Class: Litostomatea Small and Lynn, 1981
Subclass: Haptoria Corliss, 1974
Order: Pleurostomatida Schewiakoff, 1896
Family: incertae familiae
Genus: Pseudolitonotus gen. n.
Pleurostomatid in which the last left somatic kinety (LKn) is shortened and none of the right somatic kineties extend to the anterior end of the body.
Pseudolitonotus spirelis sp. n.
The genus name is a composite of the Greek prefix "Pseudo" (not genuine; sham) and the generic name Litonotus, referring to its similarity to the well-known genus Litonotus Wrzesniowski, 1870 in terms of its body shape and ciliary pattern. Masculine gender.
Pseudolitonotus spirelis sp. n. and Pseudolitonotus gracilis (Pan et al., 2015) comb. n.
This genus is distinguished from all known pleurostomatid genera by the shortened LKn and none of the right somatic kineties extending to the cell apex.
Figure 2. Pseudolitonotus spirelis gen. et sp. n. in vivo (A, D, I) and stained with protargol (B, C, E–H). A Right lateral view of a representative cell. B, C Ciliary patterns of right (B) and left (C) side of the holotype specimen, arrow indicates that the longest right somatic kinety does not extend to cell apex, asterisks mark the right somatic kineties in the leftmost region that are shortened along the perioral kineties, double arrowheads mark the shortening of the last left somatic kinety. D Extrusomes. E Ciliary pattern of anterior region of left side, arrowheads mark the right somatic kineties that do not extend to the cell apex, arrow shows the longest somatic kinety, double arrowheads point to the shortening of the last left somatic kinety. F–H Distribution of extrusomes, arrowheads show the "apical group". I Cortical granules. DB dorsal brush, LKn last left somatic kinety, LSK left somatic kinety, PK1 perioral kinety 1, PK2 perioral kinety 2, RKn1 last right somatic kinety, RKn2 penultimate right somatic kinety, RSK right somatic kinety. Scale bars: 50 μm in (A–C, F–H); 10 μm in (D, E, I)
Figure 3. Photomicrographs of Pseudolitonotus spirelis gen. et sp. n. in vivo (A–D, H, K, L) and stained with protargol (E–G*, I, J, M*). A Right lateral view of a representative cell, arrowhead marks the twisted portion at the bottom of the oral area. B, C Contracted (B) and extended (C) individuals, arrowheads point to the twisted portion at the bottom of the oral area, arrows indicate the contractile vacuole, double arrowhead marks the "apical group" of extrusomes. D Anterior part of cell, arrowhead shows the "apical group" of extrusomes. E Left view of anterior region of cell of the holotype specimen, to show perioral kinety 2 (arrow), the dorsal brush kinety (arrowhead) and the shortened somatic kineties (asterisks). G Left view of anterior region of cell, asterisks mark the shortened somatic kineties. H, I Nuclear apparatus, arrow shows the micronucleus. K, L To show the smaller (arrowhead) and larger (arrow) cortical granules. M Ciliary pattern, arrow points to the longest somatic kinety (RKn2). LKn last left somatic kinety, RKn1 last right somatic kinety, RKn2 penultimate right somatic kinety. Scale bars: 100 μm in (A–C, M); 20 μm in (D–J). *Images G and M were false-colored to reveal structures (Adobe Photoshop CS6)
Character H Min Max Mean SD n Body length 185 115 280 190.5 43.83 19 Body width 55 40 75 52.5 10.55 19 Number of RKa 13 11 15 12.2 1.31 19 Number of LKb 9 7 9 8.7 0.58 19 Number of Ma 2 2 2 2 0 19 Length of Ma 30 15 40 30.0 7.30 19 Width of Ma 10 8 20 12.4 3.50 19 Number of DB 61 46 78 59 8.53 19 Distance to RKn2c 35 25 60 36.5 9.26 19 Length of Ex 15 12 16 14.5 1.07 19 Length of Na 40 30 75 48.4 12.25 19 DB dorsal brush, Ex extrusomes, H holotype, LK left kineties, Ma macronuclear nodules, Max maximum, Mean arithmetic mean, Min minimum, n sample size, Na nematodesmata, RK right kineties, RKn2 penultimate right kinety, SD standard deviation
aPerioral kinety 2 included
bPerioral kinety 1 and dorsal brush kinety included
cDistance from anterior end to the penultimate right kinety
Table 1. Morphological characteristics of Pseudolitonotus spirelis gen. et sp. n. based on protargol impregnated specimens (all measurements in μm)
Pseudolitonotus about 160–350 µm in vivo; two macronuclear nodules; one micronucleus; 11–15 right and 7–9 left kineties; single contractile vacuole located subterminally; extrusomes bar-shaped, evenly spaced along entire ventral margin and some clustered together to form an "apical group" at anterior end of cell; bottom of oral slit invariably twisted; two types of cortical granules.
