Morphology and Molecular Phylogeny of Four Trachelocercid Ciliates (Protozoa, Ciliophora, Karyorelictea) Found in Marine Coastal Habitats of Northern China, With Description of a New Genus, Two New Species and a New Combination

The morphology of four trachelocercid ciliates, Foissnerella typica gen. nov., spec. nov., Trachelolophos monocaryon (Dragesco, 1965) comb. nov. (original combination: Tracheloraphis monocaryon Dragesco, 1965), Tracheloraphis katzae spec. nov., and Tracheloraphis colubis (Kahl, 1933) Xu et al., 2011 were studied in live and protargol-stained specimens. All samples were isolated from the intertidal zone of sandy beaches at Qingdao, China. The new genus Foissnerella can be distinguished from other trachelocercid genera mainly by the three circumoral kineties each composed of a row of dikinetids and the absence of a brosse or ciliary tuft in the oral cavity. The detailed investigation on the poorly described Tracheloraphis monocaryon (Dragesco, 1965) reveals that its oral infraciliature includes one uninterrupted circumoral kinety and a conspicuous ciliary tuft in the center of the oral cavity, which is consistent with the genus Trachelolophos rather than Tracheloraphis. Therefore, this species is transferred to Trachelolophos as Trachelolophos monocaryon (Dragesco, 1965) comb. nov. Tracheloraphis katzae spec. nov. can be recognized by the combination of its minute brownish cortical granules and 9–15 somatic kineties. The small subunit (SSU) rDNA of each species was sequenced for the first time. Phylogenetic analyses of the SSU rDNA show that Foissnerella typica gen. nov., spec. nov. clusters with Apotrachelocerca arenicola (Kahl, 1933) Xu et al., 2011 in a group that is sister to all other trachelocercids.

In the present study, the morphological data of four trachelocercids isolated from marine coastal habitats at Qingdao, China, are documented based on observations of specimens in vivo and following protargol staining. In addition, the SSU rDNA sequence of each species is provided and phylogenetic analyses are performed to assess their evolutionary relationships. Foissnerella gen. nov. is assigned to the family Trachelocercidae based on both morphological and molecular information.

Sample Collection, Observation, and Identification
Foissnerella typica gen. nov., spec. nov. and Tracheloraphis colubis (Kahl, 1933) Xu et al., 2011 were collected from the intertidal zone of Silver Beach, Qingdao (35 • 55 09 N, 120 • 11 55 E) on March 11, 2013 and May 27, 2019, respectively. The water temperature on each day of sampling was 11 and 23 • C, respectively, and the salinity was about 27 . Trachelolophos monocaryon (Dragesco, 1965) comb. nov. and Tracheloraphis katzae spec. nov. were both collected on June 24, 2019, from the intertidal zone of the No. 1 Bathing beach, Qingdao (36 • 03 24 N, 120 • 20 32 E) where the water temperature was 26 • C and the salinity was about 30 (Figure 1). For each sample, the top 5 cm of sand or sediment along with seawater from the site was collected. Ciliates were extracted from the sediment using the method from Uhlig (1968). In brief, the sand or sediment was placed in a plastic tube (4.5 cm diameter and 10 cm long, at one end of which was a tightly fitting nylon gauze (mesh size 80-90 µm)). The depth of the sediment in the tube was 5 cm. Finely crushed ice was added to fill the remainder of the tube. A glass culture dish containing about 20 ml filtered seawater was placed under the tube so that the nylon gauze was barely in contact with the seawater surface. Ciliates in the sand/sediment sample migrating downwards to escape the advancing front of meltwater were collected in the culture dish.
Cells were isolated and observed in vivo using bright field and differential interference contrast (DIC) microscopy (Olympus BX 53). The infraciliature was revealed using the protargol staining method (Wilbert, 1975). Counts, measurements, and drawings of stained specimens were performed at 1,000 × magnification. Terminology and systematics mainly follow Foissner (1996) and Lynn (2008), respectively. DNA Extraction, PCR Amplification, and Gene Sequencing DNA extraction, PCR amplification, and SSU rDNA sequencing of the four species were performed according to Wang et al. (2020). For each species, we extracted total genomic DNA from single cells using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). The primers 82F (5 -GAA ACT GCG AAT GGC TC-3 ) (Jerome et al., 1996), 18S-F (5 -AAC CTG GTT GAT CCT GCC AGT-3 ), and 18S-R (5 -TGA TCC TTC TGC AGG TTC ACC TAC-3 ) were used to amplify the SSU rDNA (Medlin et al., 1988). PCR amplification was carried out according to Wang et al. (2019Wang et al. ( , 2020 and PCR products were then sequenced bidirectionally by the Tsingke Biological Technology Company (Beijing, China).

Phylogenetic Analyses
Using MAFFT implemented in GUIDANCE 1 with default parameters (Penn et al., 2010), the newly generated SSU rDNA sequences of the four species were aligned with 54 other sequences of karyorelictean and heterotrich (outgroup) species downloaded from NCBI GenBank. The resulting alignments were manually refined by trimming both ends with Bioedit v.7.0.5 (Hall, 1999) and the final alignments were 1,802 bp.

