Bakuella (Pseudobakuella) guangdongica n. sp. (Figs. 1A–F, 2A–L, 3A–I, 4A–H, Table 1)
Table 1
Morphometric characterization of Bakuella (Pseudobakuella) guangdongica n. sp.
Character a | HT | Min | Max | Mean | Med | SD | CV | n |
Body length | 195 | 170 | 240 | 209.3 | 210 | 17.6 | 8.4 | 23 |
Body width | 70 | 55 | 100 | 72.4 | 72 | 10.1 | 13.9 | 23 |
Body length: width, ratio | 2.79 | 2.20 | 4.27 | 2.93 | 2.86 | 0.41 | 13.83 | 23 |
AZM length | 70 | 62 | 80 | 69.2 | 70 | 4.2 | 6.1 | 23 |
AZM length: Body length, ratio | 0.36 | 0.26 | 0.38 | 0.33 | 0.33 | 0.03 | 7.58 | 23 |
Adoral membranelles, no. | 37 | 35 | 42 | 37.8 | 38 | 1.8 | 4.7 | 23 |
Frontal cirri, no. | 3 | 3 | 3 | 3.0 | 3 | 0 | 0 | 23 |
Buccal cirri, no. | 4 | 3 | 5 | 4.1 | 4 | 0.5 | 13.3 | 23 |
Frontoterminal cirri, no. | 2 | 2 | 2 | 2.0 | 2 | 0 | 0 | 23 |
Midventral pairs, no. | 16 | 10 | 20 | 15.4 | 16 | 2.8 | 17.9 | 23 |
Midventral rows, no. | 2 | 2 | 2 | 2.0 | 2 | 0 | 0 | 23 |
The left midventral row, number of cirri | 5 | 3 | 11 | 4.4 | 4 | 1.8 | 40.3 | 22 |
The right midventral row, number of cirri | 11 | 8 | 16 | 12.6 | 13 | 2.2 | 17.9 | 21 |
Right marginal cirri, no. | 54 | 37 | 59 | 46.9 | 47 | 5.6 | 11.9 | 23 |
Left marginal cirri, no. | 40 | 36 | 48 | 42.0 | 43 | 3.7 | 8.8 | 23 |
Transverse cirri b, no. | 9 | 8 | 13 | 10.7 | 10 | 1.3 | 12.4 | 23 |
Dorsal kineties, no. | 4 | 3 | 3 | 3.0 | 3 | 0 | 0 | 15 |
Dorsal kinety 1, bristles, no. | 27 | 20 | 27 | 23.6 | 23 | 2.2 | 9.3 | 19 |
Dorsal kinety 2, bristles, no. | 26 | 21 | 28 | 24.2 | 24 | 5.7 | 23.5 | 19 |
Dorsal kinety 3, bristles, no. | 25 | 23 | 29 | 26.5 | 27 | 1.9 | 7.2 | 17 |
Macronuclear nodules, no. | 225 | 110 | 230 | 159.2 | 146 | 36.6 | 23 | 21 |
Micronuclei, no. | 2 | 1 | 5 | 2.6 | 2 | 1.1 | 40.9 | 14 |
a All data are based on protargol-stained specimens, measurements in µm. Abbreviations: AZM, adoral zone of membranelles; CV, coefficient of variation in %; HT, holotype; Med, median; Max, maximum; Mean, arithmetic mean; Min, minimum; n, sample size; no., number; SD, standard deviation. |
b Pretransverse ventral cirri included. |
ZooBank registration
Present work: urn:lsid:zoobank.org:pub:8BF9471D-94FF-41BA-9989-F21B93208429.
Bakuella (Pseudobakuella) guangdongica n. sp.:
urn:lsid:zoobank.org:act:825560C7-EFF0-4DF7-B895-848BED297FE3.
Diagnosis:
Body size 150–225 µm × 40–75 µm in vivo. Elongate with both ends rounded. 110–230 macronuclear nodules. Contractile vacuole positioned at about 35% of body length near left margin. Cortical granules spherical and colorless, 1 µm in diameter, arranged in small groups. Adoral zone occupies about 33% of body length and composed of 35–42 membranes. Three frontal, three to five buccal, two frontoterminal and eight to 13 transverse cirri. Midventral complex composed of 10–20 pairs and two midventral rows comprising two to 11 and eight to 16 cirri, respectively. One left and one right marginal cirral rows, composed of 36–48 and 37–59 cirri, respectively. Three bipolar dorsal kineties.
