Morphology and Molecular Phylogeny of Two New Trachelophyllid Ciliates, Monolamellophrya terricola gen. nov., sp. nov. and Trachelophyllum parapiculatum sp. nov. (Litostomatea, Haptoria), From South Korea

The morphology and molecular phylogeny of two new ciliates, Monolamellophrya terricola gen. nov., sp. nov. and Trachelophyllum parapiculatum sp. nov., discovered in South Korea, were investigated. The two species belong to the suborder Trachelophyllina, which is characterized by the presence of a mucilaginous layer containing lepidosomes covering the cortex. Monolamellophrya terricola gen. nov., sp. nov. is characterized by the presence of a single layer of type II lepidosomes, representing a new genus. Trachelophyllum parapiculatum sp. nov. has only type I lepidosomes covering the cortex, a generic character of the genus Trachelophyllum, and is distinguished from other congeners by a combination of morphological features, including the 15–24 μm long rod-shaped extrusomes, the 9–13 ciliary rows, the 7–11 and 17–25 dikinetids in brush rows 1 and 2, respectively, and the bipolar brush row 3. Furthermore, the 18S rRNA gene sequences of the two new species were provided. The phylogenetic analyses show that the sequence of M. terricola gen. nov., sp. nov. clusters with two other trachelophyllid sequences, and the sequence of T. parapiculatum sp. nov. is placed at the base of these three sequences with full support. Furthermore, the four trachelophyllid sequences that are available so far form a monophyletic clade.

They are characterized by the presence of organic epicortical scales, of unknown function called lepidosomes, embedded in a mucilaginous layer covering the cortex. The shape of the lepidosomes is considered a genus-specific character within the Trachelophyllina and can only be revealed using a scanning electron microscope, thus identification of these taxa based only on live observation and/or silver impregnation is insufficient (Foissner et al., 2002;Foissner, 2005Foissner, , 2016. So far, 12 types of lepidosomes were described in 19 well-characterized species and ten genera. Each genus is characterized by the presence of one, two, or three types of lepidosomes as follows: the genera Epitholiolus Foissner et al., 2002 and Trachelophyllum Claparéde and Lachmann, 1859 each has only a single type of lepidosomes; the genera Bilamellophrya Foissner et al., 2002, Cataphractes Foissner, 2016, Ileonema Stokes, 1884, Lingulothrix Foissner et al., 2002, Sleighophrys Foissner, 2005, and Spetazoon Foissner, 1994 each has two types of lepidosomes; and the genera Luporinophrys Foissner, 2005 and Trachelophyllides Foissner, 2016 each has three types of lepidosomes (Nicholls and Lynn, 1984;Foissner et al., 2002;Foissner, 2005Foissner, , 2016Bourland, 2017). Trachelophyllina genera belong to two different families mainly based on the dorsal brush rows organization. The family Lingulotrichidae Foissner, 2016 is characterized by the presence of more than three heteromorphic brush rows, while the family Trachelophyllidae Kent, 1881 is characterized by the presence of two dikinetidal and one monokinetidal brush rows (Foissner, 2016). Currently, the trachelophyllids are highly underrepresented in the phylogenetic trees, with only two 18S rRNA gene sequences available in the GenBank database. Unfortunately, the identification of these two sequences is doubtful because data on their lepidosomes are lacking (Vdačný et al., 2011;Jang et al., 2015Jang et al., , 2022Huang et al., 2018).
In this study, we investigate the morphology of two species discovered in South Korea. The first species is unique within the Trachelophyllidae in having a single layer of type II lepidosomes and thus representing a new genus, Monolamellophrya gen. nov., while the second species has only type I lepidosomes as other members of the genus Trachelophyllum and is distinguishable from other congeners by a combination of features, i.e., the 15-24 µm long rod-shaped extrusomes, the 9-13 ciliary rows, the 7-11 and 17-25 dikinetids in brush rows 1 and 2, respectively, and the bipolar brush row 3. Moreover, the 18S rRNA gene sequences of the two species were analyzed to determine their phylogenetic position.

