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Phylogeny of the anaerobic ciliate genus Sonderia (Protista: Ciliophora: Plagiopylea), including the description of three novel species and a brief revision of the genus

  • Special topic: Ciliatology.
  • Edited by Jiamei Li.
  • Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
  • Anaerobic protists in general, and ciliates in particular, are important components of anoxic or hypoxic environments, however, their diversity remains underestimated. Sonderia is a poorly studied genus that is distributed worldwide and is commonly found in anaerobic environments. In the present study, the taxonomy and phylogeny of three new species, namely Sonderia aposinuata sp. nov., Sonderia paramacrochilus sp. nov. and Sonderia steini sp. nov., collected from China, were investigated based on microscopic observations and SSU rRNA gene sequencing methods. Sonderia aposinuata sp. nov. is diagnosed mainly by having a relatively large body size, a crescent-shaped oral opening, numerous slender extrusomes, one suture on the ventral side and two on the dorsal side, and a buccal cavity that occupies the anterior third of the cell. Sonderia paramacrochilus sp. nov. closely resembles S. macrochilus but differs mainly by its oral opening being located closer to the anterior cell margin and its spindle-shaped extrusomes. Sonderia steini sp. nov. is a freshwater species that can be recognized by its shallow buccal cavity, sparsely distributed rod-shaped extrusomes, and having 68–79 monokinetidal somatic kineties that form sutures on both sides of the body. Phylogenetic analyses based on small subunit ribosomal RNA (SSU rRNA) gene sequence data support the monophyly of the family Sonderiidae, however, Sonderia is paraphyletic. The genus Sonderia is briefly revised and a key to the identification of species belonging to this genus is supplied.
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Phylogeny of the anaerobic ciliate genus Sonderia (Protista: Ciliophora: Plagiopylea), including the description of three novel species and a brief revision of the genus

    Corresponding author: Alan Warren, a.warren@nhm.ac.uk
    Corresponding author: Xiaozhong Hu, xiaozhonghu@ouc.edu.cn
  • 1. College of Fisheries, & Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, 266003, China
  • 2. Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao, 266003, China
  • 3. Zoology Department, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia
  • 4. Department of Life Sciences, Natural History Museum, London, SW7 5BD, UK

Abstract: Anaerobic protists in general, and ciliates in particular, are important components of anoxic or hypoxic environments, however, their diversity remains underestimated. Sonderia is a poorly studied genus that is distributed worldwide and is commonly found in anaerobic environments. In the present study, the taxonomy and phylogeny of three new species, namely Sonderia aposinuata sp. nov., Sonderia paramacrochilus sp. nov. and Sonderia steini sp. nov., collected from China, were investigated based on microscopic observations and SSU rRNA gene sequencing methods. Sonderia aposinuata sp. nov. is diagnosed mainly by having a relatively large body size, a crescent-shaped oral opening, numerous slender extrusomes, one suture on the ventral side and two on the dorsal side, and a buccal cavity that occupies the anterior third of the cell. Sonderia paramacrochilus sp. nov. closely resembles S. macrochilus but differs mainly by its oral opening being located closer to the anterior cell margin and its spindle-shaped extrusomes. Sonderia steini sp. nov. is a freshwater species that can be recognized by its shallow buccal cavity, sparsely distributed rod-shaped extrusomes, and having 68–79 monokinetidal somatic kineties that form sutures on both sides of the body. Phylogenetic analyses based on small subunit ribosomal RNA (SSU rRNA) gene sequence data support the monophyly of the family Sonderiidae, however, Sonderia is paraphyletic. The genus Sonderia is briefly revised and a key to the identification of species belonging to this genus is supplied.

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Introduction
Results
  • Class: Plagiopylea Small & Lynn, 1985.

    Order: Plagiopylida Jankowski, 1978.

    Family: Sonderiidae Small & Lynn, 1985.

    Genus: Sonderia Kahl, 1928

  • Sonderia aposinuata sp. nov. (Figs. 2AI, 3AL; Table 1)

  • Figure 2.  Sonderia aposinuata sp. nov. from life (A, B, EG) and after protargol staining (C, D, H, I). A Ventral view of a representative individual. B Distribution of bacteria between somatic kineties. C Details of postbuccal polykineties, arrowhead shows several shorter kineties located left of lower rightmost kineties, arrow shows gap. D Details of prebuccal polykineties, arrowheads indicate kinetids left of posterior ends of prebuccal polykineties, double arrowheads mark oral fibers, arrow shows gap. E Distribution of slender extrusomes. F Right lateral view showing dorsoventrally flattened body and buccal cavity. G Different body shapes. H, I Ventral (H) and dorsal (I) views of the holotype to show the somatic kineties, buccal cavity and macronucleus, arrowheads show sutures on both sides. Ba bacteria, Ex extrusome, Mi micronucleus. Scale bars: 100 μm (A, E, F, G, H, I)

    Figure 3.  Photomicrographs of Sonderia aposinuata sp. nov. from life (AG) and after protargol staining (HI). A, B Ventral view of two individuals with ingested algae, arrowhead indicates the layer of bacteria on cell surface. C Detail of posterior region of oral opening showing bacteria on cell surface. D Detailed view of the posterior end of cell, showing cytoplasmic inclusions (arrow) and bacterial layer (arrowhead). E Details of a crushed cell, showing the rod-shaped bacteria on cell surface (arrowhead). F Right lateral view of a cell. G Detail of cell showing numerous extrusomes in cytoplasm. H Anterior part of cell showing macronucleus with numerous nucleoli. I Oral region, showing postbuccal polykineties, arrowhead marks gap, arrow indicates postbuccal kineties with shorter kinety adjacent to. J Oral region, showing prebuccal polykineties, arrowhead marks gap, double-arrowheads indicate oral fibers. K Ventral view of posterior end to show suture (arrowheads). L Dorsal view of posterior end of the holotype to show sutures (arrowheads). Ma macronucleus. Scale bars: 90 μm (A, B, F)

