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Reconsideration of the taxonomy of the marine ciliate Neobakuella aenigmatica Moon et al., 2019 (Protozoa, Ciliophora, Hypotrichia)

  • Corresponding author: Chen Shao, shaochen@snnu.edu.cn
  • Received Date: 2019-07-20
    Accepted Date: 2020-01-06
    Published online: 2020-04-27
  • Edited by Jiamei Li.
  • The morphology and divisional morphogenesis in a Chinese population of Neobakuella aenigmatica Moon et al., 2019 are reinvestigated. The body size, number and arrangement of parabuccal cirri and development of nuclear nodules in this population are discussed in comparison with closely related genera. The 18S rRNA gene sequence of the Chinese population is identical to that of a Korean population, supporting a distant relationship between N. aenigmatica and the cluster of N. flava and Apobakuella fusca. We deduce that this may be caused by the differences in the pattern of the parabuccal cirri and the number of parabuccal rows which are important for the phylogeny of Bakuella-like species.
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Reconsideration of the taxonomy of the marine ciliate Neobakuella aenigmatica Moon et al., 2019 (Protozoa, Ciliophora, Hypotrichia)

    Corresponding author: Chen Shao, shaochen@snnu.edu.cn
  • 1. Laboratory of Protozoological Biodiversity & Evolution in Wetland, College of Life Sciences, Shaanxi Normal University, Xi'an 710119, China
  • 2. Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China
  • 3. College of Fisheries, Ocean University of China, Qingdao 266003, China
  • 4. College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China

Abstract: The morphology and divisional morphogenesis in a Chinese population of Neobakuella aenigmatica Moon et al., 2019 are reinvestigated. The body size, number and arrangement of parabuccal cirri and development of nuclear nodules in this population are discussed in comparison with closely related genera. The 18S rRNA gene sequence of the Chinese population is identical to that of a Korean population, supporting a distant relationship between N. aenigmatica and the cluster of N. flava and Apobakuella fusca. We deduce that this may be caused by the differences in the pattern of the parabuccal cirri and the number of parabuccal rows which are important for the phylogeny of Bakuella-like species.

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Introduction
  • Due to the variety of their ciliary patterns and nuclear apparatus, hypotrichs offer important insights for understanding the evolutionary relationships among ciliates (Berger 1999, 2006, 2008, 2011; Chen et al. 2017; Song and Shao 2017). Numerous studies have been carried out in recent years on the taxonomy, morphogenesis and molecular phylogeny of hypotrichs, thereby increasing knowledge and understanding of their systematics (Hu et al. 2019; Li F. et al. 2019; Luo et al. 2019; Lyu et al. 2018; Shao et al. 2019; Song and Shao 2017; Wang et al. 2019; Zhang et al. 2018; Zhu et al. 2019).

    The urostylid genus Neobakuella was established by Li et al. (2011) with N. flava as the type species. Neobakuella is identified as belonging to Bakuellidae (?) with a continuous adoral zone of membranelles, long undulating membranes, three frontal cirri, two or more buccal cirri, a parabuccal row, a midventral complex composed of irregularly arranged midventral pairs and/or short midventral rows in the anterior portion and several short to long midventral rows in the middle and posterior portions, two or more frontoterminal cirri, one right and two or more left marginal rows, transverse cirri present, and three bipolar dorsal kineties. Recently, a large, novel brackish water bakuellid isolated from South Korea was described by Moon et al. (2019) under the name Neobakuella aenigmatica.

    Hitherto, knowledge on the morphogenesis of Neobakuella was restricted to one early stage of N. flava, making it difficult to interpret the origin of some irregularly arranged cirri in the buccal field (Li et al. 2011). However, the same structure of irregularly arranged cirri was discovered in the genus Apobakuella Jiang et al., 2013 and interpreted according to morphogenesis of its parabuccal cirri (Jiang et al. 2013). Morphogenesis of the parabuccal cirri in Neobakuella flava probably follows a similar pattern to that in Apobakuella fusca, which was supported by recent molecular phylogenetic analyses showing that these two species cluster together in SSU rDNA gene trees (Moon et al. 2019). However, there is also a distant relationship between Neobakuella aenigmatica and the cluster of N. flava and A. fusca. Furthermore, the number and pattern of parabuccal cirri in N. aenigmatica vary greatly among individuals (Moon et al. 2019).

