Morphology and SSU rRNA gene sequence of the new brackish water ciliate, Anteholosticha pseudomonilata n. sp. (Ciliophora, Hypotrichida, Holostichidae) from Korea Author Li, Liqiong Author Khan, Sadia Nawroz Author Ji, Daode Author Shin, Mann Kyoon text Zootaxa 2011 2739 51 59 journal article 10.5281/zenodo.201911 e0179b57-7d45-477e-a178-a8f7b95a064a 1175-5326 201911 Anteholosticha pseudomonilata n. sp. ( Figs 1–3 ; Tables 1–3 ) Diagnosis. Grey coloured Anteholosticha , 110–190 µm × 40–80 µm in vivo ; contractile vacuole mid-body positioned; 29–41 adoral membranelles; 1 buccal, 3 frontal, 2 frontoterminal, 2 pretransverse and 8–11 transverse cirri; midventral complex composed of 10–16 pairs of zigzagging midventral cirri, extending posteriorly to slightly ahead of pretransverse cirri; left and right marginal row with about 28 and 29 cirri, respectively; 4 entirely long dorsal kineties; cortical granules colourless and pigment-like, 0.5 µm across, longitudinally arranged in more or less short rows on whole cortex except along dorsal kineties and cirral rows; 8–12 macronuclear nodules located left of midline. Type locality. Brackish water from the Taehwagang River (35°32ʹ56"N; 129°20ʹ11"E) near Ulsan Bay which flows into the East Sea, at Ulsan, South Korea . Type slides. Two permanent slides of protargol-impregnated specimens are deposited as a holotype and a paratype in the collection of National Institute of Biological Resources ( NIBR ), Incheon, South Korea and Department of Biology, University of Ulsan, Ulsan, South Korea and with registration numbers NIBRPR0000102719 and LLQ- 20091123 -05-02, respectively. Etymology. The species-group name pseudomonilata is a composite of the Greek adjective pseudo- (wrong, lying) and the name of the most similar congener Anteholosticha monilata which resembles our form in having the macronucleus-nodules forming a chain (see details about etymology of monilata in Berger 2006 ). The ending of the species-group name is defined (fixed) via the genus. Description. Size mostly 110–140 µm × 40–60 µm in vivo . Body shape slightly variable and elliptic-like, anterior portion conspicuously narrowed and forming a slightly cephalized appearance, while rear end broadly rounded; left margin more convex than right part ( Figs. 1 A; 2A, 2B). Body flexible, only slightly contractile. Dorsoventrally flattened about 2:1. Pellicle thin and soft. Cortical granules colourless and spherical in shape, about 0.5 µm in diameter, basically arranged in more or less longitudinal short rows on whole cortex except along cirral rows and dorsal kineties with some single ones sparsely distributed throughout the cell surface ( Figs. 1 C; 2C). Food vacuoles difficult to recognize except for many yellowish to brownish light-reflecting crystals scattered within transparent cytoplasm. Two groups of inclusions remarkably recognized, which are globular and concave like, respectively, 2–5 µm in diameter, consistently situated and give rise to darker colour in both ends of the cell ( Figs. 1 A, 1B; 2E). 8–12 colourless macronuclear nodules globular to ellipsoidal shaped, serially distributed in mid-portion of body left of midline which can be easily observed under middle to high magnification in vivo ; individual nodules, in impregnated specimens, 8–20 µm× 8–18 µm, containing small nucleoli ( Figs. 1 E; 2D, 2F). Usually two micronuclei ovoid in shape located separately among the macronuclei, 5–10 µm × 3–8 µm across after fixation ( Figs. 1 E; 2D, G). Single contractile vacuole positioned in mid-body near left margin with two long collecting canals ( Fig. 1 A). Locomotion relatively slow, but crawling without pause. Body apparently flexible, folded or twisted when crawling on bottom of Petri dish or drilling through debris. FIGURE 