Holostichides songi nov. spec. (Figs. 1A–E, 2A–M, 3A–I, 4A–H, 5A–N, Table 1)
Table 1
Morphometric characterization of Holostichides songi nov. spec.
Character a | H | Min | Max | Mean | M | SD | CV | n |
Body length | 145 | 126 | 173 | 147.9 | 146.0 | 14.2 | 9.6 | 25 |
Body width | 40 | 32 | 56 | 39.5 | 38.0 | 6.7 | 16.9 | 25 |
Body length: width, ratio | 3.6 | 2.8 | 4.3 | 3.8 | 3.9 | 0.4 | 9.5 | 25 |
Adoral zone, length | 50 | 35 | 53 | 44.2 | 44.0 | 4.6 | 10.5 | 25 |
Adoral zone length: body length, ratio | 0.34 | 0.26 | 0.35 | 0.30 | 0.30 | 0.02 | 6.3 | 25 |
Adoral membranelles, no. | 41 | 35 | 42 | 38.4 | 38.0 | 1.9 | 4.9 | 25 |
Frontal cirri, no. | 3 | 3 | 3 | 3.0 | 3.0 | 0 | 0 | 25 |
Buccal cirri, no. | 1 | 1 | 1 | 1.0 | 1.0 | 0 | 0 | 25 |
Frontoterminal cirri, no. | 13 | 9 | 15 | 11.0 | 11.0 | 1.4 | 12.3 | 25 |
Cirri below the middle frontal cirrus, no. | 3 | 1 | 4 | 2.5 | 2.0 | 0.7 | 28.8 | 25 |
Midventral pairs, no. | 8 | 5 | 8 | 6.5 | 7.0 | 0.9 | 13.4 | 25 |
Midventral rows, no. | 3 | 2 | 3 | 2.6 | 3.0 | 0.5 | 19.8 | 25 |
The left long midventral row, number of cirri | 12 | 6 | 12 | 8.1 | 8.0 | 1.6 | 20.4 | 23 |
The right long midventral row, number of cirri | 14 | 9 | 18 | 13.8 | 14.0 | 2.0 | 14.5 | 24 |
The short midventral row, number of cirri | 4 | 3 | 5 | 3.6 | 3.0 | 0.8 | 23.1 | 14 |
Cirri in right marginal row, no. | 46 | 36 | 53 | 44.2 | 43.0 | 5.4 | 12.2 | 25 |
Cirri in left marginal row, no. | 42 | 31 | 50 | 38.9 | 37.0 | 5.0 | 12.7 | 25 |
Caudal cirri, no. | 8 | 4 | 9 | 5.2 | 5.0 | 1.7 | 32.1 | 17 |
Dorsal kineties, no. | 4 | 4 | 4 | 4.0 | 4.0 | 0 | 0 | 24 |
Dorsal kinety 1, bristles, no. | 26 | 18 | 30 | 24.8 | 25.0 | 2.9 | 11.6 | 25 |
Dorsal kinety 2, bristles, no. | 30 | 19 | 31 | 26.6 | 27.0 | 2.6 | 9.6 | 24 |
Dorsal kinety 3, bristles, no. | 26 | 19 | 28 | 23.6 | 23.5 | 2.3 | 9.6 | 22 |
Dorsal kinety 4, bristles, no. | 29 | 20 | 31 | 27.8 | 28.0 | 2.6 | 9.4 | 21 |
Macronuclear nodule, no. | 34 | 27 | 46 | 34.6 | 33.0 | 5.5 | 16.0 | 25 |
Macronuclear nodule, length | 9 | 4 | 11 | 8.1 | 8.6 | 2.0 | 24.2 | 25 |
Macronuclear nodule, width | 5 | 4 | 7 | 5.1 | 5.2 | 1.1 | 21.8 | 25 |
Micronuclei, no. | 4 | 2 | 8 | 4.7 | 4.0 | 2.0 | 41.7 | 25 |
a All data is based on protargol-stained specimens, measurements in µm. Abbreviations: CV, coefficient of variation in %; H, holotype; M, median; Max, maximum; Mean, arithmetic mean; Min, minimum; n, sample size; no., number; SD, standard deviation. |
Zoobank Registration
Present work: urn:lsid:zoobank.org:pub:3D13BEB2-15AE-4186-B5AC-BA9230C99738
Holostichides songi nov. spec.:
urn:lsid:zoobank.org:act:2D0D0574-BAB4-4469-830F-7B7972B359B4
Diagnosis:
Body 150–180 µm × 45–60 µm in vivo in size, flexible and elongate-elliptical. 27–46 macronuclear nodules. Contractile vacuole positioned at about 40% of body length near left margin, collecting canals present. Cortical granules dark, spherical, about 0.5 µm across, clustered in groups and then arranged longitudinally. Adoral zone composed of 35–42 membranelles. Three frontal cirri and one to four smaller cirri below the middle frontal cirrus. One buccal and nine to 15 frontoterminal cirri. Midventral complex composed of five to eight cirral pairs and two long midventral rows. One left and one right marginal cirral rows, composed of 31–50 and 36–53 cirri, respectively. Four bipolar dorsal kineties and four to nine caudal cirri.
