Abstract
Background
As unicellular eukaryotes, ciliates are indispensable components of ecosystems, serving as an intermediate nutrition link between bacteria or algae and meiofauna. The class Nassophorea Small and Lynn, 1981 is a group of morphologically specialized ciliates characterized by a basket-like cytopharynx supported by thick microtubular rods, which are algivorous and thus act as primary consumers in ecosystems. In the present study, the morphology and molecular phylogeny of three new nassulid ciliates were studied based on morphological and small subunit ribosomal ribonucleic acid gene sequence data. The secondary structure of the V9 region of their 18S rRNA gene sequences was also analyzed to investigate their phylogenetic relationships.
Results
Based on morphological feature and phylogenetic analysis, we establish a new genus Aponassulides gen. nov. and discovered three new species, viz. Aponassulides lacustris gen. nov., sp. nov., Naxella vorax sp. nov. and Nassula paralabiata sp. nov. The new genus is distinguished from its relatives by having nassulid organelles orientated obliquely to frange axis and diagonally along the top left and bottom right directions. Aponassulides lacustris gen. nov., sp. nov. can be recognized by its large-sized and broadly rounded body and linear nassulid frange orientated by numerous nassulid organelles as well freshwater habitat. Naxella vorax sp. nov. is characterized by having a medium-sized and broadly ellipsoidal body, three or four nassulid organelles and 55–68 somatic kineties. Nassula paralabiata sp. nov. is a marine species with sigmoidal nassulid frange, three caudal cilia and two lip-shaped cytoplasmic membranes. Phylogenetic analyses revealed that three new species clusters within the family Nassulidae de Fromentel, 1874. The analysis of the secondary structure of the SSU rRNA V9 region is also consistent with the topology of the phylogenetic tree.
Conclusions
The study of the morphology of three new nassulids increases the overall knowledge about the biodiversity of this ciliate group. It also adds to the genetic data available, substantiates the paraphyly of the Nassulida, and recovers the monophylies of genera Naxella and Nassula for the first time.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12866-025-04541-z.
Keywords: Ciliature, Morphology, Nassulida, SSU rDNA
Background
The phylum Ciliophora Doflein, 1901 represents a highly diverse and evolutionarily significant group within Alveolata microeukaryotes. While research has historically emphasized dominant classes such as Spirotrichea Bütschli, 1889 and Oligohymenophorea de Puytorac et al., 1974 [1, 2], growing evidence reveals that less-studied groups, e.g., Prostomatea Schewiakoff, 1896 and Phyllopharyngea de Puytorac et al., 1974 harbor substantial undocumented diversity [2].
Ciliates of the class Nassophorea Small & Lynn, 1981 are morphologically characterized by a specialized pharyngeal basket and a set of oral polykinetids. To date, integrated morphological and molecular phylogenetic studies recognize four orders within this class: Nassulida Jankowski, 1967, Microthoracida Jankowski, 1967, Discotrichida Fan et al., 2014 and Colpodidiida et al., 2002 [1, 3–9]. With respect to ecology, most nassulids and some microthoracids are phytophagous, feeding on a wide variety of microalgae, especially some cyanobacteria [10–12]. Concurrently, they are preyed upon by diverse predators [10, 13–16].thereby playing a critical role in the structure and functioning of the microbial food web.
Within the order Nassulida, the family Nassulidae de Fromentel, 1874 comprises the greatest diversity. Its five constituent genera, viz. Nassula Ehrenberg, 1834, Nassulides Foissner et al., 2002, Naxella Fryd-Versavel et al., 1980, Obertrumia Foissner et al., 1994 and Rhinakis Deroux, 1994, collectively contain approximately 70 nominal species [1, 17–20]. Nevertheless, a majority of species remain unexamined by contemporary methodological approaches. The absence of key morphological features and genetic sequences in many historic descriptions creates taxonomic confusion, making it difficult to assign species and accurately determine intraspecific variations. Furthermore, sampling bias toward freshwater environments has left the diversity of nassulids in marine, brackish, and terrestrial realms profoundly understudied. Only Ozaki and Yagiu (1941) recorded some species in the inland sea of Japan, and Foissner et al. (2002) described 24 new terrestrial nassulids [17, 21]. Given cosmopolitan distribution of most well-known groups of ciliates, the research on the diversity of the nassulids living in different ecological environments is still insufficient.
The present study aims to: (i) reveal species diversity of poorly know groups of ciliates from under-sampling areas using an integrative approach; (ii) explore the morphological characteristics and classification basis among genera of the nassulids; and (iii) discuss the phylogenetic relationships within the family Nassulidae.
ZooBank registration
The ZooBank registration number of present work is: urn:lsid:zoobank.org:pub:14AB98CD-EF8B-4861-AF0A-B4D5A3654771
Results
Taxonomy
Class Nassophorea Small and Lynn, 1981.
Order Nassulida Jankowski, 1967.
Family Nassulidae de Fromentel, 1874.
Genus Aponassulides gen. nov.
ZooBank registration: urn:lsid:zoobank.org:act:DE58DD7F-D977-4E09-9A8F-7AA2A7E842AC
Diagnosis: Nassulidae with nassulid frange extending linearly onto dorsal side of cell. Nassulid organelles numerous and all orientated obliquely to frange axis, diagonally along the top left and bottom right directions.
Type species: Aponassulides lacustris gen. nov., sp. nov.
Etymology: Aponassulides is a composite of the Greek prefix apo- (derived from) and the genus-group name Nassulides. This indicates that the new genus has a nassulid frange pattern similar to that of Nassulides. Masculine gender.
Species assignable: Only type species.
Aponassulides lacustris gen. nov., sp. nov. (Figs. 1 and 2; Table 1)
Fig. 1.
Drawings of Aponassulides lacustris gen. nov., sp. nov. from life (A, E, F, G) and after silver-nitrate (B, D, H, I) staining, and for genera within Nassulidae distinction by the nassulid frange (C; open circle symbolizes oral opening, black rectangles mark nassulid organelles). (A) Ventral view of a representative individual, showing general morphology. (B) Detail of oral region, showing the preoral suture (SU), oral opening (OO), postoral kineties (PO), part of nassulid frange (NF) and postorganellar stripe (S). (C) The pattern of nassulid frange for genera Aponassulides (1), Nassula (2), Nassulides (3), Obertrumia (4) and Naxella and Rhinakis (5) (2–5 from Foissner et al., 2002). (D) Complete nassulid frange. (E) Undernourished individual. (F, G) Ejected (F) and (G) resting extrusomes. (H, I) Ventral side view (H) of the holotype specimen and dorsal side view of (I) another specimen showing ciliary pattern. Scale bars, 60 μm
Fig. 2.
