Species Diversity of the Pleurostomatid Ciliate Genus Amphileptus (Ciliophora, Haptoria), With Notes on the Taxonomy and Molecular Phylogeny of Three Species

Amphileptus is one of the largest genera of pleurostomatid ciliates and its species diversity has been reported in various habitats all over the world. In the present work, we review its biodiversity based on data with reliable morphological records. Our work confirms that there are 50 valid Amphileptus species, some of which have a wide range of salinity adaptability and diverse lifestyles. This genus has a high diversity in China but this might be because of the relatively intensive sampling. Phylogenetic analyses based on SSU rDNA sequence data verify the non-monophyly of the genus Amphileptus. Furthermore, two new and one poorly known Amphileptus species, namely A. shenzhenensis sp. n., A. cocous sp. n., and A. multinucleatus Wang, 1934, from coastal habitats of southern China were investigated using morphological and molecular phylogenetic methods. These three species are highly similar based on their contractile vacuoles and macronuclear nodules. However, they can be discriminated by details of their living morphology and somatic kineties. We also propose two new combinations, Amphileptus polymicronuclei (Li, 1990) comb. n. (original combination Hemiophrys polymicronuclei Li, 1990) and Amphileptus salimicus (Burkovsky, 1970b) comb. n. (original combination Hemiophrys salimica Burkovsky, 1970b).

Amphileptus Ehrenberg, 1830 is the oldest genus within the order Pleurostomatida and comprises over 60 nominal species reported from marine (Wang, 1934;Dragesco, 1965;Song, 1991;Carey, 1992;Lin et al., 2005aLin et al., ,b, 2007a, brackish waters (Pan et al., 2014;Wu et al., 2014Wu et al., , 2015b, and freshwater habitats (Wang and Nie, 1933;Wang, 1940;Curds, 1982;Song and Wilbert, 1989;Li, 1990) all over the world. Most are free-living but some live as parasites on the skin and gills of certain freshwater fishes and tadpoles (Wenrich, 1924;Chen, 1955;Mitchell and Smith, 1988;Masoumian et al., 2005). Amphileptus is generally defined by the following combination of characters: (1) a single anterior suture formed by the right somatic kineties; (2) the presence of two rows of perioral kineties [three rows were detected in a single species, A. yuianus, by Lin et al. (2005b); molecular data are needed to confirm its generic classification]; (3) extrusomes not distributed along the dorsal margin, and (4) the absence of a spoon-shaped apex in the anterior end of the body (Foissner, 1977(Foissner, , 1984Song and Wilbert, 1989;Foissner and Leipe, 1995;Lin et al., 2007a). Species of Amphileptus have a high degree of morphological similarity in vivo, and many have not been studied using modern methods such as silver staining. This has resulted in numerous examples of misidentifications and/or synonyms and homonyms within the Amphileptus-Litonotus-Loxophyllum complex, especially among the many nominal species described before the 1960s (Kahl, 1931(Kahl, , 1933Wang and Nie, 1932;Wang, 1934Wang, , 1940Dragesco, 1960;Vuxanovici, 1960Vuxanovici, , 1961. Since the ciliary pattern as revealed by silver staining is of great importance for species identification, there is an urgent need to redescribe those that are currently known only from in vivo observation. Amphileptus has long been considered to be monophyletic based on morphological information (Fryd-Versavel et al., 1975;Foissner, 1977Foissner, , 1984Corliss, 1979;Song and Wilbert, 1989). However, recent studies based on the molecular data have indicated that the molecular and morphological data are not concordant and the molecular data suggest that the genus Amphileptus is non-monophyletic (Pan et al., 2014;Wu et al., 2015b). In addition, most congeners within this genus are very similar in terms of their body shape, the number and position of contractile vacuoles, and other aspects of their living morphology. Therefore, more detailed morphological information and molecular data obtained from expanded taxon sampling are necessary.
In this paper we: (1) briefly review previous studies of the species diversity of the genus Amphileptus; (2) provide a checklist of valid species including synonyms following analyses of nomenclatural problems, and (3) reconstruct the molecular phylogeny of the family Amphileptidae Ehrenberg, 1830 and the genus Amphileptus based on all reliable small subunit (SSU) rDNA sequences from the NCBI/GenBank database. In addition, we investigate three morphologically similar Amphileptus species from coastal waters of southern China. After detailed comparisons, they were identified as Amphileptus multinucleatus Wang, 1934, Amphileptus shenzhenensis sp. n. and Amphileptus cocous sp. n.

