The Indonesian greeneye spurdog (or a dogfish shark), Squalus montalbani Whitley, 1931, is widely distributed in the warm temperate to tropical waters of Indonesia, Philippines, the island of Taiwan, and Australia. Previous studies suggested that the distribution of dogfish shark species in the South China Sea is composed of two species, Squalus mitsukurii Jordan et Snyder, 1903 and Squalus brevirostris Tanaka, 1917. In March 2020 a dogfish shark specimen was collected from the Nansha (Spratly) Islands, South China Sea. We identified it as S. montalbani based on morphology and mitochondrial DNA barcoding. Our results confirmed the presence of S. montalbani in the South China Sea, leading us to conclude that it represents a new species record of the genus Squalus in the region. Furthermore, our findings demonstrate that the combined approach is highly effective in identifying Squalus species that share similar morphological characteristics.

fish taxonomy, mitochondrial DNA barcoding, new record, South China Sea, Squalus montalbani

The family Squalidae includes 2 genera, 39 species. Among these species, 36 species represents dogfish sharks (genus Squalus) (Ziadi-Künzli et al. 2020; Ariza et al. 2022; WoRMS Editorial Board 2023). Due to difficulties in morphological characteristics between different dogfish sharks, species in Squalus have a high taxonomic complexity (Viana et al. 2016). Thus, studies related to the taxonomy, evolution, and new species records are especially meaningful. Species of the genus Squalus are mainly distributed in the continental shelf waters, upper slope waters, and underwater cracks of the Atlantic, Pacific, and Indian oceans (Viana et al. 2016).

It has been reported that there were 9 species of Squalus genus distributed in China, including Squalus acanthias Linnaeus, 1758; Squalus mitsukurii Jordan et Snyder, 1903; Squalus brevirostris Tanaka, 1917; Squalus blainville (Risso, 1827); Squalus formosus White et Iglésias, 2011; Squalus japonicus Ishikawa, 1908; Squalus megalops (MacLeay, 1881); Squalus montalbani Whitley, 1931; and Squalus suckleyi (Girard, 1855) (see Zhu et al. 1963, 1984; Cheng and Zheng 1987; Shao 2023; Zhang 1960; Zhu 1960; White and Iglesias 2011; Straube et al. 2013). Among these species, S. montalbani has been found off the island of Taiwan, Australia, Philippines, Indonesia, in eastern Indian Ocean and Western Central Pacific regions (Graham 2019). Previous reports suggested that the distribution of dogfish shark species in the South China Sea is limited to only two species, namely S. mitsukurii and S. brevirostris (see Zhu 1962, 1979, 1984; Zhang et al. 2018).

A single dogfish shark specimen, at our disposal, collected in the middle of the South China Sea prompted us to identify it using morphological methods and DNA barcoding technique. The specimen could potentially represent a new record of a dogfish species for the studied area.

A single dogfish shark specimen was collected by Jianwei Zhou, at the Dianjian Fishing Harbour Marina, Beihai (mainland China), on March 2020. The fish originated from in the Nansha Archipelago (known also as the Spratly Islands), South China Sea (09°47′57″N, 114°5′35″E). The specimen was identified based on morphological characteristics used by Last et al. (2007).

A piece of muscle tissue was cut from the specimen, stored in 95% ethanol, and DNA was extracted with a DNA Extraction Kit of Tiangen, then PCR amplification. 5′-TCGACTAATCATAAAGATATCGGCAC-3′ and 5′-ACTTCAGGGTGACCGAAGAATCAGAA-3′ were used as primer sequences for cytochrome oxidase I (COI) amplification (Ivanova et al. 2007). PCR amplifications were performed in 25 μL volume including 1 μL of forward primer (F, 10 uM · L^–1), 1 μL of reverse primer (R, 10 uM · L^–1), 2 μL of dNTPs (2.5 mM · L^–1 each), 0.15 μL of EasyTaq DNA Polymerase (5 U · μL^–1), 2.5 μL of 10 × PCR buffer (25 uM · L^–1), 1 μL of DNA template (50 ng · uL^–1). The PCR conditions consisted of use of 95°C for 5 min for initial denaturation, 35 cycles of 94°C for 35 s, annealing at 54°C for 35 s, and extension at 72°C for 35 s, with a final extension at 72°C for 10 min. The above reactions were conducted through Biometra thermal cycler (Gottingen, Germany). Finally, the PCR products were stored in 4°C environment. Agarose gel was used for electrophoresis and was sequenced. Subsequently, the COI gene sequence of this specimen was obtained and revised through DNASTAR software (DNASTAR Inc., Madison, WI, USA).

