Integrative studies on the taxonomy and molecular phylogeny of four new Pleuronema species (Protozoa, Ciliophora, Scuticociliatia)

Abstract

The ciliate genus Pleuronema comprises approximately 30 nominal species and has been reported in freshwater, brackish water, and marine habitats. Nevertheless, recent studies have indicated that there might be a large undiscovered species diversity. In the present work, four new Pleuronema species, namely P. foissneri sp. nov., P. parasmalli sp. nov., P. parasalmastra sp. nov., and P. paraorientale sp. nov., collected from Shenzhen, southern China, was investigated using taxonomic methods. The diagnosis, description, comparisons with morphologically related species and detailed morphometric data are supplied for each. The small subunit ribosomal RNA (SSU rRNA) gene of the four new species is sequenced and their molecular phylogeny is analyzed. The SSU rRNA gene tree shows that Pleuronema is polyphyletic comprising several separate clades. All four new species cluster consistently with P. orientale KF206429, P. puytoraci KF840520 and P. setigerum FJ848874 within the core Pleuronematidae + Peniculistomatidae clade. Phylogenies of Pleuronematidae-related taxa are also discussed.

Introduction

The ciliate genus Pleuronema was established by Dujardin (1841), based on its ovoidal body with a depression, long oral opening, and prominent oral cilia, in a redescription of “Paramecium chrysalis” by Ehrenberg (1838), which is very distinct from other reports on Paramecium. Dujardin (1841) also described another two Pleuronema species: P. crassa and P. marina. Today, Pleuronema is a speciose and cosmopolitan genus comprising approximately 30 nominal species which can be found in various aquatic environments (Fan and Pan 2020; Hu et al. 2019; Lynn 2008; Pan et al. 2015a, 2016; Song et al. 2009). In recent years, several new species have been discovered, suggesting there is a large undescribed diversity and a need to conduct further studies on this genus (Pan et al. 2015a, b, 2016; Wang et al. 2008a, b, 2009). Molecular phylogenetic analyses have increasingly been applied in modern taxonomic studies on ciliated protists, and the polyphyly of Pleuronema has been widely reported (Antipa et al. 2016, 2020; Gao et al. 2012, 2013, 2017; Pan et al. 2010, 2015a, b, 2016, 2020; Yi et al. 2009).

Although Pleuronema is a common genus with a long research history, there are still problems with its taxonomy. These include: (1) Incomplete morphological information: some species, especially those described in the eighteenth and nineteenth centuries, lack detailed descriptions or graphic illustrations. For example, eight species lack illustrations or photomicrographs of cells in vivo and the whole ciliature pattern remains unknown for P. grassei, P. prunulum, and P. simplex. Consequently, these poorly known taxa have not been reported since they were first described (Agatha et al. 1993; Corliss and Snyder 1986; Dragesco 1960, 1968; Fernandez-Leborans and Novillo 1994; Kahl 1926). (2) Confusion in taxonomic status: some characters from live observation (e.g., right ventrolateral side straight or convex) and silver-stained specimens (e.g., position and length of the buccal field relative to the body length, anterior position of membranelle 2b) were overlooked until the middle of the twentieth century causing unreliable identifications and synonyms/homonyms (Agamaliev 1968; Borror 1963, 1972; Dragesco 1960, 1968; Noland 1937). (3) Insufficient molecular data: SSU rRNA gene sequence data are available for only half of nominal Pleuronema species and a large proportion of these sequences lack corresponding morphological information, thus their identity needs to be confirmed (Gao et al. 2013). In addition, the large disparity in gene sequences among species within the genus suggests there is a large undiscovered species diversity of Pleuronema.

In the present study, four Pleuronema species collected from brackish and freshwater habitats in Shenzhen, southern China (Fig. 1), were investigated using modern taxonomic methods. The morphological and molecular data indicate that each of these is a new species.

Fig. 1

Geographic location of Shenzhen and photographs of sampling sites where the four new Pleuronema species were collected. A Portion of a map of China, red dot depicts the location of Shenzhen. B Map of Guangdong Province, Shenzhen is marked in pink (modified from https://d-maps.com). C–F Sampling sites and habitats of P. foissneri sp. nov. (C), P. parasmalli sp. nov. (D), P. parasalmastra sp. nov. (E) and P. paraorientale sp. nov. (F)

Results

ZooBank registration

This article: urn:lsid:zoobank.org:pub:7B92C073-DD1B-4135-8121-B1818132A52E.

Pleuronema foissneri sp. nov.: urn:lsid:zoobank.org:act:DFB07D56-DD70-4D66-813E-BF2B669A1D13.

Pleuronema parasmalli sp. nov.: urn:lsid:zoobank.org:act:1D239E98-A986-4F0D-882A-8D0A6FB86650.

Pleuronema parasalmastra sp. nov.: urn:lsid:zoobank.org:act:75533119-0DFA-4647-85F2-37882789C453.

Pleuronema paraorientale sp. nov.: urn:lsid:zoobank.org:act:5F0B617D-D758-4877-B048-373ABF75D9AC.

Taxonomy

Subclass: Scuticociliatia Small, 1967.

• Order: Pleuronematida Fauré-Fremiet in Corliss, 1956.

  • Family: Pleuronematidae Kent, 1881.

    • Genus: Pleuronema Dujardin, 1841.

Pleuronema foissneri sp. nov. (Fig. 2; Table 1)

Fig. 2

Pleuronema foissneri sp. nov. from life (A, D, F–H) and after protargol staining (B, C, E, I–N). A Left ventrolateral view of a representative cell, arrow shows the contractile vacuole. B, C Left ventrolateral (B) and right dorsolateral (C) views of the holotype specimen, arrow in B indicates membranelle 1, arrowhead in B depicts the shortened rightmost row of membranelle 3. D Left ventrolateral view of an individual in vivo, arrow points to the contractile vacuole that is not fully expanded. E Detail of oral structure. F Left lateral view of cell, arrow shows the oral cilia, arrowheads indicate the caudal cilia. G Detail of cortex, arrow depicts extrusomes below the pellicle. H Detail showing the macronucleus and the grooves on the cell surface (arrow). I, J Left ventrolateral (I) and right dorsolateral (J) views of the holotype specimen, showing the ciliature and pear-shaped macronucleus, arrow in I shows membranelle 1. K Ventral view of a silver-stained cell, revealing the ciliature and macronucleus. L, M Detail of oral region, arrowheads depict the shortened rightmost row of membranelle 3. N Showing a heart-shaped macronucleus. M1 membranelle 1, M2a membranelle 2a, M2b membranelle 2b, M3 membranelle 3, Ma macronucleus, PK preoral kineties, PM paroral membrane, PoK postoral kineties. Scale bars = 30 μm

Table 1 Morphometric data for Pleuronema foissneri sp. nov. (upper row) and Pleuronema parasmalli sp. nov. (lower row)

Diagnosis Body size in vivo approximately 60–75 μm × 30–40 μm. Anterior end slightly narrowed. Right ventrolateral side convex. 32–40 somatic kineties. Four to eight preoral kineties. Macronucleus usually with a notch in mid-portion. Membranelle 1 relatively long, approximately one-sixth of cell length. Membranelle 2a in zig–zag pattern in mid-portion, posterior portion hook-like. Membranelle 3 three-rowed, rightmost row shortened. Brackish water habitat.

