First record of a reticulated toadfish, Arothron reticularis (Tetraodontiformes: Tetraodontidae), in Korea

A single specimen of Arothron reticularis (398 mm in total length), belonging to the family Tetraodontidae, was collected in the coastal waters off Hansan island using a set net in May 2019. The morphological characteristics of the specimen are as follows: large eyes and mouth, white spots on the body, and a white line surrounding the eyes in an annular shape. A result of maximum likelihood tree showed that A. reticularis is related to A. hispidus (93.6%). We proposed the Korean name “Geu-murl-mu-nui-kkeo-kkeurl-bok,” in accordance with the characteristics of the specimen.


Background
The family Tetraodontidae (Order Tetraodontiformes) consists of 2 subfamilies, 26 genera, and 196 species worldwide (Nelson et al. 2016), of which 7 genera and 52 species occur in Japan (Nakabo and Yagishita 2013) and 5 genera and 31 species occur in Korea (MABIK 2020). Of the 196 species, the genus Arothron comprises 10 species that are present in Japan and 4 species in Korea (MABIK 2020).
The genus Arothron is distributed in the tropical regions of the Indo-Pacific, except for Arothron firmamentum (Temminck and Schlegel 1850), which is antitropical (Matsuura 2016). The reticulated toadfish, Arothron reticularis (Bloch and Schneider 1801), is distributed in the tropical waters of Okinawa, Taiwan, and the Indo-West Pacific (Masuda et al. 1984). The first report from the Red Sea was obtained in 1956, when Roux-Estève identified a specimen (197 mm in total length), which was from the island of Abulat (Abu Latt) in the Farasan Islands, southern Red Sea, as Tetraodon reticularis (Randall et al. 2012).

Materials and methods
On May 9, 2019, a single reticulated pufferfish, A. reticularis, was captured from the coastal waters off Hansan Island using a set net (Fig. 1). The methods used for the fin ray counts and morphometric characters follow those of Han (1995). All morphometric measurements were measured to the nearest 0.1 cm using Vernier calipers. Soft x-ray microscopy (LISTEM, REX-525R, Korea) was used to count the number of vertebrae and fin rays.
For genetic analysis, the total genomic DNA was extracted from the prepared part of the tissue using the QIAamp DNA Micro Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. The polymerase chain reaction (PCR) amplification was performed using the 2X TOPsimple TM PreMIX (aliquot) (Enzynomics, Daejeon, Korea) with the universal mitochondrial COI primer set, LCO1490 forward primer (5′-GGT CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 reverse primer (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) (Folmer et al. 1994). The amplified PCR product was sequenced using the Applied Biosystems 3730xl DNA Analyzer (ThermoFisher, Foster City, CA, USA). The obtained mt COI sequence (658 bp) was compared with 16 Tetraodontiformes mt COI sequences (14 Tetraodontidae species and 2 Ostraciidae species [as an outgroup]) retrieved from GenBank (Appendix 1). In total, 17 sequences were aligned using MAFFT v.7 (Katoh and Standley 2013) in Geneious v.9 (Biomatters Ltd.). The phylogenetic tree was reconstructed using the maximum likelihood (ML) method based on the GTR+ G+I model and 10,000 bootstrap replications in the raxmlGUI 2.0 program (Edler et al. 2020).

Description
The morphological trait measurements for the present specimen are shown in The fin ray counts are as follows: dorsal fin rays, 10; pectoral fin rays, 10; anal fin rays, 10; caudal fin rays, 11; and vertebrae, 18 (10 + 8).
The nostril has two fleshy flaps, formed by the bifurcation of a single base (Fig. 4). The body is elongated and covered with prickles, except for the regions around the mouth and caudal peduncle. The eyes are notably large, broad, and flat. The mouth is large with four teeth,  and the dentary is protruded further than the premaxillary. The lateral line is indistinct. The gill slit is large.

Coloration of the specimen
When fresh, there were white lines on the body that curved from around the eyes and then across the abdomen. There were no ventral extensions of the dark body color into the pale ventral part of the head or body. White spots were present from the dorsal of the head to the caudal fin. After preservation, the ventral surface turned beige, and the body turned greenish brown. There are white spots on the body and a white line surrounds the eyes in an annular shape (Fig. 3)

Distribution
The distribution occurred in the Southern Sea of Korea (present study) and Indo-West Pacific Ocean, including the South China Sea and Ryukyu Islands (Veeruraj et al. 2011).

Comparison with type species
The difference between A. reticularis and the type species (Tetraodon lineatus) are as follows: T. lineatus has 2~3 dorsal spines, 9~10 dorsal soft rays, 2 anal spines, and 7~8 anal soft rays (Fishbase 2020). There was no difference in the distribution of prickles; however, the white spots and white lines around the eyes were absent in T. lineatus.

Remarks
Although the measurements were taken in a dry state, the morphological traits of the present specimen are similar to those of Sujatha and Padmavathi (2015). There were differences in morphometric traits between A. stellatus and A. firmamentum (Table 1). The morphological classification keys of the genus Arothron fish are the spots and lines around the eyes, ventral part, and caudal fin. A. reticularis morphologically resembles the genus Arothron fish, but differs in the distribution of prickles (prickles absent on the snout and  at least the posterior half of the caudal peduncle in A. hispidus), white line around the head (white spots around the head in A. hispidus) (Randall et al. 2012), white spots scattered around the caudal fin (caudal fin has no white spots of A. carduus and A. manilensis) (Nakabo 2000), and discontinuous stripes on the body (continuous stripes on the body of A. multilineatus) (Matsuura 2016). This species has a reticulate pattern on the body. In accordance with the characteristics, a new Korean name was proposed for this species "Geu-murlmu-nui-kkeo-kkeurl-bok." In this study, a partial gene sequence of the mt COI (658 bp) of A. reticularis was obtained for the first time. Using this sequence, we created an mt COI dataset, including 16 Tetraodontiformes mt COI sequences from GenBank, and analyzed the genetic distance of A. reticularis using Kimura-2-Parameter method. Neighborjoining tree showed that A. reticularis belongs to the genus Arothron (family Tetraodontidae) and is related to A. hispidus (similarity 93.6%) (Fig. 5).

Conclusions
One specimen of reticulated toadfish was collected in the coastal waters off Hansan Island on May 9, 2019. The specimen was identified as Arothron reticularis, based on morphological traits and the first record of species in Korea.