    In November 2016, a population of N. aenigmatica was isolated from a stream in Tangdao Bay Park, Qingdao, China. In this work, we investigate the morphology and divisional morphogenesis of N. aenigmatica, and give some more information about this species. The value of the pattern of the parabuccal cirri in assessing phylogenetic relationships among Bakuella-like species is also discussed.

Results

    Morphology of Chinese population of Neobakuella aenigmatica (Figs. 1a, 2a-h; Table 1)

  • Figure 1.  Morphology and infraciliature of Neobakuella aenigmatica in vivo (a-d) and after protargol staining (e, f). a Ventral view of a representative individual. b Ventral view, to show cortical granules distributed near marginal cirri. c, d Dorsal views, to show cortical granules near the dorsal cilia (c) and lateral side (d). e, f Ventral (e) and dorsal (f) view of the same specimen showing the infraciliature and nuclear apparatus. In e, arrow shows parabuccal cirri and arrowhead marks the buccal cirri. ADK additional dorsal kineties anterior to right marginal row; AZM adoral zone of membranelles; E endoral membrane; FC frontal cirri; FT frontoterminal cirri; L1-3 the first, second and third left marginal rows, respectively; Ma macronucleus nodules; Mi micronuclei; MP midventral pairs; P paroral membrane; RMR right marginal rows, TC transverse cirri; D1-3 dorsal kineties 1-3; 1-6, midventral rows 1-6. Scale bars = 75 μm (a, e, f)

    Figure 2.  Photomicrographs of Neobakuella aenigmatica in vivo (a-h) and after protargol staining (i-p). a, b Ventral views of different individuals, showing contractile vacuoles (arrowhead in a). c Ventral view of the anterior portion of cell, showing the adoral zone. d-f Dorsal views, showing arrangement of grass-green colored cortical granules (arrowheads) and midventral cirri (arrows). g, h Ingested algae (arrowheads in g), flagellates (arrows in g) and fungal spores (arrow in h). i, j Ventral views, showing infraciliature and nuclear apparatus. Arrows mark the frontoterminal cirri; arrowhead indicates the third left marginal row. k Ventral view of anterior portion, showing buccal (arrowheads), frontal and parabuccal cirri (in circle) respectively. l Dorsal view of anterior portion, showing dorsal kineties (arrowheads). m-p Ventral views of different individuals, showing different patterns of left marginal rows (arrowheads). Note: m-p are false-colored by inverting color in Photoshop CS5. FC frontal cirri; FT frontoterminal cirri; L1, 2, left marginal rows 1, 2; PBC parabuccal cirri. Scale bars = 85 μm