Topology Testing
The phylogenetic relationships among different taxa within Karyorelictea were assessed using the approximately unbiased (AU) test (Shimodaira, 2002). Two constrained ML trees were generated by RAxML v8.2.10 (Stamatakis, 2014) with the enforced constraints (Table 1) and their topologies were compared with those of the best unconstrained ML trees implemented in CONSEL (Shimodaira and Hasegawa, 2001).  The topology constraints column refers to proposed taxonomic groups that were tested for monophyly through the approximately unbiased test (AU). Rejected monophyly (p < 0.05) is highlighted in gray.
Internal relationships in the constrained group and among the remaining taxa were unspecified.

Diagnosis
Trachelocercidae with three circumoral kineties each composed of a row of dikinetids. No brosse or ciliary tuft in oral cavity. Marine habitat.

Etymology
The new genus name is dedicated to the eminent ciliatologist, Prof. Wilhelm Foissner, Universität Salzburg, Austria, in recognition of his significant contributions to the study of ciliates.

Discussion
Foissnerella gen. nov. possesses all the diagnostic characters of trachelocercids, i.e., elongate body shape with distinct "head" and "neck" and somatic ciliature covering the body apart from longitudinal glabrous zone that is bordered by a "bristle-like" kinety. Therefore, Foissnerella gen. nov. undoubtedly belongs to the family Trachelocercidae. The shape and structure of the oral ciliature is the main character for generic classification (Foissner and Dragesco, 1996a,b;Foissner and Al-Rasheid, 1999). Foissnerella gen. nov. possesses three circumoral kineties each composed of a row of dikinetids. Consequently, it can be easily separated from its most closely related genera such as Apotrachelocerca Xu et al., 2011, which has two rows of uninterrupted circumoral kineties, Trachelocerca Ehrenberg, 1840, which has a single uninterrupted circumoral kinety composed of dikinetids, and Prototrachelocerca Foissner, 1996 has two rows of circumoral kineties interrupted by short brosse kineties ( Table 2). It is noteworthy that no anterior or posterior secant system is present in Foissnerella typica gen. nov. spec. nov., which is similar to Apotrachelocerca arenicola (Kahl, 1933) Xu et al., 2011. However, the presence/absence of the anterior

Type Specimens
A protargol-stained slide containing the holotype specimen marked with an ink circle is deposited in the Laboratory Frontiers in Marine Science | www.frontiersin.org

Etymology
This species-group name typica means that it is the representative species of this genus.
century due to the lack of knowledge of the infraciliature. Observations of both live and silver-stained specimens of the present isolate indicates that this taxon should be assigned to the genus Trachelolophos. Therefore, a new combination is suggested and a redescription and improved diagnosis are supplied.

Deposition of Voucher Materials
A voucher slide with protargol-stained specimens has been deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China (registration number: MMZ2019062406).
Locomotion by gliding on substrate, winding between sand grains and organic debris.
Entire infraciliature consisting of dikinetids (Figures 4H,  5K). Cilia about 10 µm long in vivo and arranged in longitudinal rows. Oral infraciliature consists of a single uninterrupted circumoral kinety and a conspicuous ciliary tuft located in center of oral cavity (Figures 4F-H, 5I,L). Glabrous stripe very narrow, width about equal to the gap between two adjacent somatic kineties, bordered by irregularly spaced bristle kinety (Figures 4H, 5J). Anterior and posterior secant systems formed on left side of glabrous stripe and some kineties also abut to bristle kinety (Figures 4H, 5J). Four macronuclei (in which crystals are sometimes present) and two micronuclei in a single group (Figures 4E, 5H,M).

Discussion
This organism was first reported by Dragesco (1965) under the name Tracheloraphis monocaryon and the original description was based solely on a rather schematic figure and a short description of the cell in vivo. The Qingdao population corresponds closely with the original population in several key characters such as the narrow glabrous stripe, the single nuclear group composed of four macronuclei and two micronuclei and the possession of about 40 somatic kineties. Therefore, we consider them to be conspecific. Before transferring this species to Trachelolophos, we compare the Qingdao isolate to all four species of Trachelolophos.

Type Locality
The intertidal zone of the No. 1 Bathing beach, Qingdao (36 • 03 24 N, 120 • 20 32 E), China, where the water temperature was 26 • C and the salinity was about 30 (Figure 1).

Type Specimens
A protargol slide containing the holotype specimen marked with an ink circle is deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China (No. MMZ2019062407).