    Charactera Species Min Max Mean M SD CV n
    Body, length S. aposinuata 150.0 233.0 188.7 190.0 19.7 10.3 22
    S. paramacrochilus 75.0 94.0 85.2 86.0 5.4 6.2 18
    S. steini 84.0 107.0 93.6 93 6.6 7.1 15
    Body, width S. aposinuata 54.0 96.0 77.7 78.0 11.0 14.1 22
    S. paramacrochilus 37.0 49.0 43.0 43.0 3.8 8.8 18
    S. steini 43.0 59.0 49.1 50 4.0 8.0 15
    Body length: width, ratio S. aposinuata 2.0 3.3 2.5 2.4 0.3 13.3 22
    S. paramacrochilus 1.8 2.3 2.0 2.0 0.1 7.0 18
    S. steini 1.6 2.1 1.9 2 0.1 6.9 15
    Macronucleus, length S. aposinuata 32.0 63.0 42.8 39.5 7.8 19.9 18
    S. paramacrochilus 20.0 31.0 25.5 26 3.1 12.0 18
    S. steini 19.0 29.0 23.8 24 2.4 9.9 15
    Macronucleus, width S. aposinuata 19.0 39.0 29.7 29.5 4.4 15.0 18
    S. paramacrochilus 19.0 30.0 24.3 24.5 3.4 13.7 18
    S. steini 16.0 25.0 20.7 20 3.1 15.4 15
    Prebuccal polykineties, number S. aposinuata 30.0 35.0 32.0 32.0 1.3 4.2 20
    S. paramacrochilus 19.0 22.0 20.2 20.0 0.8 4.1 18
    S. steini 27.0 31.0 28.8 29 1.4 4.9 13
    Postbuccal polykineties, number S. aposinuata 26.0 30.0 27.6 27.5 1.3 4.7 20
    S. paramacrochilus 16.0 21.0 17.8 18.0 1.1 6.0 18
    S. steini
    Ventro-frontal kineties, number S. aposinuata 25.0 31.0 27.9 28 1.6 5.8 17
    S. paramacrochilus 20.0 24.0 22.4 23.0 1.4 6.1 8
    S. steini 29.0 34.0 30.9 30 1.6 5.4 15
    Ventro-lateral kineties, number S. aposinuata 5.0 8.0 6.9 7 1.1 15.4 17
    S. paramacrochilus 3.0 4.0 3.4 3.0 0.5 16.6 8
    S. steini 5.0 7.0 6.2 6 0.8 13.9 15
    Ventral kineties, number S. aposinuata 31.0 38.0 34.9 35 2.1 5.9 17
    S. paramacrochilus 23.0 28.0 25.4 26.0 1.3 5.0 18
    S. steini 34.0 40.0 37.1 37 1.9 5.1 15
    Mid-dorsal kineties, number S. aposinuata 14.0 24.0 19.0 19 2.1 11.1 17
    S. paramacrochilus
    S. steini
    Dorso-right (Dorso-lateral) kineties, number S. aposinuata 5.0 12.0 9.2 9 1.6 17.9 17
    S. paramacrochilus 7.0 10.0 8.3 8 0.9 11.1 17
    S. steini 10.0 14.0 11.6 11.5 1.2 10.2 14
    Dorso-left kineties, number S. aposinuata 6.0 10.0 8.2 8 1.2 15.0 17
    S. paramacrochilus
    S. steini
    Dorso-frontal kineties, number S. aposinuata
    S. paramacrochilus 16.0 21.0 18.1 18 1.6 8.7 16
    S. steini 20.0 28.0 23.8 24 1.9 8.1 14
    Dorsal kineties, number S. aposinuata 33.0 42.0 36.4 36 2.2 6.0 17
    S. paramacrochilus 23.0 31.0 26.4 26 2.0 7.6 17
    S. steini 30.0 38.0 33.4 33 2.4 7.2 14
    Somatic kineties, number S. aposinuata 68.0 76.0 71.3 71 2.4 3.3 17
    S. paramacrochilus 49.0 57.0 51.8 52 2.0 3.9 17
    S. steini 66.0 78.0 70.5 70 2.8 4.0 14
    Disconnected prebuccal polykineties, number S. aposinuata 19.0 24.0 21.9 22 1.3 5.8 17
    S. paramacrochilus
    S. steini
    Gap of prebuccal polykineties, width S. aposinuata 5.8 9.5 7.1 6.7 1.1 17.1 18
    S. paramacrochilus
    S. steini
    Disconnected postbuccal polykineties, number S. aposinuata 23.0 29.0 24.9 24 1.9 8.0 17
    S. paramacrochilus
    S. steini
    Apex of cell to distal end of buccal cavity, distance S. aposinuata 68.0 99.0 76.8 75.0 8.2 10.9 18
    S. paramacrochilus 29.0 39.0 34.0 34.0 2.7 8.0 18
    S. steini 34.0 45.0 39.2 39.0 3.3 8.4 12
    CV coefficient of variation in %, M median, Max maximum, Min minimum, Mean arithmetic mean, n number of specimens examined, SD standard deviation
    aData are based on protargol-stained specimens. Measurements in μm

    Table 1.  Morphometric characteristics of Sonderia aposinuata sp. nov., Sonderia paramacrochilus sp. nov., Sonderia steini sp. nov

    Diagnosis. Body size about 180–250 × 60–90 μm in vivo with ratio of length to width about 2.3–2.8:1. About 71 somatic kineties, mainly composed of dikinetids but also with some monokinetids and trikinetids, forming one suture on ventral side and two sutures on dorsal side. 30–35 prebuccal and 26–30 postbuccal polykineties. Oral opening crescent-shaped, subapical, almost as wide as cell. Buccal cavity about 33% of cell length. Numerous slender, needle-shaped extrusomes, 22–46 µm long. Brackish or sea water biotope.

    Type locality. Sludge in intertidal zone at Liya Hill, Nantong, China (32°11′N; 121°52′E).

    Deposition of slides. The protargol slide (No. LR2020102305-01) with the holotype specimen (Figs. 2H, I, 3L) and several paratype specimens, and another slide (No. LR2020102305-02) with paratype specimens, were deposited in the Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China.

    Etymology. The species-group name aposinuata is a combination of the Greek prefix apo- (derived from) and the species-group name sinuata, which alludes to the similarity between this species and S. sinuata.

    Zoobank registration. Zoobank number of present work: urn: lsid: zoobank.org: pub: C1107D8B-C960-4F29-B632-4183735A4812. Zoobank number of Sonderia aposinuata sp. nov.: urn: lsid: zoobank.org: act: FF549A18-3E69-4C6B-846A-13A2E8DC34E2.

    Description. Body size about 180–250 × 60–90 μm in vivo. Ratio of length to width about 2.3–2.8:1 in vivo and 2–3:1 in protargol-stained individuals. Cell rigid, non-contractile, dorsoventrally flattened, elliptical in outline (Figs. 2A, F, G, 3A, B, F). Cell surface covered with a layer of rod-shaped bacteria arranged between kineties (Figs. 2B, 3AE). Endoplasm contains numerous algae of different kinds (Figs. 2A, 3A, B, D). Neither contractile vacuole nor striated band observed. Numerous slender, needle-shaped extrusomes, 22–46 µm long, distributed mainly around oral opening and randomly in endoplasm (Figs. 2E, 3G). Macronucleus about 30–60 × 20–40 µm in vivo, globular to ellipsoidal, located on right side of cell near buccal cavity, with many spherical nucleoli (Figs. 2A, G, H, 3HJ). Single globular micronucleus about 5 µm across, closely associated with macronucleus but usually not easy to observe due to abundant cell contents (Fig. 2A). Locomotion usually by swimming slowly, sometimes by crawling on debris.

    Somatic cilia 11–15 μm long, uniformly and densely distributed, arranged in 68–76 kineties (31–38 on ventral side and 33–42 on dorsal side), mainly composed of dikinetids, but also with some monokinetids and trikinetids (Figs. 2H, I, 3H, K, L). Ventral kineties of two types: 25–31 ventro-frontal kineties; 5–8 ventro-lateral kineties that form a suture at lower left side of cell (Figs. 2A, H, 3K). 33–42 dorsal kineties of three types, i.e., 6–10 dorso-left kineties, 5–12 dorso-right kineties and 14–24 mid-dorsal kineties, forming two sutures in subterminal region of cell (Figs. 2I, 3L).

    Oral opening crescent-shaped, subapically located, almost as wide as cell, parallel to anterior cell margin (Figs. 2A, E, G, H, 3A, H, I, J). Buccal cavity funnel-like, extending towards lower left, length usually about 33% of cell length, about 40% of cell length in smaller cells (Figs. 2G, H, 3A, B). Oral ciliature denser than somatic ciliature, with 30–35 prebuccal and 26–30 postbuccal polykineties (Figs. 2C, D, 3I, J). About 10 continuous prebuccal polykineties on right side, remaining 19–24 prebuccal polykineties interrupted by a gap about 6–10 μm wide. 13–18 prebuccal polykineties on left side with posteriormost 1–4 kinetids turned leftwards (Fig. 2D). Row of oral fibers present at bottom of prebuccal polykineties, about half width of buccal cavity (Figs. 2D, 3J). 23–29 postbuccal polykineties, interrupted by a relatively narrowed gap (Figs. 2C, 3I). A shorter kinety is present adjacent to and left of each of the 4–6 rightmost lower postbuccal kineties (Figs. 2C, 3I).