Dedication We dedicate this new species to Dr. Wilhelm Foissner (Salzburg University) in recognition of his significant contributions to the taxonomy of ciliates.

Type locality and habitat Brackish water from a mangrove wetland in Shenzhen Bay Park (22°31′19.8′′ N; 114°0′11.1′′ E), Shenzhen, southern China. Water temperature was approximately 20 °C and salinity was approximately 8.

Deposition of type slides The protargol slide containing the holotype specimen (Fig. 2B, C) and several paratype specimens (registration number: LMJ2016040401-1), and a second protargol slide containing paratype specimens (registration number: LMJ2016040401-2), were deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China.

Small subunit ribosomal RNA (SSU rRNA) gene sequence The sequence of Pleuronema foissneri sp. nov. was deposited in GenBank with accession number OL654416. The length and G + C content of the sequence are 1637 bp and 43.01%, respectively.

Description Cell size in vivo approximately 60–75 μm × 30–40 μm. Elliptical or oval in outline (Fig. 2A, D, F). Anterior end slightly narrowed, posterior end rounded, right ventrolateral and dorsal sides convex (Fig. 2A, D, F). Buccal field occupying 70–85% of cell length (Fig. 2A, D; Table 1). Oral cilia approximately 30 μm long. Pellicle slightly notched with shallow longitudinal grooves (Fig. 2H). Extrusomes bar-shaped, located beneath pellicle, approximately 5 μm in length (Fig. 2E). Cytoplasm colorless to greyish, containing several food vacuoles, refractile globules and crystals that usually posteriorly distributed (Fig. 2A, D, F). Single contractile vacuole dorsally located about 85% down length of cell, approximately 8–10 μm in diameter when fully expanded, pulsating at intervals of approximately 20–40 s (Fig. 2A, D, F). Somatic cilia densely packed and approximately 8–10 μm long, perpendicular to surface when cell is at rest (Fig. 2A, D, F). Approximately 15 caudal cilia, each 25–30 μm in length. Locomotion typical of genus, i.e., usually by fast swimming with body rotating continuously about longitudinal axis.

Thirty-two to 40 somatic kineties (SK) extending almost entire length of body. Each SK consisting of dikinetids in anterior three-quarters of SK and monokinetids in posterior quarter (Fig. 2B, C, I–L; Table 1). Four to eight preoral kineties located to left of buccal field, commencing at anterior end of cell and terminating posteriorly approximately two-thirds down length of body (Fig. 2B, I; Table 1). Postoral kinety usually absent, one present in five out 15 individuals examined. Single macronucleus located one-third down length of body, approximately 25–35 μm × 15–30 μm after protargol staining, generally ellipsoidal to spherical in shape; in 14 out of 24 individuals examined, macronucleus notched in mid-portion giving it a heart- or pear-shaped appearance (Fig. 2C, I–K, N; Table 1). Micronucleus not detected.

Anterior quarter of membranelle 1 (M1) three-rowed while rest of M1 two-rowed (Fig. 2B, E, I). M1 15–20% of cell length, commencing approximately one-tenth down length of body (Fig. 2B, I, K; Table 1). Anterior one-fifth and posterior quarter of membranelle 2a (M2a) conspicuously double-rowed, basal bodies in mid-portion arranged in zig–zag pattern (Fig. 2B, E, I). Length of M2a approximately 40–60% of body length, commencing near anterior end of M1 (Fig. 2B, I, K; Table 1). Posterior portion of M2a hook-like (Fig. 2B, E, I, K). Membranelle 2b (M2b) basically V-shaped, with basal bodies arranged in several single-rowed groups in zig–zag pattern (Fig. 2B, E, I). Each group with two to six basal bodies. Length of M2b approximately 13–22% of body length, commencing at same level as posterior end of M2a (Fig. 2B, E, I, K; Table 1). Membranelle 3 composed of three densely arranged rows, rightmost row shortened, length approximately 20% of two left rows (Fig. 2B, E, I, L, M). Paroral membrane double-rowed in zig–zag pattern, occupying about 70–80% of cell length (Fig. 2B, E, I, K).

Pleuronema parasmalli sp. nov. (Fig. 3; Table 1)

Fig. 3

Pleuronema parasmalli sp. nov. from life (A, E–G) and after protargol staining (B–D, H–K). A Left ventrolateral view of a representative cell, arrow shows the contractile vacuole. B, C Left ventrolateral (B) and right dorsolateral (C) views of the holotype specimen, arrowhead in B indicates that the mid-portion of membranelle 2a is single-rowed, arrow in C points to the micronucleus. D Detail of oral structure. E, F Left ventrolateral (E) and dorsal (F) views of representative individuals, arrow in E shows the contractile vacuole, arrow in F indicates the narrowed posterior end. G Detail of cortex, arrowheads point to the extrusomes below the pellicle. H Nuclear apparatus, arrow shows the micronucleus. I, J Left ventrolateral (I) and right dorsolateral (J) views of the holotype specimen. K Ventral view, showing the oral infraciliature and macronucleus, arrowheads depict the preoral kineties. M1 membranelle 1, M2a membranelle 2a, M2b membranelle 2b, M3 membranelle 3, Ma macronucleus, PK preoral kineties, PM paroral membrane, PoK postoral kineties. Scale bars = 30 μm

Diagnosis Body 55–85 μm × 25–35 μm in vivo. Right ventrolateral side basically straight. 26–32 somatic kineties. Four to six preoral kineties. Macronucleus and micronucleus spherical. Membranelle 2a single-rowed in mid-portion, posterior portion hook-like. Freshwater habitat.

Etymology Composite of the Greek word para (beside) and the species-group name smalli, indicating the similarity between the new species and Pleuronema smalli in terms of their small body size and ciliature pattern.

Type locality and habitat Shallow freshwater pond at Dameisha sandy beach (22°35′12.7′′ N; 114°18′14.9′′ E) in Shenzhen, southern China. Water temperature was approximately 24 °C.

Deposition of type slides The protargol slide containing the holotype specimen (Fig. 3B, C) and several paratype specimens (registration number: LMJ2016111701-1), and two protargol slides containing paratype specimens (registration numbers: LMJ2016111701-2, LMJ2016111701-3), were deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China.

SSU rRNA gene sequence The sequence of Pleuronema parasmalli sp. nov. was deposited in GenBank with accession number OL654417. The length and G + C content of the sequence are 1632 bp and 43.26%, respectively.