Thirteen COI sequences of the genus Squalus were downloaded from NCBI for phylogenetic study, Somniosus rostratus (Risso, 1827) (KJ083255) was selected as the outgroup to root the tree (Table 1). The genetic relation between COI sequences was analyzed by the maximum likelihood method (ML, Felsenstein 1981). Phylogenetic tree construction first used JModeltest (Posada 2008) based on AIC (Akaike 1973) to filter the best alternative model as TPM2uf+I+G, then use RAxML-NG (Kozlov et al. 2019) to construct the ML phylogenetic tree based on Bootstrap method (Felsenstein 1985). The number of Bootstraps is set to 1000 times, and finally, the online tool ITOL (Letunic and Bork 2007) was used to view and adjust the phylogenetic tree (https://itol.embl.de/). The genetic distances of species were calculated in pairs using MEGA7 (Kumar et al. 2016), and thermodynamic maps of genetic distances were constructed using the R software (Ihaka and Gentleman 1996) combination package (Wei et al. 2017).

Morphological characteristics of the studied specimen of Squalus montalbani were shown in Fig. 1. The detailed measurements (in absolute values) were included in Table 2. Those absolute values yielded the relative values presented below.

Figure 1. 

Squalus montalbani (sample_SM; length 550 mm TL); (A) Left lateral view; (B) Lateral view of the first dorsal fin; (C) Lateral view of the second dorsal fin; (D) Ventral view of the head; (E) Coloration of the caudal fin.

Diagnosis. Body elongate to robust; trunk depth 12.2% TL; pre-first dorsal length 29.0% TL; pre-second dorsal length 61.2% TL; interdorsal space 25.4% TL; low raked dorsal fins; prepectoral length 22.5% TL; pelvic-caudal space 26% TL; dark spots on upper caudal lobe showing saddle-like extension toward upper caudal lobe margin.

The above morphological characteristics basically conform to the description of the Indonesian greeneye spurdog (dogfish shark), Squalus montalbani, in the literature (Last et al. 2007). In addition, based on observations of the morphological characteristics of the sample teeth, dermal denticles tricuspidate and rhomboid were found, in agreement with those described by Viana and De-Carvalho (2018).

The COI gene (655 bp) was sequenced from our sample. The accession number for the sequence submitted to GenBank is OQ826088. The phylogenetic tree was constructed through the downloaded sequences, as shown in Fig. 2. Sample_SM belongs to the genus Squalus. Squalus acanthias, Squalus cubensis Howell Rivero, 1936, S. brevirostris, and S. blainville are clustered in one group. As an outgroup of phylogenetic tree, Somniosus rostratus is a branch alone. Sample_SM and the rest of the species clustered in another group.

Figure 2. 

Maximum likelihood phylogenetic tree based on the COI sequence. Somniosus rostratus (KJ083255) was chosen as the outgroup to root the tree.

The genetic distance thermodynamic diagram shows (Fig. 3) that the genetic distance of COI sequence between different species, the intraspecific genetic distance of Squalus genus is 0.01–0.09. The genetic distance between sample_SM and S. montalbani is 0, and that between sample_SM and Squalus chloroculus Last, White et Motomura, 2007 is 0.01, which further proves that sample_SM and S. montalbani are the same species having a close relation with S. chloroculus. Thus, both the morphological and genetic analysis strongly supports our identification of the newly found dogfish shark specimen as S. montalbani. Therefore, the presently studied specimen constitutes a new record of Squalus montalbani from the Nansha (Spratly) Archipelago, South China Sea.

Figure 3. 

