﻿A new species of the genus Cephalodella (Rotifera, Monogononta) from Korea, with reports of four additional cephalodellid species

﻿Abstract A new monogonont rotifer, Cephalodellabinoculatasp. nov., was described from a soil sample collected in Korea. The new species is morphologically similar to C.carina but is distinguished by having two frontal eyespots, a vitellarium with eight nuclei, and the shape of its fulcrum. We also described four other cephalodellid species collected in Korea; Cephalodellaauriculata, C.catellina, C.gracilis, and C.tinca. Of these four species, C.gracilis and C.tinca were newly recorded in Korea. We provided the morphological characteristics of the five Cephalodella species along with photographs of trophi observed with a scanning electron microscope. Furthermore, we provided the mitochondrial cytochrome c oxidase subunit I gene sequences of the five species.


Introduction
The genus Cephalodella Bory de St. Vincent, 1826 is one of the most species-rich taxa in the phylum Rotifera Cuvier, 1817, containing 171 species worldwide (Segers 2007;Jersabek and Leitner 2013). This taxon is easily found in various environments but is difficult to distinguish due to its morphological similarity and fragile external characteristics (Jersabek et al. 2011). Like other taxa of Rotifera, Cephalodella has been mainly studied in Europe, and their biology, ecology, and variability are not well known because of the lack of research (Nogrady and Pourriot 1995).
In this study, we identified five cephalodellid rotifers, one of which was a new species. Two species, Cephalodella gracilis (Ehrenberg, 1830) and C. tinca Wulfert, 1937 were newly recorded in Korea and two others, C. auriculata and C. catellina, have previously been recorded in Korea. However, since the first reported paper on rotifers in Korea (Turner, 1986) did not include descriptions for these two species, we have described the two Korean specimens in this study. Here, we provide the morphological characteristics of the five species along with the photographs of trophi observed with scanning electron microscope (SEM). In addition, we deciphered the mitochondrial cytochrome c oxidase subunit I (COI) gene sequences of the five species.

Materials and methods
Specimens were collected and isolated from a pond, reservoir and soil samples (Fig. 1). The rotifers inhabiting pond and reservoir were collected using a 50-µm mesh plankton net and transferred to the laboratory alive. In case of soil sample treatment, we dried the soil samples at room temperature for several weeks and rewetted them using mineral water in a plant culture dish (310100, SPL Life Science, Korea). After hatching of the rotifers, they were isolated in a new plant culture dish under a stereo microscope (SZX7, Olympus, Japan) and stored in an incubator at 20 °C. Before the observation and preservation of living rotifers, a few drops of 1% bupivacaine solution (B5274, Sigma-Aldrich, USA) were used for anesthesia. The specimens were then observed under an optical microscope (DM2500, Leica, Germany) at magnifications of ×400-1000. Photographs and videos of the specimens were obtained using a digital camera (EOS 6D Mark II, Canon, Japan) mounted on an optical microscope. Trophi were isolated using commercial bleach containing 4-5% NaClO (Yuhan-Clorox, Korea) and prepared for SEM following the methods of De Smet (1998). Two SEM instruments, SU8010 and S-4300SE (Hitachi, Japan), were used for observation at an accelerating voltage of 7-10 kV. External characteristics and trophi elements were measured using ImageJ 1.53k (https://imagej.nih.gov/ij/) (Abràmoff et al. 2004).
Morphological identification of rotifers was based on descriptions of Koste (1978), Nogrady and Pourriot (1995), and Jersabek and Leitner (2013). All specimens described in this study were deposited at the National Institute of Biological Resources (NIBR), Korea.