Type Material:
The protargol slide (registry no. WJY2018052301B) with the holotype specimen and one paratype slide (registry no. WJY2018052301C) were deposited in the Laboratory of Protozoological Biodiversity and Evolution in Wetland, Shaanxi Normal University, China.
Type Locality:
Soil from the surface of soil in Egret Wetland Park, Suzhou, China (31°16'18''N; 120°43'18''E).
Etymology:
This species is named after the eminent ciliatologist Prof. Dr. Weibo Song, Ocean University of China, Qingdao, for his tremendous contribution to the ciliatology.
Morphology (figs. a–e, A–m, Table )
Body size in vivo 150–180 µm × 45–60 µm (n = 7), and 126–173 µm × 32–56 µm after protargol staining (Table 1). Body elongate-elliptical, rather flexible, but not contractile (Figs. 1A, 2A–C). Ratio of length to width after protargol preparation about 3.8:1, dorsoventrally flattened about 2:1. 27–46 ellipsoidal or spherical macronuclear nodules, 4–11 µm × 4–7 µm in size (after protargol staining), two to eight micronuclei scattered throughout the cytoplasm (Fig. 1E, 2L). Contractile vacuole about 12 µm across when fully extended, positioned at about 40% of body length near left margin, pulsing at intervals of about 10 s, collecting canals visible during diastole (Figs. 1A, 2B, C). Pellicle thin and soft, with cortical granules dark, spherical, about 0.5 µm across, clustered in groups and then arranged longitudinally (Figs. 1B, 2D, E). Cytoplasm hyaline colorless to grayish, containing some lipid droplets (0.5–4 µm around) and food vacuoles (6–10 µm around) in posterior portion that render cell opaque and dark in posterior portion at low magnification, usually many irregular crystals located in posterior portion of cell (Fig. 2A). Locomotion by crawling slowly on plant debris or on the bottom of the Petri dish, occasionally swimming with slight rotation around long body axis.
Most cirri relatively fine, cilia about 10–12 µm long in vivo except for frontal cirri, cilia about 15 µm. Three slightly enlarged frontal cirri arranged in a transverse “pseudo” row with one to four smaller cirri longitudinally arranged below the middle frontal cirrus. Buccal cirrus located slightly anterior to intersection of endoral and paroral. Nine to 15 frontoterminal cirri locate to the right of midventral complex, start at about 1/4 of adoral zone and terminate at about proximal end of adoral zone. Midventral complex composed of five to eight cirral pairs, extending to about the proximal end of adoral zone, usually together with two or three midventral rows, from right to left comprised of nine to 18, six to 12 and three to five (if present) cirri, respectively. One right and one left marginal row composed of 36–53 and 31–50 cirri, respectively. Right marginal row starts at about 40% of adoral zone; left marginal row commences more or less at level of buccal vertex, both terminate caudally but not confluent posteriorly (Figs. 1D, 2F–K).
Invariably, four bipolar dorsal kineties, composed of 18–30, 19–31, 19–28, and 20–31 dikinetids, respectively. Bristles about 3 µm long in life. Four to nine caudal cirri with cilia about 15 µm long (Figs. 1E, 2M).
Adoral zone occupies about 30% of body length in protargol preparations, composed of 35–42 membranelles, cilia of which are about 15 µm long (Fig. 1A, D). Paroral and endoral in Oxytricha pattern, intersect optically somewhat behind level of buccal cirrus, and almost equal in length (Fig. 1C, D).