    Character Min Max Mean M SD CV n
    Body (length) 140.0 250.0 182.8 181.0 29.1 15.9 20
    Body (width) 38.0 148.0 70.3 58.5 30.8 43.9 20
    Body length:width (ratio) 1.5 4.3 2.9 3.0 0.7 25.0 20
    Anterior body end to proximal end of adoral zone (distance) 48.0 93.0 70.2 70.5 11.4 16.2 20
    Body length:length of adoral zone (ratio) 2.3 3.1 2.6 2.6 0.3 10.2 20
    Anterior body end to anterior end of paroral membrane (distance) 6.0 15.0 11.0 10.5 2.2 19.8 20
    Paroral membrane (length) 31.0 63.0 45.6 45.5 8.6 18.8 20
    Anterior body end to anterior end of endoral membrane (distance) 8.0 17.0 13.2 13.0 2.8 21.3 20
    Endoral membrane (length) 38.0 76.0 55.1 53.5 9.1 16.5 20
    Anterior body end to first buccal cirrus (distance) 12.0 31.0 21.0 20.0 4.3 20.6 20
    Anterior body end to last buccal cirrus (distance) 31.0 65.0 47.9 45.0 9.5 19.8 20
    Anterior body end to posterior end of midventral pairs (distance) 50.0 98.0 69.7 68.0 11.1 16.0 20
    Posterior body end to rearmost transverse cirrus (distance) 8.0 48.0 17.9 15.0 10.5 58.6 20
    Adoral membranelles (number) 32.0 57.0 42.1 41.5 6.4 15.2 30
    Frontal cirri (number) 3.0 3.0 3.0 3.0 0 0 20
    Buccal cirri (number) 4.0 10.0 6.4 6.0 1.5 23.4 30
    Parabuccal cirri (number) 2.0 3.0 2.1 2.0 0.2 10.9 20
    Frontoterminal cirri (number) 2.0 2.0 2.0 2.0 0 0 20
    Midventral pairs (number) 8.0 15.0 10.2 10.0 1.7 17.0 20
    Midventral rows (number) 4.0 8.0 5.7 6.0 1.0 18.1 20
    Cirri of midventral row 1 (number)a 3.0 4.0 3.1 3.0 0.2 7.3 20
    Cirri of midventral row 2 (number) 3.0 5.0 3.7 3.5 0.8 21.7 20
    Cirri of midventral row n−1 (number)a 8.0 16.0 11.9 12.0 2.2 18.2 20
    Cirri of midventral row n (number)a 9.0 18.0 13.1 13.0 2.2 16.9 20
    Left marginal rows (number) 2.0 5.0 3.3 3.0 0.7 22.2 20
    Cirri of left marginal row 1 (number)b 34.0 68.0 46.4 45.5 8.5 18.4 20
    Cirri of left marginal row 2 (number)b 5.0 22.0 11.2 10.5 4.8 43.1 20
    Cirri of left marginal row 3 (number)b 2.0 32.0 13.9 13.5 9.6 68.9 20
    Cirri of left marginal row 4 (number)b 3.0 16.0 7.8 5
    Cirri of left marginal row 5 (number)b 4.0 9.0 6.5 2
    Cirri of right marginal row (number) 42.0 74.0 53.9 54.5 7.9 14.7 20
    Dorsal bristles ahead of right marginal row (number) 2.0 2.0 2.0 2.0 0 0 20
    Transverse cirri (number) 8.0 15.0 11.1 10.5 2.3 20.4 20
    Dorsal kineties (number) 3.0 3.0 3.0 3.0 0 0 20
    Dorsal bristles of dorsal kinety 1 (number) 23.0 47.0 31.2 29.5 7.3 23.3 20
    Dorsal bristles of dorsal kinety 2 (number) 25.0 49.0 34.5 34.5 5.8 16.8 20
    Dorsal bristles of dorsal kinety 3 (number) 27.0 47.0 35.6 33.5 5.5 15.4 20
    Macronuclear nodules (number) 100.0 179.0 127.9 121.0 21.4 16.7 20
    CV coefficient of variation in %, M median, Max maximum, Mean arithmetic mean, Min minimum, n number of cells measured, SD standard deviation
    aCirral rows with more than two cirri. Midventral row 1 is the anterior most row, midventral row n is the posterior most row
    bNumbered from inside to outside

    Table 1.  Morphometric characterization of Neobakuella aenigmatica based on protargol-stained specimens (measurements in μm)

    Body size 150-200 × 50-70 μm in vivo, usually about 170 × 60 μm, ratio of length to width approximately 3:1 in vivo. Outline wide-elliptical, the anterior portion slightly curved rightwards while foraging for food; body slightly fragile, flexible, and contractile i.e., reducing in length by about 10% under mild pressure with cover glass (Fig. 2a, b); ventral side flat, dorsal side usually convex in posterior region, and dorsoventrally flattened at a ratio of approximately 2:1. Macronuclear nodules small and numerous (about 121 nodules), scattered throughout the cell; individual nodules measure about 4-8 × 3-5 μm after protargol staining, usually globular to wide-ellipsoidal, but some are elongate-ellipsoidal, each with several small nucleoli (Figs. 1f, 2i-l). Micronuclei difficult to observe either in vivo or after protargol staining, probably several scattered throughout cell smaller than macronuclear nodules. Contractile vacuole located at the level of buccal vertex near the left cell margin (Figs. 1a, 2a). Cells slightly yellow-brownish at low (× 100) magnification because of cytoplasm and food vacuoles; cortical granules about 1 × 1 μm in size, globular to ellipsoidal, grass-greenish, easy to observe in protargol preparations when the cell is insufficiently bleached (Figs. 1b-d, 2d-f). Cytoplasm is colorless, contains fat globules (ca. 2-3 μm across) and food vacuoles (ca. 10-15 μm across) with compact contents. It is omnivorous and ingests fungal spores, diatoms, flagellates, and small ciliates (Fig. 2g, h). Moves rapidly by crawling over substrate without pause.