A mangrove wetland on Techeng Island in the city of Zhanjiang, Guangdong Province, China (21°09′12′′ N, 110°25′20′′ E). Water temperature 23.8 ℃, salinity 24‰, pH 7.0.
A protargol slide with the holotype specimen circled in ink (registration no. WL2012040602-01A), and a second protargol slide with several paratype specimens (registration no. WL2012040602-01B), are deposited in the Laboratory of Protozoology, Ocean University of China (OUC), China.
The Latin adjective spirel·is, -is, -e ([m, f, n]; twist) refers to the bottom of "neck" (oral slit) invariably having a twist.
Body size about 160–350 µm × 30–45 µm in vivo. Body shape Litonotus-like, i.e., slender lanceolate, contractile, with sharply pointed posterior end and a long conspicuous neck-like region that is about half cell-length when fully extended (Figs. 2A, 3A, B). Bottom of oral area twisted to right side in all specimens (n > 20), conspicuously twisted when cell contracts (Figs. 2A, 3A, B), and can be detected in protargol-stained specimens (Fig. 3F, J). Two ovoidal macronuclear nodules, each about 20–25 µm × 10–15 µm in vivo, located in mid-body region (Figs. 2F–H, 3H, I). Single ovoidal micronucleus, located between macronuclear nodules, about 2–3 µm across (Fig. 3H, I). One contractile vacuole, about 15–20 µm in diameter, subterminally located (Figs. 2A, 3A–C). Extrusomes bar-shaped, about 15 µm long and 0.2 µm wide in vivo, densely and evenly spaced along entire ventral margin, some clustered together to form a conspicuous "apical group" at anterior end of cell (Fig. 2A, F–H) that can be detected with DIC microscopy (Fig. 3C, D). Pellicle thin with inconspicuous longitudinal furrows on right side within which ciliary rows are located (Fig. 3A). Two kinds of cortical granules: type 1 dot-like, ca. 0.2 µm across, grayish, irregularly scattered between ciliary rows (Figs. 2I, 3K); type 2 globular, ca. 1–1.5 µm across, grayish, regularly arranged in a single line between adjacent ciliary rows (Figs. 2I, 3L). Right side densely ciliated with cilia ca. 8 µm long; left side sparsely ciliated. Cytoplasm colorless to pale yellow, often with numerous refringent globules ca. 2 µm across and several food vacuoles 3–5 µm across that renders main part of body opaque (Fig. 3A, B). Locomotion by swimming or by gliding on substrate.
Ciliary pattern as shown in Figs. 2B, C, E, 3E–G, J, M. Excluding perioral kinety 2 (PK2), ten to 14 right somatic kineties (Figs. 2B, C, 3F, J) none of which extend to cell apex; penultimate right somatic kinety (RKn2) is the longest of them (Figs. 2C, E, 3E, G, M); leftmost region of RKn2 shortened along PK2; rightmost region of RKn2 shortened along dorsal brush kinety (DB) (Figs. 2B, C, E, 3E–G, J). Left side with 7–9 ciliated kineties including perioral kinety 1 (PK1) and dorsal brush kinety (DB) which extends to about 40% of cell length and is composed of narrowly spaced dikinetids (Fig. 2B, C, E); last left somatic kinety (LKn) does not extend to cell apex and is shorter than last right somatic kinety (RKn1) (Figs. 2C, E, 3E, G).