Photomicrographs of Aponassulides lacustris gen. nov., sp. nov. from life with bright field (A–D, M, N) and differential interference contrast (E, G–L) microscopy and after silver nitrate (F, O, P) staining. (A, B) Ventral views of slightly compressed cells, arrow indicates oral opening. (C) Lateral and view of the same individual as (B), arrow indicates the pharyngeal basket. (D) Undernourished individual, arrow indicates the pharyngeal basket. (E, F) Nassulid organelles in vivo (E) and after staining (F). (G) Contractile vacuole with small contributory vesicles during diastole. (H) Oral region showing the fibrous annulus and nassulid organelles (arrow). (I) Excretory pore of the contractile vacuole. (J) Optical section through cortical layer. (K) Macronucleus and two micronuclei (arrows). (L) Pharyngeal rods. (M, N) Ejected (N) and resting (M) extrusomes. (O, P) Ventral view (O) of the holotype specimen and dorsal view (P) of another specimen, showing the ciliature and the excretory pore (arrow) of the contractile vacuole. Scale bars, 60 μm
Table 1.
Morphometrics of Aponassulides lacustris gen. nov., sp. nov. CV, coefficient of variation (%); M, median value; Max, maximum value; Mean, arithmetic mean; Min, minimum value; n, number of specimens; SD, standard deviation of the mean
| Charactera | Mean | M | SD | CV | Min | Max | n |
|---|---|---|---|---|---|---|---|
| Body, length | 149.8 | 147 | 16.36 | 10.9 | 120 | 199 | 25 |
| Body, width | 137.0 | 134 | 16.49 | 12.0 | 110 | 175 | 25 |
| Length: width, ratio | 1.1 | 1.1 | 0.06 | 5.8 | 1.0 | 1.2 | 25 |
| Anterior body end to macronucleus, distanceb | 57.4 | 57 | 10.26 | 17.9 | 38 | 76 | 15 |
| Anterior body end to excretory pore, distanceb | 124.7 | 120 | 8.06 | 6.5 | 118 | 136 | 3 |
| Anterior body end to distal end of nasse, distanceb | 69.6 | 69 | 13.06 | 18.8 | 49 | 96 | 7 |
| Anterior body end to macronucleus, % of body length | 39.2 | 40.8 | 0.07 | 16.9 | 24.5 | 49.0 | 15 |
| Anterior body end to excretory pore, % of body length | 81.4 | 81.0 | 0.02 | 3.0 | 78.7 | 84.5 | 3 |
| Anterior body end to distal end of nasse, % of body length | 47.4 | 46.4 | 0.05 | 11.1 | 40.8 | 58.9 | 7 |
| Macronucleus, length | 30.9 | 31 | 2.86 | 9.3 | 27 | 39 | 15 |
| Macronucleus, width | 27.2 | 27 | 2.83 | 10.4 | 25 | 37 | 15 |
| Nasse, largest diameter | 15.7 | 16.0 | 2.35 | 15.0 | 11 | 21 | 20 |
| Nasse, length | 59.7 | 60 | 8.66 | 14.5 | 45 | 75 | 7 |
| 1 st nassulid organelle, length | 6.0 | 6.0 | 1.70 | 28.3 | 3 | 9 | 18 |
| Last nassulid organelle, length | 4.9 | 5.0 | 1.44 | 29.2 | 3 | 7 | 14 |
| Nassulid organelles, number | 18.4 | 19.0 | 2.06 | 11.2 | 15 | 21 | 12 |
| Ventral kineties, number | 61.8 | 62.0 | 6.37 | 10.3 | 50 | 71 | 16 |
| Dorsal kineties, number | 56.1 | 57 | 4.66 | 8.3 | 50 | 65 | 15 |
| Somatic kineties, number | 116.0 | 114.0 | 11.91 | 10.3 | 100 | 132 | 8 |
aAll morphometrics from fixed specimens impregnated by Chatton–Lwoff silver nitrate method
bMeasured from anterior end of cell to anterior margin of macronucleus
Zoobank registration: urn:lsid:zoobank.org:act:DF52EEC8-6C44-4A36-AD1E-6B9E2FC5A42E
Diagnosis: Cell size 135–180 × 130–160 μm in vivo; broadly rounded. Extrusomes fusiform, about 10 μm long. 100–132 somatic kineties; 15–21 nassulid organelles. Pharyngeal basket composed of 19–27 straight rods extending to middle portion of cell. Paroral membrane absent. Freshwater habitat.
Type locality: Freshwater pond in Shaohai Wetland, Qingdao, China (36°14′58″N; 120°5′3″E). The water temperature was 29 ℃.
Type specimens: A silver nitrate-stained slide (registration number: SQH2023063001-1) containing the holotype specimen (Figs. 1H, 2O, circled in ink) has been deposited in the Laboratory of Protozoology, Ocean University of China, China. Another silver nitrate-stained slide (registration number: SQH2023063001-2) has been deposited in the Marine Biological Museum, Chinese Academy of Sciences, China.
Etymology: The species-group name lacustris (Latin, living in the lake or pond) alludes to the type of habitat where the species was found.
Description: Cell size about 135–180 × 130–160 μm in vivo (n = 7), 120–199 × 110–175 μm after silver nitrate-stained (Table 1; morphologic variations due to different cell reactions to silver nitrate staining process). Length to width ratio 1–1.1:1 in vivo and 1–1.2:1 after silver nitrate staining. Body shape broadly rounded with a slight oral depression (Figs. 1A and 2A, B, C). Macronucleus usually in middle third of cell, rounded to ellipsoidal, about 42 μm in vivo. Two micronuclei near or close to macronucleus, about 5 μm across in vivo (Fig. 2K). Contractile vacuole in middle to posterior half of cell, surrounded by small contributory vesicles during diastole, about 40 μm in diameter and excretory pore about 2 μm across (Figs. 2G, I). Cytopyge slit-like and in line with excretory pore, extending to posterior body end. Cortex about 2 μm thick and bright, indistinctly separated from cytoplasm. Resting extrusomes attached almost perpendicularly to cortex, fusiform, about 10 μm long, rather numerous forming more or less distinct fringe; exploded extrusomes also fusiform but up to 50 μm long and 2 μm wide (Figs. 1F, G and 2M, N). Well-fed cells containing numerous food vacuoles, about 2–10 μm across (Fig. 2B, C). Undernourished cells colorless and slightly smaller, about 120 × 100 μm in vivo, filled with few food vacuoles and many transparent vacuoles (Figs. 1E and 2D). Locomotion by swimming while rotating about main body axis.
Somatic cilia about 8 μm long in vivo, arranged in 100–132 meridional and equidistantly spaced ciliary rows, except for more closely spaced paroral and postoral kineties; ventral kineties (on average 62) slightly denser than dorsal kineties (on average 56). Cilia in anterior pole arranged scattered forming indistinct preoral suture. Post-organellar stripe conspicuous (Fig. 1B). Nassulid frange composed of 15–21 organelles, extends in a flat bow onto dorsal side of cell and terminates near mid-body. Nassulid organelles orientated obliquely to frange axis, diagonally along the top left and bottom right directions. Each nassulid organelle consists of three rows of basal bodies, each row composed of 5–20 basal bodies. Cilia of nassulid organelles about 12 μm long in vivo (Figs. 1D and 2E, F).