    Adoral zone palpable, occupying, 32-43% of body length, on average about 38% of body length after protargol staining; composed of 42 membranelles on average, bases of the largest membranelles about 14 μm wide, cilia up to 20 μm long in vivo. Paroral arches strongly, approximately four-fifth the length of endoral which is bow-shaped, anterior ends of each at the same level, paroral and endoral optically intersect in their posterior region (Figs. 1e, 2i-k). Buccal cavity large and deep, the hyaline lip covers the proximal end of the adoral zone of membranelles (Fig. 2c).

    Three conspicuously enlarged frontal cirri, about 18 μm long in vivo, the rightmost one near distal end of adoral zone; two or three parabuccal cirri behind right frontal cirrus (Figs. 1e, 2i-k). A row of four to ten buccal cirri at the right of paroral cirri becomes progressively smaller from the anterior to the posterior (Figs. 1e, 2i-k). Constantly two fine frontoterminal cirri are seen which are anterior to the right of the anterior most midventral pair (Figs. 1e, 2i, j). Midventral complex including a row of 8-15 pairs of cirri that terminates at the level of buccal vertex, together with 4-8 oblique, short or long midventral rows; rightmost long midventral row extends to the right of the rightmost transverse cirrus (Figs. 1e, 2i, j). Eight to 15 transverse cirri, with cilia approximately 18 μm long in vivo, closely arranged in oblique rows, either does not project beyond body margin or project only slightly (Fig. 1a, e).

    Marginal and midventral cirri 9-14 μm long in vivo. Constantly one right marginal row composed of 42-74 cirri, starting at the right of frontoterminal cirri on dorsolateral surface (with two dorsal bristles ahead), and ending subterminally (Fig. 1e, f). Number of left marginal rows variable, i.e. out of 20 individuals, one had two rows, 14 had three rows, three had four rows and two had five rows. Among these left marginal rows, only left marginal row 1 (the innermost) is a complete row, comprising 34-68 cirri, starting ahead of the level of buccal vertex and terminating at the posterior end of body; left marginal rows, 2-5, commence behind the level of anterior end of innermost row, and comprise variable numbers of sparsely distributed cirri (Figs. 1e, 2i, j, m-p). Three bipolar dorsal rows, with bristles about 5 μm long in vivo; dorsal kineties 1-3 are composed of 31, 35 and 36 dikinetids on average, respectively (Fig. 1f; Table 1).

  • Morphogenesis of Neobakuella aenigmatica (Figs. 3a-i, 4a-f, 5a-u)

  • Figure 3.  Morphogenesis of Neobakuella aenigmatica after protargol staining. a, b Ventral views of cells in early stages, to show the opisthe's oral primordium (arrowheads in a) and dedifferentiation of the endoral (arrow in b). c-e Ventral views of an early middle divider, showing the development of frontoventral-transverse cirral anlagen and dedifferentiation of paroral (double-arrowhead). Note d and e, are magnified views of anlagen developing in proter and opisthe as shown in c. In c, the opisthe's oral primordium differentiates into new membranelles (arrow) and proximal membranelles of the proter are in the process of dedifferentiation (arrowhead). In d, arrow indicates that the parental midventral cirri dedifferentiate and join in the formation of the frontoventral-transverse cirral anlagen, arrowhead marks dedifferentiation of proximal membranelles in the proter, and double-arrowhead shows dedifferentiation of paroral. In e, arrow marks the development of opisthe's oral primordium, arrowheads show dedifferentiation of midventral cirri and double-arrowhead points to the appearance of undulating membrane anlage in the opisthe. f, g Ventral and dorsal view of a slightly later divider, to show the formation of dorsal kinety anlagen (arrowheads in g), formation of left and right marginal anlagen, development of frontoventral-transverse cirral anlagen (double-arrows), as well as undulating membrane anlagen starting to split longitudinally and gives rise to the leftmost frontal cirri in both daughter cells (arrows). Note that several marginal anlagen segments form to the left of the innermost left marginal anlagen (arrowheads in f). h, i Ventral and dorsal view of a middle divider, to show the development of dorsal kinety anlagen (arrowheads in i), segmentation of frontoventral-transverse cirral anlagen and the leftmost frontal cirri (arrows in h) and development of the anlagen to form the left marginal segments (arrowheads in h). FVTA frontoventral-transverse cirral anlagen; LMA left marginal anlagen; Ma macronucleus nodules; Mi micronuclei; OP oral primordium; RMA right marginal anlagen. Scale bars = 80 μm (a-c); 15 μm (d, e); 65 μm (f-i)

