DNA barcoding and morphological identification of spiny lobsters in South Korean waters: a new record of Panulirus longipes and Panulirus homarus homarus

To date, 19 species of spiny lobsters from the genus Panulirus have been discovered, of which only P. japonicus, P. penicilatus, P. stimpsoni, and P. versicolor have been documented in South Korean waters. In this study, we aimed to identify and update the current list of spiny lobster species that inhabit South Korean waters based on the morphological features and the phylogenetic profile of cytochrome oxidase I (COI) of mitochondrial DNA (mtDNA). Spiny lobsters were collected from the southern and eastern coasts of Jeju Island, South Korea. Phylogenetic analyses were performed using neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI) methods. The ML tree was used to determine the spiny lobster lineages, thereby clustering the 17 specimens collected in this study into clades A, B, C, and D, which were reciprocally monophyletic with P. japonicus, P. homarus homarus, P. longipes, and P. stimpsoni, respectively. These clades were also supported by morphological examinations. Interestingly, morphological variations, including the connected pleural and transverse groove at the third abdominal somite, were observed in four specimens that were genetically confirmed as P. japonicus. This finding is novel within the P. japonicus taxonomical reports. Additionally, this study updates the documentation of spiny lobsters inhabiting South Korean waters as P. longipes and P. homarus homarus were recorded for the first time in this region.


INTRODUCTION
Studies on the taxonomical status of spiny lobsters have been conducted throughout the Indian, Pacific, and Atlantic Oceans. Nineteen species from the genus Panulirus have been discovered in these regions, of which seven species have been found within the East China Sea (Holthuis, 1991), including P. japonicus, P. penicilatus, P. stimpsoni, andP. versicolor in South Korean waters (Kim et al., 2009). This genus can be identified based on their transverse ridges with clear-cut connections and decalcified areas on the female sternum. In male lobsters, variations can be observed in the copulatory ornamentation and setation (George, 2005).
In addition to morphological observations, genetic information, such as the mitochondrial cytochrome oxidase subunit I (COI) mitochondrial DNA (mtDNA), is used to identify unknown and novel specimens (Meyer & Paulay, 2005). Marine fauna diversity can be assessed using the COI mtDNA through a technique known as DNA barcoding. DNA barcoding has also been used for conservation purposes, such as phylogeographic analysis, invasive species detection, and forensic studies (Meyer & Paulay, 2005;Bucklin, Steinke & Blanco-Bercial, 2011;Senevirathna & Munasinghe, 2013;Sembiring et al., 2015;Leray & Knowlton, 2016). Phylogenetic studies on spiny lobsters have classified them into four (I-IV) non-formal genetic clades (Ptacek et al., 2001). In addition, phylogeographic analysis based on the P. homarus mtDNA profile has suggested discrimination between the western (parapatric isolation, secondary contact, and introgression) and eastern (active peripheral speciation) populations (Farhadi et al., 2017).
Documenting biodiversity is an essential step that could improve the management and conservation of sustainable natural resources. In South Korea, biodiversity studies have been conspicuously initiated since 2007, following the establishment of the National Institute of Biological Resources (NIBR). Consequently, several new species have been discovered in South Korea, and this number is expected to increase continuously, with a potential for recording up to 60,000 species by 2020 (Biodiversity Division, Nature Conservation Bureau& Ministry of Environment, 2014). In the case of marine fishes, Kim (2009) reported that approximately five described species are documented for the first time within the Korean Peninsula every year. Meanwhile, in 2018, 76 species of epibenthic invertebrates were documented within the southern part of the East Sea, Korea and ∼61% of these were identified as decapods (Park & Huh, 2018).
As a part of the general biodiversity documentation, this study aimed to update the existing records by identifying spiny lobster species inhabiting South Korean waters (Jeju Island). The identification was conducted by using molecular (DNA barcoding) and morphological examination. The spiny lobsters used in this study were collected from the southern part of Jeju Island, South Korea, where several branches of Kuroshio currents (Yellow Sea and Tsushima warm currents) are reported to drift. Based on the newly recorded P. homarus homarus and P. longipes as well as the new morphotype of P. japonicus discovered in this study, the list of spiny lobster species and the general species diversity record within the South Korean waters was updated. Thus, the findings of this study will facilitate appropriate regulation and management of spiny lobsters in this area.