Sample Collection and Identification
Monolamellophrya terricola gen. nov., sp. nov. was discovered in a soil sample collected near Bongnae Falls, Ulleung Island, South Korea (N 37 • 29 ′ 48.12 ′′ E 130 • 53 ′ 29.64 ′′ ) in August 2018. The soil sample was air-dried for at least 2 weeks and stored in a plastic bag. The sample was rewetted in February 2019 with mineral water (Jeju Samdasoo, Jeju Province Development Co., South Korea) to induce excystment of ciliates using the non-flooded Petri dish method (Foissner et al., 2002). Trachelophyllum parapiculatum sp. nov. was discovered in a water sample including debris collected from a temporary pond on a lawn in the Gangneung-Wonju National University, Gangneung-si, South Korea (N 37 • 46 ′ 12.4 ′′ E 128 • 52 ′ 16.5 ′′ ) after heavy rainfall in July 2020. The water sample was kept in a plant culture dish at room temperature. Attempts to establish enriched cultures were unsuccessful for both species. Living specimens were investigated using a stereomicroscope (Olympus SZ61, Tokyo, Japan) and a light microscope (Olympus BX53) with differential interference contrast at magnifications of 50-1,000×. The infraciliature was revealed by protargol impregnation and scanning electron microscopy. Protargol powder was synthesized using the methods described by Pan et al. (2013) and Kim and Jung (2017), and the protargol impregnation technique is based on 'procedure A' described by Foissner (2014). The SEM technique was conducted following the procedures described by Foissner (2014) and Moon et al. (2020). The terminology is according to Foissner et al. (2002) and Foissner (2016).

DNA Extraction, PCR Amplification, and Sequencing
Under the stereomicroscope, five cells were collected using a microcapillary from raw cultures of both species. The cells were transferred to habitat water filtered by a 0.2-µm syringe filter (Minisart R CA Syringe Filters; Sartorius, Aubagne, France), starved for at least 3 h, washed at least five times using the same filtered water to remove other eukaryotes, and then transferred to a 1.5-ml centrifuge tube each with a minimum volume of water. Genomic DNA was extracted using a RED-Extract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO, USA). The 18S rRNA gene was amplified using the primer New Euk A (5 ′ -CTG GTT GAT YCT GCC AGT-3 ′ ) , which is a slightly modified version of the primer Euk A in Medlin et al. (1988), and the primer LSU rev4 (5 ′ -GTT AGA CTY CTT GGT CCG TG-3 ′ ) (Sonnenberg et al., 2007) for M. terricola gen. nov., sp. nov. The primers New Euk A and Euk B (5 ′ -TGA TCC TTC TGC AGG  TTC ACC TAC-3 ′ ) were used to cover nearly the entire 18S rRNA gene of T. parapiculatum sp. nov. The PCR conditions for the primers New Euk A and LSU rev4 were as follows: denaturation at 94 • C for 1 min 30 s, followed by 40 cycles of denaturation at 98 • C for 10 s, annealing at 58.5 • C for 30 s, and extension at 72 • C for 3 min, and a final extension step at 72 • C for 7 min. The PCR conditions for the primers New Euk A and Euk B were as follows: denaturation at 94 • C for 90 s, followed by 40 cycles of denaturation at 98 • C for 10 s, annealing at 58.5 • C for 30 s, extension at 72 • C for 2 min, and a final extension step at 72 • C for 7 min. For purification of the PCR products, MEGAquickspin Total Fragment DNA Purification Kit (iNtRON Biotechnology, South Korea) was used. DNA sequencing was performed using an ABI 3700 sequencer (Applied Biosystems, Foster City, CA, USA). New Euk A, LSU rev4, and three internal primers [18SF790v2: 5 ′ -AAA TTA KAG TGT TYM ARG CAG-3 ′ , 18SR300: 5 ′ -CAT GGT AGT CCA ATA CAC TAC-3 ′ (Park et al., 2017), and 18SF1470: 5 ′ -TCT GTG ATG CCC TTA GAT GTC-3 ′ (Jung et al., 2018)] were used for the sequences of M. terricola gen. nov., sp. nov., and only New Euk A and Euk B primers were used for the sequences of T. parapiculatum sp. nov.