  • Sonderia paramacrochilus sp. nov. (Figs. 4AH, 5AO; Table 1)

  • Figure 4.  Sonderia paramacrochilus sp. nov. from life (AD) and after protargol staining (EH). A Ventral view of a representative individual. B Distribution of extrusomes. C Extruded extrusomes. D Different body shapes. E, F Details of prebuccal (E) and postbuccal (F) polykineties. G, H Ventral (G) and dorsal (H) views of the holoype showing somatic kineties and oral region. Arrowheads show sutures on both sides. Scale bars: 40 μm (A, B, D, G, H), 15 μm (C)

    Figure 5.  Photomicrographs of Sonderia paramacrochilus sp. nov. from life (A–G, L–O) and after protargol staining (H–K). A Ventral view of a representative individual showing body shape. B, C Oral region, showing the oral opening (B) and buccal cavity (C). D Part of striated band on dorsal side (arrowhead). E Rod-shaped bacteria on surface. F Details of posterior end of cell, arrowhead indicates caudal cilia. G Ventral view of a slightly compressed cell showing macronucleus. H, I Ventral (H) and dorsal (I) views of the holotype showing somatic kineties and sutures (arrowheads). J, K Details of oral ciliature, showing postbuccal (J) and prebuccal (K) polykineties, arrowhead indicates four or five prebuccal polykineties on right side curving rightwards at their posterior end. L Details of bacterial layer. M Spindle-like extrusomes (arrowheads). N, O Compressed (N) and uncompressed (O) cells showing fibers (arrowheads). Ma macronucleus. Scale bars: 40 μm (A, G), 30 μm (H, I)

    Diagnosis. Body size about 80–125 × 45–60 μm in vivo. Ratio of length to width about 1.8–2.2:1. 49–57 somatic kineties mainly composed of dikinetids, forming one suture on lower left of ventral side and one on dorsal side. 19–22 prebuccal and 16–21 postbuccal polykineties. Oral opening belt-shaped, subapical, extending almost to left body margin. Region above oral opening about 5–7% of cell length. Buccal cavity 38–43% of cell length. Extrusomes spindle-shaped, 7–16 µm long, randomly distributed. Multiple caudal cilia. Brackish or seawater biotope.

    Type locality. Sludge in intertidal zone at Liya Hill, Nantong, China (32°11′N; 121°52′E).

    Deposition of slides. The protargol slide (No. LR2020102301-01) with the holotype specimen (Figs. 4G, H, 5H, I) and several paratype specimens, and another slide (No. LR2020102301-02) with paratype specimens, are deposited in the Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China.

    Etymology. The species-group name "paramacrochilus" is composed of the Greek prefix "para-" (beside) and the species name "macrochilus" meaning a ciliate similar to S. macrochilus.

    Zoobank registration. Zoobank number of Sonderia paramacrochilus sp. nov.: urn: lsid: zoobank.org: act: D5818AC0-2E89-48C9-870E-ADAC8B73775C.

    Description. Body size about 80–125 × 45–60 μm in vivo, 75–94 × 37–49 μm after fixation, ratio of length to width about 1.8–2.2:1 in both living and protargol-stained specimens. Cell non-contractile, slightly dorsoventrally flattened, ellipsoidal in shape (Figs. 4A, 5A, G). Well-fed individuals with numerous ingested algae may be ovoidal due to posterior end becoming broadly rounded (Fig. 4D). A layer of rod-shaped bacteria covers cell surface (Figs. 4A, 5E, L). One globular to slightly ellipsoidal macronucleus, about 23–35 × 19–33 µm in vivo, usually located on right side of cell just below buccal cavity (Figs. 4A, 5A, C, G). Rarely, macronucleus is located on left side or in posterior region of cell (Fig. 4D). Micronucleus not observed in either living or protargol-stained specimens. Most cells with numerous variable-sized food vacuoles containing algae (Figs. 4A, 5A). Extrusomes 7–16 µm long, spindle-shaped, randomly distributed (Figs. 4B, 5M). Extruded extrusomes needle-like, approximately 30 µm long (Fig. 4C). Striated band about 2 µm wide, located at right margin of cell, commencing below oral opening and extending to dorsal side along right cell margin (Fig. 5D). Locomotion by swimming slowly or by crawling on substrate such as organic debris.

    Somatic cilia approximately 7–13 µm long, densely arranged (Figs. 4A, 5A, G). 49–57 somatic kineties (23–28 on ventral side and 23–31 dorsal side) mainly composed of dikinetids (Figs. 4G, H, 5H, I). Ventral kineties comprise 20–24 ventro-frontal kineties and 3–4 ventro-lateral kineties that form a suture at lower left side of cell (Figs. 4G, 5H), difficult to observe in some individuals due to small number of ventro-lateral kineties and angle of observation. 16–21 dorso-frontal kineties and 7–10 dorso-lateral kineties forming a suture on dorsal side (Figs. 4H, 5I). Multiple caudal cilia, 8–10 μm long, located along left posterior margin of cell (Figs. 4A, 5A, F).

    Oral opening belt-shaped, about 50–60% of cell width, subapically located, slightly tilted to left posterior, extending almost to left cell margin (Figs. 4A, B, D, G, 5A, B, H). Distance from anterior end of cell to beginning of oral opening about 5–7% of cell length (Figs. 4A, D, 5A). Buccal cavity pocket-like, wide, about 10–11 μm high in mid-region, extending posteriorly on left and right sides (Figs. 4A, D, 5A, C, G). Distance from cell apex to posterior end of buccal cavity about 38–43% of cell length (Figs. 4A, D, 5A). Right edge of buccal cavity supported by about four fibers, usually only visible in compressed cells (Fig. 5N). In uncompressed cells, only two fibers are visible, each with a crutch-like head (Fig. 5O). Oral ciliature composed of 19–22 prebuccal and 16–21 postbuccal polykineties (Figs. 4E, F, 5J, K). Posterior ends of four or five rightmost prebuccal polykineties curve rightwards, remaining prebuccal kineties extend towards lower left (Figs. 4E, 5K). Kinetosomes of postbuccal polykineties somewhat staggered (Figs. 4F, 5J).

  • Sonderia steini sp. nov. (Figs. 6AJ, 7AN; Table 1)

  • Figure 6.  Sonderia steini sp. nov. from life (A–E) and after protargol staining (F–J). A Ventral view of a representative individual. B Different body shapes and variable location of macronucleus. C Lateral view of a specimen showing elongated ellipsoidal macronucleus. D, E Resting (D) and extruded (E) extrusomes. F, G Details of prebuccal (F) and postbuccal (G) polykineties. H Macronucleus and micronucleus. I, J Ventral (I) and dorsal (J) views of the holotype specimen showing somatic kineties and oral region, arrowheads indicate sutures, arrow shows pore of contractile vacuole. Scale bars: 60 μm (A, C, I, J), 5 μm (D), 30 μm (E)