Sample collection
This study was conducted under the approval of the animal care and use committee of Jeju National University (2020-0012). Adult spiny lobsters (Panulirus spp.) were collected from three sampling sites along the southern coast of Jeju Island, South Korea. Site 1 was located around Hwasun Harbor (33 • 13 57.5 N 126 • 19 55.8 E), site 2 was around Seogwipo Harbor (33 • 13 57.0 N 126 • 33 53.2 E), and site 3 was around Pyoseon Port (33 • 19 37.2 N 126 • 50 49.9 E) ( Fig. 1). Spiny lobsters were located by scuba diving at night (8:00 to 11:00 p.m.) from August to November 2020 and caught using hand nets. The collected animals were placed in iceboxes and transported to the Jeju Tropical Seawater Research Facility at the Jeju Marine Research Center of the Korea Institute of Ocean Science and Technology, Jeju Island, South Korea. All lobsters were reared in acrylonitrile butadiene styrene tanks (20 tons) with a constant circulation of seawater (23 ± 1 • C) and fed daily at 16:00 with commercially formulated powder feed (Heukja, Kopec Ltd., Jeonla-Namdo, South Korea) until analysis.

Examination of morphological features
A total of 17 spiny lobsters were collected for morphological examination. The specimens were anesthetized on ice for 10 min prior to the examination. Body length (BL) and weight (BW) of each lobster were measured, and the detailed features of the body and appendages were photographed for examination (Fig. 2). Morphological features and color markings were examined with reference to the morphological characterization of lobsters described by George & Holthuis (1965). Species identification was done based on the examination of body color, presence of cross bands on antennal and antennular flagella, size and number of spines on the antennular plate, availability of transverse grooves on abdominal segments, existence of stridulating organs, and presence of exopods in the second and third maxillipeds.

DNA Extraction and PCR amplification
Genomic DNA was extracted from the muscle of the pereiopod of all 17 spiny lobsters. DNA was extracted using the AccuPrep R Genomic DNA Extraction Kit (Bioneer, Daejeon, South Korea), following the manufacturer's instructions. Concentration and purity of the extracted DNA were measured using a Thermo Scientific TM NanoDrop TM One microvolume UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
A polymerase chain reaction (PCR) was performed to amplify the mitochondrial COI gene region using the HCO1490/LCO2198 universal primers, specially designed for invertebrates (Folmer et al., 1994). PCR was performed using a 50 µL reaction mixture, consisting of 100 ng genomic DNA, 0.25 µL Taq polymerase (Takara Bio Inc., Shiga, Japan), 5 µL 10X Ex. Taq DNA polymerase buffer (Takara Bio Inc.), 1 µL each of 10 µM forward and reverse primers, and 4 µL (2.5 mM) dNTPs (Takara Bio Inc.). The PCR thermal profile comprised an initial step of 5 min at 94 • C, followed by 30 cycles at 94 • C for 30 s, 50 • C for 30 s, and 72 • C for 45 s, followed by a final extension at 72 • C for 5 min. The amplified PCR products were separated using 1% agarose gel electrophoresis, and target bands were purified using the AccuPrep R PCR Purification Kit (Bioneer), according to the manufacturer's instructions.

COI cloning and sequencing
Purified PCR products were ligated to the T-easy vector (Takara Bio Inc.). The ligation mixture was prepared with 2 µL ligation buffer, 3 µL T-easy vector, 1 µL T4 ligase, and 4 µL PCR water. Recombinant plasmids were transformed into Escherichia coli JM109 (DE3) (Promega, USA), cultured on Luria-Bertani agar plates supplemented with ampicillin (LB amp+) and incubated overnight at 37 • C. Subsequently, LB broth (4 mL) was inoculated with the grown colonies and incubated overnight at 37 • C, following which plasmids were extracted using the AccuPrep R plasmid extraction kit (Bioneer). Extracted plasmids were sent for sequencing at Macrogen Pvt. Ltd. (South Korea).

Alignment and phylogenetic analyses
COI sequences were edited and aligned with the reference sequences of various spiny lobster species, retrieved from the BOLD system (http://barcodinglife.org/) and the NCBI  et al., 2016). Sequence alignment was generated using a high-throughput MUSCLE method (Edgar, 2004), and subsequent phylogenetic analyses were performed using neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI) methods. NJ and ML were constructed using MEGA 7.0 (Kumar et al., 2016) and RAxML v8.2.X (Stamatakis, 2014), respectively. Both the analyses were run with 1000 bootstrap replications. In addition to ML, a priori test was performed using jModelTest 0.1.1 (Posada, 2008) to determine the best evolutionary model fitted to the current sequences, and the general time-reversible gamma distribution rate parameter (GTR+G) was used to construct the tree (Guindon & Gascuel, 2003;Posada, 2008). Finally, BI was performed for 5,000,000 generations using MrBayes 3.2.7a Ronquist et al. (2012). Additionally, pairwise mean distances between groups were calculated using MEGA 7.0 to obtain the genetic divergence information. Furthermore, DnaSP v5 (Librado & Rozas, 2009) was used to measure the nucleotide diversity and the number of polymorphic sites within each clade to which the newly recorded Jeju Island spiny lobsters belonged.