Pharyngeal basket extends obliquely to dorsal side, terminating in mid-body region, funnel-shaped, about 75 μm long in vivo, composed of 19–27 (n = 5) thin, straight, and comparatively narrowly spaced rods with shallow indentation anteriorly; dense annulus unrecognizable, fibrous annulus conspicuous in vivo (Figs. 2H, L).
Genus Naxella Fryd-Versavel et al., 1980
Naxella vorax sp. nov. (Figs. 3 and 4; Table 2)
Fig. 3.
Drawings of Naxella vorax sp. nov. from life (A, C–H) and after silver-nitrate staining (B, I, J). (A) Ventral view of a representative individual. (B) Detail of oral region showing the preoral suture (SU), oral opening (OO), paroral membrane (PM), kinety 1 (K1), kinety 2 (K2), kinety 3 (K3), postoral kineties (PO), nassulid frange (NF) and postorganellar stripe (S). (C–E) Rest position (C), contact position (D) and swallow position (E) of the pharyngeal basket during feeding movement. (F) Macronucleus and two micronuclei (arrows). (G, H) Ejected (G) and (H) resting extrusomes. (I, J) Ventral view (I) of the holotype specimen and dorsal view (J) of another specimen showing ciliary pattern. Arrow indicates the cytopyge. Scale bars, 30 μm
Fig. 4.
Photomicrographs of Naxella vorax sp. nov. from life with bright field (B–D) and differential interference contrast (A, E–I, M) microscopy, after DAPI fluorescent (L) and silver nitrate (K, N, O) staining. (A, B) Ventral views of slightly compressed cells, arrow indicates the contractile vacuole and arrowhead indicates the macronucleus. (C) Individual with irregular body shapes during feeding movement. (D) Undernourished individual, arrow indicates the contractile vacuole. (E) Oral region showing the paroral membrane (arrow), the fibrous annulus (double arrowhead) and nassulid organelles (arrowhead). (F) Excretory pore of the contractile vacuole. (G) Contractile vacuole with small contributory vesicles during diastole. (H) Pharyngeal rods. (I) Nassulid organelles in vivo. (J) Resting extrusomes. (K) Detail of oral region showing the nassulid organelles (arrow) and the paroral membrane (arrowhead). (L, M) Macronucleus and micronuclei (arrows) in vivo (M) and after fluorescent staining (L). (N, O) Ventral view (N) of the holotype specimen and dorsal side view of (O) the paratype specimen, showing the ciliature and the paroral membrane (arrow), the nassulid frange (arrowhead) and the excretory pore (double arrowhead). Scale bars, 15 μm
Table 2.
Morphometrics of Naxella vorax sp. nov. CV, coefficient of variation (%); M, median value; Max, maximum value; Mean, arithmetic mean; Min, minimum value; n, number of specimens; SD, standard deviation of the mean
| Charactera | Mean | M | SD | CV | Min | Max | n |
|---|---|---|---|---|---|---|---|
| Body, length | 81.8 | 83 | 11.77 | 14.4 | 61 | 101 | 25 |
| Body, width | 62.6 | 67 | 12.44 | 19.9 | 36 | 84 | 25 |
| Length: width, ratio | 1.3 | 1.3 | 0.13 | 10.1 | 1.1 | 1.7 | 25 |
| Anterior body end to macronucleus, distanceb | 35.5 | 39 | 6.80 | 19.1 | 23 | 48 | 11 |
| Anterior body end to excretory pore, distanceb | 63.7 | 67 | 11.70 | 18.4 | 48 | 81 | 9 |
| Anterior body end to distal end of nasse, distanceb | 35.7 | 35.0 | 3.90 | 10.9 | 31 | 41 | 6 |
| Anterior body end to macronucleus, % of body length | 44.4 | 46.2 | 0.04 | 9.2 | 37.7 | 50.6 | 11 |
| Anterior body end to excretory pore, % of body length | 75.7 | 75.3 | 0.04 | 5.9 | 68.6 | 82.5 | 9 |
| Anterior body end to distal end of nasse, % of body length | 45.8 | 45.6 | 0.04 | 9.0 | 40.0 | 52.4 | 6 |
| Macronucleus, length | 16.2 | 16 | 3.01 | 18.6 | 11 | 24 | 11 |
| Macronucleus, width | 14.8 | 14 | 3.27 | 22.1 | 10 | 24 | 11 |
| Nasse, largest diameter | 10.3 | 10.0 | 1.89 | 18.2 | 6 | 14 | 18 |
| Nasse, length | 29.5 | 32.0 | 10.23 | 34.7 | 13 | 41 | 4 |
| 1 st nassulid organelle, length | 6.3 | 6.0 | 1.30 | 20.8 | 4 | 8 | 8 |
| Last nassulid organelle, length | 3.5 | 3.0 | 0.76 | 21.8 | 3 | 5 | 6 |
| Nassulid organelles, number | 3.8 | 4 | 0.40 | 10.5 | 3 | 4 | 5 |
| Ventral kineties, number | 32.6 | 32 | 2.27 | 7.0 | 30 | 38 | 15 |
| Dorsal kineties, number | 29.4 | 29.5 | 2.55 | 8.7 | 24 | 34 | 16 |
| Somatic kineties, number | 61.7 | 62 | 4.27 | 6.9 | 55 | 68 | 11 |
| Dikinetids in paroral membrane, number | 17.9 | 17.5 | 1.70 | 9.5 | 15 | 20 | 10 |
aAll morphometrics from fixed specimens impregnated by Chatton–Lwoff silver nitrate method
bMeasured from anterior end of cell to anterior margin of macronucleus
Zoobank registration: urn:lsid:zoobank.org:act:62475413-45B8-42FF-8EE3-62B992169765
Diagnosis: Cell size 80–95 × 60–70 μm in vivo; broadly ellipsoidal. Extrusomes fusiform, about 6 μm long. 55–68 somatic kineties; 3 or 4 nassulid organelles extending behind level of oral opening. Pharyngeal basket composed of 15–19 straight rods extending to middle portion of cell. Paroral membrane conspicuous, composed of about 15–20 dikinetids.
Type locality: Freshwater pond in Shaohai Wetland, Qingdao, China (36°14′58″N; 120°5′3″E). The water temperature was 29 ℃.
Type specimens: A silver nitrate-stained slide (registration number: SQH2023063002-1) containing the holotype specimen (Figs. 3I, 4N, circled in ink) has been deposited in the Laboratory of Protozoology, Ocean University of China, China. Another silver nitrate-stained slide (registration number: SQH2023063002-2) has been deposited in the Marine Biological Museum, Chinese Academy of Sciences, China.