Two perioral kineties along oral slit. Perioral kinety 1 left of oral slit, comprises dikinetids in anterior 40% and extends posteriorly as a row of monokinetids (Figs. 2B, 3F, J). Perioral kinety 2 right of oral slit, comprises regularly spaced dikinetids in anterior 40% and monokinetids in posterior 60% (Figs. 2B, 3F, J). Perioral kineties invariably twisted towards right side (Fig. 3F, J). Nematodesmata well-developed, all originating from kinetosomes of perioral kinety and extending into cytoplasm (Fig. 2H).
The SSU rDNA sequence of Pseudolitonotus spirelis sp. n. has been deposited in GenBank with accession number, length, and GC content as follows: MT653620, 1492 bp, 43.16%. The sequence identities of the SSU rDNA between the new species and its morphologically similar and closely related species were 91.0–99.9%, i.e., 1 to 134 nucleotide site differences (Figs. 4, 5).
Figure 4. The sequence identities (upper right) and numbers of nucleotide differences (lower left) of SSU rDNA sequences between Pseudolitonotus spirelis gen. et sp. n. and morphologically similar and/or closely related species. The new species is in bold font
Figure 5. Unmatched sites from SSU rDNA sequence alignment of Pseudolitonotus spirelis gen. et sp. n. with morphologically similar and/or closely related species sequences included in the phylogenetic analyses. Numbers indicate the unmatched site positions. Missing sites are indicated by dashes (-) and matched sites are marked with dots (.)
The topologies of trees constructed using each of the two algorithms were almost identical, so only the maximum likelihood (ML) tree is shown (Fig. 6). In both analyses, the order Pleurostomatida is monophyletic. The family Protolitonotidae is divided into three clades. The first clade comprises three sequences of two Protolitonotus species (Pr. magnus and Pr. longus). The second clade, which contains Apolitonotus lynni, is a sister group to the genus Pseudolitonotus gen. n., which is represented by Ps. spirelis sp. n. and two populations of Ps. gracilis comb. n. The third clade, which consists of Protolitonotus clampi and an unidentified ciliate, is sister group to the genus Kentrophyllum with moderate to full support (ML/BI, 93/1.00). The other two families, Amphileptidae and Litonotidae, are sister groups and each is monophyletic.
Figure 6. Maximum likelihood (ML) tree inferred from 42 SSU rDNA sequences of pleurostomatid and trichostomatid (outgroup) ciliates, revealing the phylogenetic position of Pseudolitonotus spirelis gen. et sp. n. (arrow). Bootstrap values of ML analysis and the posterior probabilities of Bayesian inference analysis (BI) are given at nodes. Dashes indicate incongruity between BI and ML trees. All branches are drawn to scale. GenBank accession numbers are given after names of species. Scale bar corresponds to two substitutions per 100 nucleotide positions. *Designated as Litonotus gracilis in Pan et al. (2015)
Type locality and ecological features
Deposition of slides
Morphology and ciliary pattern
SSU rDNA sequence and phylogenetic analyses
Ciliary patterns of both the somatic and the perioral kineties are important characters for the classification of pleurostomatids (Foissner 1984; Foissner et al. 1995; Lynn 2008). Among the known genera of the order Pleurostomatida, the last left somatic kinety (LKn) extends to the anterior end of the cell whereas in Pseudolitonotus gen. n. the LKn is obviously shortened. Consequently, this new genus can be separated from other pleurostomatid genera.