1. Anteholosticha pseudomonilata n. sp. from live cells ( A–C ), and after protargol impregnation ( D, E ). ( A ) Ventral view of a typical individual. ( B ) Ventral view, arrows indicate the inclusions within cytoplasm at both cell ends. ( C ) Noting arrangement of cortical granules (arrows). ( D, E ) Ventral and dorsal views of the holotype specimen, showing the general infraciliature. Arrow in D marks the posterior end of the midventral complex. Arrowheads in E depict the “extra” dikinetids ahead of the right marginal row. AZM = adoral zone of membranelles; BC = buccal cirrus; EM = endoral membrane; FC = frontal cirri; FTC = frontoterminal cirri; LMR = left marginal row; Ma = macronuclei; Mi = micronuclei; MP = midventral pairs; PM = paroral membrane; PTC = pretransverse ventral cirri; RMR = right marginal row; TC = transverse cirri; 1-4 = dorsal kineties. Scale bars in (A) = 40 µm; in (D, E) = 30 µm. FIGURE 2. Photomicrographs of Anteholosticha pseudomonilata n. sp. from live cells ( A–E ), and after protargol staining ( F– H ). ( A, B ) Ventral view of typical individuals. ( C ) Partial of dorsal view, to show the distribution of cortical granules (arrowheads) and dorsal bristles (arrows). ( D ) Ventral view showing macronuclear nodules (arrows) and micronuclei (arrowheads). ( E ) Posterior body, arrows mark the inclusions within cytoplasm. ( F ) Ventral view of the holotype specimen (the same individual as illustrated in 1 D, E and 2 G, H ), arrow denotes anterior end of left marginal row. ( G ) Ventral view of mid-posterior portion, arrows and arrowheads show micronuclei and macronuclear nodules, respectively. ( H ) Anterior portion of ventral side, showing the buccal cirrus (arrow), frontoterminal cirri (arrowheads), and the right frontal cirrus (double-arrowheads). Scale bars = 50 µm. Adoral zone of membranelles occupied 30.3–41.7% of body length in fixed specimens ( Table 1 ), base of longest membranelles about 9 µm long. Distal end of AZM terminated at right margin of cell and bending posteriad at about anterior 1/3 of buccal field ( Figs. 1 D; 2F). Paroral and endoral membrane almost the same length, likely both composed of monokinetids, distinctly intersecting each other and terminating anteriorly at about 2/5 of buccal field ( Figs. 1 D; 2H). Single buccal cirrus situated near the intersection of undulating membranes ( Figs. 1 D; 2H). Three slightly enlarged frontal cirri lying in anterior frontal area with right one very close to the distal end of adoral zone of membranelles ( Fig. 2 H). Consistently two frontoterminal cirri near and right to the distal end of adoral zone of membranelles ( Fig. 2 H). 8–11 relatively undeveloped transverse cirri arranged in J-shaped row, ca. 15 µm long in vivo , slightly protruding beyond rear end of cell ( Figs. 1 A, 1D; 2G). Always two pretransverse ventral cirri located close to the right transverse cirrus ( Fig. 1 D). Midventral complex composed of 10–16 pairs of midventral cirri arranged in a typical zig-zag pattern, continuing with frontal cirri and terminated ahead of the level of the left-most transverse cirrus ( Figs. 1 D; 2F, 2G). The last midventral cirri often dispersedly situated other than in a zigzagging pattern ( Fig. 1 D). Two marginal rows distinctly separated posteriorly ahead of cell end, the cirri within which about 10 µm long and never protruding the margins in life; 23–33 cirri in left row, while 24–34 in right one ( Figs. 1 A, 1D; 2A, 2F). Invariably four complete dorsal kineties with dorsal cilia about 3 µm long. Usually two “extra” dorsal bristles present on the anterior right margin of the body ( Figs. 1 E; 2C). TABLE 1. Morphometrical characterization of Anteholosticha pseudomonilata n. sp. All data based on protargol-impregnated specimens. Measurements in micrometers.