Phylogenetic analyses of spiny lobster COI genes
The COI sequences ranging from 526-712 bp were successfully sequenced. Sequence editing and trimming resulted in 338 bp, which were used for phylogenetic analyses. The NCBI GenBank accession numbers obtained for the sequences are provided in Table S1. A phylogenetic tree was constructed using NJ, ML, and BI, with the values on the branch indicating the bootstrap proportion of NJ and ML, followed by the posterior probability from BI analysis. Trees constructed from ML were used to visualize the topological lineage of spiny lobsters. According to the ML tree, spiny lobsters collected from Jeju Island were grouped under four different clades: Clade A, Clade B, Clade C, and Clade D, which were monophyletic with Panulirus japonicus, P. longipes, P. stimpsoni, and P. homarus, respectively (Fig. 3).
Clade A included eight COI sequences of spiny lobsters from Jeju Island, which were closely related to P. japonicus from Japan and Taiwan. Of these, seven sequences were shown to be exclusively claded with P. japonicus specimens collected from Japan, with a high bootstrap proportion and posterior probability (NJ/ML/BI; 99/72/92) (Fig. 3). Meanwhile, only one sequence was claded with the P. japonicus specimens collected from Taiwan (NJ/ML/BI; 99/77/99). Intraspecific diversity revealed that seven haplotypes were found exclusively from Jeju Island with Pi value 5.0% ± 2.0% and 59 polymorphic sites (Table 1).
Two COI sequences were included under Clade B. As shown in Fig. 3, this clade was highly supported (NJ/ML/BI; 100/89/100) to be monophyletic with P. longipes as well as its subtypes, the P. longipes longipes and P. longipes fermorstriga. Specifically, the two spiny lobster sequences obtained in this study seemed to share a common ancestor, P. longipes from India (NJ/ML/BI; 67/67/100). However, the intraspecific diversity analysis indicated that both sequences differed slightly from each other, as indicated by the two haplotypes that were identified. The nucleotide diversity (Pi ± SD) was 2.4% ± 1.2% and five polymorphic sites were observed (Table 1).
Among the COI sequences of spiny lobsters collected in the current study, six sequences were clustered into clade C, which included P. stimpsoni from regions such as South China Sea and Hongkong. This clade was supported by 100/99 of NJ/ML bootstrap proportion and 100 posterior probability based on BI analysis (Fig. 3). In this study, four haplotypes of P. stimpsoni were found within Jeju Island. In addition, nucleotide diversity (Pi ± SD) and number of polymorphic sites were reported at 2.4% ± 1.2% and eight, respectively.
In the current study, only one specimen was claded with the P. homarus group (Clade D). The sequence of this specimen was closely related to the P. homarus homarus from Marquesas Island, French Polynesia, and the topology was strongly supported by bootstrap proportions and posterior probability (NJ/ML/BI; 100/100/100). These two sequences were distinct from the P. homarus collected from Indonesia, Oman, India, Sri Lanka, Iran, and Mozambique. As for the Jeju intraspecific variation, the analysis could not be performed due to the insufficient number of samples.  The pairwise mean distances between clades were run using Tamura 3-parameter in MEGA 7.0. The closest genetic distance was between clade C (P. stimpsoni) and clade D (P. homarus), with 25.3% differences. Meanwhile, the furthest distance was between clade A (P. japonicus) and clade C (P. stimpsoni), with 41.7% differences (Table 1).
Description: The carapace is reddish brown in color, and abdominal segments are greenish brown ( Fig. 2A). White spots present in the lateral margin of the carapace and the lateral region of the abdomen. Spines of various sizes are randomly scattered on the carapace, and majority of the spine bases are black in color. The mid-dorsal surface of the carapace bears reddish-brown hairs (Fig. 4A). The dorsal surface of the frontal horns is dark greenish-brown and white spots are present. The ventral side of the frontal horn is orange. Frontal margin of the antennular plate armed with two separate medium-sized spines. The inner dorsal side of the antennal peduncles is pinkish in color. The antennules are without cross bands. The ventrolateral margin of the carapace made a reddish-brown soft surface line with white blotches (Fig. 4A). Non-interrupted transverse grooves are visible on the dorsal surface of each abdominal segment (Fig. 5A). Posteriorly directed hair is present in both the posterior