Sample Collection, Observation, and Identification
All samples were collected from coastal waters at two sites in southern China using 250 ml wide-mouth bottles after gently stirring the water. Amphileptus multinucleatus Wang, 1934  . Each species was cultivated at room temperature (∼25 • C) in habitat water in Petri dishes with rice grains to enrich the growth of bacteria as a food source for the ciliates.
Observations of living cells were executed with bright field and differential interference contrast microscopy. The number, size and location of contractile vacuoles were recorded based on live observations. The protargol staining method according to Wilbert (1975) was used to reveal the ciliary pattern. Living cells were examined at 100-1,000 × magnifications. Measurements of stained specimens were performed at a magnification of 1,000×. Drawings of stained specimens were conducted with the help of a camera lucida at a magnification of 1,000×. Classification and terminology are according to Vd'ačný et al. (2015) and Wu et al. (2017).

DNA Extraction, Gene Amplification, and Gene Sequencing
For each species, one or several cells taken from cultures were isolated, repeatedly washed in filtered habitat water and transferred into 45 µl ATL buffer for DNA extraction. Genomic DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Shanghai, China) according to the manufacturer's protocol. SSU rDNA amplification and gene sequencing were conducted as described in Wu et al. (2013).

Phylogenetic Analyses
In total, 36 SSU rDNA sequences of the order Pleurostomatida, representing four families and including all available and reliable sequences of the family Amphileptidae, were used to conduct the phylogenetic analyses. Apart from the three new SSU rDNA sequences provided in the present study, all other sequences used in the phylogenetic analyses were obtained from the NCBI/GenBank database (see Figure 4 for GenBank accession numbers). Sequences were first aligned with CLUSTAL W and further modified manually using Bioedit v.7.0. The final alignment of 1625 characters and 40 taxa, including four haptorians as outgroup taxa, were used to construct phylogenetic trees using three different methods. Maximum likelihood (ML) analysis was carried out using RaxM-HPC2 v7.2.8 (Stamatakis et al., 2008) on CIPRES Science Gateway 1 . The reliability of internal branches came from a majority rule consensus tree by using a non-parametric bootstrap method with 1,000 replicates. Bayesian inference (BI) analysis was conducted in MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) by using the Markov chain Monte Carlo algorithm under the GTR + G + I evolutionary model indicated by MrModeltest v.2 (Nylander, 2004), which was run for 1,500,000 generations with a sample frequency of 100 generations. The first 3,750 generations were discarded as burn-in. Maximum parsimony (MP) analysis was performed with PAUP 4.0b10 (Swofford, 2002) using the tree-bisectionreconnection algorithm and bootstrapping with 1000 replicates.

Statistical Tree Topology Test
The Kishino-Hasegawa (KH) test (Kishino and Hasegawa, 1989) was used to test the hypothesis that the genus Amphileptus is monophyletic. The ML tree was generated with a constraint block, enforcing the constraint of focal group monophyly in PAUP 4.0b10 under the GTR + I + G model. The site-wise likelihoods were calculated using PAUP 4.0b10 (Swofford, 2002) for the resulting constrained and non-constrained ML topologies. The scores were then subjected to the KH test as implemented in Consel (Shimodaira and Hasegawa, 2001).

Morphological Diversity Data Collection
The species diversity of the genus Amphileptus was studied based on data from the present study and published sources, mainly monographs (Kahl, 1931;Song and Wilbert, 1989;Carey, 1992;Song et al., 2009;Vd'ačný and Foissner, 2012;Hu et al., 2019) and papers on the taxonomy and biodiversity of Amphileptus (see Tables 1, 2 for a complete list).

