene similarities with Uroleptoides magnigranulosa and Orthamphisiella breviseries with pairwise similarities 97.4–99.5%, but form a distinct clade outside of the core Urostylida.

Discussion The validity of the family Bakuellidae Berger (2006) assigned urostylids with zigzagging midventral pairs developing from more than six anlagen into four families, i.e., Holostichidae, Bakuellidae, Urostylidae, and Epiclintidae. Although most genera of the former three families were recently merged into a single large family, Urostylidae, in Lynn’s (2008) classification scheme, we prefer Berger (2006) since all genera assigned into Urostylidae sensu Lynn (2008) appear to be paraphyletic and therefore need to be addressed into more detailed taxa groups (Song et al. 2011; Yi and Song 2011). However, the monophyly of Bakuellidae has recently been challenged in a number of reports (Chen et al. 2011; Dai and Xu 2011; Yi et al. 2008a,b), and there is a need for additional data in order to outline the

Apobakuella gen. n. most likely belongs to the Bakuellidae because it possesses three frontal cirri and a midventral complex composed of pairs and row(s). In this regard it can be compared to four bakuellid genera: Bakuella, Neobakuella, Metaurostylopsis, and Parabirojimia (Table 3). Morphologically, Bakuella and Neobakuella are the most similar genera to Apobakuella since they exhibit a common infraciliature with respect to the arrangement of the midventral complex and the buccal and parabuccal cirri. Molecular data of Bakuella are still unavailable, but the close relationship between Neobakuella flava and Apobakuella fusca has been confirmed by the topology of the SSU rRNA gene trees (Fig. 7). Although Apobakuella has two or more right marginal rows, in common with the other two genera, it can be distinguished due to the lack of frontoterminal cirri (Berger 2006; Li et al. 2011). Metaurostylopsis, meanwhile, resembles Apobakuella in the shared presence of transverse cirri and the lack of caudal cirri. However, these genera can be distinguished from each other by the number of buccal and parabuccal cirri (Metaurostylopsis has just one whereas Apobakuella has more than one); by the number of frontoterminal cirri (three or more in Metaurostylopsis vs. none in Apobakuella), and the number of left marginal rows (two or more vs. one). Additionally, the midventral complex of Metaurostylopsis terminates distinctly ahead of the transverse cirri, whereas in Apobakuella it terminates close to the transverse cirri (Berger 2006; Song et al. 2001). The morphological distinction is also confirmed by the molecular analyses since Metaurostylopsis and Apobakuella are phylogenetically separated in the topology (Fig. 7). Parabirojimia resembles Apobakuella in lacking frontoterminal cirri and having one left, and two or more right marginal rows. However, it possesses a curious bipartite adoral zone

Table 3. Comparison of the two new genera with bakuellid genera sensu Berger (2006) and Neobakuella (Li et al. 2011). Genera

AZM

Buccal cirrus(i)

Number of LMR

Number of RMR

Transverse cirri

Caudal cirri

FTC

Apobakuella Parabistichella Neobakuella Bakuella Metaurostylopsis Birojimia Parabirojimia Paragastrostyla Holostichides Australothrix

Continuous Continuous Continuous Continuous Continuous Continuous Bipartite Continuous Continuous Continuous

+ + + + + + + − + +

1 1 ≥2 1 ≥2 1 1 1 1 ≥1b

≥2 1 1 1 ≥2 ≥2 ≥2 1 1 ≥2b

+ + + + + + + − − −

− − − − − + − + + +

− −a + + + + − + + −

Abbreviations: AZM, adoral zone of membranelles; FTC, frontoterminal cirri; LMR, left marginal row; RMR, right marginal row. +, present; −, absent. The two new genera established in this work are highlighted in bold. a Parabistichella lacks frontoterminal cirri; however, a very long frontoventral row is present. b Further investigations are required to distinguish midventral, ventral, and marginal rows.

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Fig. 7. Phylogeny of the hypotrichs inferred from the SSU rRNA gene sequences. New species are in bold. BP for ML tree and PP for BI tree are given near nodes, respectively. Fully supported (100%/1.00) branches are marked with solid circles. Species assigned to the bakuellids for morphological reasons are marked by a triangle. The scale bar represents two substitutions per 100 nucleotide positions.

(vs. continuous) (Chen et al. 2010c; Hu et al. 2002). In congruence with previous results (Huang et al. 2010), Parabirojimia appears rather close to Tunicothrix, which together form a markedly distant group from Apobakuella, occupying the basal branch in the Hypotrichia trees. Diaxonella shows a close relationship with Apobakuella in the SSU rRNA gene trees (Fig. 7). This result corresponds well with their morphological similarities in life and after protargol impregnation. The former, as a genus of the family Holostichidae, however, is characterized by a midventral complex that is composed of cirral pairs only (vs. pairs and rows in Apobakuella) and by having one right and more than one left marginal rows (vs. one left and more than one right marginal rows) (Berger 2006; Oberschmidleitner and Aescht 1996; Shao et al. 2007).

Apobakuella fusca sp. n. Due to the combination of two-rowed right marginal cirri, long midventral pairs of cirri, and the absence of frontoterminal cirri, our new species can be clearly distinguished. The closest apparent genera in terms of morphology and morphogenesis appear to be Neobakuella flava and Bakuella. These will be discussed here. Considering its general characteristics, its brackish biotope, and the pattern of buccal and parabuccal cirri, Neobakuella flava might initially be thought to most closely resemble Apobakuella fusca. However, it can be easily distinguished from the new species by the presence of frontoterminal cirri (vs. their absence), and the number of midventral rows (6–12 vs. 1–4) (Li et al. 2011).