Description Body size in vivo approximately 55–85 μm × 25–35 μm. Elongate-elliptical in outline when viewed from left ventrolateral aspect (Fig. 3A, E). Anterior end rounded (Fig. 3A, E), posterior end narrowed when viewed from dorsal aspect (Fig. 3F). Laterally flattened (Fig. 3F). Right ventrolateral side basically straight, dorsal side convex (Fig. 3A, E). Length of buccal field 67–80% of body length (Fig. 3A; Table 1). Oral cilia approximately 30 μm in length. Pellicle slightly notched with shallow longitudinal grooves (Fig. 3H). Bar-shaped extrusomes densely arranged beneath pellicle and approximately 5 μm in length (Fig. 3E). Cytoplasm transparent to greyish, containing several food vacuoles, refractile globules, and crystals that are usually concentrated in posterior half of cell rendering this region black when viewed at low magnification (Fig. 3A, E, F). Single contractile vacuole dorsally located 80% down length of cell, approximately 10–12 μm across when fully expanded, pulsating at intervals of approximately 15–30 s (Fig. 3A, E). Somatic cilia densely packed, 10–12 μm in length (Fig. 3A, E–G). Approximately 15–18 caudal cilia, each 20–25 μm in length. Locomotion typical of genus, i.e., usually by fast swimming with body rotating continuously about longitudinal axis.

Twenty-six to 32 somatic kineties extending almost entire body length, each with densely spaced dikinetids in anterior three-fifths, and monokinetids in posterior two-fifths (Fig. 3B, C, I, J; Table 1). Four to six preoral kineties located to left of buccal field, commencing near anterior end of cell and terminating posteriorly 65% down length of cell (Fig. 3B, I; Table 1). Thirteen out of 25 cells examined with one postoral kinety (PoK), others without PoK. Usually (in 19 out of 25 cells examined) with a single spherical macronucleus positioned at 33 to 40% down length of body, diameter approximately 15–22 μm after protargol staining (Fig. 3C, I–K); six out of 25 cells examined with two to 14 spherical macronuclei, diameter of each varying from 5–15 μm. Single spherical micronucleus, 5–6 μm in diameter, adjacent to macronucleus (Fig. 3C, H).

First quarter of membranelle 1 (M1) three-rowed while rest two-rowed (Fig. 3B, D, I, K). Length of M1 10–15% of cell length (Fig. 3B, I; Table 1). Anterior end of M1 located approximately 10% down length of cell (Fig. 3B, I). Anterior one-eighth and posterior one-third of membranelle 2a (M2a) two-rowed, mid-portion single-rowed (Fig. 3B, D, I, K). Length of M2a 40 to 50% of body length, commencing at level of 20% down length of M1 (Fig. 3B, D, I, K). Posterior portion of M2a hook-like (Fig. 3B, D, I, K). Membranelle 2b (M2b) V-shaped, portion near each end single-rowed, basal bodies in mid-portion arranged in zig–zag pattern (Fig. 3B, D, I, K). M2b occupying approximately 10% of body length. Anterior end of M2b slightly below level of posterior end of M2a (Fig. 3B, D, I, K). Membranelle 3 with three closely packed rows, posterior end of rightmost row conspicuously separated from other rows and pointing to right (Fig. 3B, D, I, K). Paroral membrane double-rowed and in zig–zag pattern, occupying about 67–80% of cell length (Fig. 3B, D, I, K; Table 1).

Pleuronema parasalmastra sp. nov. (Fig. 4; Table 2)

Fig. 4

Pleuronema parasalmastra sp. nov. from life (A, D, F–J) and after protargol staining (B, C, E, K–M). A Left ventrolateral view of a representative cell, arrow shows the contractile vacuole. B, C Ventral (B) and dorsal (C) views of the holotype specimen, showing the ciliature and nuclear apparatus; arrowhead in B depicts the single-rowed mid-portion of membranelle 2a. D Left lateral view of a representative individual, arrow shows the oral cilia. E Oral ciliature. F Cell in vivo, arrow shows the contractile vacuole. G Detail of cortex, arrowhead points to the extrusomes below the pellicle. H Posterior end of cell, showing the caudal cilia. I Lateral view of cell. J Different individuals, showing the variation in body size. K, L, Left ventrolateral (K) and right dorsolateral (L) views of the same silver-stained specimen, arrow in K indicates the gap between cell apex and oral apparatus. M Detail of cell showing the oral ciliature. M1 membranelle 1, M2a membranelle 2a, M2b membranelle 2b, M3 membranelle 3, Ma macronucleus, PK preoral kineties, PM paroral membrane, PoK postoral kineties. Scale bars = 30 μm (A, D), 50 μm (B, C, I, K, L), 45 μm (F)

Table 2 Morphometric data for Pleuronema parasalmastra sp. nov. (upper row) and Pleuronema paraorientale sp. nov. (lower row)

Diagnosis Body size in vivo approximately 90–120 μm × 40–55 μm. Right ventrolateral side straight. 37–43 somatic kineties. Four to six preoral kineties. Membranelle 1 commences about 20% down length of cell. Membranelle 2a single-rowed in mid-portion, posterior portion hook-like. Brackish water habitat.

Etymology Composite of the Greek word para (beside) and the species-group name salmastra, indicating that the new species resembles the large individuals of Pleuronema salmastra in having an entirely posterior-positioned buccal field.

Type locality and habitat Brackish water from Yelin Sand Beach (22°31′19.5′′ N; 113°59′17.2" E), Shenzhen, southern China. Water temperature was approximately 10 °C and salinity was approximately 10.

Deposition of type slides The protargol slide containing the holotype specimen (Fig. 4B, C) and several paratype specimens (registration number: LMJ2015121701-1), and two protargol slides containing paratype specimens (registration numbers: LMJ2015121701-2, LMJ2015121701-3), were deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China.

SSU rRNA gene sequence The sequence of Pleuronema parasalmastra sp. nov. was deposited in GenBank with accession number OL654418. The length and G + C content of the sequence are 1630 bp and 44.36%, respectively.

Description Body size in vivo approximately 90–120 μm × 40–55 μm. Elongate-elliptical in outline (Fig. 4A, D, F). Anterior and posterior ends rounded, right ventrolateral side straight, dorsal side convex (Fig. 4A, D, F). Length of buccal field approximately 60–75% of body length (Fig. 4A, D; Table 2). Oral cilia approximately 25 μm long. Pellicle slightly notched with shallow longitudinal grooves. Extrusomes bar-shaped and densely distributed beneath pellicle, approximately 4–5 μm long (Fig. 4G). Cytoplasm greyish to yellowish-brown, containing numerous food particles including algae, usually distributed in anterior and posterior portions of cell (Fig. 4A, D, F). Single contractile vacuole dorsally located 80% down length of cell, approximately 15 μm across when fully expanded, pulsating at intervals of approximately 60–120 s (Fig. 4A, F). Somatic cilia densely packed, radiating from cell surface, approximately 10 μm in length (Fig. 4A, D, G). Approximately 18–20 caudal cilia, each 25–30 μm in length. Locomotion typical of genus, i.e., usually by fast swimming with body rotating continuously about longitudinal axis.

Thirty-seven to 43 somatic kineties extending entire body length, each with closely arranged dikinetids in anterior 33–75% portion, and monokinetids in remaining portion (Fig. 4B, C, K, L; Table 2). Four to six preoral kineties located to left of buccal field, commencing near anterior end of cell and terminating posteriorly 75% down length of cell (Fig. 4B, K; Table 2). One postoral kinety in seven out of ten cells examined, postoral kinety lacking in remaining three cells (Fig. 4B). Usually (in 12 out of 16 cells examined) with a single ellipsoidal macronucleus, centrally positioned, approximately 23–36 μm in length after protargol staining (Fig. 4C); four out of 16 cells examined with three to six spherical macronuclei, each approximately 15–20 μm across (Fig. 4K, L). Micronucleus not detected.