Phylogenetic Analyses
The 18S rRNA gene sequences of M. terricola gen. nov., sp. nov. and T. parapiculatum sp. nov. were assembled using Geneious 9.1.5 (Kearse et al., 2012). To determine the phylogenetic position of the two new species, 18S rRNA gene sequences of 77 ciliates were retrieved from the NCBI database, including three metopids as outgroup taxa: Clevelandella panesthiae (KC139719), Metopus palaeformis (AY007450), and Nyctotherus ovalis (AJ222678). The sequences were aligned using ClustalW (Thompson et al., 1994), and both ends were manually trimmed using BioEdit 7.0.9.0 (Hall, 1999). The length of the final alignment was 1609 bp. Using jModelTest 2.1.7 (Darriba et al., 2012), the best-fit evolutionary model TVM+I+G under the Akaike information criterion (AIC) was selected. The maximum likelihood (ML) tree was constructed using IQ-Tree 1.5.3 (Nguyen et al., 2015). The reliability of internal branches was assessed using a nonparametric bootstrap method with 100,000 replicates. The number of nucleotide differences and pairwise sequence similarity was calculated using MEGA 6.06 (Tamura et al., 2013). MrBayes 3.1.2 (Ronquist et al., 2012) was used for Bayesian inference (BI) analyses with Markov Chain Monte Carlo for 3,000,000 generations at a sampling frequency of every 100 generations, and the first 25% of trees were discarded as burn-in. Phylogenetic trees were visualized using the free software package FigTree version 1.4.3 by Rambaut (2006).  Lynn, 1996Class Litostomatea Small and Lynn, 1981Subclass Haptoria Corliss, 1974Order Spathidiida Foissner and Foissner, 1988Suborder Trachelophyllina Grain, 1994Family Trachelophyllidae Kent, 1881 Monolamellophrya gen. nov.

Diagnosis
Trachelophyllidae with only one layer of type II lepidosomes. Lepidosomes with conical superstructure composed of six or seven concave meridional arcs.

Etymology
Composite of the Greek numeral mono (one), the Latin noun lamella (thin plate), and the Greek noun ophrya (eyebrow, cilia, ciliate), referring to the single type of lepidosome. Feminine gender.

Species Assignable
Only the type species.

Etymology
The Latin species-group name terricola (living in soil) refers to the habitat in which the species was discovered.
Size 100-160 × 15-25 µm in vivo and 75-137 × 11-23 µm after protargol impregnation; length:width ratio ∼ 6.7:1 in vivo and 3.9-11.4:1 (6.1:1 on average) after protargol impregnation. Body slenderly fusiform, slightly flattened dorsoventrally, with slightly (in contracted specimens) to distinctly (in extended specimens) narrow neck, ∼ 5 µm wide in protargol preparations, slightly widened in the oral region, and gradually broadened posteriorly merging into the wider, slender trunk. The anterior end (oral bulge) narrow and conical, the posterior end usually narrowly rounded, rarely broadly rounded (   Cortex thin and very flexible, covered with a very thin mucilaginous layer of lepidosomes, recognizable only in scanning electron micrographs. Mucilaginous layer composed of a single layer of tightly spaced type II lepidosomes (Figures 3C-L). Individual lepidosomes 1.0 × 0.7 × 0.6 µm on average in scanning electron micrographs, baseplate circular to broadly ellipsoidal, finely faceted, central area slightly to distinctly convex ( Figures 1H, 3I), gives rise to six or seven fine, concave arcs, forming cone-shaped superstructure in lateral view and a rosettelike structure in top view (Figures 1G-I, 3I-L).
Oral bulge inconspicuous in vivo and after protargol impregnation because of hyaline and small size (2 × 2 µm in stained cells); conical and set off from body proper, contains extrusomes. Circumoral kinety at the base of the oral bulge, composed of ∼12 dikinetids, each bearing a single cilium and inconspicuous nematodesmata extending posteriorly forming a pharyngeal basket (Figures 1A,C,E,F, 2A,B,E).