Etymology: The species-group name vorax (Latin, swallowing greedily) refers to its feeding state.
Description: Cell size about 80–95 × 60–70 μm in vivo (n = 5), 61–101 × 36–84 μm after silver nitrate-stained (Table 2; morphologic variations due to different cell reactions to silver nitrate staining process). The ratio of length to width 1.3–1.5:1 in vivo and 1.1–1.7:1 after staining. Body shape broadly ellipsoidal (Figs. 3A and 4A, B, D). Macronucleus usually in middle third of cell, rounded to ellipsoidal, about 20 μm across in vivo. Two micronuclei near or close to macronucleus, about 3 μm across in vivo and after DAPI staining (Figs. 3F and 4L, M). Contractile vacuole in middle to posterior half of cell, surrounded by small contributory vesicles during diastole, about 15 μm in diameter.; excretory pore in line with or slightly left of kinety 1 (Fig. 4F, G, N). Cytopyge slit-like and in line with excretory pore, near posterior body end, about 20 μm long (Fig. 3I). Cortex about 1 μm thick and bright, indistinctly separated from cytoplasm. Resting extrusomes attached almost perpendicularly to cortex, fusiform, about 6 μm long, rather numerous forming more or less distinct fringe; ejected extrusomes also fusiform but up to 30 μm long and 1.5 μm wide (Figs. 3G, H and 4G). Well-fed cells containing numerous food vacuoles, about 3–14 μm across (Fig. 4A). Locomotion by swimming while rotating about main body axis.
Somatic cilia about 8 μm long in vivo, arranged in 55–68 meridional and equidistantly spaced ciliary rows, except for more closely spaced postoral kineties and second (paroral, K2) and third (K3) somatic kinety; which end near mid-body; leave blank an about 3.5 μm wide post-organellar stripe underneath nassulid frange and form distinct preoral suture (Figs. 3B and 4K, N). Ventral kineties (on average 33) slightly denser than dorsal kineties (on average 29). Nassulid frange begins underneath oral opening, extends leftwards, almost orthogonal to main body axis, thus splitting somatic ciliary rows, composed of 3 or 4 nassulid organelles arranged parallel to frange axis. Each nassulid organelle consists of three rows of basal bodies, each row composed of 6–14 basal bodies. Paroral membrane rather conspicuous, continuous posteriorly with first somatic kinety (K1), commences at level of oral opening and extends in flat bow to blank stripe behind nassulid frange, composed of about 15–20 clockwise inclined dikinetids (Figs. 3B and 4I, K, N).
Pharyngeal basket extends obliquely to dorsal side, terminating in mid-body region, funnel-shaped, about 40 μm long in vivo, composed of 15–19 (n = 4) thin, straight, and comparatively narrowly spaced rods with shallow indentation anteriorly; dense annulus unrecognizable, fibrous annulus conspicuous in vivo (Figs. 4E, H). Once the top of the basket closely applied to the cyanobacteria filament the basket performs a sequence of feeding movements. When in contact with the filaments, the basket becomes elliptical in cross-section as the tops and bottoms of the rods bordering the longer sides of this ellipse become more widely spaced. Pharyngeal basket maintains this shape until it reaches one end of the filament, then the basket constricts and the organism begin feeding. The shape of cell becomes irregular due to swallowing the filament, and as the filament digested, the cell returns to its original shape (Figs. 3C, D, E and 4C).
Genus Nassula Ehrenberg, 1834
Nassula paralabiata sp. nov. (Figs. 5 and 6; Table 3)
Fig. 5.
Drawings of Nassula paralabiata sp. nov. from life (A, C, E, F) and after protargol staining (B, D, G, H). (A) Ventral view of a representative individual, arrow indicates the caudal cilia. (B) Detail of oral region showing the preoral suture (SU), paroral membrane (PM), lip-shaped cytoplasmic membranes (L), postoral kineties (PO), nassulid frange (NF) and postorganellar stripe (S). (C) Ejected and resting and trichocyst, drawn to scale. (D) Left lateral view showing the pharyngeal basket and the macronucleus. (E) Different body shapes and different positions of the contractile vacuole (hollow circle) and the macronucleus (solid circle). (F) Optical section through cortical layer and somatic cilia. (G, H) Ventral (G) and dorsal (H) view of the holotype specimen, arrow indicates the preoral suture, arrowhead indicates the cytopyge and double arrowhead indicates the first somatic ciliary row. Scale bars, 10 μm.
Fig. 6.
Photomicrographs of Nassula paralabiata sp. nov. from life with bright field (A–K) and after protargol staining (L–O). L-O from Hu et al. (2019). (A, B) Ventral views of slightly compressed cells, arrow indicates the caudal cilia. (C) Lateral and view of the same individual as B, arrow indicates the pharyngeal basket. (D) Undernourished individual. (E) The golden granules. (F) Detail of the anterior portion of the dorsal side, showing the patch of golden granules. (G, H) Ejected (H) and resting (G) extrusomes. (I) Undigested orange rod-shaped algae. (J) Oral region showing the nassulid organelles (arrow). (K) Pharyngeal basket. (L) Lateral view showing the pharyngeal basket. (M) Detail of oral region showing the paroral membrane (arrow) and the lip-shaped cytoplasmic membranes (arrowhead). (N, O) Ventral (N) and dorsal (O) view of the holotype specimen, showing the ciliature. Scale bars, 15 μm (A–D) and 5 μm (N, O)
Table 3.