Pairwise comparison of genetic differentiation between the sample in the presently reported study (sample_SM) and other 14 species of the Squaliformes based on COI gene sequence data. The genetic distance relation is expressed according to the color depth of the color block (above diagonal). The genetic distance value (below diagonal).

Due to the unique growth characteristics of the genus Squalus, its species have highly similar morphological characters that are difficult to identify, thus hindering taxonomic studies of the genus (Geraci et al. 2017), just as Squalus montalbani was once considered by Compagno (1984) to be a junior synonym of S. mitsukurii. Over the years, scholars have used ambiguous morphological diagnostic characters to distinguish between different species of the genus Squalus and have not achieved uniformity in diagnostic methods for the same species, leading to extensive taxonomic confusion and synonymization in the past (Veríssimo et al. 2017). The lack of well-preserved holotypes for many shark species, misidentifications in databases and in the literature, and challenges in retrieving representative series of specimens for comparison are top-down impediments to the proper taxonomic identification and the potential revision of genera (Veríssimo et al. 2014).

On the other hand, the slow growth, low reproductive capacity (Cortés 2000), and ease of capture by trawling and longlining, with a high proportion of bycatch, are the main reasons for the dramatic decline in the population of the genus Squalus (see Dulvy et al. 2014). Therefore, the majority of the species of the genus Squalus have been included in the IUCN Red List, and they have been classified in five categories according to their threatened level: Data Deficient, Least Concern, Near Threatened, Vulnerable, and Endangered (IUCN 2020). And the majority of the of the species of the genus Squalus in the Red List are currently classified as Data Deficient, Least Concern, Near Threatened, while eight species are classified as Vulnerable and Endangered, namely, S. acanthias, S. chloroculus, S. brevirostris, S. mitsukurii, S. japonicus, S. montalbani, Squalus hemipinnis White, Last et Yearsley, 2007, S. formosus (IUCN 2020). Squalus montalbani in Australia most of its range with light or absent fishing pressure, and the deeper parts of its depth range may provide refuge from fishing. Therefore, it is assessed as Vulnerable species (Graham 2019).

The genus Squalus has a low evolutionary rate (Hara et al. 2018) and its morphology is very similar, so species identification is often carried out by subtle morphological differences. Currently, morphological identification of the genus Squalus is based on the color and morphological characteristics of the caudal fin, the morphological characteristics of the head and trunk, and various morphological measurement parameters (Last et al. 2007). Last et al. (2007) have stated that S. montalbani and S. chloroculus have been confused, and our results show that they are genetically very close to each other; the genetic differentiation between S. montalbani and S. chloroculus is minimal, so there is a reason for their confusion. Both share the same morphological characteristics: relatively large body size, dark tail, low dorsal fin spines, and small, sloping first dorsal fin. However, there are also slight differences between the two, with the dorsal fin of S. chloroculus being smaller compared to S. montalbani, having a wider base of the fin spines, shorter adult claspers, and the upper postventral caudal margin is short relative to the lower postventral margin, having a marginally higher mean precaudal count (Last et al. 2007).

According to the data obtained in this study, the intraspecific genetic distances of the genus Squalus mainly ranged from 0.01 to 0.09, which indicates that the differentiation rate of the genus Squalus is very low, namely, the genetic expression is relatively conserved, resulting in a very similar morphology of the genus Squalus. Therefore, traditional taxonomic methods alone are not sufficient to identify species of the genus Squalus, and in recent years, molecular methods have begun to be used to supplement traditional taxonomic methods to make the identification of species of the genus more accurate, but molecular methods cannot completely replace traditional taxonomic methods at present (Schlick-Steiner et al. 2010). It is now customary to use a combination of traditional taxonomic methods and molecular methods of COI or NADH mitochondrial DNA labeling to identify the genus Squalus. This combined approach has proven to be very effective in identifying such species (Lim et al. 2022; Cerutti-Pereyra et al. 2012; Gabbanelli et al. 2018).

1. Hainan Provincial Natural Science Foundation of China (No. 320QN361).
2. National Natural Science Foundation of China (42206109).
3. Asia Cooperation Fund Project—Modern fishery cooperation between China and neighboring countries around the South China Sea.