Morphology and molecular phylogeny of two freshwater ciliates, with notes on a new species and redescription of a known species (Ciliophora, Oligohymenophorea)

Abstract

The class Oligohymenophorea comprises a diverse array of ciliates, however, the diversity and phylogeny of freshwater taxa, particularly from inland waters, remain inadequately explored. Therefore, we investigated two freshwater ciliates from Harbin, China, one of which was identified as a new species, Cyclidium orientale n. sp. The new species is characterized by a small body size (20–25 × 10–12 μm in vivo), a buccal field occupying approximately 80% of the body length, 10 somatic kineties, and a scutica composed of eight kinetosomes arranged in two groups. A redescription of the known species Lembadion bullinum is also provided, along with notes on its novel epibiotic lifestyle on the fish Pelteobagrus fulvidraco. Phylogenetic analyses based on SSU rDNA sequences revealed that Cyclidium orientale clusters with Cyclidium marinum, while L. bullinum forms a fully supported clade with its congeners, thereby corroborating their morphological identification.

Introduction

Ciliates are among the most complex and diverse microbial eukaryotes, inhabiting a wide range of environments including seawater, freshwater, and soil [37][69]. They play crucial roles in ecosystems, functioning as key components in nutrient cycling and energy flow [4, 26, 29, 32–34, 37, 54, 57, 58, 71, 78–80, 83]. Due to their tiny size, high abundance, and sensitivity to environmental changes, ciliates are considered reliable bioindicators [84]. Furthermore, they hold significant research value in aquatic ecology, biodiversity studies, and fisheries science [23, 55, 64]. Importantly, with the advancement of genomics, ciliates have emerged as key model systems for exploring fundamental eukaryotic biology [28, 82, 86].

The class Oligohymenophorea de Puytorac et al. 1974 is globally distributed and exhibits considerable morphological and biological diversity [10, 15, 30, 37, 70]. Recent studies suggest that the species richness of this group has historically been underestimated [7, 35, 51–53, 60]. Over the past decade, molecular phylogenetic approaches, particularly those utilizing SSU rDNA sequences, have been widely used to elucidate evolutionary relationships within this class [14, 16–20, 43, 44, 61–63, 73, 81, 85]. Faunistic surveys conducted in Chinese coastal waters have provided valuable data for the discovery of new or rare oligohymenophorean species [12, 13, 26, 38–40, 45, 53, 55, 74–76]. However, compared with the substantial discoveries in marine habitats, the diversity and phylogeny of oligohymenophorean ciliates in freshwater environments, particularly inland waters, have been far less explored and remain inadequately documented [35, 51–53, 74, 75]

The genus Cyclidium, originally described by Müller in 1773 with the type species C. glaucoma [37, 48]. Historically, species identification within this genus relied predominantly on morphological characteristics observed either in vivo or after silver staining. However, the minute size and considerable morphological diversity within the genus presents significant challenges for morphological-based identification. This has frequently led to misidentification, where convergent phenotypes from different lineages were erroneously lumped into the same taxon [15, 41, 65]. Following the revised generic diagnosis by Pan X. et al. [53], a clear framework for precise species delimitation has been established, which enhances the overall taxonomic precision of this genus. To date, over 40 nominal species have been reported from different habitats [1, 2, 5, 8, 22, 53, 55, 66]. However, many nominal species remain inadequately described and/or lack molecular data. Consequently, an integrative approach combining classical morphology and molecular techniques is essential to charify the phylogenetic relationships within this genus and the broader, complex class Oligohymenophorea [21] [88].

The genus Lembadion was established by Perty [56], while Bursaria bullina [47] was transferred into it and designated as the type species. A major taxonomic revision was undertaken by Kahl [30], who consolidated several previously misidentified species into Lembadion, including Thurophora lucens sensu [42] and the Hymenostoma magnum sensu [72]. Lembadion exhibits a small to medium size, oval shape, holotrichous somatic ciliation, and a conspicuously large oral apparatus that makes it easily distinguishable from other ciliates [6, 37]. Currently, the genus comprises seven nominal species [9, 11, 15, 35, 50].

In this study, a novel species, Cyclidium orientale n. sp., isolated from the Runheng Aquatic Products Market in Harbin, China, was described, along with a redescription of the known species Lembadion bullinum [47] Perty, [56] that incorporates new findings on its epibiotic lifestyle. Phylogenetic analyses based on SSU rRNA gene sequences were inferred to determine the phylogenetic positions of both species.

