A single specimen of Neoclinus (family Chaenopsidae) was collected from the East Sea, Korea, in June 2016.This is the first report of the larval stage of Neoclinus chihiroe Fukao, 1987. The lack of distinctive morphological features in fish larvae makes species identification difficult; however, the species was identified through DNA barcoding. Analysis of mitochondrial DNA cytochrome oxidase subunit I revealed that the larval specimen corresponds to an adult of N. chihiroe reported from Korea in 2021. Morphologically, the larval specimen of N. chihiroe is characterized by the posterior tip of the maxilla being located below the middle of the eye, two multifid cirri on the orbit, dorsal fin with 13 spines and 15 soft rays, anal fin with 2 spines and 27 soft rays, and 13 pectoral fin rays. In the pigmentation pattern, there are three pairs of melanophores on the top of the head (one pair in each of the interorbital, occipital, and nape regions), several melanophores in a row from the isthmus to the anus, six pale melanophores on both sides of the dorsal contour, and many melanophores along the ventral contour on the anal fin base.

larval fish, Neoclinus chihiroe, DNA barcoding, identification, East Sea

Accurate identification of fish larvae is important and essential for understanding early life history and ecology and supports biodiversity research (Smith et al. 2018; Marancik et al. 2020; Appleyard et al. 2022). However, identifying fish larvae is very difficult because of a lack of diagnostic morphological characters or their shape information is unknown (Hulley et al. 2018; Pozzobon et al. 2024). Therefore, recent research on larval fish identification has incorporated both morphological and genetic analyses (Ko et al. 2013; Azmir et al. 2017; Xing et al. 2022).

The fringehead genus Neoclinus Girard, 1858 (family Chaenopsidae) consists of benthic marine fishes that inhabit rocky near shore areas or areas with a sandy bottom below the low tide line (Froese and Pauly 2025). Eleven species of the genus occur in coastal areas of the Pacific Ocean (Hubbs 1953; Murase et al. 2010), of which eight species occur on the western side of the ocean and three on the eastern side. Of the eight western species, four have been reported from Korea (Myoung and Kwun 2022): Neoclinus bryope (Jordan et Snyder, 1902), Neoclinus chihiroe Fukao, 1987, Neoclinus lacunicola Fukao, 1980, and Neoclinus nudiceps Murase, Aizawa et Sunobe, 2010. Research on this genus has consisted mostly of taxonomic reviews, descriptions of new species, and documentation of distribution patterns (Jordan and Snyder 1902; Hubbs 1953; Fukao 1980, 1987; Murase et al. 2010; Hongjamrassilp et al. 2020). There have been no studies of the larval stage of the genus except for the life history of N. bryope and larval descriptions of Neoclinus blanchardi Girard, 1858, Neoclinus okazakii Fukao, 1987, Neoclinus stephensae Hubbs, 1953, and Neoclinus uninotatus Hubbs, 1953 (see Shiogaki and Dotsu 1972; Watson 1996, 2009; Lindquist 1981; Okiyama 2014).

In June 2016, a larval stage of Neoclinus was collected from the eastern coast of Ulleung Island, East Sea (otherwise known as the Sea of Japan), and was identified as N. chihiroe by means of molecular analysis. The larva of this species has not previously been reported, so a morphological description is provided for the first time.

A single specimen of a Neoclinus chihiroe larva was collected in June 2016 from near Juk Island, which is located off the eastern coast of Ulleung Island, East Sea, Korea (Fig. 1), and fixed as a whole body in 99% ethanol. All counts and measurements (to the nearest 0.1 mm) were made following Kendall (2011) using a stereomicroscope camera and associated imaging software (Leica Application Suite V.4.13; Leica, Germany). The specimen is deposited in the National Marine Biodiversity Institute of Korea.

Figure 1. 

Sampling location (red dot) of Neoclinus chihiroe larva off Ulleung Island, East Sea.

Genomic DNA was extracted from the right eye of the specimen with a DNeasy Blood and Tissue Kit (Qiagen, Germany). The mitochondrial DNA Cytochrome Oxidase subunit I (COI) gene was amplified with a fish universal primer set (VF2_t1 and FishR2_t1) (Ward et al. 2005). The nucleotide sequence of the larval specimen and comparative species were deposited in the DDBJ/EMBL/GenBank databases (accession numbers: N. chihiroe larva, PV715913; N. bryope, PV715914; Neoclinus nudus Stephens et Springer, 1971, PV715915; N. okazakii, PV715916; N. lacunicola, PV715917; and N. chihiroe, PV715918) (Table 1), but sequence data for N. nudiceps (one of the four species reported in Korea) is not publicly available and could not be included. Sequence for outgroup species was downloaded from the National Center for Biological Information database. The molecular analysis was conducted following Kwun and Kang (2023).

Material examined. BDT-722: 1 specimen, 16.0 mm standard length (SL), 17 June 2016, Juk Island (37°31.54′N, 130°56.23′E), Ulleung-eup, Ulleung-gun, Gyeongsangbuk-do, Korea.