Anterior 20% of membranelle 1 (M1) three-rowed, posterior 80% two-rowed (Fig. 4B, E, M). Length of M1 10–13% of cell length (Fig. 4K; Table 2). M1 commencing approximately 15 to 25% down length of cell (Fig. 4B, K). Anterior 20% and posterior 25% of membranelle 2a (M2a) two-rowed, remaining portion single-rowed (Fig. 4B, E, M). Length of M2a occupying approximately 33 to nearly 50% of body length, commencing slightly lower than anterior end of M1 (Fig. 4B, E, M). Posterior portion of M2a hook-like (Fig. 4B, E, K, M). Membranelle 2b (M2b) V-shaped, first quarter on right side single-rowed, basal bodies in remaining portion arranged in zig–zag pattern (Fig. 4B, E, M). M2b occupying approximately 10% of body length (Fig. 4B, K). Anterior end of M2b and posterior end of M2a at approximately same level (Fig. 4B, E, M). Membranelle 3 three-rowed and closely packed (Fig. 4B, E, M). Paroral membrane double-rowed in zig–zag pattern, occupying about 60–75% of cell length (Fig. 4B, E, K).

Pleuronema paraorientale sp. nov. (Fig. 5; Table 2)

Fig. 5

Pleuronema paraorientale sp. nov. from life (A, E–H) and after protargol staining (B–D, I–L). A Left ventrolateral view of a representative cell, arrow shows the contractile vacuole. B, C Left ventrolateral (B) and right dorsolateral (C) views of the holotype specimen, showing the ciliature and nuclear apparatus; arrowhead in B indicating that membranelle 2b commences significantly above the level of the posterior end of membranelle 2a. D Details of oral ciliature. E Left lateral view of an individual in vivo. F, H Caudal cilia on ventral (F, arrow) and dorsal (H, arrowheads) side of cell. G Detail of pellicle, showing the grooves along the ciliary rows. I, J Left ventrolateral (I) and right dorsolateral (J) views of the holotype specimen. K Detail of cell showing the oral ciliature. L Details of nuclear apparatus (color-inverted by Adobe Photoshop), arrowheads point to the multiple micronuclei adjacent to macronucleus. M1 membranelle 1, M2a membranelle 2a, M2b membranelle 2b, M3 membranelle 3, Ma macronucleus, PK preoral kineties, PM paroral membrane, PoK postoral kineties. Scale bars = 50 μm

Diagnosis Body size in vivo approximately 95–115 μm × 55–70 μm. Right ventrolateral side convex. 52–62 somatic kineties. Three to five preoral kineties. Two to ten spherical micronuclei. Membranelle 2a hook-like in posterior portion, basal bodies in mid-portion arranged in zig–zag pattern. Membranelle 2b commences significantly above level of posterior end of membranelle 2a. Brackish water habitat.

Etymology Composite of the Greek word para (beside) and the species-group name orientale, indicating that this species resembles Pleuronema orientale in body size, shape and ciliature pattern.

Type locality and habitat Brackish water from a mangrove wetland on the west coast of Shenzhen Bay (22°30′8.2′′ N; 113°57′10.7′′ E), Shenzhen, southern China. Water temperature was approximately 24 °C, salinity was approximately 12.

Deposition of type slides The protargol slide containing the holotype specimen (Fig. 5B, C) and several paratype specimens (registration number: LMJ2017010601-1), and one protargol slide containing paratype specimens (registration number: LMJ2017010601-2), were deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China.

SSU rRNA gene sequence The sequence of Pleuronema paraorientale sp. nov. was deposited in GenBank with accession number OL654419. The length and G + C content of the sequence are 1639 bp and 43.50%, respectively.

Description Body size in vivo approximately 95–115 μm × 55–70 μm. Broad-elliptical in lateral view (Fig. 5A, E). Anterior and posterior ends rounded (Fig. 5A, E). Right ventrolateral and dorsal sides convex (Fig. 5A, E). Length of buccal field approximately 60–80% of body length (Fig. 5A; Table 2). Oral cilia approximately 40 μm in length. Pellicle slightly notched with shallow longitudinal grooves (Fig. 5G). Extrusomes bar-shaped, approximately 5 μm in length, densely distributed beneath pellicle. Cytoplasm grey to yellowish, making cells appear brown in color at low magnification (Fig. 5E). Several food vacuoles, refractile globules and crystals usually concentrated in posterior part of cell (Fig. 5E). Single contractile vacuole dorsally located approximately 80% down length of cell, diameter 10–12 μm when fully expanded, pulsating at intervals of 40–60 s (Fig. 5A, E). Somatic cilia densely arranged, radiating from cell surface, 10–12 μm long (Fig. 5A, E). Twenty to 30 caudal cilia, each 30–40 μm in length (Fig. 5F, H). Locomotion by fast swimming with body rotating continuously about longitudinal axis.

Fifty-two to 62 somatic kineties (SK) extending almost entire body length, with slender glabrous area at anterior end of cell (Fig. 5B, C, I, J; Table 2). Each SK with close-set dikinetids in anterior 80%, and monokinetids in posterior 20% (Fig. 5B, C, I, J). Three to five preoral kineties located to left of buccal field, commencing near anterior end of cell and terminating posteriorly approximately 65% down length of cell (Fig. 5B, I; Table 2). Zero to three postoral kineties (PoK) located near right side of posterior end of SKn (first kinety on left of buccal field): among 26 examined cells, one cell without PoK, 21 cells with one PoK, three cells with two PoK, and one cell with three PoK, (Fig. 5B, I; Table 2). Usually (in 23 out of 25 cells examined) with a single ellipsoidal macronucleus positioned 40% down length of cell, approximately 25–45 μm in length after protargol staining (Fig. 5C, I, J); occasionally (in two out of 25 cells examined) with three or 12 spherical macronuclei, each approximately 15–20 μm in diameter. Two to ten (usually four) spherical micronuclei, each approximately 3–6 μm across, usually adjacent to macronucleus (Fig. 5L; Table 2).

Membranelle 1 (M1) three-rowed in anterior 15 to 20% portion, two-rowed in remaining portion (Fig. 5B, D, I, K). Length of M1 8–13% of cell length (Fig. 5B, I; Table 2). M1 commencing about 12% down body length (Fig. 5B, I). Anterior 15% and posterior 33% of membranelle 2a (M2a) two-rowed, basal bodies in remaining portion arranged in a zig–zag pattern (Fig. 5B, D, I, K). M2a occupying approximately 40–60% of body length, commencing near level of anterior end of M1 (Fig. 5B, I; Table 2). Posterior portion of M2a hook-like, located approximately 60% down length of cell (Fig. 5B, D, I, K). Membranelle 2b (M2b) V-shaped, basal bodies in right anterior 20% arranged in a continuous single row, those in remaining portion arranged in several single-rowed groups, each group composed of two to six basal bodies and linked with other groups (Fig. 5B, D, I, K). M2b occupying approximately 15 to 28% of body length, commencing significantly above level of posterior end of M2a (Fig. 5B, D, I, K). Membranelle 3 three-rowed and closely packed, posterior end of rightmost row slightly separated from other rows by diverging rightwards (Fig. 5B, D, I, K). Paroral membrane (PM) double-rowed, basal bodies arranged in zig–zag pattern, occupying approximately 60–80% of cell length (Fig. 5B, D, I, K).

