Figure 2. Amphileptus paracarchesii sp. nov. from life (A–G) and after protargol impregnation (H, I). A Left view of a representative individual, red arrow denotes the curved and twisted anterior body end, red arrowhead shows the lateral fossa (groove). B Shape variants, red arrows denote the anterior group of extrusomes. C Oral extrusomes. D Frontal view, showing cortical granules (arrowheads) of the left side. E A contracted individual, red arrow points to the apical group of extrusomes, red arrowhead marks the lateral groove. F Detail showing the lateral groove (arrowhead). G Nuclear apparatus. H Ciliary pattern of the left side of the holotype specimen. I Ciliary pattern of right the side of the holotype specimen, red dashed line shows the anterior suture. DB dorsal brush, LSK left somatic kineties, PK1 perioral kinety 1, PK2 perioral kinety 2, RSK right somatic kineties. Scale bars = 100 μm (A, B, E, H, I), 5 μm (C)
Figure 3. Amphileptus paracarchesii sp. nov. from life (A–H, J, L–M) and after protargol impregnation (I, K, N). A, B Right side view, arrows point to the groove, arrowhead shows the curved anterior body end. C Nuclear apparatus, arrowheads denote the four macronuclear nodules. D, G, J Shape variants, arrowheads mark the macronuclear nodules, arrow denotes the posterior groove. E Detail showing the apical group of extrusomes (arrow). F Detail showing the lateral groove situated in the posterior body region (arrow). H Contractile vacuoles (arrowheads). I Detail of the oral apparatus, showing a single perioral kinety right and left of the oral slit. K, N Detail of the anterior body portion, showing the ciliary pattern of the right and left sides of the holotype specimen. L A contracted individual, arrow shows the curved anterior body end. M Cytoplasmic extrusomes (arrowheads). Abbreviations: PK1, perioral kinety 1. PK2, perioral kinety 2. Scale bars = 100 μm
Character HT Min Max Mean Median SD CV n Body length (μm) 233 184 334 239 233 52.91 22.3 22 298 215 359 278 283 40.80 13.4 20 190 162 290 213 208 57.04 15.8 28 Body width (μm) 55 47 74 59 60 6.44 11.0 22 84 59 91 72 69 9.63 13.4 20 55 30 85 48 46 12.33 25.8 28 Number of right kinetiesa 47 44 50 47 47 1.74 3.7 54 37 35 42 38 38 2.03 5.4 21 33 31 35 33 33 1.29 3.9 31 Number of left kinetiesb 6 4 6 5 5 0.50 9.5 55 27 22 31 27 28 2.23 8.3 20 4 4 5 4 4 0.57 15.9 30 Number of dorsal brush dikinetids 78 59 103 77 75 11.01 14.3 51 95 66 165 125 130 24.50 19.7 21 56 47 74 63 65 7.85 12.5 22 Number of macronuclear nodules 4 4 4 4 4 0 0 38 7 4 14 8 8 2.27 27.4 21 2 2 2 2 2 0 0 30 Length of macronuclear nodule (μm) - 27 71 40 39 9.27 23.0 36 36 25 42 33 34 4.30 13.0 21 50 32 67 43 42 8.88 20.8 29 Width of macronuclear nodule (μm) - 14 46 30 29 7.28 24.4 36 24 21 32 26 26 3.36 12.7 21 3 20 50 30 27 7.57 25.4 29 CV coefficient of variation (%), HT holotype, Max maximum, Min minimum, n number of specimens investigated, SD standard deviation
aPerioral kinety 2 included
bPerioral kinety 1 and dorsal brush kinety included
Table 1. Morphometric characteristics of Amphileptus paracarchesii sp. nov. (upper line), Amphileptus pilosus sp. nov. (middle line) and Amphileptus orientalis sp. nov. (lower line). Data based on protargol-impregnated specimens
Diagnosis. Body lanceolate, about 185–380 × 50–90 μm in vivo; a lateral fossa (groove) in posterior body portion; four macronuclear nodules; contractile vacuoles distributed along dorsal margin; extrusomes very narrowly ovate to clavate, arranged in an apical group and scattered throughout cytoplasm; cortical granules dot-like and colorless; 4–6 left and 44–50 right kineties; right anterior suture; perioral kinety 1 dikinetid in anterior one-third of body, monokinetid in posterior two-thirds; freshwater habitat.
