Ontogenetic comparison of larvae and juveniles of Diaphus garmani and Benthosema pterotum (Myctophidae, Pisces) collected from Korea

During June 2017, we collected two postflexion larvae (6.01 and 7.56 mm in standard length [SL]) and two juveniles (7.72 and 9.62 mm SL) belonging to Myctophidae in the waters of Jejudo Island. Those four individuals were identified as Diaphus garmani, which had not been reported in Korea. They were distinguished from Benthosema pterotum by melanophores in the abdominal cavity (absent in D. garmani vs. present in B. pterotum) and the development of photophores (developed in D. garmani vs. rudimentary in B. pterotum) when shorter than 10.0 mm SL. Analysis of 16S rRNA sequences showed that the sequences of four individuals matched those of adult D. garmani (Kimura 2-parameter distance: 0.6–0.8%). This is the first record of larvae and juveniles of D. garmani in Korean waters, and we propose a new Korean name, Gar-ma-ni-sat-bi-neul-chi.

The genus Diaphus in the family Myctophidae comprises 77 recognized species worldwide (Nelson et al. 2016; Froese and Pauly 2019) and has not been reported in Korea. The species in the genus Diaphus are classified into the Diaphus A and B groups depending on the presence and absence, respectively, of photophores on the suborbital organ (So), mouth morphology of early larvae, position of the anus, expression of melanophores and photophores, and timing of metamorphosis (Javadzadeh et al. 2012). Of which, Diaphus garmani is a small fish (~60 mm total length) belonging to the Diaphus B group and thus lacks photophores on the So (Nafpaktitis 1978;Nelson et al. 2016). Some studies of Diaphus have examined its early life history (Bineesh et al. 2010) and the first recorded species (Sassa et al. 2003). In Korea, studies of mesopelagic fish have examined the early life history of Maurolicus muelleri (Kim and Yoo 1999;Kim et al. 2007) and a new record for Sigmops gracilis (Lee and Kim 2013).
As the Myctophidae larvae collected in this study had similar morphological features to those of Benthosema pterotum, we confirmed the correctness of species identification by using 16S rRNA sequences. Afterward, indepth morphological data related to ontogenetic features of these two species were compared with each other, which are barely distinguishable at the larval stage.

Sampling
Four larval specimens (PKUI 618-621) of Diaphus garmani were collected from the southern Jejudo Island in June 2017 by a research vessel from the National Institute of Fisheries Science using a bongo net (mouth diameter 80 cm; mesh size 500 μm). The collected specimens were immediately fixed in 5% formalin for 1 h on the vessel and then washed before being placed in a 1L polyethylene container, where they were fixed in 99% alcohol. Four Benthosema pterotum larvae (PKUI 614-617) were also collected from the western Jejudo Island to the East Sea between July and December 2018 (Fig. 1). After the study, the specimens were deposited at Ichthyoplankton Laboratory of Pukyong National University (PKUI).

Molecular analysis
Total DNA was extracted from eyeballs removed from the right side of the larvae using 150 μL Chelex 100 resin (Bio-Rad Laboratories, USA). The mitochondrial 16S rRNA region was amplified using the primers 5′-CGC CTG TTT ATC AAA AAC AT-3′ and 3′-CCG GTC TGA ACT CAG ATC ACG T-5′ (Ivanova et al. 2007). The polymerase chain reaction (PCR) conditions were as follows: initial denaturation at 95°C for 11 min; 35 cycles of denaturation at 94°C for 1 min, annealing 52°C for 1 min, extension at 72°C for 1 min; and a final extension at 72°C for 5 min. The same conditions were used for PCR analysis of B. pterotum, except that the annealing temperature was 54°C. The sequences were aligned using ClustalW in the program BioEdit (ver. 7.0.5.3;Hall 1999), and genetic distances were calculated using the Kimura 2-parameter model (Kimura 1980) using Mega X (ver. 10.0.5). The genetic relationships were analyzed by constructing a neighbor-joining tree using Mega X (ver. 10.0.5) with 1000 bootstrap replications. The 16S rRNA sequences of D. garmani (KR231737) and B. pterotum (JX133756) were also obtained from the National Centre for Biology Information database to compare eight specimens of Myctophidae larvae between the two species. The 16S rRNA sequence of B. pterotum and D. garmani used in this study was assigned the following registration number from NCBI(MT242581-242587).