Material and method

Sample collection, morphological methods, and identification

Cyclidium orientale n. sp. and Lembadion bullinum [47] Perty, [56] were isolated from the skin of Pelteobagrus fulvidraco. The host fish was obtained from the Runheng Aquatic Products Market, Hulan District, Harbin, Heilongjiang Province, China (45°50′31.0" N, 126°30′56.6" E). Following isolation, clonal cultures were established and maintained at room temperature (approximately 24 °C) in Petri dishes containing filtered habitat water, with sterilized fish tissue provided as a food source.

The species were observed and photographed in vivo via differential interference contrast microscopy (Zeiss Axio Imager A2, Germany). The infraciliature and nuclear apparatus of Cyclidium orientale n. sp. were revealed using the protargol staining method [77], while those of Lembadion bullinum were examined with the silver carbonate staining method [38]. Observations and measurements were carried out in vivo at 40–1,000 × magnification. For stained cells, measurements and counts were performed at 1,000 × magnification, and drawings were produced by employing a camera lucida [52], while freehand sketches and photomicrographs served as the basis for drawings of live cells. The classification and terminology were based on Lynn [37].

DNA extraction, PCR amplification and sequencing

Approximately three to five cells from clonal cultures of each species were repeatedly washed with distilled water to remove contaminants. Genomic DNA was subsequently extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer's instructions.

The SSU rDNA of Lembadion bullinum was amplified using primers 82 F (5′–GAA ACT GCG AAT GGC TC–3′) and 18S-R (5′–TGA TCC TTC TGC ACG TTC ACC TAC–3′) [90, 91]. For Cyclidium orientale n. sp., amplification was performed with primers Euk A (5′–AAC CTG GTT GAT CCT GCC AGT–3′) and Euk B (5′–TGA TCC TTC TGC AGG TTC ACC TAC–3′) [91] .The PCR conditions were as follows: initial denaturation at 94 °C for 5 min; 5 cycles of 94 °C for 30 s, 55 °C for 105 s, and 72 °C for 120 s; 30 cycles of 94 °C for 45 s, 58 °C for 105 s, and 72 °C for 120 s; and a final extension at 72 °C for 8 min. Bidirectional Sanger sequencing was performed by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) via an ABI PRISM 3730xl Genetic Analyzer (Applied Biosystems, USA).

Phylogenetic analyses

Phylogenetic analyses were conducted based on newly obtained SSU rDNA sequences. The sequence of Cyclidium orientale n. sp. was aligned with 40 congeners and related taxa obtained from GenBank, while that of Lembadion bullinum was aligned alongside 72 such sequences. The accession numbers were provided after the species names in the phylogenetic trees. SSU rDNA sequences were aligned with the ClustalW algorithm integrated in BioEdit 7.0.1 [25], with ambiguous regions manually adjusted. Phylogenetic analyses were conducted via maximum likelihood (ML) and Bayesian inference (BI) methods. The ML trees were constructed with RAxML-HPC2 v8.2.12 (Stamatakis, 2014), and BI trees were performed with MrBayes v3.2.7 [59] via the CIPRES Science Gateway [46]. The best-fit nucleotide substitution model, GTR + I + G, were selected by the Akaike information criterion (AIC) in MrModeltest v2.2 [49]. For ML, rapid bootstrap analysis with 1,000 replicates were applied. For BI, four Markov chains were run for 10 million generations, sampling every 10,000 generations, with a 25% burn-in. Nodal support was interpreted as follows: ML bootstrap values < 70% or BI posterior probabilities < 0.94 were considered low; 70–94% (ML) or 0.94 (BI) as moderate; and ≥ 95% (ML) or ≥ 0.95 (BI) as high [3, 27]. The final trees were visualized via MEGA 7.0 [31].

Results

Class Oligohymenophorea de Puytorac et al., 1974.

Subclass Scuticociliatia Small, 1967.

Order Pleuronematida Fauré-Fremiet in Corliss, 1956.

Family Cyclididae Ehrenberg, 1838.

Genus Cyclidium Müller, 1773.

Cyclidium orientale n. sp. (Fig. 1 and Table 1)

Fig. 1.