Type material. A protargol slide with the holotype specimen circled by black ink, and two further slides with protargol-stained paratype specimens, have been deposited in Laboratory of Protozoology, Ocean University of China, with registration numbers ZGAT2020120701, ZGAT2020120702, and ZGAT2020120703, respectively.
Type locality. A touring boat port of Lake Weishan, China (N34°34′40.80″, E117°23′52.80″).
ZooBank registration number. Urn: lsid: zoobank.org: act: 5324DEE9-57C2-4086-AE91-10314ABB2AE1.
Etymology. Composite of the Greek adjective "para-" (beside, near) and the species-group name carchesii, indicating the high morphological similarity of the new species to A. carchesii Stein, 1867.
SSU rDNA sequence. The SSU rDNA sequence of A. paracarchesii sp. nov. has been deposited in GenBank (accession no. OL828281). The sequence is 1563 nucleotides long and has a GC content of 42.48%.
Description. Body about 185–380 × 50–90 μm in vivo, typically lanceolate in lateral view, anterior end curved and twisted clockwise from right to left (Figs. 2A, B, E, 3A, L); highly contractile (Fig. 3A, L); neck region conspicuous occupying almost 1/4 of cell length, posterior region narrowed and tail-like occupying about 7% of cell length; fossa (groove) in posterior portion of left side, about 37–41 μm long (Fig. 3A, B, D, G, J). Nuclear apparatus in center of trunk region. Macronucleus invariably consists of four nodules; individual nodules ellipsoidal, about 15–25 × 8–12 μm in size in vivo; nucleoli globular to irregular, small to medium-sized, evenly distributed in macronuclear nodules (Figs. 2A, B, G, 3B–D). Micronuclei not observed. About 10 contractile vacuoles arranged in a row along dorsal body margin, 10–14 μm in diameter during diastole, pulsating every 30 s (Fig. 2A, B, H). Extrusomes very narrowly ovate, sometimes slightly curved, about 11.0–15.0 × 1.2‒1.5 μm in vivo; 2‒4 extrusomes attached to oral slit forming an apical group, numerous other extrusomes scattered throughout cytoplasm; impregnated deeply with protargol method used (Figs. 2A–C, E, 3E, M). Cortex very flexible; cortical granules dot-like, colorless, about 0.5 μm across, ordinarily spaced between adjacent left somatic kineties (Fig. 2D). Cytoplasm grayish, contains numerous granules (ca. 0.5–1.0 μm across) rendering cell opaque (Figs. 2A, E, 3A, L). Swims slowly while rotating about longitudinal body axis; feeds by attaching to stalk of sessile peritrichs using fossa as a sucker (Figs. 2F, 3A, B, F, G).
Somatic cilia about 10–13 μm long in vivo, very densely arranged on right side (Fig. 2A, E), sparsely distributed on left side and hence undetectable in vivo. Ciliary pattern as shown in Figs. 2H, I, 3I, K, N. About 44–50 right kineties including perioral kinety 2; intermediate kineties progressively shortened anteriorly forming a suture (Figs. 2H, I, 3K, N); 4‒6 left kineties including perioral kinety 1 and dorsal brush (Figs. 2H, 3N). Fossa lined by cilia that very likely have a thigmotactic function. Dorsal brush kinety composed of densely spaced dikinetids in anterior body third and of monokinetids in posterior two-thirds (Fig. 2H).
Oral slit extends over two-thirds down length of body, marked by dikinetids of perioral kineties. Perioral kinety 1 runs along left margin of oral slit, consists of densely spaced, oblique dikinetids in anterior body third and monokinetids in posterior two-thirds. Perioral kinety 2 extends along right margin of oral slit, consists of densely spaced, oblique dikinetids in anterior body half and monokinetids in posterior half (Figs. 2H, 3I). Nematodesmata not recognizable either in vivo or and after protargol impregnation.