Etymology
Composite of the Greek prefix para-(besides, like, resembling) and the species-group name apiculatum, referring to the similarity to T. apiculatum (Perty, 1852) Claparéde and Lachmann, 1859.
Micronuclei near or attached to macronuclear nodules, spherical ( Figures 4A,B,D, 5C,E, 6A,B). Contractile vacuole in posterior body end and connects with single terminal excretory pore by a distinct excretory canal (Figures 4A, 5C,E,F). Extrusomes straight or slightly curved rod-shaped, 15-24 µm long in vivo, form bundles in the oral region and extend into the oral bulge and scattered or form bundles in the cytoplasm, do not impregnate with protargol (Figures 4A,J, 5A,E,G). Cytoplasm colorless, contains 1-3 µm across lipid droplets and up to 10 µm across food vacuoles containing flagellates and small ciliates (Figures 4A, 5B,C,E). Usually swims slowly and rarely glides on the bottom of the culture dish. Cortex thin and very flexible, contains small and irregularly arranged cortical granules (Figure 5I), covered by a mucilaginous layer of lepidosomes, recognizable in vivo and scanning electron micrographs. Mucilaginous layer composed of tightly spaced type I lepidosomes (Figures 4A, 5F,G, 6D-H). Individual scales 1.0-2.1 × 0.7-1.7 × 0.3-0.5 µm (1.4 × 1.1 × 0.4 µm on average; n = 12) in scanning electron microscopes, baseplate circular to elliptical finely faceted, perforations in baseplate margin hardly recognizable, superstructure hemispherical composed of an average of 11 polygons (Figures 4F-I, 6D-H).
Cilia in vivo ∼8 µm in length, rather widely spaced. Ciliary rows meridional and equidistant; two of them (dorsal brush rows) form dikinetids anteriorly and continue posteriorly as ordinary somatic ciliary rows. Brush rows 1 and 2 composed of 7-11 and 17-25 dikinetids, respectively; row 1 approximately half the length of row 2, both bearing 3-4 µm long immobile, parallel bristles; bristles laying down and possibly covered by epicortical scale layer and hardly recognizable both in vivo and in scanning electron micrographs (Figures 4C,E). Brush row 3 extends to the posterior body end, composed of widely spaced monokinetids bearing 2-3 µm long immobile bristles, recognizable both in vivo and after protargol impregnation (Figures 4A, 5D,H, 6B,C,D; Table 1). In a few specimens, the row right of brush row 3 commences with shortened cilia as long as brush bristles and continues posteriorly as ordinary somatic ciliary rows.
Oral bulge conspicuous in vivo because of extrusomes, 4 × 5 µm after protargol impregnation; cylindrical and set off from body proper and not covered by lepidosomes. Circumoral kinety at the base of the oral bulge, composed of ∼11 dikinetids, each bearing a single cilium and inconspicuous  nematodesmata extending posteriorly, forming a pharyngeal basket (Figures 4C,E, 5A,D,G, 6C-E,I).

Phylogenetic Analyses
The 18S rRNA gene sequences of M. terricola gen. nov., sp. nov. and T. parapiculatum sp. nov. are 1,490 base pairs long each, having a GC content of 40.15% and 40.27%, and are available under GenBank accession numbers ON212658 and ON212659, respectively. The phylogenetic trees using ML and BI analyses show rather similar topologies, thus only the ML tree was used (Figure 7). In the phylogenetic tree, the trachelophyllid sequences form a monophyletic clade with full supports. The sequence of M. terricola gen. nov., sp. nov. exhibits a similarity of 99.86% (2 nucleotides difference) with both T. brachypharynx and an unidentified Trachelophyllum sp., and clusters with both sequences with high (93% ML) and low (0.78 BI) support forming a polytomy (Figures 7, 8; Table 2). The sequence of T. parapiculatum sp. nov. is placed as a sister branch to these three sequences with full support and shows a similarity of 99.39% (9 nucleotides difference) with both M. terricola gen. nov., sp. nov. and T. brachypharynx and shows a similarity of 99.46% (8 nucleotides difference) with Trachelophyllum sp. (Figure 8; Table 2).