    Figure 7.  Photomicrographs of Sonderia steini sp. nov. from life (A–D, H–L) and after protargol staining (E–G, M, N). A–C Ventral (A, B) and right lateral (C) views of different individuals, showing the variation in body shape, arrowheads show macronucleus. D Oral region, showing the oral opening (arrowhead). E Macronucleus and slightly enveloped micronucleus (arrowhead). F Oral ciliature, showing prebuccal polykineties, arrowhead marks several discontinuous polykineties curving rightwards at their posterior end. G Oral ciliature, showing postbuccal polykineties, arrowhead shows extrusome. H Ventral view of a slightly compressed cell, arrowhead indicates bacterial layer. I Oral region, showing the buccal cavity (arrowhead). J Right-anterior region of cell showing crutch-like fibers (arrowhead). K Rod-shaped extrusomes (arrowheads). L Right margin of cell showing striated band (arrowheads). M, N Ventral (M) and dorsal (N) views of the holotype specimen showing the somatic kineties and sutures (arrowheads). Ma macronucleus. Scale bars: 60 μm (A–C, F–H, M, N)

    Diagnosis. Body roughly ellipsoidal, right side almost straight. Cell size about 115–155 × 60–90 μm in vivo. Ratio of length to width about 1.8–2.1:1. Extrusomes rod-shaped, 4–8 µm long, distributed randomly beneath pellicle. 68–79 monokinetidal somatic kineties forming one suture on lower left ventral side and one on dorsal side. Oral opening belt-shaped, distance from apex of cell to beginning of oral opening about 7–10% of cell length. Buccal cavity shallow on right, about 7–13 µm high in mid-region, 37–43% of cell length. 27–31 prebuccal polykineties. Postbuccal polykineties arranged in no discernable pattern. Freshwater habitat.

    Type locality. A freshwater pond in Baihuayuan Garden, Qingdao, China (36°07′N; 120°34′E).

    Deposition of slides. The protargol slide (No. LR20200619-01) with the holotype specimen (Figs. 6I, J, 7M, N) and several paratype specimens, and another slide (No. LR2020061901-02) with paratype specimens, are deposited in the Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China.

    Etymology. We dedicate this new species to the Czech protozoologist Prof. Friedrich Stein in recognition of his contributions to the taxonomy of ciliates.

    Zoobank registration. Zoobank number of Sonderia steini sp. nov.: urn: lsid: zoobank.org: act: E7D2A48B-BA49-424A-B104-CDBB73987C02.

    Description. Body size about 115–155 × 60–90 µm in vivo, ratio of length to width about 1.8–2.1:1. Cell outline approximately elliptical, right side almost straight (Figs. 6A, 7A, B). Body non-contractile, dorsoventrally flattened about 3:5 (Figs. 6C, 7C). A layer of rod-shaped bacteria covers cell surface (Figs. 6A, 7H). Macronucleus about 20–30 × 15–30 µm in vivo, ellipsoidal in shape, elongate-ellipsoidal in lateral view (Figs. 6B, C, H, 7A, C, EG). Position of macronucleus variable, usually in mid-region of cell but in either anterior or posterior part of cell in some individuals (Figs. 6B, 7A, F, G). Single globular micronucleus, about 2–4 µm in diameter in stained specimens, slightly enveloped by macronucleus (Figs. 6H, 7E). Numerous food vacuoles containing ingested algae, slender algae distributed almost throughout cell endoplasm in some individuals (Figs. 6A, 7AC, H). Extrusomes 4–8 µm long, rod-shaped, distributed randomly in endoplasm (Figs. 6D, 7G, K). Extruded extrusomes needle-like, about 30 µm long (Fig. 6E). Contractile vacuole (CV) close to posterior end of cell, difficult to observe because of abundant inclusions; CV pore present on dorsal side (Fig. 6J). Striated band located at right margin of cell, about 3 µm wide, commencing just below oral opening (Fig. 7L). Locomotion by swimming slowly while rotating about main body axis.

    Body densely ciliated, somatic cilia about 10–14 µm long; 68–79 monokinetidal somatic kineties in total, 34–40 on ventral side, 32–39 dorsal side (Figs. 6A, I, J, 7M, N). Kinetosomes of each kinety densely arranged (Figs. 6I, J, 7M, N). About 29–34 ventro-frontal kineties and 5–7 ventro-lateral kineties, forming conspicuous suture at lower left side of cell (Figs. 6A, I, J, 7M, N). 18–27 dorso-frontal kineties and 10–14 dorso-lateral kineties forming posterior suture on dorsal side (Figs. 6J, 7N).

    Belt-shaped oral opening, subapically located, slightly tilted to left posterior and terminating close to left margin of cell (Figs. 6A, B, I, 7A, B, D, M). Distance from apex of cell to beginning of oral opening about 7–10% of cell length (Figs. 6A, B, I, 7A, B, M). Buccal cavity pocket-like, narrow, about 7–13 µm high in mid-region, extending to about 37–43% of cell length (Figs. 6A, B, I, 7A, I). Right margin of buccal cavity supported by crutch-like fibers (Figs. 6A, 7J). Oral ciliature more densely arranged than somatic ciliature (Figs. 6A, B, I, 7A, I). About 27–31 prebuccal polykineties extending to lower left side apart from four or five rightmost polykineties the posterior ends of which turn rightwards (Figs. 6F, 7F). Several postbuccal polykineties on right side arranged without discernable pattern around an arc-like intrusion and, therefore, difficult to enumerate (Figs. 6G, 7G).

Molecular data and phylogenetic analyses (Fig. 8)
  • Figure 8.  Maximum likelihood (ML) tree based on the small subunit rRNA gene sequences showing the positions of the three new sequences in the present study (red font). Numbers near nodes represent bootstrap values of ML and posterior probabilities of Bayesian inference (BI), respectively. Black circles indicate full support (ML/BI, 100/1.00) in both analyses. Disagreements between ML and BI are shown by asterisks. The scale bar corresponds to two substitution per 100 nucleotide positions

    The three new SSU rRNA gene sequences were deposited in GenBank with accession numbers, length and GC contents as follows: Sonderia aposinuata sp. nov (OL986005, 1716 bp, 43.65%), Sonderia paramacrochilus sp. nov. (OL986006, 1727 bp, 43.31%), Sonderia steini sp. nov. (OL986007, 1712 bp, 43.22%). The maximum likelihood (ML) and Bayesian inference (BI) analyses based on SSU rRNA gene sequence data generated phylogenetic trees with similar topologies, therefore only the ML tree is shown here with nodal support provided from both algorithms (Fig. 8). Sonderiidae groups with Plagiopylidae with maximal support, which together cluster with Trimyemidae. Epalxellidae occupies the basal position within the class Plagiopylea. Within Sonderiidae there are three main clades (Clade Ⅰ, Clade Ⅱ, and Clade Ⅲ). Clade Ⅱ (ML/BI, 95/1.00) and Clade Ⅲ (ML/BI, 100/1.00) cluster together to form a group that is sister to Clade Ⅰ (ML/BI, 70/1.00). Clade Ⅰ comprises Parasonderia vestita and two uncultured taxa (KT346294, AB505461). In Clade Ⅱ, S. paramacrochilus sp. nov. and S. steini sp. nov. group together (sequence similarity 94.2%) forming a fully supported branch that is sister to a subclade comprising Sonderia aposinuata sp. nov. and two unidentified Sonderia species (MK543437, MK543438). Clade Ⅲ comprises Sonderia vorax and three uncultured taxa (EF526738, EF526757, EF526974) (Fig. 8).

Discussion
  • The three species studied in this work correspond well with the diagnostic characters of the genus Sonderia in having an ellipsoidal, dorsoventrally flattened body shape, dense ciliation, a subapical oral opening, a pocket-like buccal cavity, and a layer of bacteria covering the cell surface (Jankowski 2007; Kahl 1928, 1931; Modeo et al. 2013). They can be easily excluded from closely related genera such as Plagiopyla Stein, 1860, which has a slit-like oral opening that is well-separated from the cell apex and a tube-like buccal cavity (Kahl 1928; Nitla et al. 2019), and Parasonderia Jankowski, 2007, which has an oval to circular oral opening (Fauré-Fremiet 1973; Jankowski 2007; Kahl 1931; Xu et al. 2013).