Genomic DNA was extracted using a LaboPass TM Tissue Genomic DNA Isolation Kit Mini (Cosmo Genetech, Korea). Partial COI gene was amplified using the 30F/885R primers (Zhang et al. 2021). The PCR amplifications were conducted in a final volume of 25µL under the following conditions: 2 min at 95 °C for the initial denaturation, followed by 40 cycles of 95 °C for 15 s, 51 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 5 min. In the case of C. auriculata and C. tinca, the primer sets mlCOIintF/ jgHCO2198 (Leray et al. 2013) and LCO1490/HCO2198 (Folmer et al. 1994) were used at an annealing temperature of 45 °C. PCR products were visualized by 1% agarose gel electrophoresis, and purified using a LaboPass TM PCR Purification Kit (Cosmo Genetech). DNA sequencing was performed at Macrogen (Korea), and the sequences were trimmed and aligned using Geneious ver. 8.1.9 (https://www.geneious.com). Genetic distance was calculated using MEGA ver. 11 with the Kimura 2-parameter model (K2P) (Tamura et al. 2021). All the extracted DNAs of the five species were deposited at the NIBR, and COI sequences were uploaded to GenBank.
The maximum-likelihood (ML) tree was inferred based on the partial COI gene sequences of 11 notommatid species and one euchlanid species (Table 1). The ML tree was constructed using IQ-TREE ver. 1.6.12, with the GTR+I+G model and 1000 replicates (Nguyen et al. 2015;Kalyaanamoorthy et al. 2017).

Results and discussion
In the present study, we identified five cephalodellid species in Korea; C. auriculata, C. binoculata sp. nov., C. catellina, C. gracilis, and C. tinca. The new species, C. binoculata sp. nov., was distinguished from other cephalodellid species by a combination of the following characteristics: two distinct frontal eyespots, short tail and toes, vitellarium with eight nuclei, and the shape of the trophi components. Two species, C. gracilis and C. tinca were newly recorded in Korea. Cephalodella gracilis is a common species worldwide. However, the morphological characteristics of C. gracilis have been reported to exhibit high morphological variation (Nogrady and Pourriot 1995), and it is necessary to re-examine these characteristics through morphological redescription and molecular analysis. Cephalodella tinca is probably a cosmopolitan species and has been recorded in the Australian, Neotropical, Oriental, and Palearctic regions (Segers 2007). The remaining two species, C. auriculata and C. catellina were recorded in Korea by Turner (1986) as a species list without description. Therefore, we described the Korean specimens of the two species and provided photographs of the trophi observed using SEM.
In this study, we obtained partial COI sequences from each of the five species and constructed an ML tree using the sequences of 11 notommatid rotifers and one euchlanid rotifer. The sequence of Euchlanis dilatata Ehrenberg, 1830 was used as the outgroup. The final length of the sequence alignment was 561 bp, and the genetic distance between the notommatid species was 0.172-0.412 (Table 2). The species in the genus Cephalodella formed a monophyletic group, with a support value of 100 (Fig. 7). The new species, C. binoculata sp. nov., formed a clade with C. auriculata and C. gracilis and was located closest to C. auriculata. However, the phylogenetic relationships between species within the Cephalodella was not clearly revealed when compared using morphological characteristics. Although more than 170 morphospecies of the genus Cephalodella have been recorded worldwide (Nogrady and Pourriot 1995;Segers 2007;Jersabek and Leitner 2013), only seven sequences from two species, C. forficula and C. gibba, have been registered in GenBank. For the phylogenetic study of cephalodellid rotifer species, further acquisition and analysis of the COI sequences and nuclear gene sequences such as 18S ribosomal RNA or internal transcribed spacer (ITS), is required.  Wulfert, 1959 in terms of frontal eyes, type B virgate trophi, dorsally curved toes, total length/toe length ratio, and short tail. The new species, however, was distinguished from C. carina by the following characteristics: (1) the new species has two distinct eyespots, whereas C. carina has one small eyespot; (2) the vitellarium of the new species contains eight nuclei, while that of C. carina contains six; and (3) the fulcrum of the new species is straight and without extension at the distal end, while the fulcrum of C. carina is thicker at the distal end.