margins of the somites and transverse grooves. Transverse grooves are curved at the lateral end of the second, third, and fourth somites, and interconnected with the corresponding pleural grooves in the first and third somites (Figs. 6A, 6B). The transverse groove in the second somite ends up too close to its pleural groove, making a pseudo connection. Both second and third maxillipeds bear exopods (Figs Description: Base color of the carapace is reddish brown, and abdominal segments are greenish brown. Lateral margin of the carapace and lateral region of the abdomen contain white spots. Spines which are varying in sizes with black bases are scattered on the carapace. The mid-dorsal surface of the carapace bears reddish-brown hairs (Fig. S1). There are no interconnections of transverse grooves with corresponding pleural grooves in the second and third somites (Fig. S2). Both second and third maxillipeds bear exopods. Description: The carapace and front part of the abdominal region are brownish to greenish in color. Antennal and antennular peduncles, walking legs, posterior part of the abdominal region, peduncles of the uropod, and base of the telson are greenish in color (Fig. 2B). The body consists of numerous white spots, which are apparent in the posterior part of the abdomen. Both antennular peduncles and antennular flagella contain white cross bands. The distal part of each antennular peduncle contains white blotches. There are four well-separated spines on the antennular plate and randomly scattered small spines between  (Fig. 2C). Both carapace and abdominal regions are covered with randomly scattered white and orange spots. The base of the antennal peduncle is purple, and its dorsal inner side is pinkish brown. The frontal margin of the antennular plate is armed with two separate small spines. Frontal horns are brown, while the tips are orange, and there are two conspicuous medium-sized spines behind the frontal horns. The carapace bears numerous spines of different sizes; some are black, while some have orange tips with white bases (Fig. 4C). Antennular flagella are cross-banded in white. Legs bear orange and white spots at the end of each segment. Orange lines on the lateral and ventral legs are broken into irregular blotches. Transverse grooves are present in every abdominal segment and connect laterally to the pleural grooves (Fig. 5C, Figs. 6E, 6F). Exopods are present in the second and third maxillipeds (Figs. 7G, 7H, 7I).
Material examined: P. stimpsoni ; Female; TL: 28 cm; BW: 765 g; Site 02, Seogwipo Harbor, Seogwipo, Jeju Island, South Korea; 33 • 13 57.0 N, 126 • 33 53.2 E; April 06, 2020; ∼15 m depth Description: Body color is brown to olive green. The posterior part of the cervical groove on the carapace is darker in color (Fig. 2D). Frontal horns are reddish brown in color, tips are pale yellow, and four cross lines of the same color are present. The lateral margin of the carapace forms a white line, and another white line starting from the anterior part of the cervical groove extends over it (Fig. 4D). The lateral region between these two lines is light orangish yellow. The antennule plate is armed with four well-separated spines projected among the hairs, and the frontal pair of spines is larger than that at the posterior region. Antennules are reddish brown with white cross bands. The distal part of each antennule peduncle shows white blotches. Pereiopods are reddish brown with longitudinal white lines and blotches in the propodus, carpus, and merus. The abdominal segments are olive green in color. Reddish brown and pale-yellow speckles are present in each segment. Transverse grooves are not present in any of the abdominal segments. Instead, slight depressions associated with firm hairs are present in the middle of the second, third, and fourth somites. These grooves were disturbed in the middle and became broad on the lateral sides (Fig. 5D). A white spot was present at the top-notch of the triangular plate in the first somite. The same conspicuous white spots are present in the anterolateral region of each somite, except in the second somite, where it is present as a posteriorly interrupted white line (Figs. 6G, 6F). The posterior margin of each pleuron bears four conspicuous teeth associated with hairs, arranged in descending order of size. The second maxilliped bears an exopod, but the third maxilliped does not (Figs. 7J, 7K, 7L). The major comparative morphological features used for the identification of spiny lobsters are shown in Table 2.