The ciliary pattern on the right side of the cell is one of the most important characters for the identification of pleurostomatids at family and/or genus level (Lynn 2008; Vďačný et al. 2015; Wu et al. 2017). Hitherto, the order Pleurostomatida was divided into four families based mainly on the ciliary pattern of the right side, i.e., Amphileptidae (single-suture), Epiphyllidae (double-suture), Protolitonotidae (semi-suture), and Litonotidae (no suture) (Vďačný et al. 2015; Wu et al. 2017). The right somatic kineties of Pseudolitonotus gen. n. do not extend to the anterior end of cell but instead are progressively shortened from the middle to both sides, thus distinguishing it from all known pleurostomatid genera. Pseudolitonotus gen. n. cannot to be assigned to any of the four pleurostomatid families based on their current diagnostic characters (Vďačný et al. 2015; Wu et al. 2017). However, we are hesitant to suggest that the shortening of the right somatic kineties and/or the LKn is a diagnostic character at family level. Therefore, we regard Pseudolitonotus gen. n. as incertae familiae at this time.
Litonotus gracilis was originally described by Pan et al. (2015) who also sequenced its SSU rRNA gene. In their phylogenetic analyses, two populations of Litonotus gracilis clustered with Kentrophyllum rather than with its congeners or other litonotids (Pan et al. 2015). Although Pan et al. (2015) noted that some right somatic kineties (RSKs) of L. gracilis are shortened along the oral slit in the typical Litonotus pattern, they overlooked that none of the RSKs extend to the apical region of the cell (see Fig. 2G in Pan et al. 2015). We re-examined the type specimens of Litonotus gracilis and discovered that all of the RSKs are shortened, either along the dorsal margin or along the perioral kineties (Fig. 7B–D). Furthermore, Pan et al. (2015) did not recognize that the LKn, does not extend to the cell apex (Fig. 7A, E, F). The patterns of both the RSKs and LKn in L. gracilis are diagnostic characters of Pseudolitonotus gen. n. In addition, the two populations of Litonotus gracilis group with Pseudolitonotus spirelis sp. n. in the SSU rDNA tree to form a well-supported clade (ML/BI, 93/1.00) (Fig. 6). Hence, we suggest that Litonotus gracilis Pan et al., 2015 should be assigned to Pseudolitonotus gen. n. as a new combination, i.e., Pseudolitonotus gracilis (Pan et al., 2015) comb. n. (original combination: Litonotus gracilis Pan et al., 2015), and its diagnosis and ciliary pattern are improved based on original and current observations of the holotype and paratype specimens (Fig. 7).
Figure 7. Pseudolitonotus gracilis (Pan et al., 2015) comb. n. stained with protargol (A–F). A B Ciliary patterns of right (B) and left (A) side of the holotype specimen, double arrowhead points to the shortened last left somatic kinety (LKn) and arrow marks the longest right somatic kinety (RKn2) not extending to cell apex. C, D Left view of anterior region of cell, to show the dorsal brush (arrow) and the last left somatic kinety (arrowhead). E, F Right view of anterior region of cell, to show the last right somatic kinety (double arrowhead), the penultimate right somatic kinety (arrow) and perioral kineties (arrowhead). DB dorsal brush, LKn last left somatic kinety, PK1 perioral kinety 1, PK2 perioral kinety 2, PK3 perioral kinety 3, RKn1 last right somatic kinety, RKn2 penultimate right somatic kinety. Scale bars: 100 μm in (A, B); 10 μm in (C–F)
Body about 200–350 µm in vivo, with conspicuous neck that is up to 50% of body length when fully extended; usually four macronuclear nodules; one contractile vacuole subterminally located; bar-shaped extrusomes arranged along oral silt; cortical granules arranged in honeycomb-like pattern; 5–9 left and 12–18 right kineties.