Description. Counts are provided in Table 2. Proportions as % SL: head length 25.6, body depth 13.8, snout length 5.0, caudal peduncle depth 8.1, caudal peduncle length 7.5, predorsal length 22.5, prepectoral length 25.6, prepelvic length 21.3, preanal length 40.0. Proportions as % head length (HL): eye diameter 26.8, upper jaw length 31.7, pectoral fin length 53.7, pelvic fin length 34.1.

Body slender, elongated and compressed. Head slightly pointed. Snout short and its anterior tip blunt. Mouth terminal and moderately large. Posterior tip of maxilla reaching middle of eye vertically. Both lips thick. Two pairs of nostrils on snout, tubular anteriorly and round posteriorly. Head with cirri, one on anterior nostril and one on orbit. Both gill membranes continuous across and free from isthmus. Eye large, interorbital region slightly wide and convex. Anus located in front of middle of body. Single dorsal fin, and its origin located behind nape. Origin of anal fin located just behind anus. Dorsal and anal fins height low, extending to caudal peduncle. Caudal fin truncate, separated from posterior end of dorsal and anal fins. Pectoral fin fan-like, rounded, and posterior tip reaching level of anus. Pelvic fin slender, widening posteriorly, and posterior tip not reaching anus.

Coloration. When preserved in ethanol, head and body opaque yellowish white. Varying sizes of melanophores around eye, cheek, upper jaw, lower jaw angle, anterior region of branchiostegal membrane, preopercular area, and opercular area (Fig. 2A). Three pairs of asterisk-shaped melanophores on upper head, first pair medium in size and located on interorbital region, second pair large and on occipital, and third pair small and on nape (Fig. 2B). Six large pale asterisk-shaped melanophores sparsely separated on both sides of dorsal contour, and pale melanophores on mediolateral area of posterior body and middle of caudal peduncle. On ventral side, several melanophores aligned in row on isthmus, origin of pelvic fin, and abdomen (Fig. 2C). Many dark melanophores along ventral contour between each spinous and soft rays of anal fin. All fins semi-transparent.

Figure 2. 

Neoclinus chihiroe, larva, BDT-722, 16.0 mm (SL), Ulleung Island, Gyeongsangbuk-do, Korea. A: lateral view; B: dorsal view; C. ventral view. Scale bars = 5 mm.

Molecular analysis. Based on an analysis of the mitochondrial DNA COI sequence (537 bp), the larval specimen corresponded to the adult specimen of N. chihiroe at a genetic distance of 0.002 but differed from the other four Neoclinus species at genetic distances of 0.138–0.197. On a neighbor-joining tree, the larval specimen clustered closely with the adult specimen of N. chihiroe rather than with the other Neoclinus species (Fig. 3). This finding was supported by high bootstrap values.

Figure 3. 

Neighbor-joining tree of mitochondrial DNA Cytochrome Oxidase subunit I for five species of the genus Neoclinus. Number of branches (>80) correspond to bootstrap probabilities in 10 000 bootstrap replications. Bar indicates genetic distance of 0.10.

Neoclinus chihiroe is only known to occur in Korea and Japan (Fukao 1987; Myoung et al. 2021), and its early life stages have previously been unknown. The early life stages of Neoclinus in general are poorly known, with the exception of N. bryope. A single specimen collected from the eastern coast of Ulleung Island, East Sea, was assigned to the genus Neoclinus based on the body shape (compressed, elongate, and preanal length short) and the presence of cirri on the head (Watson 2009; Okiyama 2014). Although this specimen is in the postflexion stage, morphological identification could not be made due to difficulties in distinguishing external characters among congeneric species and insufficient comparative data; however genetic analysis allowed the larval specimen to be identified as N. chihiroe. This is the first time that a N. chihiroe larva has been confirmed anywhere in the world.

The available information for Neoclinus chihiroe, N. bryope, and N. okazakii indicates that their larval stages are similar. Neoclinus chihiroe differs from N. bryope and N. okazakii in the distribution of melanophores on the body: there are one or two pale melanophores on the mediolateral area of the posterior body in N. chihiroe (presently reported study); many small melanophores along the mediolateral area of the body in N. bryope; and several large melanophores along the mediolateral area of the body in N. okazakii (see Okiyama 2014). Furthermore, despite the interspecific similarity in fin-ray counts within the genus Neoclinus, the larval N. chihiroe (presently reported study) was distinguished from the seven species reported in the western Pacific Ocean by its combination of dorsal fin spines and soft rays (Table 3).

The adult N. chihiroe specimen reported from Korea was collected at Dokdo, East Sea (Myoung et al. 2021), and the larval specimen was collected at Ulleung Island, East Sea. Dokdo and Ulleung Island are geographically far from the Korean peninsula and have unique environmental and biological characteristics (Park et al. 2024). The fact that adults and larvae of coastal fishes are found together on islands far from land suggests that the species is adapted to and settled in these areas.

I thank Prof. Hiroyuki Motomura (Kagoshima University Museum) for providing tissues of comparative specimens. This work was supported by National Marine Biodiversity Institute of Korea Program (2025M00200).