Cyclidium orientale n. sp. in vivo (A–D, F–I) and after protargol staining (E, J–Q). A, B Ventral views of a representative individual, to demonstrate general body shape, arrow in B marks caudal cilium; (C, D, F–I) Lateral views of different individuals, showing the caudal cilium (arrows in C, D, H), paroral membrane (double-arrowhead in C, I), contractile vacuole (arrowheads in C, F, G), macronucleus (arrowhead in D), and somatic cilia (arrow in G); (E, K) Different shapes of macronucleus; (L) Infraciliature in ventral view of the holotype specimen; (J, Q) Showing the somatic kinety, arrowhead marks dikinetids and arrow marks monokinetids; (M, N) Showing the details of scutica; (O, P) Oral ciliature, to show the membranelles 1–3. Cc: caudal cilium; M1–3: membranelles 1–3; Ma: macronucleus; PM: paroral membrane; Sc: scutica. Scale bars: 5 μm (K), 10 μm (A, O), 20 μm (B–D, F–J, L)

Table 1.

Morphological data of Cyclidium orientale n. sp. based on protargol-stained specimens, measurements in μm. Abbreviations: CV, coefficient of variation (%); M, median; Max, maximum; Mean, arithmetic mean; Min, minimum; n, number of samples examined; SD, standard deviation

Character	Min	Max	Mean	M	SD	CV	n
Body length	35.5	54.5	43.2	42.4	5.4	12.7	20
Body width	23.4	47.5	33.9	34.8	5.9	17.0	20
Buccal field, length	24.6	37.8	29.8	29.2	3.8	13.1	20
Buccal field, width	6.6	15.6	11.2	10.9	2.8	25.2	19
Somatic kineties, number	10	10	10.0	10	0.0	0.0	11
Number of longitudinal rows in membranelle 1	2	2	2.0	2	0.0	0.0	11
Macronucleus length	4.3	6.8	5.6	5.4	0.8	14.8	19
Macronucleus width	3.3	5.3	4.3	4.3	0.4	10.1	22
Macronucleus, number	1	2	1.6	2.0	0.5	24.4	18

Diagnosis

Body about 20–25 × 10–12 µm in vivo. Buccal field about 80% of body length; 10 somatic kinetids; somatic kinety n (SK_n) consisting of 10 or 11 kineties. One or two macronuclei. Membranelle 1 (M1) composed of two longitudinal rows of monokinetids; membranelle 2 (M2) triangle-shaped; scutica comprises eight kinetosomes divided into two groups. Freshwater habitat.

Type locality

Skin, internal organs, and gills of Pelteobagrus fulvidraco.

Holotype

A slide containing the holotype specimen, marked with an ink circle, was deposited in the Key Laboratory of Biodiversity of Aquatic Organisms, Harbin Normal University (slide number: PMM–20191012–01).

Paratypes

CHINA – Heilongjiang Province; same collection data as holotype; PMM–20191012–02.

Etymology

“Orientale” means “of the East,” which refers to its discovery in Heilongjiang Province, Northeastern China.

ZooBank registration

urn:lsid:zoobank.org:pub:59cB0c40-708B-42C0-95CE-EDDAC346C5D2.

Description

Body size 20–25 × 10–12 µm in vivo, rod-shaped with a tapered anterior end and a slightly rounded posterior end, featuring a truncation zone (Fig. 1A, B). Buccal field about 80% of body length, buccal area about 12 µm long (Fig. 1A–D). One or two macronuclei, approximately 5.6 µm in diameter (Fig. 1K, O, P); micronucleus not observed. Cytoplasm colourless, containing several to many large food vacuoles (approximately 2 µm in diameter) and refringent granules (Fig. 1A–D). Contractile vacuole located near posterior half of the cell, approximately 3 µm in diameter (Fig. 1A–C, F, G), single caudal cilium about 20 µm long (Fig. 1A–D, F-I). Somatic cilia about 20 µm long. Locomotion by swimming fast.

Ten somatic kineties extend from the anterior to the posterior end, with approximately the anterior 40% composed of dikinetids and the remainder of monokinetids (Fig. 1E, J, L, Q). Somatic kinety n (SK_n) comprises 10 or 11 kinetids (Fig. 1E, J, L). Paroral membrane L- shaped, with two monokinetids rows (Fig. 1E, J, L, M, P). M1 has two longitudinal rows of monokinetids; M2 triangle-shaped, with four longitudinal and six horizontal rows (Fig. 1L, O); Membranelle 3 consists of four longitudinal rows of kinetids (Fig. 1E, J, L, O). Scutica composed of eight kinetosomes arranged in two groups (Fig. 1E, L, M, N).