Molecular data and phylogenetic analyses (Figs. 6, 7; Supplementary Table S1)

Fig. 6

Maximum likelihood (ML) tree based on SSU rRNA gene sequence data, showing the phylogenetic positions of the four new Pleuronema species (in bold). Numbers at nodes denote ML bootstrap values/Bayesian inference (BI) posterior probabilities. Asterisks (*) indicate mismatch in topologies between ML and BI analyses. Fully supported (100%/1.00) nodes are marked with solid circles. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. All branches are drawn to scale. The systematic classification mainly follows Gao et al. (2016). Pleuronema coronatum JX310014 (marked with red triangle) deviates from the other three P. coronatum sequences, unfortunately, available information is not sufficient to determine its identity. Main morphological features of Pleuronema species and the number of unmatched nucleotides within each Pleuronema clade are both provided. “Exceeding” in anterior end of M2b means that M2b commences above level of posterior end of M2a, while “not exceeding” means it commences below level of posterior end of M2a. BW brackish water, FW freshwater, M2a membranelle 2a, M2b membranelle 2b, MW marine water

Fig. 7

Comparison of SSU rRNA gene sequences of four new species with their most closely related congeners. A SSU rRNA gene comparison, showing the positions (given at the top of each column) of unmatched columns in the alignment. Insertions and deletions are compensated by introducing alignment gaps (–). B Matrix showing the percentage of sequence identity (below the diagonal) and the number of unmatched nucleotides (above the diagonal)

The topologies of the maximum likelihood (ML) and Bayesian inference (BI) trees were similar, therefore, only the ML tree, with support values from both algorithms, is shown (Fig. 6). The order Pleuronematida is monophyletic with high support (97% ML/1.00 BI). Within the Pleuronematida, the families Ctedoctematidae and Eurystomatidae form a fully supported clade that branches first, followed by the family Histiobalantiidae. The family Peniculistomatidae is nested within the family Pleuronematidae forming a single clade (59% ML/0.88 BI). Within this clade, members of the genera Schizocalyptra, Mytilophilus, and Peniculistoma are scattered among members of Pleuronema, making the genus Pleuronema polyphyletic.

All four newly sequenced Pleuronema species group with P. puytoraci KF840520, P. orientale KF206429 and P. setigerum FJ848874 with high support (96% ML/1.00 BI) (Fig. 6). Among these, P. paraorientale sp. nov. OL654419 clusters with P. orientale KF206429 and P. puytoraci KF840520 (87% ML/1.00 BI), followed by P. foissneri sp. nov. OL654416 and P. parasalmastra sp. nov. OL654418. In the ML tree, P. parasmalli sp. nov. OL654417 clusters with P. setigerum FJ848874 but with very low support (41% ML), forming a sister clade to the P. puytoraci + P. parasalmastra clade, whereas in the BI tree, P. parasmalli sp. nov. OL654417 is sister to the P. puytoraci + P. parasalmastra clade with low support (0.54 BI).

A comparison of all SSU rRNA gene sequences of Pleuronema species (Supplementary Table S1) shows that the intraspecific sequence identities are 94.7–100% with 0–85 unmatched nucleotides (for four P. coronatum sequences) and 91.6% with 135 unmatched nucleotides (two P. setigerum sequences). In contrast, the interspecific sequence identities range from 88.8 to 99.8%, with two (P. puytoraci KF840520 and P. orientale KF206429) to 180 (P. setigerum FJ848874 and P. parawiackowskii KT033423) unmatched nucleotides (Supplementary Table S1).

The SSU rRNA gene sequence Pleuronema foissneri sp. nov. OL654416 largely resembles those of P. puytoraci KF840520, P. paraorientale sp. nov. OL654419, and P. orientale KF206429, with 43–45 unmatched nucleotides and sequence identities of 97.2–97.3% (vs. 62–151 unmatched nucleotides and 90.6–96.1% sequence identities when compared with other Pleuronema sequences) (Fig. 7; Supplementary Table S1).

Pleuronema parasmalli sp. nov. OL654417 is most similar to P. parasalmastra sp. nov. OL654418, although the number of unmatched nucleotides is 82 (sequence identity 94.9%) (Fig. 7; Supplementary Table S1). In addition, P. parasmalli sp. nov. OL654417 seems to be more divergent from other sequences of the P. puytoraci + P. setigerum FJ848874 clade (with 82–135 unmatched nucleotides). In contrast, the other three new sequences (OL654416, OL654418, OL654419) differ from that clade by 19–125 unmatched nucleotides (Fig. 7).

The SSU rRNA gene sequence Pleuronema parasalmastra sp. nov. OL654418 is most similar to P. puytoraci KF840520 and P. orientale KF206429, with 51 and 53 unmatched nucleotides (96.8% and 96.7% sequence identity), respectively. It differs from other Pleuronema sequences in 62–151 nucleotides (90.6–96.1% sequence identity) (Fig. 7; Supplementary Table S1).

Pleuronema paraorientale sp. nov. OL654419 is most similar to P. puytoraci KF840520 and P. orientale KF206429, with 19 and 21 unmatched nucleotides (98.8% and 98.6% sequence identity), respectively, while there are 44–154 different nucleotides (90.4–97.2% sequence identity) compared with sequences of other congeners (Fig. 7; Supplementary Table S1).

Discussion

Comments on Pleuronema coronatum Kent, 1881

Pleuronema coronatum is one of the most common and well-studied species of Pleuronema. Since its establishment based on a freshwater population (Kent 1881), several populations isolated from freshwater, brackish water or marine water, mainly collected from Europe, North America, Africa, and East Asia, have been reported under the name “P. coronatum” (Agamaliev 1968; Borror 1963, 1972; Chorik 1968; Dragesco 1960, 1968; Dragesco and Dragesco-Kernéis 1986; Foissner et al. 1994; Kahl 1926, 1931; Noland 1937; Small and Lynn 1985; Song 2000; Wang et al. 2008a). This species, which was originally called “Pleuronema coronata”, is shorter and thicker than the morphologically related congener “Pleuronema chrysalis”, and has extrusomes and caudal cilia, both of which are absent in the latter (Ehrenberg 1838; Kent 1881). In addition, the former has a straight right ventrolateral side and a spherical macronucleus according to the original drawing of a cell in vivo (Kent 1881), which we consider to be diagnostic features of P. coronatum.