The order Pleurostomatida is consistently recovered as a monophyletic group in morphological cladistic analyses and SSU rDNA phylogenies (Chi et al. 2021; Gao et al. 2008; Pan et al. 2020; Rajter and Vd'ačný 2017; Vd'ačný et al. 2011a, b, 2014, 2015; Wu et al. 2017, 2022; Zhang et al. 2012, 2022b; present study). Based on morphological and molecular data, pleurostomatids are currently divided into five families: Amphileptidae, Epiphyllidae, Litonotidae, Paralitonotidae, and Protolitonotidae. In the SSU rDNA tree, each family is monophyletic apart from Protolitonotidae, which is paraphyletic as Protolitonotus clampi clusters with Epiphyllidae rather than Protolitonotidae (Fig. 10).
As concerns the family Amphileptidae, the name-bearing genus Amphileptus is paraphyletic due to Pseudoamphileptus macrostoma (AY102173) nesting within it. The Amphileptidae consists of four subclades. The first subclade includes Amphileptus sp. (FJ870086), Pseudoamphileptus macrostoma and possibly A. dragescoi although the statistical support for the position of latter is low. The second subclade comprises A. paracarchesii sp. nov., A. weishanensis, A. parapleurosigma, and A. procerus. Interestingly, all members of the second subclade share an apical group of extrusomes. The third subclade comprises six species (A. multinucleatus, A. shenzhenensis, A. cocous, A. spiculatus, A. aeschtae, and A. litonotiformis) whose close kinship is morphologically supported by the possession of a row of contractile vacuoles along the ventral margin, an unusual feature in pleurostomatids. In the fourth subclade, which is made by A. pilosus sp. nov., A. orientalis sp. nov., and A. bellus, the former species has a comparatively long branch. Interestingly, A. pilosus sp. nov. has a peculiar ciliary pattern, i.e., the presence of the anterior and posterior sutures on the right side, an anterior suture on the left side, and a postoral semi-suture on the right side. Nonetheless, SSU rDNA phylogenies suggest that this deviating and complex ciliary pattern might be a species-level rather than a genus-level character (Fig. 10). This hypothesis is also corroborated by the rather high variability in the presence/absence of the right posterior suture within the genus Amphileptus (Table 2). Since there are some further peculiarities in the somatic ciliary pattern, the branch leading to A. pilosus sp. nov. is comparatively long and the statistical support for its position is variable, we cannot exclude the possibility that it represents a separate genus. However, we retain A. pilosus sp. nov. within the genus Amphileptus pending the availability of greater taxon sampling and sequences of more gene markers.
Species RAS RPS LAS RVS Number of PK Type of PK1 Habitat Reference A. paracarchesii sp. nov + – – – 2 Type 1 FW Present work A. orientalis sp. nov + – – – 2 Type 1 FW Present work A. pilosus sp. nov + + + + 2 Type 2 FW Present work A. aeschtae Lin et al., 2007 + – – – 2 Type 1 MW  A. affinis Song and Wilbert, 1989a + + a – – 2 Type 1 FW  A. agilis (Penard, 1922) Song and Wilbert, 1989a + – – – 2 Type 1 FW  A. bellus Wu et al., 2015 + – – – 2 Type 1 BW  A. cocous Wu et al., 2021 + + a – – 2 Type 1 BW  A. eigneri Lin et al., 2007 + – – – 2 Type 1 MW  A. ensiformis Song and Wilbert, 1989a + + – – 2 Type 1 FW  A. falcatus Song and Wilbert, 1989a + + – – 2 Type 1 FW  A. fusidens (Kahl 1926) Song and Wilbert 1989a + + – – 2 Type 1 FW  A. fusiformisa + + – – 2 Type 1 FW  A. gui Lin et al., 2005 + – – – 2 Type 1 MW  A. litonotiformis Song, 1991a + + – – 2 Type 1 MW  A. marinus (Kahl, 1931) Pan et al., 2014 + – – – 2 Type 1 MW  A. meiianus Song and Wilbert, 1989a + – – – 3 Type 1 FW  A. mutinucleatus Wang, 1934 + – – – 2 Type 1 BW  A. parafusidens Song and Wilbert, 1989a + + – – 3 Type 1 FW  A. parapleurosigma Zhang et al., 2022 + – – – 2 Type 1 FW  A. pleurosigma (Stokes, 1884) Foissner, 1984a + + – – 2 Type 1 FW  A. proceroformis Song and Wilbert, 1989a + + – – 2 Type 1 FW  A. procerus (Penard, 1922) Song and Wilbert, 1989a + + – – 2 Type 1 FW  A. puncatatus (Kahl, 1926) Foissner, 1984 + – – – 2 Type 1 FW  A. shenzhenensis Wu et al., 2021 + – – – 2 Type 1 FW  A. songi Pan et al., 2014 + – – – 2 Type 1 MW  A. spiculatus Wu et al., 2015 + – – – 2 Type 1 BW  A. yuianus Lin et al., 2005 + – – – 3 Type 1 FW  A. weishanensis Zhang et al., 2022 + – – – 2 Type 1 FW  A. wilberti Pan et al., 2014 + – – – 2 Type 1 MW  BW brackish water, FW freshwater, LAS left anterior suture, MW marine water, PK perioral kinety, RAS right anterior suture, RPS right posterior suture, RVS right ventral semi-suture