Monolamellophrya gen. nov. as a New Genus
Members of the family Trachelophyllidae are characterized by the two dikinetidal and one monokinetidal dorsal brush rows and the presence of lepidosomes. Currently, 16 wellcharacterized trachelophyllid species belonging to eight genera are assigned to the family, mainly based on the types of the lepidosomes (Nicholls and Lynn, 1984;Foissner, 1994Foissner, , 2005Foissner, , 2016Foissner et al., 2002;Bourland, 2017). Only two genera have a single type of lepidosomes each, namely, Epitholiolus with only type III lepidosomes and Trachelophyllum with only type I lepidosomes. Each of the other six genera has two or three types of lepidosomes. The new genus, Monolamellophrya gen. nov., is unique in having only one layer of type II lepidosomes. According to Foissner (2005Foissner ( , 2016 and Foissner et al. (2002), type II lepidosomes have four rather different shapes, and all of them are characterized basically by the cone-shaped superstructure and are found only in the genera Bilamellophrya and Luporinophrys. The first shape is found only in Bilamellophrya australiensis Foissner et al., 2002 and is characterized by the presence of ∼10 concave, unevenly spaced arcs rising from the baseplate and fusing together at their ends forming a cone and connected to each other by three transverse rings. The second shape is found in B. hawaiiensis Foissner et al., 2002 andB. fraterculus Foissner, 2016 and is characterized by the presence of 12-16 scattered polygons in the cone-shaped superstructure. The third shape is found only in Luporinophrys micelae Foissner, 2005, and it has a larger size than other type II lepidosomes (4 × 4 µm) and its superstructure is formed by ∼20 fine arcs (fibers) in two to five groups rising from the baseplate and fusing together at their ends forming a narrow, curved dome. The fourth shape is simple and consists of approximately seven arcs rising from the baseplate and fusing together forming a cone-shaped superstructure and found in B. etoschensis Foissner et al., 2002. The lepidosomes of M. terricola gen. nov., sp. nov. are similar to the fourth shape of type II in the shape of the superstructure but slightly differ in the thickness of the arcs (very thin vs. thick) and the shape of the baseplate (slightly to distinctly convex in middle portion vs. obconical). However, the convexity in the middle portion of the baseplate resembles the obconical baseplate described by Foissner et al. (2002) but is more shallow probably due to the preparation procedure. However, this variability in lepidosomes, especially  Foissner et al., 2002Foissner et al., 2002Foissner et al., 2002Foissner, 2016 type II, suggests that the trachelophyllids are an underestimated group and more studies are needed to reveal its true diversity.
Comparison of Monolamellophrya terricola gen. nov., sp. nov. With Closely Related Species In vivo, M. terricola gen. nov., sp. nov. is hardly distinguishable from most trachelophyllid species because they have similar appearances and their sizes usually overlap. In addition, the mucilaginous layer of M. terricola gen. nov., sp. nov. is not recognizable in vivo and after protargol impregnation and thus could be misidentified as a non-trachelophyllid (without mucilaginous layer) haptorid. After protargol impregnation, many trachelophyllid species look similar and even have a similar number of ciliary rows and nuclear apparatus patterns. Thus, the scanning electron microscope is indispensable for the proper identification of such taxa. M. terricola gen. nov., sp. nov. is very similar to three Bilamellophrya species, namely, B. etoschensis, B. fraterculus, and B. hawaiiensis especially in body size and shape, the number of somatic kineties, and the overlapping numbers of dikinetids in dorsal brush rows (Table 3). However, M. terricola differs from these species mainly by the types of lepidosomes (one type vs. two types). Moreover, M. terricola gen. nov., sp. nov. is similar to B. hawaiiensis in having acicular extrusomes, but B. hawaiiensis has an extra type of extrusomes (rod-shaped).