Phylogenetic position of Cyclidium orientale n. sp. (Fig. 2)

Fig. 2.

Maximum likelihood tree based on SSU rDNA sequences, showing the position of Cyclidium orientale n. sp. (highlighted in bold). The numbers at nodes represent the bootstrap values of ML out of 1,000 replicates and the posterior probability of BI. Fully supported (100%/1.00) branches are marked with solid circles. * Indicates the disagreement in topology between ML and BI trees. The scale bar corresponds to five substitutions per 100 nucleotide positions

The SSU rDNA sequence of Cyclidium orientale n. sp. has been deposited in the GenBank database with the following accession numbers, length, and DNA G + C contents: MT001894, 1748 bp, and 44.22%. The ML and BI trees presented nearly identical topologies; thus, only the ML tree is presented here with support values from both methods (Fig. 2). In the phylogenetic tree, Cyclidium orientale n. sp. forms a clade with C. marinum, albeit with low support (68%ML, 0.77BI). Cyclidium varibonneti branches outside this clade (85%ML, 1.00BI). Subsequently, this clade was robustly placed as sister to a clade comprising Ancistrum spp., Boveria subcylindrica, and Protocyclidium spp. (Fig. 2).

Class Oligohymenophorea de Puytorac et al., 1974.

Subclass Peniculia Fauré–Fremiet in Corliss, 1956.

Order Peniculida Fauré–Fremiet in Corliss, 1956.

Family Lembadionidae Jankowski in Corliss, 1979.

Genus Lembadion Perty, 1849.

Lembadion bullinum [47] Perty [56] (Fig. 3 and Table 2)

Fig. 3.

Lembadion bullinum from life (A–G) and after silver carbonate impregnation (H–M). A, B Ventral views, arrows indicate the buccal area, arrowhead marks the food vacuole; (C–E) Lateral and ventral views of typical individuals, arrowhead indicates the buccal area; (F) Dorsal view, double arrowhead indicates the network of rectangles; (G) Ventral side, showing the cytoplasm, arrowhead indicates the food vacuole and arrow marks the crystal. H–M Dorsal (H, J, K) and ventral (I, L, M) views of stained cells, showing the infraciliature and nuclear apparatus, arrows in H, J, K indicate somatic kineties, double arrowhead in I marks the caudal cilia. Ma: macronucleus; Mi: micronucleus. Scale bars: 70 μm (A, G), 100 μm (J)

Table 2.

Morphometric data of the Harbin population of Lembadion bullinum [47] Perty, [56] on the basis of silver-stained samples, measurements in μm. Abbreviations: CV, coefficient of variation (%); M, median; Max, maximum; Mean, arithmetic mean; Min, minimum; n, number of samples examined; SD, standard deviation

Character	Min	Max	Mean	M	SD	CV	n
Body length	200.3	281.8	249.9	248.9	19.7	7.9	19
Body width	141.6	244.2	182.7	178.6	26.8	15.0	15
Buccal field, length	88.8	162.0	123.8	122.5	22.0	18.0	15
Buccal field, width	48.2	117.1	84.6	81.9	18.9	23.1	15
Somatic kineties, number	55	76	63.0	62	7.6	12.3	11
Macronucleus length	122.0	217.5	180.8	175.4	24.7	14.1	14
Macronucleus width	59.5	157.2	116.3	113.4	25.8	22.8	14

Improved diagnosis

Body size 96–200 × 60–106 µm in vivo. Outline circular to elongated oval. Buccal apparatus large and broad, occupying about 80% of body length. Single contractile vacuole located on the right margin. Fifty to seventy-six somatic kineties; one macronucleus, kidney-formed or elongated oval. Approximately 10–16 caudal cilia; double arrowhead; freshwater habitat.

Deposition of voucher slides

The slide containing the specimen marked with an ink circle has been deposited in the Key Laboratory of Biodiversity of Aquatic Organisms, Harbin Normal University (slide number: PMM–20180416).