Type material:
The protargol slide (registry no. WJY2016032301B) with the holotype specimen and one paratype slide (registry no. WJY2016032301C) were deposited in the Laboratory of Protozoological Biodiversity and Evolution in Wetland, Shaanxi Normal University, China.
Type locality:
Soil from Seaside Garden (21°43'15''N; 112°14'39''E), Yangjiang, Guangdong, China.
Etymology:
The species-group name “guangdongica” refers to the geographic location where the species was discovered.
Morphology (Figs 1A–F, 2A–L, Table 1)
Size 150–225 µm × 40–75 µm in vivo. Body elongate with both ends rounded and both margins almost straight; dorsoventrally flattened at a ratio of about 2:1, highly flexible but not contractile (Figs. 1A, 2A, B). 110–230 macronuclear nodules, oval to elongate in shape, scattered throughout cytoplasm, difficult to observe in vivo, one to five micronuclei (Table 1, Fig. 1F). Contractile vacuole about 16 µm across when fully extended, positioned at about 35% of body length near left margin, pulsing at intervals of about 10 s (Figs. 1A, 2B). Pellicle thin and soft, with colorless spherical cortical granules, 1 µm in diameter, arranged in small groups (Figs. 1B–D, 2C, D). Cytoplasm colorless to grayish, usually contains numerous lipid droplets (2–6 µm across) and food vacuoles (5–25 µm across) in middle portion that render cell opaque and dark in middle portion at low magnification (Fig. 2B, E). Locomotion by fast crawling over debris, sometimes rotating slowly around main body axis when swimming.
Consistently three relatively enlarged frontal cirri, with cilia 16 µm long in vivo. Three to five buccal cirri arranged alongside paroral, cilia about 12 µm in vivo. Two frontoterminal cirri slightly ahead of the level of cirrus III/2. Midventral complex composed of 10–20 pairs of cirri that extending to about two thirds of body length, together with two midventral rows comprising three to 11 (left one) and eight to 16 (right one) cirri respectively and both of which extending posteriad to transverse cirri. Eight to 13 transverse cirri with cilia ca. 20 µm long in vivo. One left and one right marginal cirral rows, composed of 36–48 and 37–59 cirri, respectively, cilia about 12 µm long in vivo. Left marginal row commences left of proximal portion of adoral zone of membranelles (AZM) while right marginal row commences near anterior end on dorsal side, posterior ends of both terminate caudally and nearly confluent. Most somatic cirri relatively fine with cilia about 12 µm long in vivo (Figs. 1E, F, 2H–K).
Three dorsal kineties extending almost entire length of cell, composed of 20–27, 21–28, and 23–29 dikinetids, respectively, with bristles about 5 µm long in vivo (Figs. 1F, 2L).
Adoral zone occupies about 33% of body length after protargol impregnation, composed of 35–42 membranes, with cilia 18–20 µm long in vivo. Paroral strongly while endoral slightly curved and the former slightly shorter and lower than the latter.
Morphogenesis during binary fission and physiological regeneration (Figs. 3A–I, 4A–H, 5A–R)
Stomatogenesis
Stomatogenesis commences with the apokinetal formation of several groups of closely spaced basal bodies near the left cirri of midventral pairs, which is the oral primordium of the opisthe (Figs. 3A, B, 5A). No old structures join in the formation of the oral primordium. Then, these basal bodies increase in number and the oral primordium elongates to reach the level of the cytostome (Figs. 3A, B, 5B). Subsequently, an anarchic field of closely arranged basal bodies originates de novo on the dorsal side of the buccal cavity. Meanwhile, the old undulating membranes begin to dedifferentitate (Figs. 3C, 5D, E). With the proliferation of more basal bodies, the oral primordium of the opisthe differentiates new membranelles posteriad and the oral primordium of the proter grows rapidly within a wedge-shaped, and the undulating membrane anlagen appear on the right of the oral primordia both in opisthe and proter (Fig. 3D, F). We failed to obtain specimens in early stage and were therefore unable to determine the origin of the undulating membrane anlagen both in opisthe and proter. The parental undulating membranes probably contribute to the formation of the undulating membranes anlage in proter. Meanwhile, the proximal portion of old AZM becomes disordered (Fig. 3D, F). Later, the new membranelles continue to differentiate in both the proter and opisthe, and the undulating membranes anlagen develop and give rise to the leftmost frontal cirrus in each the proter and opisthe (Figs. 3F, H, 4A, 5G, I). In late stages, the differentiation of membranelles is almost complete, forming the new structures for the proter and opisthe. Anterior ends of the new adoral bend to the right, and the undulating membranes anlagen split longitudinally into the endoral and paroral in each daughter cell. The parental AZM has been almost completely resorbed and the newly formed AZM eventually replaces the old one (Figs. 4A, C, E, 5L, N, P, Q).