Geographic Distribution of the Genus Amphileptus
Amphileptus has been found in a wide variety of habitats worldwide. To date, 50 valid species of this genus have been reported from marine (Carey, 1992;Lin et al., 2005aLin et al., ,b, 2007a, brackish (Pan et al., 2010(Pan et al., , 2014Chen et al., 2011), freshwater (Wang and Nie, 1933;Wang, 1940;Song and Wilbert, 1989), and terrestrial (Foissner, 1984) habitats worldwide. In freshwater habitats, species of Amphileptus are most commonly reported from lakes (Song and Wilbert, 1989;Li, 1990), rivers (Stokes, 1884), wastewater treatment plants (Foissner, 1984), and as parasites on the body surface and gills of certain freshwater fishes and tadpoles in North America, Asia and Europe (Wenrich, 1924;Chen, 1955;Mitchell and Smith, 1988;Masoumian et al., 2005). In marine and brackish water habitats, species are most commonly reported from mangrove wetlands (Pan et al., 2010(Pan et al., , 2013(Pan et al., , 2014Chen et al., 2011;Wu et al., 2013Wu et al., , 2014Wu et al., , 2015aWu et al., ,b, 2017 this study), mariculture ponds (Song, 1991;Lin et al., 2005aLin et al., ,b, 2007aSong et al., 2009;Pan et al., 2014), the intertidal zones of beaches (Pan et al., 2014), and coastal marine waters (Kahl, 1931;Wang, 1934;Borror, 1963;Dragesco, 1965;Al-Rasheid, 1996). The vast majority of Amphileptus species are free-living although a few are reported as parasites on the skin and gills of fish (Chen, 1955;Masoumian et al., 2005), or tadpoles (Wenrich, 1924). Of the known Amphileptus species more than one-third have been found in the coastal waters of China Hu et al., 2019). These include eight species from mariculture ponds in the coastal waters of the Bohai and Yellow seas of northern China and 11 species (12 populations) from coastal waters of the South China Sea, seven of which were isolated from mangrove wetlands. We have listed the references to reliable morphological descriptions of Amphileptus in Table 1. Species no longer assigned to the genus Amphileptus, and species of Amphileptus originally assigned to other genera, are listed in Table 2 along with their current names and taxonomic status.

Voucher Material
One voucher slide with protargol-stained specimens is deposited in the Laboratory of Protozoology, OUC, China, with registration number WL2011121901.

SSU rDNA Sequence
The SSU rDNA sequence of Amphileptus multinucleatus is deposited in the GenBank database with the accession number, length, and GC content as follows: MT653624, 1560 bp, 43.40%.

Morphological Description Based on Daya Bay Population
Body size highly variable in vivo, about 200-450 µm long; body shape fairly stable, generally elongate-pyriform with bluntly pointed; in all individuals (n > 20) observed in vivo, posterior portion perpetually twisted from left side to right side beginning at mid-dorsal region; conspicuous "neck" region (about 25%   Figures 1A,G,H).
Extrusomes thick bar-shaped, straight or slightly curved, about 10 µm long, densely arranged along anterior part of buccal area, some scattered in cytoplasm (Figures 1B,C,I). Pellicle thin with small (<0.5 µm across), densely spaced, grayish, dot-like cortical granules between ciliary rows on both sides of cell (Figures 1D,J). Cytoplasm colorless to pale yellow, often with numerous tiny, refringent globules (1-3 µm across) that render main part of body opaque (Figures 1G,H). Locomotion usually by gliding slowly on substrate, or swimming with a slow clockwise rotation about longitudinal axis. Ciliary pattern as shown in Figures 1E,F,L,M. Eight to twelve left kineties (mean 9.9; median 10), including perioral kinety 1 and dorsal brush kinety (DB) which extends to about anterior 2/5 of cell-length and is composed of regularly spaced dikinetids (Figures 1E,L). Right side with 29-38 (mean 33.0; median 33) ciliated kineties including perioral kinety 2; intermediate somatic kineties are shortened forming a distinct anterior single-suture on right side (Figures 1F,M).