The body length in vivo of two populations of P. coronatum described by Wang et al. (2008a) range from 55 to 170 μm, the extremes of which deviate significantly from the original description (approximately 90 μm in length), suggesting that these populations may have included multiple species. It is noteworthy that Wang et al. (2008a) regarded Pleuronema balli, P. borrori, and P. smalli as synonyms of P. coronatum since they share some similar characteristics. After reinvestigating those descriptions, we agree that P. balli is a synonym of P. coronatum, since the former matches well with the original and other reliable populations of the latter in terms of body size, ciliature pattern, and macronucleus shape (Borror 1963; Chorik 1968; Dragesco 1968; Foissner et al. 1994; Kent 1881; Small and Lynn 1985; Song 2000). As a result, both P. balli populations (Dragesco 1968; Small and Lynn 1985) should be considered as P. coronatum populations. However, we disagree that Pleuronema borrori and P. smalli are synonyms of P. coronatum (Wang et al. 2008a). Pleuronema borrori has a much wider body (70–77 μm after silver staining vs. usually 25–65 μm in P. coronatum) and a smaller ratio of buccal field/body length (approximately 0.55 in P. borrori vs. 0.64–0.75, data measured from the drawings of silver-stained cells) than P. coronatum (Borror 1963; Dragesco 1968; Foissner et al. 1994; Small and Lynn 1985; Song 2000). Pleuronema smalli has fewer somatic kineties than P. coronatum (28–36 vs. 35–48) and an ellipsoid macronucleus (vs. spherical in P. coronatum) (Borror 1963, 1972; Chorik 1968; Dragesco 1968; Foissner et al. 1994; Kent 1881; Small and Lynn 1985; Song 2000). Hence, we recognize P. borrori and P. smalli as valid species.

In summary, Pleuronema coronatum can be characterized as follows: body size in vivo usually 60–125 μm × 30–60 μm; right ventrolateral side straight; 35–48 somatic kineties; three to seven preoral kineties; single spherical macronucleus; one to three micronuclei; membranelle 2a usually arranged in a zig–zag pattern in mid-portion, posterior region hook-like; freshwater and marine habitat (Borror 1963; Chorik 1968; Dragesco 1968; Foissner et al. 1994; Kent 1881; Small and Lynn 1985; Song 2000).

Comments on Pleuronema foissneri sp. nov. (Fig. 8; Table 3)

Fig. 8

Comparison of the oral ciliature of the four new species (yellow blocks) with the related Pleuronema species (grey blocks, redrawn from previous studies). A P. coronatum (from Song 2000). B P. salmastra (from Dragesco and Dragesco-Kernéis 1986). C P. arctica (from Agatha et al. 1993). D P. glaciale (from Corliss and Snyder 1986). E P. smalli (from Dragesco 1968). F P. paucisaetosum (from Pan et al. 2015a). G P. setigerum (from Pan et al. 2010). H P. borrori (from Dragesco 1968). I P. orientale (from Pan et al. 2015a). J P. puytoraci (from Groliere and Detcheva 1974). K–N The four new species described in the present work. Scale bars = 30 μm (scale bar in J is inferred from the average length of membranelle 1 in Groliere and Detcheva 1974)

Table 3 Morphological comparison of Pleuronema foissneri sp. nov. with related congeners

Based on the presence of approximately 30–60 somatic kineties, a single spherical or ellipsoidal macronucleus, posterior end of M2a (hook-like), the position of the anterior end of the V-shaped M2b slightly above or at the same level as the posterior end of M2a, and the three-rowed membranelle 3, two Pleuronema species should be compared with P. foissneri sp. nov., namely P. coronatum Kent, 1881, and P. salmastra Dragesco & Dragesco-Kernéis, 1986 (Fig. 8; Table 3).

Pleuronema foissneri sp. nov. can be distinguished from P. coronatum mainly in body shape (anterior end slightly narrowed, right ventrolateral side convex in the former vs. anterior end rounded, right ventrolateral side straight in the latter) and the longer M1 relative to the body length (0.14–0.21 vs. 0.08–0.11 in P. coronatum) (Borror 1963; Chorik 1968; Dragesco 1968; Foissner et al. 1994; Kent 1881; Small and Lynn 1985; Song 2000).

Pleuronema foissneri sp. nov. resembles P. salmastra in the slightly narrowed anterior end and in the body size after silver staining. The former, however, differs from the latter by having fewer somatic kineties (32–40 vs. 43–63 in P. salmastra), fewer preoral kineties (four to eight, five on average vs. six to ten, eight on average in P. salmastra), and a longer M1 relative to the body length (0.14–0.21 vs. 0.09–0.10 in P. salmastra) (Dragesco and Dragesco-Kernéis 1986).

Like Pleuronema foissneri sp. nov., P. glaciale and P. arctica both have a narrowed anterior end. However, they can be easily separated from P. foissneri sp. nov. by their larger body size (longer than 120 μm after silver staining vs. 60–90 μm in P. foissneri sp. nov.) and in having more somatic kineties (44–58 and 39–61, respectively, vs. 32–40 in P. foissneri sp. nov.). Furthermore, in P. glaciale, the length of M1 is about half (vs. one-third in P. foissneri sp. nov.) of M2a length, and the M3 is double-rowed (vs. three-rowed in P. foissneri sp. nov.) (Agatha et al. 1993; Corliss and Snyder 1986). For further details of comparisons between P. foissneri sp. nov. and its congeners, see Table 3.

Comments on Pleuronema parasmalli sp. nov. (Fig. 8; Table 4)

Table 4 Morphological comparison of Pleuronema parasmalli sp. nov. with related congeners

Based on its body size and shape (50–90 μm in length, right ventrolateral side straight), number of somatic kineties (20–40), shape of the posterior portion of M2a (hook-like), position of the anterior end of the V-shaped M2b (not above the level of the posterior end of M2a) and three-rowed M3, Pleuronema parasmalli sp. nov. should be compared with P. smalli Dragesco, 1968, P. paucisaetosum Pan et al., 2015, and P. setigerum Calkins, 1902 (Fig. 8; Table 4).

Pleuronema parasmalli sp. nov. closely resembles P. smalli in its small body size after silver staining and similar ciliature pattern. The former, however, can be distinguished from the latter by having a single micronucleus (vs. two in P. smalli) and its freshwater habitat (vs. brackish and marine water in P. smalli). Morphological information of P. smalli in vivo is not available, therefore, a comparison with P. parasmalli sp. nov. in vivo cannot be performed (Borror 1972; Dragesco 1968).

Pleuronema parasmalli sp. nov. has a similar body size, shape, and oral structure to P. paucisaetosum, but the former differs from the latter in having more somatic kineties (26–32 vs. 21–23), a single micronucleus (vs. one to five, two on average), and a freshwater (vs. brackish water) habitat (Pan et al. 2015a).

Pleuronema setigerum can be separated from P. parasmalli sp. nov. by having a shorter and more slender body (25–50 μm × 10–30 μm in the former vs. 55–85 μm × 25–35 μm in the latter), a ring-like (vs. hook-like in the latter) posterior portion of M2a, fewer caudal cilia (9–13 vs. 15–18), fewer somatic kineties (12–25 vs. 26–32) and a marine (vs. freshwater) habitat (Borror 1963; Calkins 1902; Jung 2021; Kahl 1931; Noland 1937; Pan et al. 2010, 2015b).