Morphometrics of Nassula paralabiata sp. nov. CV, coefficient of variation (%); M, median value; Max, maximum value; Mean, arithmetic mean; Min, minimum value; n, number of specimens; SD, standard deviation of the mean
| Charactera | Mean | M | SD | CV | Min | Max | n |
|---|---|---|---|---|---|---|---|
| Body, length | 41.9 | 43 | 3.92 | 9.4 | 34 | 49 | 17 |
| Body, width | 25.9 | 26 | 3.02 | 11.6 | 20 | 30 | 17 |
| Length: width, ratio | 1.6 | 1.6 | 0.12 | 7.4 | 1.5 | 2.0 | 17 |
| Anterior body end to macronucleus, distanceb | 19.5 | 19 | 2.57 | 13.2 | 16 | 26 | 17 |
| Anterior body end to excretory pore, distanceb | 33.2 | 33.5 | 2.37 | 7.1 | 28 | 37 | 14 |
| Anterior body end to distal end of nasse, distanceb | 20.1 | 20 | 2.38 | 11.9 | 16 | 25 | 15 |
| Anterior body end to macronucleus, % of body length | 46.5 | 46.7 | 0.04 | 8.5 | 39.1 | 54.8 | 17 |
| Anterior body end to excretory pore, % of body length | 72.2 | 77.8 | 0.20 | 27.1 | 71.4 | 83.3 | 14 |
| Anterior body end to distal end of nasse, % of body length | 48.2 | 48.9 | 0.05 | 9.4 | 39.5 | 55.6 | 15 |
| Macronucleus, length | 12.8 | 13 | 1.58 | 12.3 | 10 | 15 | 17 |
| Macronucleus, width | 11.8 | 12 | 1.35 | 11.5 | 10 | 14 | 17 |
| Nasse, largest diameter | 2.8 | 3 | 0.38 | 13.5 | 2 | 3 | 17 |
| Nasse, length | 15.5 | 15 | 1.71 | 11.0 | 13 | 18 | 15 |
| 1 st nassulid organelle, length | 2.8 | 3 | 0.55 | 19.7 | 2 | 4 | 17 |
| Last nassulid organelle, length | 1.3 | 1 | 0.46 | 35.2 | 1 | 2 | 17 |
| Nassulid organelles, number | 8.0 | 8 | 0.49 | 6.1 | 7 | 9 | 17 |
| Ventral kineties, number | 19.8 | 20 | 1.95 | 9.8 | 16 | 23 | 17 |
| Dorsal kineties, number | 15.6 | 15 | 2.03 | 13.0 | 12 | 20 | 15 |
| Somatic kineties, number | 35.5 | 37 | 2.50 | 7.0 | 31 | 39 | 15 |
| Paroral membrane, length | 12.7 | 12.5 | 1.03 | 8.1 | 11 | 15 | 12 |
aAll morphometrics from fixed specimens impregnated by protargol staining method
bMeasured from anterior end of cell to anterior margin of macronucleus
Syn. Nassula cf. labiata (Hu et al., 2019; pages 124 and 125)
Zoobank registration: urn:lsid:zoobank.org:act:E48CF08F-5388-47C3-AC0C-4512BCB12932
Remark: This species was mentioned as Nassula cf. labiata in the ciliate atlas by Hu et al. (2019), but has never been described yet.
Diagnosis: Cell size 32–60 × 20–32 μm in vivo; broadly ellipsoidal. Extrusomes fusiform, about 5 μm long. Usually 31–39 somatic kineties. Three slightly longer caudal cilia. Nassulid frange sigmoidal, extending behind level of oral opening terminating near left body margin, composed of on average eight organelles. Oral opening with two lip-shaped cytoplasmic membranes. Paroral membrane conspicuous, including about 14 dikinetids.
Type locality: Intertidal zone of the South China Sea coast near Daya Bay, Guangdong, China (22°46′27″N; 114°40′31″E). The salinity was 30‰ and the water temperature was 22°C.
Type specimens: A protargol slide (registration number: LMJ2016122501-1) containing the holotype specimen (Figs. 5G, H, 6N, O, circled in ink) has been deposited in the Laboratory of Protozoology, Ocean University of China, China. A second protargol slide (registration number: LMJ2016122501-2) with paratypes has been deposited in the Marine Biological Museum, Chinese Academy of Sciences, China.
Etymology: The species-group name paralabiata is a composite of Greek prefex para- (beside) and the species-group name labiata alluding to the similarity of the new species and Nassula labiata.
Description: Cell size about 32–60 × 20–32 μm in vivo (n = 6), 34–49 × 20–30 μm in protargol-stained specimens (Table 3; morphologic variations due to different cell reactions to protargol staining process). Length to width ratio 1.6–2:1 in vivo and 1.5–2:1 after protargol staining. Body shape broadly ellipsoidal (Figs. 5A and 6A, B, C). Macronucleus usually in the middle or posterior of cell, rounded to ellipsoidal, about 13 μm across in vivo. Contractile vacuole in the middle third of the right side of cell, slightly closer to the anterior part than macronucleus, surrounded by small contributory vesicles during diastole, about 5–7 μm in diameter (Fig. 5E). Cytopyge slit-like and in line with paroral membrane, near posterior body end, about 10 μm long. Cortex about 0.5 μm thick and bright, indistinctly separated from cytoplasm. Resting extrusomes attached almost perpendicularly to cortex, fusiform, about 4–5 μm, rather numerous forming more or less distinct fringe; ejected extrusomes also fusiform but up to 20 μm long and 1 μm wide (Figs. 5C, F and 6G, H). Undernourished cells colorless, golden and greasily shining granules about 0.5 μm-sized accumulating subapically on dorsal side forming two distinct, golden patches in anterior and posterior of cell (Figs. 5D). Well-fed cells orange and/or golden due to undigested orange rod-shaped algae and more golden granules scattered in cytoplasm (Figs. 6B, C, E, F). Locomotion by swimming while rotating about main body axis.
Somatic cilia about 6–7 μm long in vivo, arranged in 31–39 meridional and equidistantly spaced ciliary rows, except for more closely spaced postoral kineties, which end near mid-body; leaving blank an about 3 μm wide post-organellar stripe behind nassulid frange and forming distinct preoral suture (Fig. 5B). Ventral kineties (on average 20) slightly denser than dorsal kineties (on average 16). Nassulid frange begins behind oral area, extends sigmoidally across ventral side terminating left body margin, thus splitting somatic ciliary rows, composed of 7–9 nassulid organelles, the innermost organelle curved, others straight. Each nassulid organelle consists of three rows of basal bodies, each row composed of 4–7 basal bodies (Figs. 5B and 6J). Paroral membrane rather conspicuous, continuous with first somatic ciliary row, commences at level of oral opening and extends in flat bow to blank stripe underneath nassulid frange, composed of about 14 clockwise inclined dikinetids. Three caudal cilia slightly longer than somatic cilia, about 9–10 μm long (Figs. 5B, G and 6M).
Oral opening in anterior quarter of cell, with two lip-shaped cytoplasmic membranes distinct after protargol staining (Figs. 5B and 6M). Pharyngeal basket extends obliquely to dorsal side, terminating in mid-body region, funnel-shaped, about 15 μm long in vivo, composed of about 18 thin, straight, and comparatively narrowly spaced rods with shallow indentation anteriorly (Figs. 5D and 6C, K).
Sequence data and phylogenetic analyses
The three new SSU rRNA gene sequences were deposited in GenBank under the accession numbers, length and GC contents as follows: Aponassulides lacustris gen. nov., sp. nov. (1718 bp, 43.83%, PX281933), Naxella vorax sp. nov. (1687 bp, 43.63%, PX281934), Nassula paralabiata sp. nov. (1645 bp, 45.17%, PX281935). The maximum likelihood (ML) and Bayesian inference (BI) trees based on SSU rRNA gene sequences share similar topologies, and thus only the ML tree, with support values from both algorithms, is presented (Fig. 7). The class Nassophorea is divided into three highly or maximally supported clades, viz., Discotrichida, Microthoracida, and Nassulida + Colpodidiida. Within the latter clade, Nassulidae and Colpodidiidae are sister groups, with Furgasoniidae branching basally. The family Nassulidae consists of three robust clades in both ML and BI analyses. Clade I includes only Rhinakis faurei and Obertrumia georgiana. In Clade II, Aponassulides lacustris clusters with a subclade composing Naxella vorax, Naxella paralucida, and two uncultured eukaryotes (AB695449 and AB695498). Clade III contains a subclade consisting Nassula paralabiata and Nassula labiata (KC832949), which occupies a sister position to an unidentified Nassula form.