Molecular identification
A 479 bp of 16S rRNA sequence was obtained from each of the four Myctophidae larvae for comparison with the sequence from adults of the candidate species D. garmani (KR231737). The genetic distances ranged from 0.6 to 0.8%, indicating close consistency. We also compared the 16S rRNA gene sequences of the four B. pterotum larvae with that of the adult B. pterotum (JX133756) and found complete consistency, with a genetic distance of 0%. The D. garmani larvae, juveniles, and adult were clearly separated from the four Diaphus spp. and B. pterotum in the neighbor joining tree (Fig. 3).

Morphological features
Diaphus garmani Gilbert, 1906 (Fig. 4 The 6.01 mm standard length (SL) post-flexion larva of D. garmani (Fig. 4a) has large eyes with a pair of small nostrils situated in front. The mouth is large and slopes, with the rear end of the upper jaw reaching the posterior rim of the eye. There is a row of small sharp teeth in the jaws. The anus is positioned slightly behind the middle of the body. The end of the operculum tapers to a triangular shape. The myomeres show a clear M shape. Br 2 photophores are found on the middle part of the lower jaw, and only a trace of PO 5 photophore remain in front of the pelvic fin. The larva has an adipose fin, 13 dorsal fin rays, 14 anal fin rays, nine pectoral fin rays, and seven pelvic fin rays (Table 1). A few melanophores are present on the opercular organ, a series of three melanophores on the ventral side, one star-shaped melanophore on the anus, and one large dot-shaped melanophore at the posterior end of the anal fin base.
The 7.56 mm SL post-flexion larva of D. garmani (Fig.  4b) has a heavier head. Br 2 photophores are observed on the middle part of the lower jaw, a line of PO 1235 photophores is arranged along the ventral side of the abdominal cavity, and a raised PO 4 photophore is situated between PO 3 and PO 5 . A PVO 12 photophore is observed between the base of the opercular organ and the pectoral fin, a VO 1 photophore on the base of the pelvic fin, and VO 45 photophores on the anus. There are 14 dorsal fin rays, 15 anal fin rays, 10 pectoral fin rays, and 8 pelvic fin rays (Table 1). The occurrence of melanophores is similar to that of the 6.01 mm SL larva, but with the addition of two star-shaped melanophores in front of the pectoral fin and one and two on the upper and lower lobes, respectively, of the caudal fins.
The 7.72 mm SL juvenile of D. garmani (Fig. 4c) has a sharper snout than that at the previous stage, with the anus shifted forward toward the head. Br 123 photophores are arranged in a line in the lower jaw. OP 2 is recognized in the opercular organ under the eye. PLO is situated posterior to the operculum between the lateral line and pectoral fin. PO 1235 photophores are aligned in the abdominal cavity. The PO 4 photophore is raised between PO 3 and PO 5 . The PVO 12 photophore is situated obliquely between the base of the opercular organ and the pectoral fin. VLO is raised slightly from the middle between the base of the pelvic fin and the lateral line. VO 145 photophores are arranged in a line behind the pelvic fin. The VO 2 photophore is raised between VO 1 and VO 4 . AOa 23 photophores are lined on the anal fin. There are 14 dorsal fin rays, 15 anal fin rays, 13 pectoral fin rays, and 8 pelvic fin rays (Table 1). Several melanophores are present on the opercular organ. Two star-shaped melanophores are aligned on the abdominal cavity. One star-  shaped melanophore is present on the anus. A dot-shaped melanophore is present at the end of the anal fin base. There are one and two melanophores dotted on the upper and lower lobes, respectively, of the caudal fin. The 9.62 mm SL juvenile of D. garmani (Fig. 4d) has a similar morphology to that of the previous developmental stage. A D n photophore is present in front of the eye. Br 123 photophores are lined in the lower jaw. A OP 2 is located under the