Comments on Pleuronema parasalmastra sp. nov. (Fig. 8; Table 5)

Table 5 Morphological comparison of Pleuronema parasalmastra sp. nov. with related congeners

Based on its body shape (right ventrolateral side straight and dorsal side convex), number of somatic kineties (approximately 30–60), shape of macronucleus (spherical to ellipsoidal), shape of the posterior portion of M2a (hook-like), anterior end of the V-shaped M2b (located at approximately the same level as the posterior end of M2a), and three-rowed M3, three species should be compared with P. parasalmastra sp. nov., namely P. salmastra Dragesco & Dragesco-Kernéis, 1986, P. borrori Dragesco, 1968, and P. coronatum Kent, 1881 (Fig. 8; Table 5).

Pleuronema parasalmastra sp. nov. can be distinguished from P. salmastra by having a larger body size in silver-stained specimens (95–160 μm × 45–77 μm vs. 50–116 μm × 22–40 μm in P. salmastra), fewer somatic kineties (37–43, 40 on average vs. 43–63, 53 on average in P. salmastra) and fewer preoral kineties (4–6 vs. 6–10 in P. salmastra). Furthermore, the distance between the cell apex and the anterior end of M1 occupies a larger portion of the body length in P. parasalmastra sp. nov. (0.16–0.25 of body length) than that in P. salmastra (0.08–0.18 of body length, data inferred from the drawings of silver-stained specimens) (Dragesco and Dragesco-Kernéis 1986).

Pleuronema borrori was established by Dragesco (1968) based only on silver-stained specimens. This species can be characterized by its large body size with a relatively small buccal field. There have been no redescriptions of P. borrori. Pleuronema parasalmastra sp. nov. resembles P. borrori in the large body size with a small and posteriorly positioned buccal field, but it differs from P. borrori by having fewer somatic kineties (37–43 vs. 41–46 in P. borrori) and a relatively slender body after silver staining (45–75 μm, 57 μm on average in width vs. 70–77 μm, 74 μm on average in P. borrori). In addition, the buccal field in P. parasalmastra sp. nov. occupies a larger portion of the body length (0.57–0.74 vs. 0.55 in P. borrori, inferred from the drawing of a silver-stained cell) (Dragesco 1968).

When compared with Pleuronema coronatum, P. parasalmastra sp. nov. has a larger body size in silver-stained specimens (95–160 μm in length vs. 50–115 μm in P. coronatum) and a more posteriorly positioned buccal field (distance between cell apex to anterior end of M1 in P. parasalmastra sp. nov. is 0.16–0.25 of body length vs. 0.09–0.21 in P. coronatum). In addition, the mid-portion of M2a is clearly single-rowed in P. parasalmastra sp. nov., whereas it has a zig–zag pattern in P. coronatum (Borror 1963; Chorik 1968; Dragesco 1968; Foissner et al. 1994; Kent 1881; Small and Lynn 1985; Song and Wilbert 2000).

Comments on Pleuronema paraorientale sp. nov. (Fig. 8; Table 6)

Table 6 Morphological comparison of Pleuronema paraorientale sp. nov. with related congeners

Based on its body size in vivo (approximately 100–150 μm in length), body shape (right ventrolateral and dorsal sides convex), number of somatic kineties (approximately 30–60), shape of macronucleus (spherical to ellipsoidal), posterior portion of M2a (hook-like), anterior end of the V-shaped M2b (significantly above the posterior end of M2a), and three-rowed M3, there are two species that should be compared with Pleuronema paraorientale sp. nov., namely P. orientale Pan et al., 2015 and P. puytoraci Groliere & Detcheva, 1974 (Fig. 8; Table 6).

Pleuronema paraorientale sp. nov. most closely resembles P. orientale in its body size, shape, and oral structure. The former, however, is different from the latter mainly by having more somatic kineties (52–62 vs. 42–50 in P. orientale), more preoral kineties (3–5 vs. 2–3 in P. orientale) and more micronuclei (2–10, four on average vs. 1–3, one on average in P. orientale) (Pan et al. 2015a).

Pleuronema paraorientale sp. nov. mainly differs from P. puytoraci by having more somatic kineties (52–62 vs. 28–29 in P. puytoraci) and more preoral kineties (3–5 vs. 1–3 in P. puytoraci) (Groliere and Detcheva 1974; Pan et al. 2011).

Phylogenetic analyses

Phylogenetic relationships between Pleuronema and Schizocalyptra

Studies on the molecular phylogeny of Pleuronema began with the sequencing of the large subunit rRNA gene of P. marinum (Baroin-Tourancheau et al. 1992). Then the SSU rRNA gene and internal transcribed spacer 2 region data of P. coronatum were added and phylogenetic analyses showed that Pleuronema was closely related to Schizocalyptra and Cyclidium (Lynn and Strüder-Kypke 2005; Miao et al. 2008).

According to the phylogenetic analysis in Yi et al. (2009), the genus Pleuronema is polyphyletic since two Schizocalyptra sequences nest within it forming a fully supported clade. The polyphyly of Pleuronema caused by Schizocalyptra was subsequently confirmed in most phylogenetic studies based on the SSU rRNA gene (Antipa et al. 2016, 2020; Gao et al. 2012, 2013, 2017; Pan et al. 2010, 2015a, b, 2016, 2020). However, in phylogenetic analyses based on nuclear or mitochondrial data, the genus Schizocalyptra falls outside of Pleuronema (Gao et al. 2014; Lu et al. 2021; Zhang et al. 2019). Consistent with previous studies based on SSU rRNA gene sequence data, our ML tree shows that Schizocalyptra sequences nest within Pleuronema (Fig. 6). However, the support values are very low and the phylogenetic position of Schizocalyptra is unstable, which may be due to the inclusion of insufficient taxa in the analyses. Considering the low support values and inconsistency of the phylogenetic position of Schizocalyptra based on previous and present studies, the relationship between Pleuronema and Schizocalyptra is still uncertain, and more morphological and molecular data are needed to further clarify their positions.

Phylogenetic relationships between the families Peniculistomatidae and Pleuronematidae

Based on SSU rRNA gene data, Antipa et al. (2016) was the first to reveal that the monophyletic family Peniculistomatidae falls within the Pleuronema spp., resulting in the polyphyly of Pleuronematidae. In subsequent studies, the polyphyly of Pleuronematidae caused by Peniculistomatidae was verified (Antipa et al. 2020; Lu et al. 2021; Zhang et al. 2019). In the present study, the family Peniculistomatidae nests within the Pleuronematidae in both the ML and BI trees, albeit with low support, supporting previous finding.