Etymology
Named after Shenzhen, where this species was first isolated.
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SSU rDNA Sequence
The SSU rDNA sequence of Amphileptus shenzhenensis is deposited in the GenBank database with the accession number, length, and GC content as follows: MT653621, 1534 bp, 43.22%. All measurements in µm. Data based on protargol-stained specimens. CV, coefficient of variation in%; Max, maximum; Mean, arithmetic mean; Min, minimum; n, sample size; SD, standard deviation. a Perioral kineties 2, 3 included. b Perioral kinety 1 and dorsal brush kinety included.

Etymology
The Latin adjective "cocous" (rice-shaped) refers to the shape of cortical granules.

Type Slides
One protargol slide with the holotype specimen (circled in black ink) and several paratype specimens is deposited in the Laboratory of Protozoology, Ocean University of China (OUC), Qingdao, China, with registration number WL20111027-01.

SSU rDNA Sequence
The SSU rDNA sequence of Amphileptus cocous is deposited in the GenBank database with the accession number, length, and GC content as follows: MT653622, 1533 bp, 43.25%.
Two perioral kineties around cytostome: PK1 left of oral slit, composed of closely spaced dikinetids in anterior 2/5 and continues posteriorly as a row of closely spaced monokinetids (Figures 3F,O); PK2 right of oral slit, formed of closely spaced dikinetids in anterior 2/5 and continues posteriorly as a row of closely spaced monokinetids (Figures 3G,P).

Molecular Phylogenetic Analysis of Amphileptus
Phylogenetic trees conducted using Bayesian inference (BI) and maximum likelihood (ML) had identical topologies so the two trees were combined ( Figure 4A). The topology of the MP tree differed slightly from that of the ML/BI tree as shown in Figure 4B. The genus Amphileptus forms a polytomy with two clades in the ML/BI tree and three clades in the MP tree, and the three newly sequenced species form a clade with another four Amphileptus spp. (clade a in Figure 4A; clade 1 in Figure 4B) with poor to moderate support (60% ML, 0.71 BI, 79% MP). Within clade a/clade I, Amphileptus cocous groups with A. spiculatus which together group with A. shenzhenensis with high to maximum support (94% ML, 99% MP, 1.00 BI). These three species cluster with A. multinucleatus with poor to moderate support (86% ML, 0.77 BI, 59% MP), and this subclade groups with A. aeschtae with high support (100% ML, 1.00 BI, 99% MP), forming a clade that is sister to A. litonotiformis with high support (97% ML, 1.00 BI, 98% MP). In the second clade in ML/BI tree (clade b in Figure 4A), the remaining three Amphileptus spp. (Amphileptus sp., A. dragescoi and A. procerus) group with Pseudoamphileptus macrostoma with weak support (0.69 BI, 65% ML). In the MP tree, however, A. procerus clusters with A. dragescoi (81% MP) to form the second clade (clade II in Figure 4B), and the remaining two species (Pseudoamphileptus macrostoma and Amphileptus sp.) form the third clade (clade III in Figure 4B) with high support (99% MP).