eye. PO 1235 photophores are arranged in a line in the abdominal cavity. PO 4 is raised between PO 3 and PO 5 . The PVO 12 photophore is situated obliquely between the base of the opercular organ and the pectoral fin. PLO is situated between the base of the pectoral fin and the lateral line. VLO is raised slightly from the middle between the base of the pelvic fin and the lateral line. The VO 145 photophores are arranged in a line behind the pelvic fin. The VO 23 photophores are situated obliquely between VO 1 and VO 4 . The SAO 123 photophores are gradually raised behind VO 5 . AOa 1 is slightly raised from the anal fin base and follows the line of AOa 23456 photophores arranged along the anal fin base. AOa 7 is raised more. Pol is near the lateral line at the end of the anal fin base. The AOp 1234 photophores are arranged in a line along the caudal peduncle base behind the anal fin base. A line of Prc 123 photophores is near the caudal peduncle, and Prc 4 is observed under the lateral line of the caudal peduncle. There are 14 dorsal fin rays, 14 anal fin rays, and 8 pelvic fin rays (Table 1). Of the melanophores, one is observed above the left side of the eye, a few are recognized on the top of the head and the opercular organ with two starshaped melanophores nearby. One melanophore is dotted on the anus, and one on the upper lobe and two dotted on the lower lobe of the caudal fin.
Benthosema pterotum (Alcock, 1890) (Fig. 5) 5a) has a small body depth and large head depth, with the head depth decreasing from the nape. At this stage of development, the larva has large eyes and a sharp snout. The anus is positioned slightly behind the middle of the body. A Br 2 photophore is found in the lower jaw under the eye and Bu under the rear of the eye. The adipose fin is present. There are 10 dorsal fin rays, 14 anal fin rays, 11 pectoral fin rays, and 7 pelvic fin rays (Table  2). Observed melanophores include one on the end of the lower jaw snout, a line of melanophores in the lower jaw, one on the central upper part of the gut, many melanophore dots around the anus, and a line of very short, indistinct melanophores on the anal fin.
The 7.27 mm SL juvenile of B. pterotum (Fig. 5b) has a Dn photophore in front of the eye, Br 2 on the lower jaw under the eye, and Bu under the rear of the eye. There are 12 dorsal fin rays, 18 anal fin rays, 11 pectoral fin rays, and 8 pelvic fin rays (Table 2). Observed melanophores include one on the end of the lower jaw, one around the nostrils, a line of four melanophores at the base of the opercular organ, in addition to irregularly distributed small dots of melanophores, and a few on the anus. During this stage of development, existing melanophores disappear from the central anus and central muscle of the anal fin.
The 8.07 mm SL juvenile of B. pterotum (Fig. 5c) has a Dn photophore in front of the eye, Br 2 on the lower jaw under the eye, and Bu under the rear of the eye. There are 12 dorsal fin rays, 18 anal fin rays, 11 pectoral fin rays, and 8 pelvic fin rays (Table 2). Observed melanophores include one at the tip of the lower jaw snout, one under the nostrils, a line of dark dots on the base of the opercular organ, and a large melanophore dot on the anus.
The 9.81 mm SL juvenile of B. pterotum (Fig. 5d) has a Dn photophore in front of the eye, Br 2 on the lower jaw under the eye, Cp on the opercular organ, a series of PO 1234 photophores followed by a raised PO 5 in the isthmus, PVO 1 in front of the base of the pectoral fin behind the operculum organ, VO 1 under the pelvic fin followed by a raised VO 2 , AOa 12 on the anal fin, and Prc 2 below the lateral line of the anal fin. There are 10 (?) dorsal fin rays, 18 anal fin rays, 11 pectoral fin rays, and 8 pelvic fin rays (Table 2). Observed melanophores include a series of dots under the operculum organ, one dot on the anus, one melanophore each on the upper and lower lobes of the base of the caudal fin.