Development of the frontoventral-transverse cirri
Development of the somatic ciliature begins with the formation of the frontoventral-transverse cirral anlagen (FVT-anlagen). In an early stage, the parental buccal cirri dedifferentiate, and possibly contribute to the formation of the proter’s FVT-anlage (Fig. 3C). Later, several streaks are formed to the right of the oral primordium and develop into the FVT-anlagen. Some cirri of the midventral complex join in the construction of FVT-anlagen (Fig. 3D). Subsequently, with further basal bodies proliferation, two sets of FVT-anlagen are formed and become organized into about 20 oblique streaks posteriad (Figs. 3F, H, 5E–G).
In middle to late stages, the FVT-anlagen for both daughters fragmentize and differentiate into new cirri (Figs. 3H, 4A, 5I). Later, the segregation of cirri from the FVT-anlagen is almost complete (Fig. 4A). Anlage II provides the second frontal cirrus and buccal cirri, anlage III produces the rightmost frontal cirrus and cirrus III/2, and midventral pairs develop from anlagen IV to n–2, two midventral rows formed from anlagen n and n–1. The anterior portion of anlage n forms two frontoterminal cirri additionally, while anlagen n–12 (or n–11 to n–7, deduced from morphometric data) to n develop transverse cirri (Figs. 4C, 5I, L).
Finally, the cell begins to elongate, the daughters separate and new ciliary structures move further apart and migrate towards their final position. Two frontoterminal cirri migrate anteriorly and the parental structures are resorbed (Figs. 4E, 5N, P, Q).
Development of marginal rows and dorsal kineties
As is usual for most hypotrichs, marginal and dorsal kineties anlagen are formed intrakinetally both in the proter and the opisthe. These anlagen increase in size by further proliferation basal bodies and subsequently elongate and gradually replace the parental structures. Caudal cirri not formed (Figs. 3E, F–I, 4A–F, 5H, J, L, O–Q).
Division of nuclear apparatus
The nuclear apparatus divides in the usual way for urostylids, i.e. all macronuclear nodules fuse to form a single mass during the ontogenetic process and then divide into many nodules. Micronuclei were observed to divide mitotically during the morphogenesis (Figs. 3E, G, I, 4B, D, F, 5K, M, R).
Physiological reorganisation (Fig 4G, H)
Only one physiological regeneration stage was observed. From the data available, the main features of cortical development in reorganiser are similar to those in the proter, i.e.: (1) the parental undulating membranes are completely replaced by the new structure developing from undulating membranes anlage; (2) the undulating membranes anlage (= anlage I) gives rise to the leftmost frontal cirrus; (3) the number of frontoventral-transverse cirral anlagen about 20; and (4) the marginal rows and the dorsal kineties originate and develop in the same way as in cell division.
Phylogenetic analyses based on SSU rDNA gene sequences (Fig. 6)
The SSU rDNA sequence of Bakuella (Pseudobakuella) guangdongica n. sp. was deposited in GenBank with the accession number MT435536. The length and GC content of the new sequence are 1853 bp and 44.47%, respectively. Phylogenetic trees inferred from the SSU rDNA sequences, using two different methods (ML and BI) show similar topologies; therefore, only the ML tree (Fig. 6) is presented with bootstraps and posterior probabilities from both algorithms.
Consistent with previous studies [16–19], the urostyloids are non-monophyletic in our phylogenetic tree. Bakuella (Pseudobakuella) guangdongica n. sp. is placed within the core urostylids and clusters together with Bakuella (Bakuella) granulifera, Neobakuella aenigmatica, Anteholosticha antecirrata and Urostyla grandis with full support. This group is sister to a clade formed by three Bakuella spp., two Diaxonella spp., Neobakuella flava and Apobakuella fusca (ML/BI, 97/1.00).