aData from illustrations
+, present; −, absent
Type 1: PK1 begins with dikinetids and continues posteriorly with monokinetids
Type 2: PK1 terminates above the mid-portion of cell with dikinetids
References:  Lin et al. (2007);  Song and Wilbert (1989);  Wu et al. (2015);  Wu et al. (2021a);  Lin et al. (2005);  Song (1991);  Pan et al. (2014);  Zhang et al. (2022a);  Foissner et al. (1995);  Song et al. (2004)
Table 2. Comparison of Amphileptus species with respect to their somatic and oral ciliary patterns and habitat
Hitherto, 30 Amphileptus species have been studied using protargol impregnation and, therefore, their ciliary pattern is known. These species consistently exhibit a right anterior suture, which was traditionally considered a generic character (Foissner and Leipe 1995; Vd'ačný et al. 2015). Only 12 species (including A. pilosus sp. nov.) have, in addition, a right posterior suture (Table 2). Interestingly, members of the family Epiphyllidae also possess both an anterior and a posterior suture on the right side of the body. Given their molecular phylogenies, the possession of two sutures on the right side is very likely a homoplastic character that evolved convergently in amphileptids and ephiphyllids. Another homoplastic feature of amphileptids might be the number of perioral kineties. According to Foissner and Leipe (1995), the family Amphileptidae was defined, inter alia, by having two perioral kineties. However, three Amphileptus species (A. meiianus, A. parafusidens and A. yuianus) have three perioral kineties, similar to members of the family Litonotidae (Lin et al. 2005; Song and Wilbert 1989). Due to the lack of molecular data, the generic affiliation of these three species could not be tested and remains questionable. It is noteworthy that A. pilosus sp. nov. also differs from its congeners by its oral ciliary pattern, i.e., its perioral kinety 1 terminates above the mid-portion of the cell and is entirely built from dikinetids, whereas it continues to the posterior end of the body as monokinetds in all other congeners (Table 2). Nevertheless, the molecular data support the classification of A. pilosus sp. nov. within the family Amphileptidae (Fig. 10).
It is well known that the SSU rDNA sequence does not necessarily carry a species-specific signal, i.e., distinct species could share an identical SSU rDNA sequence (e.g., Doerder 2019; Lynn and Strüder-Kypke 2006; Rataj and Vd'ačný 2021). This is the case both for A. paracarchesii sp. nov. and A. parapleurosigma, and for A. orientalis sp. nov. and A. bellus. However, A. paracarchesii sp. nov. distinctly differs from A. parapleurosigma by the nuclear apparatus (4 vs. 2 macronuclear nodules), the contractile vacuole pattern (dorsal row vs. dorsal and ventral rows of vacuoles), and the number of the right somatic kineties (44–50 vs. 19–24) (Zhang et al. 2022a). Amphileptus orientalis sp. nov. can be clearly distinguished from A. bellus by its extrusome pattern (extrusomes attached to the anterior 20%–25% of the oral slit vs. along the entire oral slit and tail), the number of left somatic kineties (4–5 vs. 6–7), and the habitat (freshwater vs. brackish). These findings support the ascertain that 100% identity of SSU rDNA sequences does not necessarily correlate with morphospecies conspecificity. Consequently, SSU rDNA does not appear to be an appropriate barcode for members of the genus Amphileptus and species identities need to be confirmed by morphological analyses and/or by faster evolving molecular markers such as ITS2 and COI gene sequences.