Peniculistoma and Mytilophilus are two endocommensal genera of Peniculistomatidae. Both are characterized by an irregular oval-shaped outline when viewed from the lateral aspect, the cytostome lying at the bottom of a deeply concaved depression, and having numerous somatic kineties, which clearly differentiates these genera from Pleuronema (Antipa and Dolan 1985; Antipa et al. 2016; Dolan and Antipa 1985; Fenchel 1965). The oral structures (a long and prominent paroral membrane and three membranelles, with M2 bipartite) and stomatogenesis of Peniculistoma and Mytilophilus, however, are generally similar with those in Pleuronema (Antipa and Dolan 1985; Dolan and Antipa 1985; Fenchel 1965). Furthermore, their phylogenetic positions in the SSU rRNA gene tree reflect the morphological similarities between the families Peniculistomatidae and Pleuronematidae (Fig. 6).

Phylogeny of the family Histiobalantiidae

In the phylogenetic analyses by Foissner et al. (2009) (when Histiobalantiidae sequences were first used in molecular phylogeny) and by Antipa et al. (2016, 2020), Histiobalantiidae clustered with Schizocalyptra, which was nested within the Pleuronematidae, whereas in other phylogenetic analyses, including the present study (Fig. 6), Histiobalantiidae is invariably placed outside the Pleuronematidae with full support (Antipa et al. 2016; Gao et al. 2012, 2013, 2014, 2017; Lu et al. 2021; Pan et al. 2010, 2015a, b, 2016, 2020; Zhang et al. 2019). In consideration of the insufficient sampling of Pleuronema sequences in Foissner et al. (2009) and the relatively low support for the position of Schizocalyptra in Antipa et al. (2016, 2020), it is more credible that the phylogenetic position of Histiobalantiidae is outside the Pleuronematidae + Peniculistomatidae clade.

Materials and Methods

Sampling and cultivation

Four Pleuronema species were collected at Shenzhen, China. Pleuronema foissneri sp. nov. was collected from a mangrove wetland in Shenzhen Bay Park (22°31′19.8′′ N; 114°0′11.1′′ E) on 4th April, 2016, with a water temperature of approximately 20 °C and salinity of approximately 8. Pleuronema parasmalli sp. nov. was collected from a shallow freshwater pond (the pond was approximately 15 cm deep) at Dameisha sandy beach (22°35′12.7′′ N; 114°18′14.9′′ E) on 17th November, 2016, with a water temperature of approximately 24 °C. Pleuronema parasalmastra sp. nov. was isolated from Yelin Sand Beach (22°31′19.5′′ N; 113°59′17.2′′ E) on 17th December, 2015, with a water temperature of approximately 10 °C and a salinity of approximately 10. Pleuronema paraorientale sp. nov. was collected from a mangrove wetland on the west coast of Shenzhen Bay (22°30′8.2′′ N; 113°57′10.7′′ E) on 6th January, 2017, where the water temperature was approximately 24 °C and the salinity was approximately 12.

For Pleuronema foissneri sp. nov. and P. paraorientale sp. nov., water samples were collected from naturally formed small puddles during ebb tide. An approximately 200 ml water sample with wilted leaves and sediment was placed into a 400 ml sampling bottle using bottle caps, and stirring was avoided. For P. parasmalli sp. nov., an approximately 200 ml volume of well-stirred freshwater sample with humus was placed into a 400 ml bottle. For P. parasalmastra sp. nov., several holes were excavated in the sand to a depth of approximately 10 cm. After water gradually seeped into the holes, water and sand at the bottom of the holes were mixed and collected. An approximately 200 ml water sample was placed into a 400 ml bottle. In all cases, samples were transferred to the laboratory and stored at room temperature (~ 25 °C).

After gently mixing the samples in the bottles, water with sediment of each sample was poured into five 90 mm Petri dishes to establish the initial cultures at ~ 25 °C. No food source was added to these Petri dishes. Three to five cells from the initial cultures were then isolated with micropipettes and transferred into a 35 mm disposable Petri dish with 0.22 µm-filtered water in situ for pure cultivation. Rice grains were added to promote the growth of bacterial food for the ciliates.

Morphological studies

Living cells were isolated with micropipettes and observed using bright-field and differential interference contrast microscopy at × 100–1000 magnification. The protargol staining method was used to reveal the ciliature and nuclear apparatus (Wilbert 1975). Counts and measurements were performed according to Bai et al. (2020). Drawings of living cells were produced according to Wu et al. (2020). Drawings of silver-stained cells were made using Adobe Photoshop based on photomicrographs of the holotype specimen of each species. Terminology and systematics follow Pan et al. (2016) and Gao et al. (2016), respectively.

DNA extraction, PCR amplification, and sequencing

Six to ten cells of each species were selected from pure cultures and washed five times with filtered in situ water (0.22 µm, Millex-GP filter unit) to exclude contamination. For each species, cells were then distributed in three Eppendorf tubes (Axygen, USA) with one, three, and multiple individuals. Genomic DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, Germany) following the optimized manufacturer’s protocol, modified such that 1/4 of the suggested volume was used for each solution. The primers 18S-F, 18S-R (Medlin et al. 1988) and 82F (Jerome et al. 1996) were used for PCR amplifications of the SSU rRNA gene. To minimize the errors caused by PCR, Q5 Hot Start High-Fidelity 2 × Master Mix (New England BioLabs, USA) was used as DNA polymerase. The PCR parameters were utilized according to Jiang et al. (2021). After amplification, PCR products were sequenced bidirectionally by the Tsingke Biological Technology Company (Qingdao, China).

Phylogenetic analyses

The SSU rRNA gene sequences of the four Pleuronema species in the present work were combined with 64 sequences of related taxa downloaded from GenBank, forming the dataset for phylogenetic analyses (for accession numbers, see Fig. 6). Ten sequences from Philasterida were selected as the outgroup. All sequences were aligned using the MAFFT algorithm on GUIDANCE2 Server (http://guidance.tau.ac.il) with default parameters (Penn et al. 2010; Sela et al. 2015). The resulting alignment was manually edited using the program BioEdit version 7.0.5.2 (Hall 1999), and both ends of the alignment were trimmed. The final alignment, including 1802 positions, was used to construct the phylogenetic trees.

Maximum likelihood (ML) analysis with 1000 bootstrap replicates was performed using RAxML-HPC2 on XSEDE 8.2.10 (Stamatakis 2014) under the GTRGAMMA model at CIPRES Science Gateway (http://www.phylo.org/sub_sections/portal) (Miller et al. 2010). Bayesian inference (BI) was performed with MrBayes 3.2.6 on XSEDE 3.2.6 (Ronquist and Huelsenbeck 2003) at the CIPRES Science Gateway with the best-fit model GTR + I + G, selected under the Akaike Information Criterion using MrModeltest 2 (Nylander 2004). Markov chain Monte Carlo (MCMC) simulations were run with two sets of four chains for 10,000,000 generations at a sampling frequency of 100 and a burn-in of 10,000 trees (10%). Convergence of the MCMC analyses was confirmed in that the average standard deviation of split frequencies was well below 0.01. All remaining trees were used to calculate posterior probabilities (PP) using a 50% majority rule consensus. Tree topologies were visualized using SeaView version 4 (Gouy et al. 2010). The SSU rRNA gene comparison of all Pleuronema sequences in the present work was performed by BioEdit version 7.0.5.2 (Hall 1999). The alignment length had 1623 positions after trimming both ends.