Morphogenesis (figs. a–i, A–h, A–n)
Stomatogenesis
Stomatogenesis commences with the intrakinetally formation of several groups of closely spaced basal bodies near the left midventral row, which is the oral primordium of the opisthe (Figs. 3A, 5A). Then, these groups merge by further proliferation of basal bodies forming a single anarchic field that becomes wider and wedge-shaped (Figs. 3B, C, 5B, C). Meanwhile, in the proter, a spherical field of basal bodies appears apokinetally to the left of the undulating membranes and seems to develop on the dorsal wall of the buccal cavity (Figs. 3C, 5D). Then the oral primordium of opisthe continues to grow by further proliferation of basal bodies and the new membranelles differentiate posteriad. Simultaneously, the old undulating membranes begin to dedifferentiate, which along with the spherical field of basal bodies to form the undulating membranes anlage for the proter (Figs. 3D, E, 5E, F). Subsequently, the undulating membranes anlagen appear on the right both in opisthe and proter, interestingly, another spherical field of basal bodies appears at the proximal end of old adoral zone of membranelles, which will form the new membranelles for the proter (Figs. 3F, 5H). In the middle stage, the undulating membranes anlage splits longitudinally to form two streaks from which the endoral and paroral derive, and gives rise to the leftmost frontal cirrus in each the proter and opisthe. The proximal membranelles in the proter are replaced by newly formed structures (Figs. 3H, 5L). In the late stages, the differentiation of membranelles is almost complete, forming the new structures for both the daughter cells, anterior end of the new adoral in opisthe bends to the right. Initially, the newly formed endoral and paroral initially lie close together in parallel, but later they separate, arch, and intersect in each filia product (Fig. 4A, C, E, G).
Development Of The Frontoventral-transverse Cirri
In the early stage, some cirri in the midventral complex dedifferentiate to join in the construction of the frontoventral-transverse cirral anlagen (FVT-anlagen) (Figs. 3D, E, 5F). Subsequently, the FVT-anlagen form and lengthen by increasing the number of basal bodies, to the right of the undulating membranes anlagen in both the opisthe and proter (Figs. 3E, F, 5G, J). In the middle stage, these anlagen broaden and break apart (Figs. 3H, 5L). In late stages, the FVT-anlagen commence to fragmentize (Figs. 4A, C, 5N). Later, the segregation of cirri from the FVT-anlagen is almost complete and, the old structures are almost resorbed. New cirri migrate to their final positions as distinct cirri (Fig. 4E, G). Consequently, anlage I provides the left frontal cirrus; anlage II forms the middle frontal cirrus, the cirri below the middle frontal cirrus and the buccal cirrus; anlage III produces the rightmost frontal cirrus and the cirrus III/2; midventral pairs develop from anlagen IV to n–4 (in some individuals, n–3) and midventral rows are formed from anlagen n–3 (in some individuals, n–2) to n–1; anlage n forms the frontoterminal cirri (Fig. 4E, G).
Development Of Marginal Rows And Dorsal Kineties
The marginal and dorsal kineties anlagen are formed intrakinetally both in the proter and the opisthe (Figs. 3F, G, 5J). These anlagen subsequently elongate and gradually replace the parental structures (Figs. 3H, I, 4A–H, 5K, N). Four to nine caudal cirri are formed at the posterior ends of dorsal kineties (Fig. 4B, D, F, H).
Division Of Nuclear Apparatus
The nuclear apparatus divides in the usual way for urostylids. Briefly, the two macronuclear nodules fuse to form a single mass during the mid-divisional stage and then divide twice prior to cytokinesis. Micronuclei were observed to divide mitotically (Figs. 3G, I, 4B, D, F, H, 5I, M).
Phylogenetic analyses based on SSU rDNA gene sequences (Fig. 6)
The SSU rDNA sequence of Holostichides songi nov. spec. was deposited in GenBank with the accession number MT557692. The length and GC content of the new sequence are 1651 bp and 45%, respectively.
Phylogenetic trees using two different methods (ML and BI) generated nearly congruent relationships. Therefore, only the ML tree is presented with support values from both algorithms at the nodes (Fig. 6). In both analyses, Holostichides songi nov. spec. clusters with its congeners, namely H. chardezi, H. heterotypicus and H. obliquocirratus, with full support. The similarities of the SSU rDNA sequence of H. songi nov. spec. to H. chardezi, H. heterotypicus and H. obliquocirratus are 97.4%, 94.9% and 97.6%, respectively.