    Stomatogenesis: In the opisthe, stomatogenesis starts with several groups of closely spaced basal bodies originating apokinetally around several of the posterior left cirri of the parental midventral rows (Figs. 3a, 5a). These groups subsequently become larger, merge and form the oral primordium (Figs. 3b, 5c). The oral primordium develops further and the anterior portion then gradually differentiates into new adoral membranelles (Figs. 3c, 5f). By this stage, some posterior left cirri of the midventral complex have been resorbed and others are disintegrating, indicating that the parental basal bodies are incorporated into the oral primordium (Figs. 3c, e, 5f). At the same time, the undulating membrane anlage is formed to the right of the oral primordium (Figs. 3c, e, 5f). Later, the leftmost frontal cirrus is generated from the anterior end of the undulating membrane anlage which splits longitudinally into the endoral and paroral, and the anterior end of the newly built adoral zone of membranelles bends to the right (Figs. 3f, h, 5i, n).

    In the proter, the endoral begins to disorganize from anterior to posterior (Figs. 3b, 5b). Very soon, the paroral begins to dedifferentiate (Fig. 5d). As this process continues, the undulating membrane anlage is formed (Figs. 3c, d, 5e). Meanwhile, the posterior end of the parental adoral zone of membranelles begins to disorganize (Figs. 3c, d, 5e). Later several proximal membranelles are replaced by new ones (Figs. 3f, h, 5g, m). The development of the undulating membrane anlage follows a similar pattern to that in the opisthe (Figs. 3f, h, 4a, e, 5g, i, m-p, s).

    Figure 4.  Morphogenesis (a, b, e, f) and reorganization (c, d) of Neobakuella aenigmatica after protargol staining. a, b Ventral (a) and dorsal (b) view of a late divider, showing that anlage II gives rise to the buccal cirri (arrowhead in a), anlage III produces the parabuccal cirri (arrow), the rightmost anlage forms two frontoterminal cirri (red stars in a), left marginal segments are formed to the left of the innermost left marginal anlagen (double-arrowhead) and the dorsal kinety anlagens are in the process of development (arrowheads in b). c, d Ventral (c) and dorsal (d) view of a reorganizer, showing the frontoventral-transverse cirral anlagen (double-arrowhead), anlagen for left marginal rows (arrows), segments (arrowheads in c), and dorsal kineties (arrowheads in d). e, f Ventral (e) and dorsal (f) view of a very late divider, showing the buccal cirri (arrowheads in e), parabuccal cirri (arrows), frontoterminal cirri (red stars), left marginal segments (double-arrowheads), transverse cirri (in circle) and dorsal kinety anlagen (arrowheads in f). LMA left marginal anlagen; Ma macronucleus nodules; Mi micronuclei; RMA right marginal anlagen; TC transverse cirri. Scale bars = 80 μm

    Figure 5.  Photomicrographs of Neobakuella aenigmatica during morphogenesis (a-t) and reorganization (u) after protargol staining. a Ventral view of an early divider, to mark the oral primordium (arrowheads). b, c Ventral views of the same early divider, showing the dedifferentiation of endoral (arrows) and buccal cirri (arrowheads), and opisthe's oral primordium. d Ventral view of an early divider, showing the dedifferentiation of the buccal cirri (double-arrowheads), undulating membrane (arrowheads) and frontoventral-transverse cirral anlagen (arrows). e, f Ventral views of the same divider, showing undulating membrane anlage in proter (arrowhead in e), the parental midventral cirri (arrowheads in f) which contributes to the formation of the frontoventral-transverse cirral anlagen and the oral primordium which develops and differentiates into new membranelles (arrow). g-l Ventral (g-k), and dorsal (l) views of the same specimen, to show the undulating membrane anlagen (arrowheads in g and i), the leftmost frontal cirri (arrows in g and i), several marginal anlagen segments formed to the left of the innermost left marginal anlage which then develop into new short left marginal rows (arrows in h, j and k), and the dorsal kinety anlage (arrowheads in l). m, n Ventral views of a middle divider, to mark the leftmost frontal cirri in both daughter cells (arrows). o-r Ventral views of a late divider. o, p showing buccal cirri (double-arrowheads), two frontoterminal cirri (arrows) and transverse cirri (arrowheads). q Demonstrating the micronuclei and fusion of the macronuclear nodules. Arrow in r marks the newly formed left marginal segment. s, t Ventral views of a divider in very late stage, to show the frontoterminal cirri (arrow in s) and left marginal segment (arrow in t). u Ventral view of a middle reorganizer, showing the leftmost frontal cirrus (arrowhead) and the left marginal segment (arrow). Ma macronucleus nodules; Mi micronuclei; OP oral primordium. Scale bars = 25 μm (a-d); 40 μm (e, f); 45 μm (g-t); 90 μm (u)