DISCUSSION
A Brief Summary of the Genus Amphileptus Ehrenberg, 1830 The genus Amphileptus was established by Ehrenberg (1830) and another pleurostomatid genus, Hemiophrys, was established by Wrzesniowski (1870). Until the mid-twentieth century, descriptions of Amphileptus-Hemiophrys species were exclusively based on observations of live species (Ehrenberg, 1830;Kahl, 1931). Canella (1960) carried out the first detailed investigation of the ciliary pattern of these two genera using silver staining and revealed that the somatic kineties on the right side form a suture in the mid-to-anterior region of the cell in both genera. Furthermore, other morphological differences were regarded as species-level rather than genus-level characters, suggesting that Hemiophrys is a junior synonym of Amphileptus (Canella, 1960). This recommendation was accepted by Fryd-Versavel et al. (1975). Consequently, the genus Hemiophrys was merged into the genus Amphileptus, and the diagnosis of genus Amphileptus was emended, i.e., the formation of a single suture by the somatic kineties of the right side was considered to be the key diagnostic character. The emended diagnosis of the genus Amphileptus and the submersion of Hemiophrys were accepted by subsequent investigators (Foissner, 1984;Aescht, 2001;Lynn, 2008).
Species of the genus Amphileptus are easily identified by the pattern of right somatic kineties, i.e., the somatic kineties shortened to form a single suture in the median area, and this is the main differentiating feature for certain pleurostomatid genera, e.g., Amphileptus and Apoamphileptus within the order Pleurostomatida. Therefore, the presence of a single suture is thought to be an important genus-level, and possibly even a family-level character (Lin et al., 2005b;Wu et al., 2017).
The first molecular phylogenetic study of Amphileptus was that of Gao et al. (2008) who sequenced the SSU rDNA of A. procerus (Penard, 1922) Song and Wilbert, 1989and A. aeschtae Lin et al., 2007a. Pan et al. (2014 added another new sequence and reported the monophyly of the family Amphileptidae and the paraphyly of the genus Amphileptus with Pseudoamphileptus nested within it. These findings have been both confirmed and rejected in subsequent studies (Wu et al., 2015b(Wu et al., , 2017Pan et al., 2015) (see molecular analyses below). Including the three species described in the present study, there are nine identified and one unidentified SSU rDNA sequences in the NCBI/GenBank database.
Comments on Amphileptus multinucleatus Wang, 1934 Amphileptus multinucleatus was originally described by Wang (1934) who gave a good description based on live observations, although the ciliary pattern was not mentioned. It was characterized mainly as follows: body 150-250 µm in length, with 6-10 contractile vacuoles lying in ventral posterior half, numerous (40-70) macronuclear nodules scattered in cytoplasm, and posterior end twisted to one side. In particular, it was noted that "the twisted posterior portion is very constant in all observed individuals and may be considered as one of the specific characteristics" (Wang, 1934). This specific feature was also observed in all observed individuals of the Daya Bay population (n > 20) and has not been recorded in any other species of Amphileptus. We conclude that the twisted posterior portion of the body should be considered as a diagnostic character of this species. In addition, our form was identified as A. multinucleatus based on the distribution of extrusomes along the oral slit and with some scattered in the cytoplasm. One significant difference between the original description and the Daya Bay population of A. multinucleatus is the number of macronuclear nodules, the former having 40-70 and the latter 80-300. It should be noted, however, that the number of macronuclear nodules in the original description was based on observations of specimens fixed in Schaudinn's fluid and stained with iron-alum-hematoxylin which may not show the outline of the macronuclear nodules as clearly as the protargol stain. In addition, the body size of the Daya Bay population is considerably larger than that of the original population (200-400 vs. 150-250 µm in length), which may be another reason for the higher number of macronuclear nodules. We therefore conclude the Daya Bay population is conspecific with the original population of A. multinucleatus described by Wang (1934).
Comments on Amphileptus shenzhenensis sp. n. and A. cocous sp. n.
The most important characters for species identification and circumscription in the genus Amphileptus include the number of kineties, the number and positions of contractile vacuoles, the number of macronuclear nodules, the shape and distribution of extrusomes, the presence or absence and the shape of cortical granules, and the body shape in vivo Lin and Song, 2004;this study).
Among all the nominal species of Amphileptus, only three congeners are reported to have contractile vacuoles arranged along the ventral margin of the cell and four or more macronuclear nodules, i.e., A. multinucleatus, A. quadrinucleatus and A. aeschtae (Wang, 1934;Dragesco and Dragesco-Kernéis, 1986;Lin et al., 2007a; Table 4). The two new forms and the three described species strongly resemble each other in body size, position and number of macronuclear nodules, shape of cortical granules, and the number of kineties. However, A. multinucleatus is the only species in this genus with a posterior end constantly twisted to one side (Wang, 1934), therefore it can be clearly distinguished from the other species.
Amphileptus cocous sp. n. resembles A. aeschtae in having the same number of left kineties (7-9) and almost the same number of right kineties (27-34, mean 30 vs. 29-34, mean 31). However, the former can be separated from the latter by the different shape of cortical granules (rice-shaped vs. dot-like), the distribution of contractile vacuoles (in the posterior half of the body vs. in the posterior 2/3 of the body) and the elongated blade-shaped (vs. ellipsoidal) body (Lin et al., 2007a; Table 4).