The new species also resembles C. gibboides Wulfert, 1951 andC. graciosa Wulfert, 1956. However, it is distinguished from C. gibboides by the shape of its manubrium and tail length. The manubrium of C. gibboides has a bump in the middle with no basal lamellae, whereas the new species has basal lamellae in the manubrium and no bumps in the middle. The shape of the distal end of the manubrium also differed between the two species. The tail of C. gibboides covers the foot, whereas that of the new species is short. The new species is distinguished from C. graciosa in several morphological characteristics as follows: (1) the trophi of the new species is symmetrical, while that of C. graciosa is asymmetrical; (2) the manubrium of the new species has basal lamellae, while that of C. graciosa does not; (3) the new species has two eyespots, while C. graciosa has one eyespot; and (4) the new species has eight nuclei in the vitellarium, while C. graciosa has six.
Description. Female. Body moderately elongated and not laterally compressed (Figs 2, 3A, B). Dorsal and ventral margins slightly convex; posterior third of trunk gradually tapered to the foot. Lorica soft, transparent, and comprised of three body plates. Dorsal and ventral plates separated by narrow lateral sulci. Tail short and rounded. Head large, almost one-quarter of the total length. Head and trunk clearly distinguished by the neck fold. Corona oblique, convex, without lips. Dorsal antenna located near the junction of the head and trunk. Foot trapezoidal shape and moderate size, approximately 15% of the total length. Foot widest at the front and narrowed toward the back. Caudal setae absent. Short tail covered only part of the foot. Toes symmetrical and short, accounting for 16-17% of the total length. Toes smoothly tapered to posterior end, without any spines. In the lateral view, toes curved dorsally. In the dorsal view, toes always curved outwards. Saccate large brain extending over the neck fold. No retrocerebral organ. Two distinct red eyespots located in front of the head (Fig. 3C). Distance between the two eyespots far and clear. Mastax large, with elongated salivary gland. Esophagus thin, passing between the brain and mastax. Gastric glands large, oval shaped, containing round granules, and located in the antero-dorsal part of the stomach (Fig. 3D). Stomach colorless and indistinctly separated from the intestine. Anus located near the posterior end of the foot. Bladder round and large when fully  (Fig. 4A). Rami with no alulae on posterior end (Fig. 4C). Basal chamber of rami wide, left side relatively larger than right at distal end. Shape of the subbasal chamber foramina also slightly asymmetrical; both foramina oval shaped, but the right foramen larger in length. Inner  margin of rami with two distinct teeth and several comb-like teeth (Fig. 4C). Fulcrum long and straight in the ventral view (Fig. 4A). Terminal end of fulcrum simple, without any thickening or expanded shape. In the lateral view, the ventral margin straight and relatively thick (Fig. 4B). No basal apophysis on fulcrum. Uncus with a large, single tooth. Manubria symmetrical. Each manubrium with a basal lamella, the length of which was approximately half of that of the manubrium (Fig. 4D). Middle part of the manubrium with oblong-shaped foramina. Shaft of the manubrium thick and straight in lateral view; while terminal end curved inward in ventral view. Terminal end crutchshaped, dorsal side stubby, ventral side pointed and curved upwards (Fig. 4B, D). Characteristics of male and eggs remain unknown. Measurement. Total length 134-155 µm, toe 26-29 µm, trophi 24-28 µm, ramus 8-9 µm, fulcrum 15-17 µm, manubrium 14-17 µm.
Remarks. The morphological characteristics of the Korean specimens generally corresponded to those reported in a previous study (Nogrady and Pourriot 1995). The body was soft and stout, 110-130 µm in length (Fig. 5C). The head was large and as wide as the body. Foot was short and wide. The toes were short, 23-28 µm in length. The two toes were equal in length and curved ventrally. One red cerebral eye was located at the posterior end of the saccate brain. The vitellarium had eight nuclei. Trophi was symmetrical and virgate type A, 30 µm in length (Fig. 6A). The fulcrum was long and straight. The manubrium was thin and curved. The rami of Korean specimen had blunt teeth at the apical part, whereas the specimens of previous studies had no teeth at the apical part (Koste and Shiel 1991;Nogrady and Pourriot 1995).