Discussion
We performed a molecular identification of Myctophidae larvae and juveniles collected in the waters around Jeju Island and the East Sea in June 2017 and found that four of the larvae had 99.2-99.4% identity to the sequence of a D. garmani adult and the other four larvae 100% identity to the sequence of a B. pterotum adult. Diaphus garmani has not been recorded in Korea, and its morphological features and measurements according to developmental stage obtained in this study were found to be generally consistent with previous reports (Gilbert 1906;Kawaguchi and Shimizu 1978;Sassa et al. 2003;Richards 2006;Bineesh et al. 2010;Okiyama 2014). Okiyama (2014) suggested that the completion of metamorphosis and photophore development occurs later in B. pterotum (12-13 mm SL) than in D. garmani (11 mm SL), which is consistent with our results. In this study, a juvenile D. garmani (PKUI 621,9.62 mm BL) developed AOa 1-7 . This result is consistent with that in the original descriptive paper (Gilbert 1906), but differs from the results of Nakabo (2013) and Bineesh et al. (2010) which described only AOa 1-6 . In addition, D. garmani juveniles in this study had a Prc 4 photophore under the lateral line inside of the caudal fin, which is consistent with Bineesh et al. (2010) but clearly different than that present under the lateral line outside of the caudal fin from Nakabo (2013). It is thought that the discrepant results for the AOa photophores are due to individual variations, but further study will be needed with regard to the Prc4 photophore location. The species D. garmani and B. pterotum share the characteristics of  body shape, eye size, and photophore arrangement, but the two species can be differentiated by the photophore development process and relative growth of individuals of similar size. In terms of photophore development, B. pterotum first develops Dn in front of the eye, whereas D. garmani first develops OP 2 at the base of the opercular organ. B. pterotum has Bu under the right eye and Cp on the isthmus, which are not present in D. garmani.
The two species can also be differentiated by the position of the PO photophores; D. garmani has PO 4 at the highest raised position, whereas B. pterotum has PO 5 as the highest PO photophore. The arrangement of VO photophores can also clearly differentiate the two species: VO 3 is located at the top in D. garmani versus VO 2 in B. pterotum. AOa 1 is positioned higher than the other AOa photophores in D. garmani, whereas the AOa photophores are arranged horizontally in B. pterotum.
In the adult stage, the two species can be clearly identified by the number of Prc photophores: four in D. garmani and two in B. pterotum (Nakabo 2013). We also found four Prc photophores in D. garmani and one in B. pterotum. Comparing the body ratios of the two species at each developmental stage, the ratios of head depth, head length, preanus length, eye diameter, and pre-anal fin length to SL differentiated the two species (Fig. 6a-e), except the ratio of pre-dorsal fin length to SL (Fig. 6a-f). Okiyama (2014) suggested that in D. garmani, the ratios of pre-anal fin length, head length, and body depth to SL increase linearly until the larvae grow to 7 mm SL, after which the ratios gradually decrease until they reach the juvenile stage. In contrast to that previous study, we showed that the pre-anal fin length increased with SL in D. garmani, but with no change in head length. This result may be related to the trivial difference in genetic distance (0.6-0.8%) in terms of the 16S rRNA sequence between Japanese D. garmani adults and the D. garmani larvae collected in Korean waters, which requires further study.

Conclusions
Based on mitochondrial DNA 16S rRNA sequences, two larvae and two juveniles were identified as Diaphus garmani, which is the first record in Korean waters. Among myctophid species previously recorded in Korea, Benthosema pterotum seems to be very similar to D. garmani in external morphology. The two species were distinguished by melanophores in the abdominal cavity (absent in D. garmani vs. present in B. pterotum) and the development of photophores (developed in D. garmani vs. rudimentary in B. pterotum) when shorter than 10.0 mm standard length. We propose a new Korean name Gar-ma-ni-sat-bi-neul-chi, a combination of the specific name garmani and the Korean name of the family Myctophidae Sat-bi-neul-chi.