Fig. 7.
Maximum likelihood tree based on SSU rRNA gene sequences, showing the position of three newly sequenced species (red font) within the class Nassophorea. Numbers at nodes show ML bootstrap values and posterior probabilities (ML/BI). Black circles indicate full support (ML/BI, 100/1.00) in both analyses. Red asterisk indicates sequence KC832949, which was submitted to NCBI under the name Nassula labiata, a proposed synonym of Nassula paralabiata sp. nov. The scale bar corresponds to 5 substitutions per 100 nucleotide positions.
The putative secondary structures of the SSU rRNA gene V9 region of nine species (seven nassulids and two colpodidiids) were predicted and compared (Fig. 8). These taxa shared a similar secondary structure; the general model is formed by a long helix, with some median loops and a terminal loop. Within the family Nassulidae, variable nucleotide positions were confined to the 44–62 nt field, which comprises loops and a double-stranded region of helices. Aponassulides lacustris, Naxella vorax, Nassula paralabiata, and Apocolpodidium etoschense from the order Colpodidiida share similar secondary structure. Obertrumia georgiana, Rhinakis faurei, and Naxella paralucida possess another type of secondary structure, which differs from the former in an additional loop in variable nucleotide positions.
Fig. 8.
A putative secondary structure of V9 region of small subunit ribosomal RNA (SSU rRNA) gene of 9 representative species
Discussion
Establishment of the new genus
The large Qingdao species investigated in the present study undoubtedly belongs to the family Nassulidae, as it has the typical nassulid organelles composed of three ciliary rows, forming distinct nassulid frange extending leftwards and splitting somatic ciliary rows. Currently, only five genera are diagnosed within the Nassulidae (as mentioned above). The structure of the nassulid frange might be the best feature, at least at light microscopic level, to distinguish genera within Nassulidae [17]. However, this current form cannot be assigned to any known nassulid genus. It exhibits a distinct ciliary pattern compared to Nassula, Obertrumia, and Naxella-Rhinakis in the shape of nassulid frange (linear vs. sigmoidal in Nassula and bipartite in Obertrumia) and the number of nassulid organelles (on average 18 vs. 2–8 in Naxella-Rhinakis pattern). Furthermore, the most significant difference lies in the arrangement of the nassulid organelles, viz. nassulid organelles orientated obliquely to frange axis in the present form vs. orientated parallel to frange axis in genera Nassula, Obertrumia and Naxella-Rhinakis [17, 19]. This species resembles Nassulides in possessing numerous nassulid organelles orientated obliquely to frange axis. However, the former can be separated from the latter by the orientation of nassulid organelles (all diagonally along the top left and bottom right directions vs. partly along the top right and bottom left directions). Meanwhile, the nassulid organelles of Nassulides overlap proximally; the first organelle is slightly long and curved, while the others are short and straight. In contrast, the organelles in the current form are all straight, of irregular lengths, and exhibit no overlap [17]. Therefore, a new genus, Aponassulides gen. nov. has to be established for the present form.
Comparison of Aponassulides lacustris gen. nov., sp. nov. with related species
Based on the large size, nearly round body shape, and numberous nassulid organelles, three species should be compared with Aponassulides lacustris gen. nov., sp. nov. namely, Nassulides labiatus (Kahl,1933) Foissner et al., 2002, Nassulides pratensis (Czapik & Jordan, 1976) Foissner et al., 2002 and Nassula deroxi Njine, 1979 [17, 22–26]. The most significant difference among these four species are the orientation of nassulid organelles and the shape of nassulid frange (see above). Furthermore, Aponassulides lacustris can be distinguished from Nassulides pratensis by its body length (135–180 μm vs. 300–400 μm); and it can be separated from Nassula derouxi (which may need to be transferred to Nassulides according to the arrangement of nassulid organelles) by the body length (135–180 μm vs. 250 μm), the number of somatic kineties (100–132 vs. 200), and the paroral membrane (absent vs. present). Aponassulides lacustris and Nassulides labiatus are similar in the number of nassulid organelles (15–21 vs. 12–18) and the lack of paroral membrane. Nevertheless, the former differs from the latter mainly in habitat (freshwater vs. marine or brackish water), the slightly lower number of somatic kineties (100–132, on average 116 vs. 130–170, on average 146 in Portugal population and 129 in Namibia population), and the absence of (vs. presence) of lip-shaped cytoplasmic membranes [17, 23, 24].
Comparison of Naxella vorax sp. nov. with related species
Based on its fusiform extrusomes, conspicuous pharyngeal basket, linear nassulid frange composed of few nassulid organelles, and conspicuous paroral membrane, the small Qingdao species investigated here can be assigned to the genus Naxella [17]. So far, there are nine nominal species in the genus, namely, N. lateritia (Claparède & Lachmann, 1859) Fryd-Versavel et al., 1980, N. minuta Dragesco & Dragesco-Kernéis, 1986, N. australis Foissner & O’Donoghue, 1990, N. faurei (Fauré-Fremiet, 1967) Foissner & O’Donoghue, 1990, N. lucida (Reuter, 1961) Foissner et al., 2002, N. rosea (Tucolesco, 1962) Foissner et al., 2002, N. viridis (Dingfelder, 1962) Jankowski, 2007, N. latericollaris (Wang, 1930) Jankowski, 2007, and N. paralucida Liao et al., 2018.
Considering the three or four nassulid organelles, Naxella vorax can be clearly distinguished from its congeners with two (N. lucida and N. paralucida) or five to eight (N. minuta) nassulid organelles [17, 24, 27]. Naxella australis, N. faurei, and N. viridis all have an egg-shaped body with a moderately deep oral depression, which is rather different from Naxella vorax, which has a broadly ellipsoidal body shape [28–30]. Naxella lateritia has two contractile vacuoles while Naxella vorax has only one. The extrusomes are absent in Naxella latericollaris but present in Naxella vorax [18, 30–32]. Naxella rosea differs from N. vorax by having two kinds of fusiform extrusomes (vs. only one in Naxella vorax) [17, 33].