Comparison of Trachelophyllum parapiculatum sp. nov. With Closely Related Species
The trachelophyllid genus Trachelophyllum is characterized by having a mucilaginous layer composed only of type I lepidosomes (Foissner et al., 2002;Foissner, 2016). At present, ∼31 species are assigned to the genus, most of which probably belong to different litostomatean families (Stokes, 1888;Levander, 1894;Penard, 1922;Dumas, 1930Dumas, , 1937Kahl, 1930;Gajewskaja, 1933;Lepsi, 1960;Tucolesco, 1962;Grolière, 1977;Foissner, 1983Foissner, , 1984Foissner et al., 2002). However, the lepidosomes of only six species, namely, T. apiculatum, T. africanum Foissner et al., 2002, T. costaricanum Foissner et al., 2002, T. lineare Lepsi, 1960, T. pannonicum Foissner et al., 2002, and T. tachyblastum Stokes, 1884, are characterized using the scanning electron microscope, and thus their assignment is doubtless. When comparing T. parapiculatum sp. nov. with other congeners, we recognized that the genus Trachelophyllum is divided into two groups based on the length of dorsal brush row 3: the first group consists of species with bipolar (extends to or near to posterior body end) brush row 3, namely, two populations of T. apiculatum, T. costaricanum, T. hyalinum Foissner, 1983, T. pannonicum, T. clavatum Stokes, 1886, and T. parapiculatum sp. nov.; and the second group consists of all other species and populations either with dorsal brush row 3 similar or slightly longer than row 2 or with brush row 3 of unknown morphology but with special morphological characters such as the coloration, the body shape, or the nuclear apparatus (Foissner et al., 1995(Foissner et al., , 2002Foissner, 2016). Several populations were described as T. apiculatum, but they differed from each other by at least a single important character ( Table 4), suggesting that they should be classified as distinct species. The Venezuelan neotype population of T. apiculatum (population 8 in Table 4) designated by Foissner et al. (2002) has a short dorsal brush row 3, a character that was underestimated by the authors at that time, and differs also from T. parapiculatum sp. nov. by the number of dikinetids in dorsal brush row 1 (10-24 vs. 7-11). Trachelophyllum parapiculatum sp. nov. could be confused with two different populations described as T. apiculatum with long brush row 3: (1) the population described by Foissner (1983) (population 4 in Table 4) differs from T. parapiculatum sp. nov. in the extrusomes length (13 vs. 15-24 µm) and the number of dikinetids in dorsal brush row 2 (∼8 vs. 17-25); and (2) population II in Foissner (1984) (population 6 in Table 4), which is distinguishable from T. parapiculatum sp. nov. by the number of somatic ciliary rows (22-25 vs. 9-13) and the number of dikinetids in dorsal brush row 1 (16-26 vs. 7-11) ( Table 4). Trachelophyllum parapiculatum sp. nov. is also morphologically very similar to T. costaricanum and T. pannonicum and can be separated from each of them mainly by the types and shapes of the extrusomes, i.e., T. parapiculatum sp. nov. has only one type of rod-shaped extrusomes, while T. costaricanum and T. pannonicum each has two types of acicular and rod-shaped extrusomes. T. hyalinum, which was studied only in vivo and after silver impregnation and without morphometric data, differs from T. parapiculatum sp. nov. in having longer extrusomes (∼30 µm vs. 15-24 µm long) that do not extend into the oral bulge. T. clavatum is also characterized by long brush row 3 (i.e., extends to two-thirds of the body length) and differs from other congeners by the presence of a single macronuclear nodule (Stokes, 1888;Foissner, 1983;Foissner et al., 2002). Trachelophyllum tachyblastum resembles T. parapiculatum sp. nov. in body shape and size but differs mainly in the length of dorsal brush row 3 (short vs. bipolar), the types and shapes of the extrusomes (two types of rod-shaped and oblong vs. one type of rod-shaped extrusomes), and the numbers of dikinetids in brush rows 1 and 2 (4-7 and 6-15 vs. 7-11 and 17-25, respectively). T. lineare, which was described as Lepidotrachelophyllum fornicis by Nicholls and Lynn (1984) and Lynn and Nicholls (1985) and as L. lineare by Foissner (1994) and Foissner et al. (1999), has a large body size [up to 600 µm according to Lepsi (1960) and 40-350 µm according to Nicholls and Lynn (1984)] and a higher number of somatic ciliary rows ( Table 4). T. brachypharynx Levander, 1894, which was studied recently by Jang et al. (2015), is a large species (330-445 × 35-45 µm) with filiform extrusomes ∼30 µm long and 20-25 somatic ciliary rows. However, the lepidosomes of T. brachypharynx were observed only in vivo and described as hat-shaped. Thus, the assignment to the genus Trachelophyllum is questionable, as noted by Jang et al. (2015).

Phylogenetic Analyses
The new phylogenetic tree shows that the family Trachelophyllidae is monophyletic. The trachelophyllid clade comprises only four sequences: M. terricola gen. nov., sp. nov., T. parapiculatum sp. nov., T. brachypharynx, and an unidentified Trachelophyllum sp. However, like the unidentified Trachelophyllum sp., the assignment of T. brachypharynx is doubtful because the shape of the lepidosomes was not studied using a scanning electron microscope. According to Jang et al. (2015), the lepidosomes of T. brachypharynx in vivo look very similar to type V lepidosomes with their hat-shaped structure as described by Foissner (2005) and, up to date, found only in the genus Sleighophrys. Furthermore, the new phylogenetic tree shows a close relationship between the families Trachelophyllidae and Enchelyodontidae (Foissner et al., 2002) based on morphological similarities as discussed by Jang et al. (2015). Clearly, the two families are underrepresented in the phylogenetic tree and obtaining more trachelophyllid and enchelyodontid sequences would be of great help, especially from properly identified species (Foissner et al., 2002;Foissner, 2005Foissner, , 2016Jang et al., 2015;Bourland, 2017).

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: https://www.ncbi.nlm. nih.gov/genbank/ [GenBank accession numbers: ON212658 and ON212659].