Comparison of Sonderia aposinuata sp. nov. with its congeners
  • Sonderia aposinuata sp. nov. is most similar to the original population of S. sinuata in having a relatively large body (180–250 μm vs. 240–250 μm in the latter), a crescent-shaped subapically located oral opening, and a ventral suture at the left posterior region of the cell (Kahl 1931). However, it can be separated from the latter by the shorter distance between the apical cell margin and the bottom of the buccal cavity relative to the body length (about 30–40% vs. 50% in S. sinuata), numerous slender extrusomes (vs. few in S. sinuata), the macronucleus and micronucleus located at the anterior third of the cell (vs. in the mid-region of the cell in S. sinuata), and the oval (vs. slightly oboval in S. sinuata based on the original illustration) body outline (Kahl 1931).

    Sonderia sinuata has been redescribed based on populations from Sweden (Dragesco 1968), America (Borror 1972), the Baltic Sea (Czapik and Jordan 1976), Benin (Dragesco and Dragesco-Keneis 1986), Spain (Sola et al. 1989) and the Red Sea (Al-Rasheid 2001). However, the identity of several of these populations are in doubt. Borror (1972) described an American population with an oral opening that is belt-shaped (vs. crescent-shaped) and is separated from the cell apex by a distance of approximately 13% of the body length (vs. 3% based on the original illustration) (Kahl 1931). The description of the French population is brief and mainly focuses on the habitat and mode of locomotion (Czapik and Jordan 1976). Furthermore, the cell length of the French population is only 140–150 μm (vs. 240–250 μm in the original population) (Czapik and Jordan 1976; Kahl 1931). Sola et al. (1989) reported a Spanish population of S. sinuata from sediments of a freshwater lagoon (vs. seawater habitat in the original description). The Red Sea population described by Al-Rasheid (2001) is much smaller than the original population (90–120 μm vs. 240–250 μm) and has a belt-shaped (vs. crescent-shaped) oral opening (Kahl 1931). These discrepancies suggest that these populations were probably misidentified.

    Other congeners can be distinguished from the new species by their much smaller cell size, belt-shaped oral opening, or the absence of ventral suture (Fauré-Fremiet and Tuffrau 1955; Jankowski 2007; Kahl 1931; Kirby 1934; Small and Lynn 1985).

Comparison of Sonderia paramacrochilus sp. nov. with its congeners
  • Among the eight species of Sonderia that were recognized before the present study, S. pharyngea, S. labiata and S. paralabiata can be clearly separated from S. paramacrochilus sp. nov. by the absence (vs. presence) of a ventral suture (Fauré-Fremiet and Tuffrau 1955; Jankowski 2007; Kahl 1931; Kirby 1934; Small and Lynn 1985). Sonderia vorax and S. sinuata can be distinguished from S. paramacrochilus sp. nov. by their crescent-shaped (vs. belt-shaped) oral opening (Kahl 1931; Modeo et al. 2013). Sonderia megalabiata differs from S. paramacrochilus sp. nov. in having significantly more somatic kineties (100–120 vs. 49–57) (Alekperov and Asadullayeva 1996). Sonderia schizostoma can be separated from S. paramacrochilus sp. nov. by: the oral opening extending to cell mid-line (vs. close to left cell margin in S. paramacrochilus) which is a diagnostic feature for species identification in Sonderia; the distance between the oral opening and the apical end of the cell relative to the cell length which is 5–7% in S. paramacrochilus (vs. 17% in S. schizostoma based on the original illustration); and the shape of the buccal cavity which extends posteriorly on both sides forming a convex curve on each, whereas in S. schizostoma the buccal cavity extends down the left side only with the right side flat (Kahl 1931).

    Sonderia macrochilus has not been recorded since Kahl's (1931) first report of a German population (salinity 15). Sonderia paramacrochilus sp. nov. resembles S. macrochilus in body size (80–125 μm vs. 130 μm), the shape and orientation of the oral opening (both have belt-shaped oral opening that extends close to the left cell margin), and the presence of a ventral suture on the lower left side, but differs in the distance above oral opening relative to the cell length (5–7% vs. 14% in Kahl's illustration), the height of the mid-region of the buccal cavity (10–11 μm vs. 18 μm), the presence (vs. absence) of spindle-shaped extrusomes, and the habitat (salinity 24 vs. 15) (Kahl 1931).

    Sonderia aposinuata sp. nov. is clearly different from S. paramacrochilus sp. nov. in having a larger body size (180–250 μm vs. 80–125 μm), a crescent-shaped oral opening (vs. belt-shaped in the latter), and two sutures on dorsal side (vs. one).

Comparison of Sonderia steini sp. nov. with its congeners
  • Considering its body shape and size, belt-shaped oral opening extending almost to the left cell margin, and the presence of a suture on both the ventral and the dorsal side, S. steini sp. nov. should be compared with S. macrochilus Kahl, 1930 and S. paramacrochilus sp. nov.

    Sonderia macrochilus can be separated from S. steini sp. nov. by its habitat (brackish water, salinity 15 vs. freshwater), the distance above the oral opening relative to the body length (14% in Kahl's illustration vs. 7–10%), the height of the mid-region of the buccal cavity (18 μm vs. 7–13 μm), the shape of the buccal cavity (right and left sides extending downward to about the same level vs. right side extending only slightly downwards), and the absence (vs. presence) of extrusomes (Kahl 1931).

    Sonderia paramacrochilus sp. nov. differs from S. steini sp. nov. in having fewer somatic kineties (49–57 vs. 68–79), the shape (spindle- vs. rod-shaped) and length (7–16 μm vs. 4–8 μm) of the extrusomes, the degree of inclination of several rightmost prebuccal polykineties (angled slightly to left vs. sharply to left), and the habitat (brackish water vs. freshwater).

Phylogenetic analyses
  • In the SSU rRNA gene tree, each family within the class Plagiopylea is monophyletic, which is consistent with most previous studies (Fernandes et al. 2018; Modeo et al. 2013; Nitla et al. 2019; Xu et al. 2013). However, Li et al. (2021a) reported that the family Sonderiidae is paraphyletic, because two gene sequences of Sonderia, both lacking morphological information or vouchered specimens (MK543438, MK543437), branch off basally within the Sonderiidae-Plagiopylidae assemblage. Members of Trimyemidae form a fully supported clade that is sister to the Sonderiidae + Plagiopylidae clade, and Epalxellidae is the basal lineage within the class Plagiopylea, which is consistent with previous analyses (Fernandes et al. 2018; Modeo et al. 2013; Nitla et al. 2019; Xu et al. 2013).