In addition to its congeners, four similar Nassula species, namely, Nassula gutturata Gajewskaja, 1927, Nassula pluvialis Dingfelder,1962, Nassula minima Minkiewicz, 1899 and Nassula pussilla Kahl, 1930, should also be compared with the new species in terms of their ellipsoidal body shape. Compared with Naxella vorax, Nassula pusilla has more (about 40 vs. 15–19) nematodesmal rods; Nassula pluvialis is significantly larger (190 μm vs. 80–95 μm); Nassula minima possesses two (vs. one) contractile vacuoles; and Nassula gutturata has dense annulus in the pharyngeal basket (vs. absent in Naxella vorax) [17, 29, 34].
Comparison of Nassula paralabiata sp. nov. with related species
Nassula cf. labiata sensu Hu et al., 2019 can be unambiguously classified into the genus Nassula considering its conspicuous pharyngeal basket and sigmoidal nassulid frange [17]. But it was identified as a new species rather than Nassulides labiatus (Kahl,1933) Foissner, 2002 (basionym: Nassula labiata) because they can be easily distinguished in vivo by their significant difference in body size (on average 130 × 100 μm in Nassulides labiatus vs. 40 × 25 μm in Nassula paralabiata) and the shape of nassulid frange (orientated obliquely and overlapping proximally in Nassulides labiatus vs. sigmoidal and orientated parallel in Nassula paralabiata). Besides, Nassula paralabiata also can be separated from Nassulides labiatus in the body shape (broadly ellipsoidal in Nassula paralabiata vs. rounded in Nassulides labiatus) and the presence of caudal cilia in Nassula paralabiata (vs. absence in Nassulides labiatus). However, they both live in marine habitat and have lip-shaped cytoplasmic membranes, which may led to the misidentification. In addition, a SSU rRNA gene sequence of so-called Nassula labiata isolate LWW09052701 (accession numbers: KC832949) was deposited in GenBank. But it was only used in phylogenetic analysis and no morphological information available justifies its identity [6]. This sequence is completely identical to the SSU rRNA gene sequence of Nassula paralabiata investigated in the present study. Besides, both samples were collected from the coast near Daya Bay. Therefore, we speculate that the isolate LWW09052701 should actually be identified as Nassula paralabiata rather than Nassula labiata, (now an invalid species).
Containing approximately 50 nominal species, Nassula is the most species-rich genus in the Nassulidae [17, 21, 29, 30, 34–39]. However, only four nominal species have been reported from marine habitat(Ozaki and Yagiu 1941), viz. Nassula caudata Ozaki & Yagiu, 1941, Nassula renalis Ozaki & Yagiu, 1941, Nassula terminalis Ozaki & Yagiu, 1941 and Nassula longissima Ozaki & Yagiu, 1941. Among them, only Nassula caudata closely resembles Nassula paralabiata in body size (60–70 × 21–25 μm in the former and 32–60 × 20–32 μm in the latter), body shape (long ellipsoid), the presence of fusiform extrusomes, and caudal cilia of similar length (11 μm in the former and 10 μm in the latter). Nevertheless, Nassula caudata differs from Nassula paralabiata in the length to width ratio (about 3:1 vs.1.6–2:1), the length of somatic cilia (3 μm vs. 6–7 μm), the shape of macronucleus (long ovoid vs. round), and the position of contractile vacuole (farther vs. closer to the anterior part than macronucleus). Moreover, no lip-shaped cytoplasmic membranes were observed in Nassula caudata [21].
Additionally, several morphologically similar species exist in both freshwater and terrestrial habitats, e.g. Nassula exigua Kahl, 1931, N. longinassa Foissner, 1980 and N. minima Minkiewicz, 1899. In addition to differences in habitat, Nassula exigua can also be separated from Nassula paralabiata by the absence of extrusomes (presence in the latter) and lower number of nassulid organelles (on average 4 in Nassula exigua vs. 8 in Nassula paralabiata). Nassula longinassa differs from Nassula paralabiata by the absence of caudal cilia (presence in the latter), lower number of somatic kineties (on average 28 in Nassula longinassa vs. 36 in Nassula paralabiata), and fewer nassulid organelles (on average 6 in Nassula longinassa vs. 8 in Nassula paralabiata). Though described on the basis of live observation only, Nassula minima possesses two contractile vacuoles, which represents a unique feature among the genus [34–36].
Phylogenetic analyses
68 sequences has been used for the phylogenetic analyses, which contains 37 recognizable species and 31 undetermined species. The final alignment used to construct the phylogenetic trees contained 1816 sites. In the current SSU rRNA gene tree, the class Nassophorea is non-monophyletic and could be divided into three separate clades, corresponding to orders Disoctrichida, Microthoracida and Nassulida + Colpodiida, which is consistent with previous studies [3–5, 8, 9].
Our analysis further supports the monophyly of the family Nassulidae. Notably, the newly established genus Aponassulides gen. nov. lacks a paroral membrane, a feature also inferred to be absent in Clade I [17]. The patchy distribution of the paroral membrane, viz., absent in Aponassulides and Clade I but present in other nassulids (Naxella spp., Furgasonia blochmanni and Nassula paralabiata) as well as members of Colpodidiida and Discotrichida, implies that this structure was likely plesiomorphic in Nassophorea [7, 8, 17, 27, 40]. Its absence in these two lineages is most parsimoniously explained by at least two independent evolutionary losses. When considering the habitat of the members of these three clades, it is noteworthy that two species identified to species in Clade III are both discovered in the marine habitat, which is rare within Nassulida, suggesting that Clade III may represent an independent marine evolutionary branch.
The putative secondary structure of V9 region of SSU rRNA gene of nine representative species also confirms the phylogenetic relationship in Nassulida + Colpodiida clade. Within Nassulidae, members of Clade II shares similar secondary structure except for Naxella paralucida, which resembles species in Clade I. Compared with Nassulidae, Apocolpodidium etoschense shares the same structure with Clade II, and Colpodidium caudatum also has a high similarity. Additionally, the secondary structure of Furgasonia blochmanni differs from other species obviously, which is consistent with its phylogenetic position as sister to all other Nasssulida.
Molecular data are presently available for only a handful of morphotypes (e.g., Rhinakis, Obertrumia, Aponassulides), while detailed morphological redescriptions using modern methods are lacking for the majority of nominal species [17, 21, 29, 30, 34–38]. Consequently, an extensive sampling of the nassulid genera is warranted to deduce and verify the character evolving process in this order.
Conclusions
In this study, we establish a new genus Aponassulides gen. nov. and described three novel ciliates, viz. Aponassulides lacustris gen. nov., sp. nov., Naxella vorax sp. nov. and Nassula paralabiata sp. nov. The distinct genus and species is supported by combined evidence from morphology and ribosomal gene sequences. The absence of the paroral membrane in Aponassulides and Rhinakis-Obertrumia clade suggests that the paroral membrane was present in the most recent common ancestor of the class Nassophorea and was independently lost at least twice. Furthermore, only six species within the family Nassulidae have reliable molecular data. This study contributes to our understanding of biodiversity and enriches the database of nassulids. Further efforts should focus on discovering more species to better elucidate the biodiversity and systematics of this group.