  • Development of somatic ciliature
  • Initially a few basal bodies appear on the right of the old buccal cirri in the proter and the buccal cirri dedifferentiate simultaneously, which will become the frontoventral-transverse cirral anlagen (Figs. 3c, 5d). Then, the frontoventral-transverse cirral anlagen grows by increasing their numbers of basal bodies which become organized into numerous nascent oblique streaks as a ladder-like structure in the proter (Figs. 3c, d, 5e). Meanwhile, several nascent oblique streaks for the opisthe are formed to the right of the opisthe's oral primordium, with a few parental cirri from the midventral rows contributing to their formation (Figs. 3c, e, 5f).

    In some middle to late stages, the frontoventral-transverse cirral anlagen for both daughter cells continue to develop and differentiate into many cirri (Figs. 3f, h, 4a, e, 5g, i, m-p, s). Anlage Ⅱ (the undulating membrane anlage is frontoventral-transverse cirral anlage Ⅰ) gives rise to the middle frontal cirrus and four to ten buccal cirri; anlage III produces the rightmost frontal cirrus and one or two parabuccal cirri. In the proter of Fig. 4a, anlagen IV-VII form a midventral pair each; anlagen Ⅷ-Ⅺ produce a midventral pair and a transverse cirrus each; the last anlage (the rightmost anlage) develops two frontoterminal cirri, one midventral row and one transverse cirrus; Other anlagen (anlagen XII-XV) form one midventral row and one transverse cirrus. Different specimens have different numbers of anlagen. Finally, the cell elongates and the new ciliary structures move further apart as they migrate towards their final positions. Note that the two anterior frontoterminal cirri from the last anlage migrate anteriorly and the parental structures are resorbed gradually (Figs. 4e, 5s). Meanwhile, the development of the cytostome is completed and the daughter cells begin to separate (Fig. 4e).

  • Marginal and dorsal anlagen
  • Within the right and innermost left parental marginal rows of both daughter cells, a few cirri near the anterior end, and a few others below the mid-body, differentiate to form two separate anlagen (Fig. 3f, h). These anlagen then increase in size by adding basal bodies and develop into cirri which gradually replace the parental structures (Fig. 4a, e). It is noteworthy that several small anlagen are formed de novo or intrakinetally to the left of the anlage to form the innermost left marginal rows, and then these short anlagen elongate and develop into the other short left marginal rows/segments of each daughter cell (Figs. 3f, h, 4a, e, 5h, j, k, r, t). The parental left marginal rows will eventually be resorbed (Figs. 4a, e, 5r, t).

    Morphogenesis of the dorsal kineties is like most urostylids as they develop by intrakinetal basal body proliferation, i.e. two anlagen develop in each parental row (Figs. 3g, i, 5l). These anlagen subsequently elongate and the parental structures are either incorporated or resorbed (Fig. 4b, f). During the division process, neither surplus anlagen nor caudal cirri are formed.

  • Division of the nuclear apparatus
  • The nuclear apparatus divides in the usual way for urostylids and hence needs no detailed comment. The most striking feature of the macronuclear division process is that all the macronuclear nodules fuse into a single mass (Figs. 3g, i, 4b, 5a, m, n, q). The micronuclei divide independently (Figs. 3i, 4b, f, 5q).

  • Reorganization of Neobakuella aenigmatica (Figs. 4c, d, 5u)

  • Only one physiological regeneration stage was observed for this species. According to the available data, the processes of reorganization during physiological regeneration and morphogenesis during binary fission are similar (Figs. 4c, d, 5u).

  • The SSU rDNA sequences of Chinese population

  • The SSU rDNA sequence of N. aenigmatica (Qingdao population) is deposited in the GenBank database with the accession number MN326296. The length and G + C content are 1656 bp and 44.26%. The sequences of the Qingdao and Korean populations are identical.