Etymology
We dedicate this new species to our eminent colleague, Prof. Laura Katz, Smith College, United States, in recognition of her great contributions to ciliate research.
Tracheloraphis hamatus Wright, 1982 resembles the novel form in body size, width of the glabrous stripe and the number of macronuclei. It can be distinguished from the latter, however, by having a different type of cortical granules (globular, less than 0.5 µm in diameter vs. ellipsoidal, about 0.2 µm × 0.5 µm) (Figure 8H). Tracheloraphis africanus Dragesco, 1965 resembles T. katzae spec. nov. in body size and the number of macronuclei. However, it differs from the latter by the absence (vs. presence) of cortical granules although according to Dragesco (1965) the 3 µm long rod-shaped "granules" scattered in the superficial cytoplasm may be bacteria ( Figure 8C).
Tracheloraphis gracilis Dragesco, 1960 has a similar number of macronuclei compared to Tracheloraphis katzae spec. nov. and both species have oval cortical granules, but it can be separated from the latter by its tail which has a rounded (vs. wedge-shaped) end ( Figure 8I; Dragesco, 1960).
Based on the original illustration, the nuclear apparatus of Tracheloraphis enigmaticus Dragesco, 1960 consists of five macronuclei and two micronuclei, but these are not clustered in a nuclear group as they are in T. katzae spec. nov. Furthermore, the figure of T. enigmaticus shows that it has short subuliform tail, whereas the new species has a narrow tail with a wedge-shaped end (Figure 8J; Dragesco, 1965).
The original descriptions of Tracheloraphis striatus Raikov, 1962 andT. remanei Dragesco, 1960 are based solely on stained specimens, and no information on their live morphology is available. Nevertheless, both can be separated from T. katzae spec. nov. by having a narrower glabrous stripe (about double distance between two adjacent somatic kineties, and one-third of the body width, respectively vs. about as wide as the body) (Figures 8D,E; Dragesco, 1960;Raikov, 1962).
Although no information on the infraciliature of Trachelocerca schulzei Dragesco, 1960 is available, based on the original illustration, it can be separated from T. katzae spec. nov. by its larger ratio of body length to body width (about 35:1 vs. about 20:1) in fully extended cells (Figure 8F; Dragesco, 1960). (Kahl, 1933) Xu et al., 2011Figures 9, 10 and Table 3) This species was originally reported by Kahl (1933) and redescribed in detail by Xu et al. (2011a). Our population matches both descriptions very well, therefore redescription and an improved diagnosis based on the present and previous populations are provided here.
Locomotion by gliding, winding between sand grains and organic debris.

Discussion
There have been several redescriptions of Tracheloraphis colubis since it was first reported (Kahl, 1933;Raikov, 1963;Xu et al., 2011a). Kahl (1933) and Raikov (1963) both assigned this species to the genus Trachelocerca due to its curved posterior end and narrow glabrous stripe. However, the shape and structure of the oral ciliature are the most important characters for generic classification (Foissner and Dragesco, 1996a,b;Foissner and Al-Rasheid, 1999). Therefore, Xu et al. (2011a) assigned this species to the genus Tracheloraphis. The present population matches the population described by Xu et al. (2011a), so we have no doubt that these two populations are conspecific.
The ML and BI trees have similar topologies, therefore only the ML tree is presented (Figure 11). The family Trachelocercidae is a well-supported monophyletic group (89% ML, 1.00 BI) that is sister to the family Loxodidae (65% ML, 1.00 BI). Within Trachelocercidae there are four main clades (Clades I, II, III, and IV). Tracheloraphis colubis is sister to several Tracheloraphis and Trachelocerca species. Tracheloraphis katzae spec. nov. groups with T. dragescoi within Clade I. Prototrachelocerca fasciolata shows a close relationship with Tracheloraphis similis which together occupy the basal position within Clade I. Tracheloraphis species interdigitate with those of Trachelocerca resulting in the non-monophyly of both genera, which is consistent with previous studies (Xu et al., 2014;Yan et al., 2016). Clade II consists of three Tracheloraphis species. Two Trachelolophos species group together forming a sister branch to Clades I and II. Clade III comprises Trachelolophos monokaryon and T. quadrinucleatus. Kovalevaia sulcata is sister group to the assemblage of Clades I-III. Clade IV comprises Foissnerella typica and two species of Apotrachelocerca.
Based on the combination of morphological features and molecular evidence, Yan et al. (2016) suggested that Apotrachelocerca, which possesses two uninterrupted rows of circumoral kineties, is the closest relative to the common ancestor of trachelocercids, followed by Kovalevaia, Trachelolophos and Prototrachelocerca and that Trachelocerca, Tracheloraphis and possibly Sultanophrys derived from Prototrachelocerca. Our results mainly support this hypothesis except that Kovalevaia forms a sister branch with Clades I-III (of which Clade III comprises two Trachelolophos species) rather than grouping directly with Trachelolophos (Figure 11). In addition, the early branching of Foissnerella gen. nov. within Clade IV provides further evidence that multiple rows of uninterrupted circumoral kineties is probably an ancestral feature.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the online repositories.

AUTHOR CONTRIBUTIONS
YY and YW conceived to the study. MM, YL, and YY carried out the live observation, protargol staining, DNA extraction, and data analyses. All authors contributed to the writing of the manuscript. YY, MM, and AW contributed to the revision and all authors approved the final version.