    The family Sonderiidae consists of three main clades. Clade Ⅰ comprises two uncultured taxa (KT346294, AB505461) collected from cold-seep sediments (Japan) and the deep sea (USA), respectively (Pasulka et al. 2016; Takishita et al. 2010), that together form a sister group to Parasonderia vestita, which is consistent with the findings of Nitla et al. (2019). Clade Ⅱ comprises the three species investigated in the present study and two undefined species of Sonderia (MK543437, MK543437). Clade Ⅲ comprises Sonderia vorax, which lacks sutures on the dorsal surface, and three environmental sequences collected from the anoxic Framvaren Fjord (Norway) (Behnke et al. 2010). These findings suggest that the genus Sonderia is non-monophyletic. When considering the morphology of the members of these three clades, it is noteworthy that the somatic kineties of three new species in Clade Ⅱ all form sutures on both sides of the cell. Sonderia paramacrochilus sp. nov. and S. steini sp. nov. have one suture on the dorsal side while S. aposinuata sp. nov. has two on the dorsal side. Furthermore, S. paramacrochilus and S. steini both have a belt-shaped oral opening, whereas the oral opening of S. aposinuata is crescent-shaped which is consistent with the branching pattern within this clade. Among the three sonderiid clades, Clades Ⅱ and Ⅲ cluster together, with Clade Ⅰ as the outgroup, which is consistent with certain morphological features of their members, i.e., oral opening oval to circular in Parasonderia (Clade Ⅰ) vs. belt- or crescent-shaped in Sonderia (Clades Ⅱ and Ⅲ). Parasonderia vestita, the only available sequence in this genus, has a close relationship with Plagiopylidae according to Xu et al. (2013) but appeared to be more closely related to S. vorax after Modeo et al. (2013) acquired the first sequence of Sonderia. The addition of the new sequences here supports a clearer separation between Parasonderia and Sonderia than suggested in previous studies (Fernandes et al. 2018; Modeo et al. 2013; Nitla et al. 2019; Xu et al. 2013).

    Despite the addition of three new sequences, evolutionary relationships among species of Sonderia remain uncertain due to under-sampling and low nodal support in the phylogenetic tree. Furthermore, the relationships recovered to date are not strongly related to morphological or habitat characteristics. Therefore, more samples should be collected and more gene markers analyzed to clarify the systematics of Sonderia.

Generic revision

    Genus Sonderia Kahl, 1928

  • Improved diagnosis. Body ovoidal to ellipsoidal, dorsoventrally compressed about 1:2–4, non-contractile. A gelatinous layer is usually present on the cell surface within which bacteria are embedded. Oral opening narrow, subapical. Wide, pocket-like buccal cavity extending towards lower left of cell. Somatic kineties densely and uniformly arranged. Oral kineties are extensions of somatic kineties without any clear differentiation. Striated band located at right margin of cell, beginning below oral opening and extending onto dorsal side along right cell margin. One globular to ellipsoidal macronucleus and one globular micronucleus, usually compact type. Right edge of buccal cavity supported by 4 or 5 fibers.

    Type species. Sonderia vorax Kahl, 1928

Sonderia vorax Kahl, 1928
  • 1928 Sonderia vorax Kahl, Arch Hydrob, 19: 93–95, Fig. 20 (a).

    1931 Sonderia vorax Kahl, Tierwelt Dtl, 21:269, Fig. 45 (14).

    1933 Sonderia vorax Kahl, Lepzig: Akademische Verlagsgesellschaft Becker & Erler, 74, Fig. 9.9.

    1992 Sonderia vorax Carey, Marine Interstitial Ciliates: An Illustrated Key, 103–104; Fig. 359. (revision).

    2007 Sonderia vorax Jankowski, Russian Acad Sci, 789. (revision).

    2013 Sonderia vorax Modeo et al., BMC Microbiol, 13:1–23; Figs. 8–9.

    Modeo et al. (2013) redescribed this species in detail using modern methods, with the following diagnosis.

    Diagnosis. Body size about 130 × 69 μm in vivo. Body shape ovoidal to ellipsoidal with rounded ends. About 56 monokinetidal somatic kineties forming one suture on ventral side, including 17 ventro-frontal and 11 ventro-lateral kineties. Depth of buccal cavity never more than 33% of body length. 25–30 prebuccal and 18–20 postbuccal polykineties. Striated band commencing near right side of buccal cavity and terminating near cell posterior end. Numerous long needle-shaped extrusomes, a second smaller type visible only by TEM. Single contractile vacuole in posterior region on dorsal side of cell. One spherical macronucleus and one compact type micronucleus.

    Habitat. Seawater, brackish water and hypersaline water.

    Distribution. Found in seawater in Denmark (Fenchel et al. 1969, 1977), Black Sea (Azovsky and Mazei 2003), and White Sea (Esaulov et al. 2016). Found in brackish water in Germany (Kahl 1928, 1931), Baltic Sea (Fenchel and Jansson 1966; Mironova et al. 2009), Italy (Modeo et al. 2013; Mori et al. 1996, 1998), White Sea (Burkovsky and Mazei, 2012; Mazei and Burkovsky 2003, 2005), and France (Dietz 1964). Found in hypersaline water in Romania (Tucolesco 1961).

    Gene sequences. 18S rRNA gene, HF547270.

  • Remarks.

  • Resembles S. sinuata and S. labiata, but with a shorter buccal cavity.

Sonderia schizostoma Kahl, 1931
  • 1931 Sonderia schizostoma Kahl, Tierwelt Dtl, 21: 269, Fig. 45 (16).

    1933 Sonderia schizostoma Kahl, Lepzig: Akademische Verlagsgesellschaft Becker & Erler, 74, Fig. 9 (11).

    1992 Sonderia schizostoma Carey, Marine Interstitial Ciliates: An Illustrated Key, 103–104, Fig. 364. (revision).

    2007 Sonderia schizostoma, Jankowski, Russian Acad Sci, 790. (revision).

    Although Sonderia schizostoma has been known for a long time, only a schematic figure and a brief morphological description of living cells are available. Based on the published information, the diagnosis is provided as follows.

    Diagnosis. Body length about 140–150 μm in vivo, roughly ovoidal in shape. Oral opening belt-shaped, extending towards lower left, terminating approximately at mid-line of cell. Buccal cavity short and shallow, about 10 μm high in mid-region. Fibers at right edge of buccal cavity conspicuous. Extrusomes about 12 μm long, few in number. One macronucleus and one micronucleus. Caudal cilia present. Gelatinous layer conspicuous.

    Habitat. Seawater or brackish water.

    Distribution. Found in seawater in Germany (Kahl 1931, 1933), Denmark (Fenchel et al. 1969) and Black Sea (Azovsky and Mazei 2003). Found in brackish water of the Baltic Sea (Fenchel and Jansson 1966; Mironova et al. 2009).

    Gene sequences. Absent.

  • Remarks.

  • Kahl (1931) referred to this species as "Sonderia schizostoma Kahl, 1930". However, this appears to be an error since S. sinuata was first described in 1931 as no relevant articles were published in 1930. Furthermore, Kahl (1931) reported a small individual (85 μm) with an oral opening that extends leftwards beyond the cell mid-line. This individual resembles S. macrochilus, so we believe this was a misidentification.

Sonderia macrochilus Kahl, 1931
  • 1931 Sonderia macrochilus Kahl, Tierwelt Dtl, 21: 269, Fig. 45.17.

    1933 Sonderia macrochilus Kahl, Lepzig: Akademische Verlagsgesellschaft Becker & Erler, 74, Fig. 9 (12).

    2007 Sonderia macrochilus Jankowski, Russian Acad Sci, 789. (revision).

    Although Sonderia macrochilus has been known for a long time, only a schematic figure and a brief morphological description of living cells are available. Based on the available information, the diagnosis is provided as follows.

    Diagnosis. Body about 130 μm long in vivo, ellipsoidal in shape. Oral opening belt-shaped, extending towards lower left, terminating almost at left cell margin. Buccal cavity long and high, about 18 μm high in mid-region. Extrusomes absent. Ventral suture present, located close to lower left side of cell.

    Habitat. Seawater and brackish water.