Morphological description of the Harbin population

Body size about 120–170 × 60–106 µm in vivo, outline elongated oval in ventral view, posterior end obtusely rounded, anterior end gradually narrowed; body shape similar to pear fruit, length: width is about 2:1 (Fig. 3A–E). Buccal field large and wide, occupied about 90% of body length. Buccal cilia well developed (Fig. 3A–E). Cytoplasm contains numerous crystal and food vacuoles (Fig. 3G). Somatic cilia short, with densely packed cross-striated roots (Fig. 3F). Contractile vacuole on right body margin. Locomotion by slow forward swimming along the longitudinal axis. Freshwater habitat.

Somatic kineties 55–76, each somatic kineties composed of dikinetids in middle portion and monokinetids at both ends (Fig. 3H–M). About 38 somatic kineties observable dorsally; four to six dikinetids in middle somatic kinety, and the number of dikinetids gradually increasing from middle somatic kineties, in both left and right directions, to about 8–12 dikinetids (Fig. 3H, J, K). Single macronucleus kidney-shaped or elongated oval (Fig. 3H, K, M). Ten to twelve basal bodies of caudal cilium arranged into a single row, distributed at posterior end of the cell (Fig. 3I).

Buccal apparatus typical of genus, containing two paroral membranes (PMs) and one adoral membranelle (AM) (Fig. 3I, L, M). Adoral membranelle composed of seven rows of densely packed basal bodies (Fig. 3I, L, M). Inner three rows (excluding SK_n) almost identical in length while outer rows progressively shorter. A small barren area located posterior to the buccal apparatus, between SK_n and SK₁ (Fig. 3I, L).

Phylogenetic position of Lembadion bullinum (Fig. 4)

Fig. 4.

Maximum likelihood tree based on SSU rDNA sequences, showing the position of Lembadion bullinum (highlighted in bold). Numbers at nodes represent the bootstrap values of ML out of 1,000 replicates and the posterior probability of BI. Fully supported (100%/1.00) branches are marked with solid circles. * Indicates the disagreement in topology between the ML and BI trees. Scale bar represents 5 substitutions per 100 nucleotide positions

The SSU rDNA sequence of Lembadion bullinum has been deposited in the GenBank database with the accession number, length, and DNA G + C content as follows: MT010296, 1742 bp, 44.31%. The topologies of the SSU rDNA trees reconstructed using BI and ML analyses were almost identical; therefore, only the ML tree is presented here with support values from both algorithms (Fig. 4). The present study reveals that the subclass Peniculia is nonmonophyletic, as Lembadiondae groups with a clade comprising Stokesiidae, Frontoniidae, and Parameciidae. Despite the addition of the new species, the family Lembadiondae is still monophyletic. Within the genus Lembadion, two population of L. bullinum form a weakly supported sister clade (46% ML, 0.66 BI), L. lucens (MF072398) and Lembadion sp. (KM222113) groups outside of them with full support (100% ML, 1.00 BI).

Discussions

Comparison of Cyclidium orientale n. sp. with congeners (Fig. 5)

Fig. 5.

Various Cyclidium species in vivo (A, D, J, M) and after silver nitrate (F, G) and protargol staining (B, C, E, H, I, K, L, N, O). A–C Cyclidium vorax (from [55]); (D, E) C. orientale n. sp. (present study); (F–I) C. glaucoma; (J–L) C. sinicum (from [53]); (M–O) C. varibonneti (from [65]. Scale bars: 5 μm (H), 10 μm (D, F–G, I–L, O), 15 μm (M, N), 20 μm (A–C, E)

Key morphological characteristics for species discrimination in Cyclidium including body size, relative buccal field length, infraciliature (especially the structure of membranelles 1–3 and scutica), number of somatic kineties and macronucleus, termination pattern of posterior kineties, and habitat [15, 53, 68]. Based on the structure of the scutica and membranelles M1–M2, Cyclidium orientale n. sp. can be distinguished from most congeners. Following these criteria, Cyclidium orientale n. sp. should be compared with four morphologically similar species: C. glaucoma [47], C. sinicum [53], C. varibonneti [65], and C. vorax [55](Fig. 5).

Cyclidium orientale n. sp. differs from C. vorax in its smaller body size (20–25 × 10–12 µm vs. 35–40 × 18–20 µm in C. vorax), relatively longer buccal field (80% vs. 50% in C. vorax), and M3 composed of three rows of kinetids (vs. two rows in C. vorax) ([55]; Fig. 5A–C).