Discussion

    Identification of Neobakuella aenigmatica

  • Neobakuella aenigmatica was first described by Moon et al. (2019), based on a Korean population. The population investigated here closely resembles the Korean population except for the following minor differences: body length in vivo (150-200 μm vs. 185-300 μm), width in vivo (50-70 μm vs. 55-105 μm), numbers of adoral membranelles (48-93 vs. 36-73), frontal cirri (3 vs. 3-4), buccal cirri (4-10 vs. 5-10), parabuccal cirri (2-3 vs. 1-6), frontoterminal cirri (2 vs. 1-3), midventral pairs (8-15 vs. 8-22), midventral rows (4-8 vs. 4-10) and left marginal rows (2-5 vs. 2-4) (Moon et al. 2019). As there are so many overlapping data, we believe that these two populations are congeneric. The Qingdao population is, therefore, identified as N. aenigmatica with a high degree of certainty.

  • Morphological comparison of Neobakuella with similar genera

  • Neobakuella Li et al., 2011 is assigned to the family Bakuellidae as a result of possessing three frontal cirri and a midventral complex composed of cirral pairs and row(s). With respect to the presence of buccal and transverse cirri, the possession of a continuous adoral zone of membranelles and the absence of caudal cirri, at least three bakuellid genera should be compared with Neobakuella, namely Bakuella Agamaliev and Alekperov, 1976, Metaurostylopsis Song et al., 2001 and Apobakuella Jiang et al., 2013. Neobakuella can, however, be easily distinguished from these genera by the number of left marginal rows (two and more rows in Neobakuella vs. only one row in Bakuella and Apobakuella) (Agamaliev and Alekperov 1976; Jiang et al. 2013) and the number of right marginal row(s), i.e., only one row in Neobakuella vs. two or more rows in Metaurostylopsis (Song et al. 2001) (Fig. 6).

    Figure 6.  Comparison of some bakuellid genera (Apobakuella, Bakuella, Metaurostylopsis and Neobakuella). BC buccal cirrus(i); CC caudal cirri; DK dorsal kineties; FC frontal cirri; FTC frontoterminal cirri; FTVA IV frontoventral-transverse cirral anlage IV; LMR left marginal row; MA macronucleus nodules; MC midventral complex; MP midventral pair; MV midventral row; PBR parabuccal cirral row(s); RMR right marginal row; TC transverse cirri

  • Phylogenetic analyses

  • Neobakuella and Apobakuella show a close relationship based on phylogenetic analyses of their morphology and morphogenesis (Fig. 6). However, comparison of their SSU rDNA sequences shows a distant relationship between N. aenigmatica and the cluster of N. flava and A. fusca. Hence, the systematic position of N. aenigmatica should be reconsidered. Due to the lack of morphogenetic data for N. flava, two indistinct groups of cirri (groups I and II) were not interpreted in detail by Li et al. (2011). However, the irregular pattern of cirri in the buccal area of A. fusca was interpreted precisely by Jiang et al. (2013) following an investigation of its morphogenesis. These indistinct groups of cirri (groups I and II, sometimes group III is also present) originate from frontoventral-transverse cirral anlagen IV and V, or from IV to VI. We believe that the parabuccal cirri in N. flava probably developed in the same way as those in A. fusca. The pattern of development of parabuccal cirri in N. aenigmatica, however, differs as they form from anlage III in the Qingdao population and probably from anlage III or anlagen III to V in the Korean population (Moon et al. 2019). We deduce that the number of parabuccal rows is important in the phylogeny of Bakuella-like species. Differences in the arrangement of parabuccal cirri among N. aenigmatica, N. flava and Apobakuella are shown in Fig. 7.