    Distribution. Found in seawater in Black Sea (Azovsky and Mazei 2003) and Azerbaijan (Alekperov et al. 2017). Found in brackish water in Germany (Kahl 1931), Azerbaijan (Alekperov and Tahirova 2019; Mansimova and Alakbarov 2020), and Baltic Sea (Mironova et al. 2009).

    Gene sequences. Absent.

  • Remarks.

  • Kahl (1931) referred to this species as "Sonderia macrochilus Kahl, 1930". However, this appears to be an error, since S. macrochilus was first described in 1931 as no relevant articles were published in 1930. Furthermore, Kahl (1931) observed only five or six individuals and noted that S. macrochilus is probably often overlooked in favour of the more the numerous S. vorax.

Sonderia sinuata Kahl, 1931
  • 1931 Sonderia sinuata Kahl, Tierwelt Dtl, 21: 269, Fig. 45 (15).

    1933 Sonderia sinuata Kahl, Lepzig: Akademische Verlagsgesellschaft Becker & Erler, 74, Fig. 9 (8).

    1968 Sonderia sinuata Dragesco, 1: 81–85, Figs. 2 (4).

    1972 Sonderia sinuata? Borror, Acta Protozool, 10: 47–48, Figs. 27, 28, 30.

    1976 Sonderia sinuata? Czapik & Jordan, Acta Protozool, 15: 437.

    1986 Sonderia sinuata Dragesco & Dragesco-Kernéis, Faune Trop, 26: 220, Figs. 23 (f, g).

    1989 Sonderia sinuata? Sola et al., Arch Protistenkd, 137: 1–8, Figs. 8–11.

    1992 Sonderia sinuata Carey, Marine Interstitial Ciliates: An Illustrated Key, 103–104, Fig. 361. (revision).

    2001 Sonderia sinuata? Al-Rasheid, Trop Zool, 14: 138, Figs. 6 (51).

    2007 Sonderia sinuata Jankowski, Russian Acad Sci, 790, Fig. 355. (revision).

    Although Sonderia sinuata has been redescribed several times, the identities of some of these populations are in doubt. Therefore, the diagnosis provided here is based on the original description.

    Diagnosis. Body size relatively large and invariable, about 240–250 μm long. Elongate-ellipsoidal in shape, length to width ratio about 2.5:1. Oral opening crescent-shaped, wide, subapical. Buccal cavity funnel-shaped, extending to mid-region of cell. Extrusomes about 20 μm long, thick, sparsely distributed. Striated band present but difficult to observe. Single macronucleus.

    Habitat. Freshwater, brackish water, seawater and hypersaline water.

    Distribution. Found in freshwater in Spain (Sola et al. 1989). Found in seawater in Germany (Kahl 1931, 1933), Denmark (Fenchel et al. 1969), Black Sea (Azovsky and Mazei 2003), Sweden (Dragesco 1968), USA (Borror 1972), Baltic Sea (Czapik and Jordan 1976), and Azerbaijan (Alekperov et al. 2017). Found in brackish water in Azerbaijan (Alekperov and Tahirova 2019; Mansimova and Alakbarov 2020), Baltic Sea (Mironova et al. 2009), Sweden (Dragesco 1968), Benin (Dragesco and Dragesco-Kernéis 1986), and USA (Elliott and Bamforth 1975). Found in hypersaline water in Red Sea (Al-Rasheid 2001) and Romania (Tucolesco 1961).

    Gene sequences. Absent.

  • Remarks.

  • Kahl (1931) referred to this species as "Sonderia sinuata Kahl, 1930". However, this appears to be an error since S. sinuata was first described in 1931 as no relevant articles were published in 1930. Sonderia sinuata is the most commonly reported species of Sonderia. However, the populations from USA (Borror 1972), Baltic Sea (Czapik and Jordan 1976), Spain (Sola et al. 1989) and Red Sea (Al-Rasheid 2001) may be misidentified as they differ significantly from the original description of S. sinuata (Kahl 1931).

Sonderia pharyngea Kirby, 1934
  • 1934 Sonderia pharyngea Kirby, Archiv Protistenknd, 82: 116–119, Figs. 82, 5.

    1992 Sonderia pharyngea Carey, Marine Interstitial Ciliates: An Illustrated Key, 103–104, Fig. 363. (revision).

    2007 Sonderia pharyngea Jankowski, Russian Acad Sci, 789. (revision).

    Although Sonderia pharyngea has been known for a long time, only a schematic figure and a brief morphological description of living cells are available. Based on this information, the diagnosis is provided as follows.

    Diagnosis. Body size about 84–110 × 48–56 μm. Ovoidal to ellipsoidal in shape. Bristle-like caudal cilia. Oral opening belt-shaped. Buccal cavity tubular in shape, long, extending obliquely towards lower left, terminating near or beyond mid-body. One spherical macronucleus, usually in anterior region of cell. Single micronucleus located close to macronucleus. Few extrusomes scattered in cytoplasm, 7–9 μm long. No suture observed.

    Habitat. Hypersaline water and brackish water.

    Distribution. Found in hypersaline water in USA (Kirby 1934) and in brackish water in Baltic Sea (Mironova et al. 2009).

    Gene sequences. Absent.

  • Remarks.

  • The most distinctive feature of S. pharyngea is its buccal cavity which is tubular in shape and long, reaching or extending beyond the mid-region of the cell.

Sonderia labiata Fauré-Fremiet and Tuffrau, 1955
  • 1955 Sonderia labiata Fauré-Fremiet & Tuffrau, Hydrob, 7: 210–218, Figs. 85.

    1992 Sonderia labiata Carey, Marine Interstitial Ciliates: An Illustrated Key, 103, Fig. 360. (revision).

    2007 Sonderia labiata Jankowski, Russian Acad Sci, 789. (revision).

    Sonderia labiata was first recorded by Fauré-Fremiet and Tuffrau (1955) with a detailed description based on observations of live and silver-stained specimens.

    Diagnosis. Body ellipsoidal in shape, dorsoventrally flattened, 160–180 μm long, with length to width ratio about 2.5:1. Oral opening crescent-shaped, subapical. Buccal cavity extends to about mid-region of cell. 70–80 somatic kineties consisting of dikinetids or trikinetids. Dorsal kineties form a suture. Striated band about 1/2 of cell length. Cytoproct 10–40 μm long, located on right of striated band, terminating posteriorly near contractile vacuole pore. Numerous extrusomes, rod-shaped, approximately 20 μm long. Cytoplasm contains large lamellar storage granules of polysaccharide. One discoidal macronucleus located on right side of buccal cavity. Caudal cilia absent.

    Habitat. Brackish water (Fauré-Fremiet and Tuffrau 1955).

    Distribution. France (Fauré-Fremiet and Tuffrau 1955).

    Gene sequences. Absent.

Sonderia paralabiata Small and Lynn, 1985
  • 1985 Sonderia paralabiata Small & Lynn, An Illustrated Guide to the Protozoa, 564, Fig. 8b.

    2007 Sonderia paralabiata Jankowski, Russian Acad Sci, 789. (revision).

    Sonderia paralabiata is a rare species, and Small & Lynn (1985) provided only a brief morphological description of living and stained cells.

    Diagnosis. Body ellipsoidal in shape. Striated band located at right margin of cell. Macronucleus ovoidal. Somatic kinetids comprise groups of 3, 4 or 5 kinetosomes.

    Habitat. Seawater and brackish water.

    Distribution. Found in seawater in Azerbaijan (Alekperov et al. 2017) and in brackish water in Azerbaijan (Alekperov and Tahirova 2019; Mansimova and Alakbarov 2020).

    Gene sequences. Absent.