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The main morphogenetic features of Apobakuella fusca also show great similarity with species of Bakuella: (1) the oral primordium of the opisthe is formed de novo; (2) numerous streaks (frontoventral-transverse cirri anlagen) appear, and the posterior one(s) develop into the midventral row(s); (3) marginal cirral and dorsal kineties anlagen are formed intrakinetally (Eigner and Foissner 1992; Mihailowitsch and Wilbert 1990; Song et al. 1992). The main difference exists in the fate of the parental adoral zone of the membranelles: in Bakuella, the proximal portion is reorganized, whereas in Apobakuella it is entirely replaced by a newly formed one. In addition, the anterior cirri, originating from the rightmost streak of the frontoventral-transverse cirri anlagen, do not migrate forward in Apobakuella like those in Bakuella, resulting in the absence of frontoterminal cirri.

Parabistichella gen. n. Bistichella resembles Parabistichella in having three frontal cirri, long ventral and frontoventral rows, one left and one right marginal row, and no caudal cirri. However, the former has two short frontal rows (vs. cirri arranged in pairs), and more than one buccal cirrus (vs. one) (Berger 2008). Furthermore, the frontoventral cirral pattern of Bistichella is possibly formed in the conventional six-anlagen mode while Parabistichella forms more cirral streaks during division. Apoamphisiella also shows an apparent similarity with Parabistichella in regard to its cirral pattern. However, whereas the former is characterized by a fragmented dorsal kinety, dorsomarginal kineties, and a postoral ventral cirrus, all of these features are absent in the latter (Berger 1999; Foissner 1997). Tunicothrix differs from Parabistichella in having a cortical alveolar layer (vs. absent), a bipartite adoral zone of membranelles (vs. continuous), and a higher number of right marginal rows (two vs. one) (Lin and Song 2004; Xu et al. 2006). So far, Tunicothrix is the only one among these morphological relatives for which the SSU rRNA gene sequence is available. The morphological distinction between Parabistichella and Tunicothrix is clearly confirmed by the distant separation of these two genera in the molecular trees. Tunicothrix and Parabirojimia form a clearly divergent, monophyletic clade, basal to other hypotrichs and distant from the core urostylids. However, the relationship of Parabistichella to Bistichella or Apoamphisiella is unclear.

Parabistichella variabilis sp. n. This new species can be clearly distinguished by the midventral pairs that are restricted to the buccal area, the long frontoventral row, the absence of frontoterminal cirri, and the unique origin of the frontoventral and midventral row anlagen during binary division.

The SSU rRNA gene sequence of Uroleptoides magnigranulosus showed the highest similarity with that of Parabistichella variabilis and their close relationship is highly supported in the trees. Morphologically, U. magnigranulosus resembles P. variabilis in the pattern of cortical granules, marginal rows and dorsal infraciliature, and in having the frontal cirri (anterior midventral cirri) arranged in pairs. However, the former has a typical amphisiellid median cirral row (vs. a long ventral and a frontoventral row in P. variabilis) (Foissner 1988). The morphogenetic process of P. variabilis is characterized by the following features: (1) the posterior adoral zone of membranelles reorganizes in situ, and the anterior membranelles are maintained; (2) more than six frontoventral-transverse cirri streaks appear; (3) the midventral row anlage develops intrakinetally in the parental frontoventral row; (4) the frontoventral row anlage probably originates from the midventral row anlage; (5) each frontoventral-transverse streak gives rise to a transverse cirrus at the rear end; (6) the marginal rows and the dorsal kineties develop within the parental structures; and (7) the macronuclear nodules fuse into a single mass in the middle of division.

Familial classification of Parabistichella variabilis Parabistichella variabilis has morphological characteristics typical for Bakuellidae, including three frontal cirri and a midventral complex composed of pairs and row(s) (Berger 2006; Lynn and Small 2002). Some morphogenetic features, i.e., the presence of seven or more frontoventral-transverse streaks during division, also suggest that Parabistichella variabilis might be a “bakuellid-like” hypotrich. However, the midventral row may comprise at least two parts similar to the median ventral row in amphisiellids (Fig. 6F). Also, the frontoventral row in Parabistichella variabilis might possibly be regarded homologous to a very long non-migrating “anterior portion of the amphisiellid median cirral row”. Either way, the origin of its midventral row – intrakinetally from the parental frontoventral row – is unlike that of any urostylid or amphisiellid (Berger 2006, 2008). The evidently close phylogenetic relationship between Parabistichella, Uroleptoides and Orthamphisiella fails to provide a useful basis for explaining the ambiguous morphological features of the latter two (Berger 2008, 2011). Additionally, the familial assignment is difficult to comment upon, given the present state of knowledge, since the classification is based on only a limited number of taxa (Uroleptoides/Parabistichella/Orthamphisiella) and is, in any case, far from being settled given the low bootstrap values in the phylogenetic trees. Meanwhile, the sequences of many key taxa, especially bakuellids, are not available. For these reasons Parabistichella must be considered incertae sedis in the basal hypotrichs. Further investigations with

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increasing taxon cohorts and multiple molecular markers may enlighten the classification and evolution of Parabistichella.

Acknowledgements This work was supported by the Nature Science Foundation of China (project nos. 31172041, 31030059), and by the King Saud University, Saudi Arabia. Many thanks are given to Prof. Weibo Song, Xiaozhong Hu (Ocean University of China) and Dr. Xiaofeng Lin (South China Normal University), for their institutional support and for their helpful discussions during the preparation of the draft.

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