    Figure 7.  The morphology and infraciliature of three species (Neobakuella aenigmatica, Neobakuella flava and Apobakuella fusca). a1-a3N. aenigmatica (from present work and Moon et al. 2019). b1-b3N. flava (from Li et al. 2011). (c1-c3) Apobakuella fusca (from Jiang et al. 2013). FC frontal cirri, FTC frontoterminal cirri; a3, b3, c3, arrows show the parabuccal cirri. Scale bars = 100 μm (a1, a2, c1, c2); 80 μm (b1, b2)

  • Morphogenetic comparison of Neobakuella with similar genera

  • Three other genera in the order Urostylida, namely Bakuella Metaurostylopsis and Apobakuella, resemble Neobakuella in possessing three frontal cirri, midventral rows, buccal cirri and transverse cirri. Furthermore, morphogenesis in these three genera resembles that in Neobakuella. Differences among them are as follows: (1) the number of buccal cirri formed from the undulating membrane anlage (only one in Metaurostylopsis vs. more than one in the other three genera); (2) the number of left marginal rows formed (only one in Bakuella and Apobakuella vs. more than one in Neobakuella and Metaurostylopsis); (3) the number of right marginal rows formed (only one in Neobakuella and Bakuella vs. more than one in Apobakuella and Metaurostylopsis); (4) frontoterminal cirri formed from an anlage n (absent in Apobakuella, present in the other three genera); and (5) the number of parabuccal cirral rows formed (only one in Bakuella and Metaurostylopsis vs. two or more in Apobakuella, one or more in Neobakuella) (Jiang et al. 2013; Li et al. 2011; Moon et al. 2019; Song et al. 2001).

    For N. aenigmatica, all key morphogenetic characters except the division of the nuclear apparatus were reported in the Korean population (Moon et al. 2019). The present study of the Qingdao populations shows that the numerous macronuclear nodules fuse into a single mass before dividing.

Materials and methods

    Sample collection and cultivation

  • On 5 November 2016, a mixture of water and rotten leaves was collected from a brackish water stream in Tangdao Bay Park, Qingdao (35°56′18″N; 120°12′44″E), when the water temperature was 13 ºC, and salinity was 6‰. The sample was processed within two hours. Individuals of N. aenigmatica were isolated using micropipettes under the stereoscope and clonal cultures were established using artificial water (salinity 6‰) at room temperature (23 ºC). Diatoms were isolated from the same sample and cultivated in Petri dishes in a light-dark cycle incubator at a temperature of (25 ºC). These diatoms were used as a food source for N. aenigmatica.

  • Morphology and morphogenesis

  • Living cells were observed with bright field and differential interference contrast microscope (Olympus BX53, Tokyo, Japan). The infraciliature was revealed by protargol staining (Wilbert 1975). Counts, measurements and line diagrams of stained cells were performed at × 1000 magnification. To illustrate the changes occurring during morphogenesis, old (parental) ciliary structures are depicted by contour whereas new structures are shaded black. For general and specific terms, see Berger(2006, 2008). For the designation of the frontoventral-transverse cirri and frontoventral-transverse cirral anlagen, the numbering system of Wallengren (1900) is used.

  • DNA extraction, PCR amplification and sequencing

  • One or more cells were isolated from the single-cell cultures and washed four times with Fuwick mineral water (boiled and cooled) to remove potential contaminants. The SSU rDNA was amplified according to Gao et al. (2016), using the primers 18S-F (5′-AAC CTG GTT GAT CCT GCC AGT-3′) and 18S-R (5′-TGATCCTTCTGC AGGTTCACCTAC-3′) (Medlin et al. 1988). To minimize the possibility of amplification errors, Q5® Hot Start High-Fidelity DNA Polymerase (New England BioLabs, USA) was used (Huang et al. 2016). Sequencing was performed bidirectionally on an ABI 3700 sequencer (Applied Biosystems).

  • Comparison of the SSU rDNA sequences

  • The 18S rRNA gene sequence of N. aenigmatica (Qingdao population) was aligned with that of N. aenigmatica (Korean population) acquired from the GenBank database (accession number: MN204486) using the Clustal W implemented in Bioedit 7.2.6 (Hall 1999). The alignments were then modified manually by deleting both ends with BioEdit 7.2.6. The final alignment included 1560 bp and had a GC content of 44.55%.

    Acknowledgements This work was supported by the Natural Science Foundation of China (No. 31872190). Many thanks are given to Prof. Weibo Song (OUC) for his many kind suggestions during drafting of the manuscript.

    Author contributions Shao and Zhang wrote the manuscript. Zhang, Dong, Cheng and Duan collected the samples and performed the staining.

  • Compliance with ethical standards

  • Conflict of interest The authors declare that they have no confict of interest.

    Animal and human rights statement We declare that all applicable international, national, and/or institutional guidelines for sampling, care, and experimental use of organisms for the study have been followed and all necessary approvals have been obtained.

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