  • Remarks.

  • The original description supplied by Small & Lynn (1985) was only brief. Sonderia paralabiata resembles S. labiata after protargol staining.

  • Sonderia megalabiata Alekperov and Asadullayeva, 1996

  • 1996 Sonderia megalabiata, Alekperov & Asadullayeva, Zool Zh, 75: 768, Fig. 1.

    2007 Sonderia megalabiata, Jankowski, Russian Acad Sci, 789. (revision).

    Sonderia megalabiata is a rare species, and Alekperov and Asadullayeva (1996) supplied only a brief morphological description of both living and stained cells.

    Diagnosis. Body about 80–100 μm long, oval in outline. With a deep wedge-shaped incision at anterior end of cell forming two powerful lips and a deep shell-shaped mouth. 100–120 somatic kineties composed of dikinetids; kineties in posterior region forming a short suture. Endoplasm colorless and transparent.

    Habitat. Seawater, brackish water and hypersaline water.

    Distribution. Found in seawater in Azerbaijan (Alekperov and Asadullayeva 1996; Alekperov et al. 2017) brackish water in Azerbaijan (Alekperov and Tahirova 2019; Mansimova and Alakbarov 2020) and hypersaline water in Romania (Tucolesco 1961).

    Gene sequences. Absent.

Sonderia aposinuata sp. nov.
  • Diagnosis. See above.

    Type locality and distribution. See above.

    Gene sequences. See above.

Sonderia paramacrochilus sp. nov.
  • Diagnosis. See above.

    Type locality and distribution. See above.

    Gene sequences. See above.

Sonderia steini sp. nov.
  • Diagnosis. See above.

    Type locality and distribution. See above.

    Gene sequences. See above.

    Based on data available, a user-friendly guide to the identification of the 11 valid species of Sonderia is supplied here.

Materials and methods

    Sample collection and observation of morphology

  • Sonderia aposinuata sp. nov. and S. paramacrochilus sp. nov. were collected from black, sulfide-containing sludge from the intertidal zone at Liya Hill, Nantong, China (32°11′N; 121°52′E) on 23 October 2020 (Fig. 1). The water temperature was about 19 ℃ and the salinity 24. Sonderia steini sp. nov. was collected from a freshwater pond in Baihuayuan Garden, Qingdao, China (36°07′N; 120°34′E) on 19 June 2020, when the water temperature was 24 ℃. Samples were kept in anaerobic jars with oxygen-scavenging chemicals (Thermo Scientific Oxoid AnaeroGen) at room temperature (about 24 ℃). Autoclaved rice grains were added to promote the growth of bacterial food for the ciliates.

    Living cells were isolated with a micropipette and observed at 100–1000 × magnifications using a light microscope (Zeiss AXIO Imager. D2) equipped with differential interference contrast illumination. The infraciliature was revealed using the protargol staining method (Wilbert 1975). Counts, measurements, and drawings of living and stained specimens were from photomicrographs. Terminology is according to Modeo et al. (2013), Xu et al. (2013) and Lynn (2008).

DNA extraction, PCR amplification, and sequencing
  • For each species, one or two cells were picked out from original samples using micropipettes and washed five times in sterile-filtered in situ water before DNA extraction (Wang et al. 2021a, b). Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD) following the manufacturer's instructions (Wu et al. 2020). Q5 Hot Start high fidelity DNA polymerase (NEB, Ipswich, MA), and the PCR primers 18S F9 (5'-CTGGTTGATCCTGCCAG-3') and 18S R1513 Hypo (5'-TGATCCTTCYGCAGGTTC-3') (Nitla et al. 2019), were used to amplify the SSU rRNA gene with the cycling parameters according to Wang et al. (2020). PCR products were then sequenced bidirectionally by the Tsingke Biological Technology Company (Beijing, China).

Phylogenetic analyses
  • The SSU rRNA gene sequences of the three new species were aligned with another 57 sequences of plagiopyleans and prostomateans (outgroup: Prorodon teres X71140, Prorodon viridis U97111, Cryptocaryon irritans AF351579, Coleps spetai AM292312, Coleps hirtus KF177278, Levicoleps biwae AB354737, Tiarina fusa FJ858217, Apocoleps magnus FJ858213, Nolandia sp. FJ858215) retrieved from the GenBank database (for NCBI accession numbers, see Fig. 8) using MUSCLE on the webserver GUIDANCE (http://guidance.tau.ac.il/ver2/) with default options (Penn et al. 2010) and then manually modified by trimming both ends using BioEdit 7.0 (Hall 1999). The final alignment of 1, 685 positions was used to construct phylogenetic trees. Maximum likelihood (ML) analysis with 1, 000 bootstrap replicates was performed using RAxML-HPC2 on XSEDE 8.2.9 (Stamatakis 2014) on CIPRES Science Gateway (http://www.phylo.org) with GTRGAMMA as the optimal model selected by Modeltest v.3.4 (Posada and Crandall 1998). Bayesian inference (BI) analysis was carried out using MrBayes 3.2.6 on XSEDE (Ronquist et al. 2012) with the GTR + I + G model selected by Akaike Information Criterion in MrModeltest v2.2 (Nylander 2004). Markov chain Monte Carlo simulations were run for 1, 000, 000 generations with a sampling frequency of every 100 generations. The first 25% of trees were discarded as burn-in. MEGA 5.0 (Tamura et al. 2011) was used to view the tree topologies.

Terms
  • Ventro-lateral kineties: Several kineties on the left ventral side that turn rightwards in their posterior region, forming one or more sutures with the ventro-frontal kineties.

    Ventro-frontal kineties: Parallel ventral kineties that commence below the oral opening, extend to or almost to the posterior end of the cell, and are interrupted by ventro-lateral kineties.

    Dorso-right kineties (= Dorso-lateral kineties): Several right dorsal kineties that turn leftwards in their posterior region, forming one or more sutures with the mid-dorsal kineties or dorso-frontal kineties.

    Dorso-frontal kineties: Parallel dorsal kineties that extend to or almost to the posterior end of the cell and are interrupted by the dorso-right kineties.

    Dorso-left kineties (for individuals with two sutures): Left dorsal kineties that turn rightwards in their posterior region, forming a suture with the mid-dorsal kineties.

    Mid-dorsal kineties (for individuals with two sutures): Parallel dorsal kineties that terminate near the posterior end of the body and are interrupted by dorso-left and dorso-right kineties.

    Prebuccal polykineties: Extension of somatic kineties that are located at the upper wall of the buccal cavity into which they extend.

    Postbuccal polykineties: Extension of somatic kineties that are located at the lower wall of the buccal cavity into which they extend.

  • Acknowledgements

  • This work was supported by the National Natural Science Foundation of China (project number: 41976086) and the King Saud University, Saudi Arabia (Project No. RSP2022R7). We thank Prof. Weibo Song, Ocean University of China for his comments on the manuscript.

  • Author contributions

  • XH conceived and guided the study. RL, WZ, and XF organized sampling and performed experiments. RL identified the species, analyzed the phylogeny and wrote the original manuscript. AW, XH., WZ, XF and SAA reviewed and edited the manuscript. All authors read and approved the final version of the manuscript.

  • Data availability

  • All data generated or analyzed during this study are included in the manuscript and supporting files.

Declarations

    Conflict of interest

  • The authors declare that they have no conflict of interest. Author Alan Warren is one of the Editorial Board Members, but he was not involved in the journal's review of, or decision related to, this manuscript.

  • Animal and human rights statement

  • All applicable international, national, and institutional guidelines for the care and use of animals were followed by the authors.

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