Molecular data. Partial COI sequence was obtained from one Korean specimen (NIBR deposit number, NIBRGR0000649738; GenBank accession number, ON898533).
Remarks. Korean specimens of C. catellina had morphological characteristics that were generally consistent with those reported in previous studies (Koste and Shiel 1991;Nogrady and Pourriot 1995). The body was short and stout, and 100 µm in length (Fig. 5D). The posterior end of the body bulging. The head was large and approximately one-third of its total length. The foot and toes were located ventrally. The two toes were short and symmetrical, 12-16 µm in length (Fig. 6C). The two frontal eyes were red. The vitellarium had eight nuclei. The salivary glands were located under the mastax. Trophi was asymmetrical, virgate type C, and 25 µm in length. The fulcrum was straight and long, with a slightly expanded distal end. The manubria were asymmetrical and curved inward. The right manubrium was larger than left manubrium. Distal ends of both manubria had incomplete loop. The right ramus had tooth-like alula.
Molecular data. Partial COI sequence was obtained from one Korean specimen (NIBR deposit number, NIBRGR0000649739; GenBank accession number, ON898532).
Remarks. The body size of the Korean specimens was 120-125 µm in length (Fig. 5B). The soft body was elongated and compressed laterally. The head was clearly distinguished from the body by a neck fold. The foot was conical in shape, short, and half the length of the toes. The length of the toes was 20-25 µm, less than one-fifth of the total length. The two toes were equal in length and slightly curved dorsally. One red eye was located at the front of the head. The vitellarium was large and contained four nuclei. The large trophi was symmetrical, virgate type B, and had a length of 20 µm (Fig. 6B). The fulcrum was long and straight without expansion at the end. The manubrium was long and crutched, with a bulge in the middle. The uncus had one tooth and was less than half the length of the manubrium. The rami were denticulated.
Cephalodella gracilis has been reported to have high morphological variation in the shape of the toes and trophi. The Korean specimen had dorsally curved toes that gradually tapered toward the end. The trophi shape of the Korean specimen did not correspond to a specific specimen but was most similar to that described by Jersabek et al. (2003) in that it had a straight, slender fulcrum without expansion and a crutched manubrium end. However, this species can be regarded as a species complex, based on its morphological diversity and cosmopolitan distribution. Therefore, it is necessary to re-examine it through morphological redescription and molecular analysis.

Cephalodella tinca
Remarks. The body was 200-220 µm long, elongated, and laterally compressed (Fig. 5A). The dorsal and ventral margins were slightly bulbous in the lateral view. The  Wulfert, 1937. Scale bars: 10 µm. lorica was flexible and transparent. The head was large, approximately one-fourth of the total length, and clearly distinguished from the body by the neck fold. The tail was rounded and as long as the foot. The toes were equal in length, slightly curved dorsally, and approximately one-fourth of the total length. A pair of red eyes was located at the front of the head. The mastax was large and had a salivary gland. The gastric glands were round and contained several granules. The vitellarium was large and had eight nuclei. The trophi was virgate type D, and symmetrical (Fig. 6D). The fulcrum was straight and slightly spatulated at the posterior end. The manubrium was thick and had basal lamellae. The tip of the manubrium expanded and curved inward. The uncus had one large tooth.
Morphological characteristics of Korean C. tinca specimens corresponded well to the original description except for the size of body length. The body length of the Korean specimen was 200-220 µm, which was slightly smaller than the original description (260-280 µm) (Wulfert 1937).
Molecular data. Partial COI sequence was obtained from one Korean specimen (NIBR deposit number, NIBRGR0000649740; GenBank accession number, ON898534).