Methods
Sample collection and morphological studies
Aponassulides lacustris gen. nov., sp. nov. and Naxella vorax sp. nov. was collected from a freshwater pond in Shaohai Wetland, Qingdao, China (36°14′58″N; 120°5′3″E) on 30th June, 2023 where the water temperature was 29 ℃ at the time of sampling (Fig. 9A, B). Nassula paralabiata sp. nov. was collected from the intertidal zone of the South China Sea coast near Daya Bay, Huizhou, China (22°46′27″N; 114°40′31″E) on 25th December 2016, when the salinity was 30‰ and the water temperature 22 ℃ (Fig. 9A, C). Approximately 200 ml water samples were collected in clean plastic bottles and transported to the laboratory immediately for further processing. Despite several attempts, a thriving pure culture could not be established. Consequently, cells were isolated and studied directly from the raw samples.
Fig. 9.
Maps showing the location and photographs showing the appearance of the sampling sites. (A) location of Qingdao, Huizhou and sampling sites. (B) Photograph of a freshwater lake in Shaohai Wetland where Aponassulides lacustris gen. nov., sp. nov. and Naxella vorax sp. nov. were collected. (C) Photograph of intertidal area in Daya Bay where Nassula paralabiata sp. nov. was collected. S, salinity; T, temperature.
Living cells were observed using Zeiss (AXIO Imager D2) and Olympus (BX53) microscopes equipped with bright field and differential interference contrast illumination. The ciliary pattern was revealed using the protargol staining method [8, 41] and the Chatton–Lwoff silver nitrate staining method [3, 42]. The macronucleus and micronucleus were detected using fluorescence staining according to Wang et al. (2023) [43]. Measurements and counts were performed at 1000× magnification. Drawings of live specimens were based on free-hand sketches and micrographs, while those of stained specimens were made with the aid of a drawing device. Terminology is according to Foissner et al. (2002) and Lynn (2008) [1, 17].
DNA extraction and gene sequencing
Genomic DNA extraction and PCR amplification were conducted using the methods described in Feng et al. (2024) [44]. The SSU rRNA genes of Aponassulides lacustris and Naxella vorax were amplified using the primers Euk A (5′-AAC CTG GTT GAT CCT GCC AGT-3′) and 5.8SR (5′-TAC TGA TAT GCT TAA GTT CAG CGG-3′). The SSU rRNA gene of Nassula paralabiata was amplified using the primers 82 F (5′-GAA ACT GCG AAT GGC TC-3′) and 18SR (5′-GTA GGT GAA CCT GCA GAA GGA TCA-3′) [45, 46]. After amplification, PCR products were bidirectionally sequenced by the Tsingke Biological Technology Company (Qingdao, China).
Phylogenetic analyses
In addition to these three new sequences, 68 sequences were retrieved from the GenBank database for the phylogenetic analyses. Four gene sequences belonging to the class Litostomatea (accession numbers: EF123708, U57765, U57767, and KC493570) were selected as the outgroup taxa. MAFFT version 7 algorithm was used to align all sequences (https://mafft.cbrc.jp/alignment/server/). The resulting alignment was refined using BioEdit 7.0.5.2 [47], and both ends were trimmed manually. The final alignment used to construct the phylogenetic trees contained 1816 sites.
Maximum likelihood (ML) analysis was performed using IQ-TREE 2.0 with 10,000 ultrafast bootstrap replicates. Bayesian inference (BI) analysis was performed using MrBayes on XSEDE 3.2.6 [48] available on CIPRES Science Gateway. GTR ་＋ I ＋ G was selected as the best-fit model using MrModeltest 2 [49] under the Akaike Information Criterion. Markov chain Monte Carlo simulations were run for 10,000,000 generations at a sampling frequency of 100 and a burn-in of 25,000 trees. All remaining trees were used to calculate posterior probabilities according to the majority consensus rule. Tree topologies were visualized using Seaview 4.0 [50].
The secondary structures of the partial V9 region of the 18S rRNA molecule of some species in Nassulida + Colpodiida clade were predicted using the online application Mfold ver. 3.0 (http://www.unafold.org/) [51]. To facilitate comparison, partial corresponding primary sequences (about 102 bp) were used to form the V9 regions. The V9 region of nassulids and colpodidiids from a previous study [52] was considered to adjust the current secondary structures. To investigate inter-species variability, their 2D layouts were visualized with Varna ver. 3.93 [53].
Supplementary Information
Authors’ contributions
XH conceived and guided the study. QS and ML carried out the live observation and protargol and silver nitrate impregnation. QS and LJ did DNA extraction and PCR amplification and sequencing, and the molecular phylogenetic analyses. QS drafted the manuscript, and LJ and XH made further revisions. All authors read and approved this manuscript.
Funding
This work was supported by the National Natural Science Foundation of China (project number: 42276094), and the China Postdoctoral Science Foundation Grant (project number: 2025M772631).
Data availability
Sequence data are available in GenBank (Accession numbers: *Aponassulides lacustris* : PX281933; *Naxella vorax* : PX281934; *Nassula paralabiata* : PX281935). Three permanent slides (registration number: *Aponassulides lacustris* : SQH2023063001-1; *Naxella vorax* : SQH2023063002-1; *Nassula paralabiata* : LMJ2016122501-1) containing the holotype specimen, circled with black ink on the back of the slide have been deposited in the Laboratory of Protozoology, Ocean University of China, China. Three paratype slides (registration number: *Aponassulides lacustris* : SQH2023063001-2; *Naxella vorax* : SQH2023063002-2; *Nassula paralabiata* : LMJ2016122501-2) has been deposited in the Marine Biological Museum, Chinese Academy of Sciences, Qingdao, China.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Limin Jiang, Email: liminjiang@ouc.edu.cn.
Xiaozhong Hu, Email: xiaozhonghu@ouc.edu.cn.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Sequence data are available in GenBank (Accession numbers: *Aponassulides lacustris* : PX281933; *Naxella vorax* : PX281934; *Nassula paralabiata* : PX281935). Three permanent slides (registration number: *Aponassulides lacustris* : SQH2023063001-1; *Naxella vorax* : SQH2023063002-1; *Nassula paralabiata* : LMJ2016122501-1) containing the holotype specimen, circled with black ink on the back of the slide have been deposited in the Laboratory of Protozoology, Ocean University of China, China. Three paratype slides (registration number: *Aponassulides lacustris* : SQH2023063001-2; *Naxella vorax* : SQH2023063002-2; *Nassula paralabiata* : LMJ2016122501-2) has been deposited in the Marine Biological Museum, Chinese Academy of Sciences, Qingdao, China.