Cyclidium orientale n. sp. can be separated from C. glaucoma by its larger size (20–25 × 10–12 µm in C. orientale vs. 12–16 × 7–11 µm), more macronucleus (one or two in C. orientale vs. one), relatively longer caudal cilium (approximately body length vs. 2/3 body length), and freshwater habitat (vs. marine) ([66, 68], Fig. 5F–I).

Cyclidium orientale n. sp. can be clearly distinguished from C. sinicum by the number of somatic kineties (10 in C. orientale vs. 11 in C. sinicum) and the relative caudal cilium length (100% vs. 50% of body length) ([53]; Fig. 5J–L).

Cyclidium orientale n. sp. resembles C. varibonneti in its relative buccal length and the appearance of the buccal apparatus. However, they differ in the number of macronucleus (one or two in C. orientale vs. one) and habitat (freshwater in C. orientale vs. marine) ([65, 67]; Fig. 5M–O).

Comparison of the Harbin population of Lembadion bullinum with congeners (Table 3)

Table 3.

Morphological comparison of the Harbin population of Lembadion bullinum with similar congeners

Species	Body length in vivo, μm	Body shape	Somatic kineties, number	Caudal cilia number/length, μm	Macronucleus	Reference
L. bullinum	120–170	oval to elliptical	55–76	10–12/-	kidney-shaped/elongated oval	Present study
L. bullinum	120–200 (usually 140)	shuttle-shaped	50–60, each with diki netids anteriorly	17 in two rows/cilia 40–50 μm long	kidney-formed	[15]
L. gabonensis	100–120	shuttle-shaped	about 50	-/-	elongated	[9]
L. magnum	100–200 (usually 100–130)	shuttle-shaped	45–60, each with monokinetids anteriorly	approximately 20 in number arranged into two rows/-	kidney-formed	[15]
L. lucens	45–75	oval to elliptical	25–35, with monokinetids at both ends	7–10 kinetids in two rows/about 20–30	kidney-formed	[15, 35, 87]
L. planus	128–183 × 85–102	diamond shaped, with tapered front and rear	63–70	two rows, 5–7 dorsally and 15–18 ventrally/about 17–18	elongated oval	[50]
L. curvatum	75–125	twisted anteriorly; body tapers from the oral to the posterior end of the cell	42–45	12–17 basal bodies arranged into two groups/-	elliptical	[11]
L. bullinum arenicola	about 125	shuttle-shaped	52 on average	5–7 caudal cilia/about 55 μm in length	elliptical	[11]

We compared the Harbin population of L. bullinum with six congeners based on these characteristics (Table 3). The Harbin population generally corresponds well with the original description of L. bullinum [47] Perty, [56], however, two morphological discrepancies were noted: the Harbin population possesses a single row of 10–12 caudal ciliary kinetids, whereas the original description reported two rows of 17 caudal cilia, the body shape also differs slightly (oval to elliptical vs. shuttle-shaped), In terms of other key characteristics, that is, the body size, the number of somatic kineties, and the shape of macronuclear, the Harbin population corresponds very well with known populations of L. bullinum. Therefore, we consider that the Harbin population is conspecific with previous populations [10, 15, 24].

Lembadion bullinum is much larger than L. gabonensis (with a body length of about 120–170 µm vs. 100–120 µm) and exhibits an oval to elliptical body shape (vs. shuttle-shaped). Moreover, L. bullinum possesses 10–12 caudal cilia, whereas L. gabonensis was described as lacking them [9].

Lembadion bullinum closely resembles L. magnum in body shape, number and composition of somatic kineties, and body size in vivo. However, it differs in the shape of the macronucleus (kidney-shaped or elongated oval-shaped in L. bullinum vs. kidney-shaped in L. magnum) and the number and arrangement of caudal cilia (10–12 kinetids in one row vs. approximately 20 in number arranged into two rows) [10, 15, 24, 42].

Lembadion bullinum differs from L. lucens in the number of caudal cilia (10–12 kinetids in one row vs. 7–10 kinetids in two rows in L. lucens). In addition, L. bullinum is larger (with a body length of about 120–170 µm vs. 45–75 µm in L. lucens) and possesses more SK_S (55–76 vs. 25–35 in L. lucens) [9, 10, 15, 24, 42].