DISCUSSION
This study was conducted to update the records of spiny lobster species in South Korean waters, based on the specimen collection in Jeju Island. A previous study identified four spiny lobster species from the genus Panulirus sporadically distributed in South Korean waters: P. japonicus, P. penicilatus, P. stimpsoni, and P. versicolor. However, only P. stimpsoni has been reported in Jeju Island waters (Kim et al., 2009).
In the current study, spiny lobsters were identified using DNA barcoding and morphological analysis. A maximum likelihood tree of COI marker was constructed by including the spiny lobster sequences available in the Genbank and the BOLD system. The results confirmed that the spiny lobsters collected in this study belong to clade A, B, C, and D, which are reciprocally monophyletic with P. japonicus, P. longipes, P. stimpsoni, and P. homarus, respectively. Among the four clades, the closest genetic distance was observed between intra Jeju Island specific spiny lobsters from clade C (P. stimpsoni) and D (P. homarus). On the other hand, the furthest distance was found between clade A (P. japonicus) and C (P. stimpsoni). These results provided additional evidence supporting the previous studies which state that spiny lobsters (Genus: Panulirus) can be morphologically and phylogenetically diversified into two major lineages (Ptacek et al., 2001;George, 2005).
Intraspecific Jeju diversity analysis revealed that clade A appears to have the highest divergence rate based on the number of haplotypes, nucleotide diversity (Pi) and polymorphic sites. Notably, among the collected specimens, majority of the sequences were observed in this clade and thus, were genetically identified as P. japonicus. Among Table 2 The comparative morphological identification features of spiny lobster species collected from Jeju Island and other two species which were previously reported from South Korea. these sequences, one sequence was exclusively sub-claded with P. japonicus from Taiwan, while the remaining were sub-claded with those from Japan. Therefore, it can be assumed that the P. japonicus specimens found in Jeju Island originated from the same population source as those from Japan and Taiwan. Our findings were consistent with those of previous studies, which state that P. japonicus across populations within the Japan, Taiwan, and southern Chinese waters have originated from the same larval pool that mixed within the Kuroshio counter-current region (Inoue et al., 2007;Chan, Yang & Wakabayashi, 2019). Furthermore, this research partially supported the previous hypothesis regarding non-existence of sub-divisions in the P. japonicus population within its spatial distribution (Chan, Yang & Wakabayashi, 2019). Despite the monophyletic lineages, two morphotypes of P. japonicus were found in the current study, which will be addressed as original type (George & Holthuis, 1965) and Jeju type. Four of the eight P. japonicus specimens found in the current study appeared to be Jeju type based on the variations observed in its morphological features compared to the original type (George & Holthuis, 1965;Kim et al., 2009). The Jeju type specimens had a connected pleural and transverse groove at the third abdominal somite, unlike the holotype from Japan (George & Holthuis, 1965). Moreover, the transverse groove ends close to the pleural groove in the second abdominal somite, forming a pseudo connection, which creates a noticeable gap in the previously described specimens. Morphological variations in spiny lobsters have been discovered in the species P. homarus and P. longipes (Sekiguchi, 1991;Lavery et al., 2014). In addition to this, the evolutionary divergence and phenotypic adaptation that results from geographical dispersal were shown in the different colorations and abdominal patterns among P. homarus morphotypes observed in this study. The phylogenetic clusters clearly supported these apparent variations based on population; thus, each was introduced as a different subspecies (Lavery et al., 2014). In the case of P. japonicus from Jeju Island, each morphotype does not indicate a distinct genetic cluster or an intra spatial subdivision. It is likely that the Jeju type variation occurred as part of an adaptive response due to selective forces and/or environmental restrictions during developmental and settlement stages (George, 2005;Vieira et al., 2016).
This study confirmed that the Jeju Island spiny lobsters in clade B were P. longipes, based on morphological and phylogenetic species concepts (George & Holthuis, 1965;Ravago & Juinio, 2002). The intraspecific diversity of mtDNA COI revealed that, unlike its sister clade (clade A), clade B had a relatively low divergence based on its Pi value and polymorphic sites. In the genus Panulirus, P. longipes is one of the two spiny lobsters that have subspecies because of its morphotype variations (George & Holthuis, 1965). Around the East China Sea region, the subspecies P. longipes longipes and P. longipes femoristriga are distributed throughout southwestern Japan, including Okinawa and Yaeyama Island, through the northern side of Taiwan (Sekiguchi, 1991). The discovery of P. longipes in Jeju Island has updated the records of spiny lobsters inhabiting South Korean waters in general.
In clade C, six spiny lobsters had a well-supported monophyletic relation with P. stimpsoni from Hong Kong and the South China Sea. Compared to the other spiny lobsters in the current study, the phylogenetic analysis of P. stimpsoni remains understudied. This might partially be due to the limited distribution of this species within the East and the