Character Body, length	Min 113	Max 185	Median 150.0	Mean SD 147.7 15.4	SE 2.8	CV 10.4	n 30
Body, width Body length:width, ratio	45 2.1	80 3.2	56.0 2.5	59.2 9.5 2.5 0.3	1.7 0.1	16.0 12.0	30 30
Length of adoral zone	42	61	52.5	53.5 5.1	0.9	9.5	30

Body length:length of adoral zone, ratio 2.4	3.3	2.7	2.8	0.2	0.0	7.1	30
Number of membranelles 29 Number of frontal cirri 3	41 3	37.0 3.0	36.4 3.0	2.8 0.0	0.5 0.0	7.7 0.0	30 30
Number of buccal cirri 1 Number of frontoterminal cirri 2	1 2	1.0 2.0	1.0 2.0	0.0 0.0	0.0 0.0	0.0 0.0	30 20
Number of midventral pairs 10 Number of cirri in right marginal row 24	16 34	13.0 29.0	13.2 29.4	1.3 2.2	0.3 0.4	9.8 7.5	27 29
Number of cirri in left marginal row 23 Number of pretransverse cirri 2 Number of transverse cirri 8 Number of dorsal kineties 4 Number of macronuclei 8 Number of micronuclei 1	33 2 11 4 12 3	28.0 2.0 9.0 4.0 10.0 2.0	27.7 2.0 9.5 4.0 9.4 2.0	2.7 0.0 0.8 0.0 1.0 0.4	0.6 0.0 0.2 0.0 0.2 0.1	9.7 0.0 8.4 0.0 10.6 20.0	22 27 27 21 30 26

Abbreviations: CV = coefficient of variation in %, Max = maximum, Mean = arithmetic mean, Median = median value, Min = minimum, n = number of individuals examined, SD = standard deviation, SE = standard error of arithmetic mean. Comparison. Currently, over 40 morphotypes have been included in the genus Anteholosticha , most of which need further studies and redescription ( Berger 2006 ). As one of the most significant criteria for species circumscription, nuclear apparatus (number, arrangement) usually can be easily detected and described even in the cursory data, furthermore, the diversity of which is quite high in Anteholosticha , therefore the congeners resemble our form in having a series of macronuclear nodules should be compared here. Morphologically, the most similar congener Anteholosticha monilata ( Kahl, 1928 ) Berger, 2003 ( type species) resembles A. pseudomonilata n. sp. in several features (e.g. body shape, contractile vacuole, ciliary arrangement, the number of membranelles and transverse cirri), however, differs from the latter in having 6 or more dorsal kineties (vs. 4), 19–27 midventral pairs (vs. 10–16), 4–23 macronuclear nodules (vs. 8–12), extrusomes (vs. absent), and no cortical granules (vs. present) thus both forms can be distinctly separated (e. g. Augustin & Foissner 1992 ; Berger 2006 ). Likewise, other similar Anteholosticha spp., namely, A. distyla ( Buitkamp, 1977 ) Berger, 2003 , A. xanthichroma ( Wirnsberger & Foissner, 1987 ) Berger, 2003 , A. australis ( Blatterer & Foissner, 1988 ) Berger, 2003 , A. mancoidea ( Hemberger, 1985 ) Berger, 2003 , A. randani ( Grolière, 1975 ) Berger, 2003 , A. sphagni ( Grolière, 1975 ) Berger, 2003 , A. sigmoidea ( Foissner, 1982 ) Berger, 2003 , and A. extensa ( Kahl, 1932 ) Berger, 2003 can be easily distinguished from A. pseudomonilata n. sp. by a number of features including habitat, body shape, size, the position of the contractile vacuole, cortical granules, and morphometric data (details see Table 2) ( Berger 2003 , 2006 ; Blatterer & Foissner 1988 ; Borror & Wicklow 1983 ; Buitkamp 1977 ; Foissner 1982 ; Grolière 1975 ; Hemberger 1985 ; Kahl 1932 ; Wirnsberger & Foissner 1987 ). SSU rRNA gene sequence analysis: The length of the complete SSU rRNA gene sequence of Anteholosticha pseudomonilata n. sp. is 1772 bp; the nucleotide sequence has been given the accession number HM568416 . The GC content is 44.75%, which is within the range of other ciliates. Among the dataset of 1795 total positions, a total of 244 mismatched nucleotides are revealed from the alignment of six Anteholosticha species ( Fig. 3 ). Of these, 24 positions are unique to A. pseudomonilata . SSU rRNA gene sequence similarity among A. pseudomonilata and the other five species are listed in Table 3 . The SSU rRNA gene sequence comparison study clearly exhibits a considerable inconsistency of Anteholosticha pseudomonilata from other five congeners. Pairwise sequence similarities between A . pseudomonilata and its congeners range from 92.38% to 96.56% ( Table 3 ). The comparatively high sequence discrepancy (3.44%) with the most similar morphotype A. monilata strongly suggests our form as a distinct species. Anteholosticha monilata is the type species indicating that the new species is a true member of the genus. By contrast, some other species seem to be not congeneric as indicated by the high differences in the structural similarity ( Table 3 ). This supports the assumption by Berger (2003 , 2006 ) that members of the genus Anteholosticha are likely not to be a monophyletic group. These higher ranges of molecular divergence also coincide with the statement of morphological analysis to establish A. pseudomonilata as an individual species of the genus Anteholosticha . TABLE 3. The structural similarities (%) of the SSU rRNA gene sequences of six Anteholosticha species determined according to Elwood et al . (1985). A. pseudomonilata A. monilata A. parawarreni A. scutellum A. multistilata A. monilata 96.56 A. parawarreni 93.07 93.12 A. scutellum 93.11 92.88 97.37 A. multistilata 94.67 94.55 94.09 94.58 A. manca 92.38 93.15 91.32 91.52 93.45