Comments on the Phylogeny of the Genus Amphileptus
The family Amphileptidae is characterized by the presence of a single anterior suture on the right side, thereby differentiating it from the other three families within the order Pleurostomatida Wu et al., 2017). The Amphileptidae, comprises five genera, namely Amphileptus (the type genus), Pseudoamphileptus, Amphileptiscus, Apoamphileptus, and Opisthodon, but molecular data are available only the former two genera. Traditionally, the genus Amphileptus has been considered to be monophyletic based on morphological (Foissner, 1977(Foissner, , 1983(Foissner, , 1984Corliss, 1979;Lin et al., 2005aLin et al., ,b, 2007aLynn, 2008) and some molecular studies (Pan et al., 2010(Pan et al., , 2013Zhang et al., 2012;Vd'ačný and Foissner, 2013;Wu et al., 2013Wu et al., , 2015bWu et al., , 2017Vd'ačný, 2015;Vd'ačný et al., 2015). However, the monophyly of this Amphileptus has been questioned by several other molecular phylogenetic studies (Gao et al., 2008;Vd'ačný et al., 2011Vd'ačný et al., , 2015Pan et al., 2014Pan et al., , 2015Wu et al., 2014Wu et al., , 2015b. In our SSU rDNA trees (Figure 4) which include three new sequences of Amphileptus, the non-monophyly of the genus Amphileptus was supported, since it was divided into two and three groups in ML/BI and MP tree, respectively. Furthermore, the possibility of the monophyly of Amphileptus was also rejected (p = 0.004 < 0.05) by the KH test (Table 5). Although some previous studies have shown members of the genus Amphileptus to group together in phylogenetic trees (Pan et al., 2010(Pan et al., , 2013Zhang et al., 2012;Vd'ačný and Foissner, 2013;Wu et al., 2013Wu et al., , 2015aWu et al., , 2017Vd'ačný, 2015;Vd'ačný et al., 2015), these convergent topologies are based on insufficient taxon sampling and have only low nodal support (e.g., 76% ML, 0.65 BI in Pan et al., 2013;17% ML, 20% MP in Wu et al., 2015a;50% ML, 0.64 BI, 57% MP in Vd'ačný et al., 2015;51% ML, 0.78 BI, 67% MP in Vd'ačný, 2015). Therefore, the molecular data strongly question the morphology-based relationship of the genus Amphileptus, and even the family Amphileptidae, and indicate that this group is paraphyletic. To date, however, only two genera of the family Amphileptidae have molecular data, namely Amphileptus and Pseudoamphileptus. It is noteworthy that Pseudoamphileptus is represented by a single sequence that clusters with sequences of Amphileptus (Figure 4). Furthermore, no morphological characters can be identified as plesiomorphic or apomorphic, so the question of whether the genus Amphileptus and/or the family Amphileptidae is monophyletic will remain unresolved pending the availability of more morphological and molecular data with expanded taxon sampling.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI GenBank (accession: MT653621, MT653622, and MT653624).

AUTHOR CONTRIBUTIONS
LW and XL conceived and designed the manuscript. LW carried out the live observation and protargol staining. LW, JL, AW, and XL wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
Many thanks to Ms. Wenping Chen, Mr. Chunxu Hu, and Mr. Xudong Cui, the graduates of our laboratory, SCNU, for their help in sampling. We are also grateful to the two reviewers and associate editor HP for their constructive comments and helpful suggestions for improving our manuscript.