Lembadion planus [50] differs from L. bullinum in body shape (diamond shaped, with tapered front and rear vs. kidney-formed or elongated oval). It also possesses a generally higher number of SK_S (63–70 vs. 55–76) and a greater number of caudal cilia, which are notably short (five to seven dikinetids on the dorsal side and 15–18 on the ventral side vs. 10–12 kinetids in one row in L. bullinum) [10, 15, 24, 42, 50].

Lembadion curvatum [11] can be easily distinguished from L. bullinum by its body shape, which is widened and twisted anteriorly, slender behind the oral region, and tapered toward the posterior end of the cell, forming a sinusoidal shape in outline (vs. oval to elliptical in L. bullinum). In addition, L. curvatum possesses more caudal cilia (about 12–17 basal bodies arranged into two groups vs. 10–12 kinetids in one row in L. bullinum) [10, 11, 15, 24, 42].

Lembadion arenicola [9] can be distinguished from L. bullinum by its body shape (shuttle-shaped vs. oval to elliptical in L. bullinum) and number of caudal cilia (five to seven vs. 10–12 in L. bullinum) [89, 10, 15, 24, 42].

Phylogenetic analyses

Phylogenetic analyses of Cyclidium orientale (Fig. 2)

The newly obtained SSU-rDNA sequence of C. orientale n. sp. exhibits the highest similarity to that of C. marinum (KY886367), with a molecular similarity of approximately 99.31%. Nevertheless, 12 nucleotide differences were detected between them, supporting the validity of C. orientale as a distinct species. Phylogenetic analysis revealed that the novel species, C. orientale n. sp., clustered with C. varibonneti and C. marinum with moderate support (85%ML, 1.00BI), which corroborates its generic assignment based on morphological characteristics. This clade is sister to a clade comprising Protocyclidium and four Thigmotrichide species (Ancistrum sp., Ancistrum crassum, and Boveria subcylindricaii). In contrast, other members of the genus Cyclidium, namely C. vorax (MNS24102), C. glaucoma (EU032356), and Cyclidium sp. (KX853100), formed a separate, distinct clade. This topology confirms the non-monophyly of the genus Cyclidium, as reported in previous studies [36, 53, 55]. Furthermore, the phylogenetic tree revealed that the families Ctedectematidae, Eurystomateliidae, Histiobalantiidae, and Pleuronematidae are monophyletic, which is consistent with the results of previous studies [19, 53] (Fig. 2).

Phylogenetic analyses of the Harbin population of Lembadion bullinum (Fig. 4)

The two populations of Lembadion bullinu clusters together on the phylogenetic tree, albeit with low support (46%ML, 0.66BI). Two other Lembadion species groups outside of the Lembadion bullinu clade with fully support clade (100%ML, 1.00BI), supporting the monophyly of the family Lembadionidae. Notably, the newly sequenced L. bullinum (MT010296) differed by three nucleotides from the Canada population (AF255358). However, a direct morphological comparison was not feasible due to the absence of morphological data associated with Canada population. Even so, the morphology of the Harbin population corresponds well with both the original and previous morphological descriptions of L. bullinum, confirming the accuracy of its identification. In conclusion, the SSU rDNA sequence of L. bullinum (MT010296), validated by detailed morphological characterization, can serve as a reliable molecular reference for this species.

Authors’ contributions

Zhao Junyi, Pu Kexin and Qilei Yin: Contributed to manuscript preparation; Pan Mengmeng: Substantively contributed to sample collection, all experiments, data organization, and manuscript writing. Pan Xuming: provided full-process technical guidance for the experiments, assisted in manuscript writing, and secured funding. Song Yumeng completed the initial draft, which was reviewed collectively by all the authors.

Funding

This work was supported by the National Natural Science Foundation of China (Grant Nos. 32270544, 32570595, and 32500386), the Excellent Youth Fund of Heilongjiang Province (YQ2023C033), and the 2024 Natural Science Foundation of Heilongjiang Province (PL2024F007).

Data availability

The information of the new species in this study has been uploaded to ZooBank. Cyclidium orientale n. sp.: urn:lsid:zoobank.org:pub:59cB0c40-708B-42C0-95CE-EDDAC346C5D2. The SSU rDNA sequences have been deposited in GenBank under the accession numbers MT001894 (Cyclidium orientale) and MT010296 (Lembadion bullinum). All other data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

The animal experiments were conducted following the Guide for the Care and Use of Laboratory Animals, the protocol of which was approved by Harbin Normal University.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.