In terms of the body size and/or shape, seven pleurostomatid species resemble Pseudolitonotus spirelis sp. n., including: Pseudolitonotus gracilis (Pan et al., 2015) comb. n. (Fig. 7A, B); Protolitonotus clampi Pan et al., 2020 (Fig. 8C, D); Apolitonotus lynni Pan et al., 2020 (Fig. 8A, B); Litonotus duplostriatus (Maupas, 1883) Kahl, 1931 (Fig. 8G, H); L. blattereri Lin et al., 2008 (Fig. 8K, L); L. gongi Lin et al., 2009 (Fig. 8E, F); and L. guae Lin et al., 2009 (Fig. 8I, J) (Table 2). Pseudolitonotus spirelis sp. n. has two traits by which it can be distinguished from similar species: (1) extrusomes clustered together to form an "apical group" at the anterior end of the cell; and (2) the anterior part of the body twisted to the right at the bottom of the oral slit (Table 2). In addition, Pseudolitonotus spirelis sp. n. differs from Ps. gracilis (Pan et al., 2015) comb. n. by having fewer macronuclear nodules (2 vs. 4) and by the distribution patterns of extrusomes (along the entire ventral margin and with an "apical group" vs. along the oral slit only) (Pan et al. 2015).
Figure 8. Morphology of species related and/or morphologically similar to Pseudolitonotus spirelis gen. et sp. n. A, B Apolitonotus lynni, from Pan et al. (2020). C, D Protolitonotus clampi, from Pan et al. (2020). E, F Litonotus gongi, from Lin et al. (2009). G, H Litonotus duplostriatus, from Pan et al. (2015). I, J Litonotus guae, from Lin et al. (2009). K, L Litonotus blattereri, from Lin et al. (2008). Scale bars: 50 μm in (A–D); 100 μm in (E–L)
Species BLa (μm) RKb /LKc n-MAd pr-APe pr-Tf d-Exgc n & po-CVh Data source Pseudolitonotus spirelis 260–350 11–15/7–9 2 Yes Yes Entire ventral 1, S Present work Pseudolitonotus gracilis 200–450 12–18/5–9 4 No Yes Oral slit 1, S Pan et al. (2015) Protolitonotus clampi 80–130 9–11/5 or 6 2 No No Oral slit 1, T Pan et al. (2020) Apolitonotus lynni 100–180 5–7/4 or 5 2 No No Scatter 1, S Pan et al. (2020) Litonotus duplostriatus 90–315 11–14/5 or 6 2 No No Oral slit 1, S Pan et al. (2015) Litonotus blattereri 100–180 15–20/10–14 2 No No Entire ventral 1, T Lin et al. (2008) Litonotus gongi 150–300 11–17/8–10 2 No No Oral slit 2–4, D Lin et al. (2009) Litonotus guae 100–200 11–18/6–9 2 No No Oral slit 3–7, D & V Lin et al. (2009) AP apical group, BL body length, CV contractile vacuoles, d distribution, D dorsal, Ex extrusomes, LK left kineties, MA macronuclear nodules, n number, po position, pr presence, RK right kineties, S subterminal, T terminal, V ventral
aBody length in vivo
bPerioral kineties 2 and/or 3 included
cPerioral kinety 1 and dorsal brush kinety included
dNumber of macronuclear nodules
ePresence of apical group
fPresence of twist at the bottom of oral area
gDistribution of extrusomes
hNumber and position of contractile vacuoles
Table 2. Comparison of Pseudolitonotus spirelis gen. et sp. n. with morphologically similar species
In the SSU rDNA tree, Apolitonotus lynni is the sister group to Pseudolitonotus gen. n. with full support (Fig. 6). This close relationship is unexpected considering the differences in their morphology. For example, Pseudolitonotus gen. n. can be clearly separated from Apolitonotus by a combination of: (1) the ciliary pattern on the right side (all right somatic kineties shortened vs. presence of several full-length right somatic kineties); (2) the presence (vs. absence) of extrusomes in the oral region; (3) the number of perioral kineties (2 vs. 3); and (4) the shortened (vs. not shortened) last left somatic kinety (Pan et al. 2020). The morphological characters of Pseudolitonotus gen. n. and Apolitonotus seem insufficient to reveal the phylogeny of either genus. Therefore, greater taxon sampling and more information on the species of Apolitonotus and Pseudolitonotus gen. n., including morphogenetic data and sequences of multiple gene markers, are needed to reveal their evolutionary relationships and to determine the family assignment of Pseudolitonotus gen. n.