First Genetic Data of Nebalia koreana (Malacostraca, Leptostraca) with DNA Barcode Divergence among Nebalia Species

Abstract

We determined the cytochrome c oxidase subunit 1 (CO1) sequences of Nebalia koreana Song, Moreira & Min, 2012 (Leptostraca) collected from five locations in South Korea, and this represents the first genetic data of this species. The maximum intra-species variation was 1.2% within Nebalia hessleri Martin, Vetter & Cash-Clark, 1996, while inter-species variation ranged from 9.0% (N. hessleri and Nebalia gerkenae Haney & Martin, 2000) to 34.8% (N. hessleri and Nebalia pseudotroncosoi Song, Moreira & Min, 2013). This result is well agreed with the interspecific relationships among Nebalia species based on morphological characteristics. In conclusion, this study showed the usefulness of CO1 sequences as a DNA barcode within the genus Nebalia Leach, 1814.

INTRODUCTION

The order Leptostraca Claus, 1880 is the only extant order in the subclass Phyllocarida Packard, 1879, and is considered by many researchers to be the most primitive group in the class Malacostraca Latreille, 1802 (Claus, 1888; Manton, 1934; Hessler and Newman, 1975; Barnes, 1980; Dahl, 1987; Walker-Smith and Poore, 2001). The genus Nebalia Leach, 1814 is the largest member of the order Leptostraca (38 of 60 valid species), and so far, the following three species have been reported in South Korea: Nebalia dolsandoensis Song & Min, 2017, Nebalia koreana Song, Moreira & Min, 2012, and Nebalia pseudotroncosoi Song, Moreira & Min, 2013 (see Song et al., 2012, 2013; Song and Min, 2017). Nebalia koreana is distributed in Eastern, Western and Southern coastal waters of Korea, while the other two species were found restrictively at their type localities (Song, 2013). Nebalia koreana is diagnosed by the following characteristics: antennule fourth article with up to five short distal spines; pleonites 3-7 with rounded denticles on posterior dorsal margins; and pleopod 4 protopod lacking serrations on posterior margin.

Among the mitochondrial genes already examined in most animal phyla, including Crustacea, the cytochrome c oxidase subunit 1 (CO1) gene has proved to be a particularly useful taxonomic marker (Meyran et al., 1997; Wares, 2001; Hajibaei et al., 2006; Clare et al., 2007; Elsasser et al., 2009; Zemlak et al., 2009). However, the usefulness of the CO1 sequences in Nebalia species has never been proven. In this study, we firstly determined the CO1 sequence of N. koreana (Fig. 1), and attempted to verify its usefulness.

Fig. 1. Nebalia koreana collected from Sokcho, lateral view.

RESULTS AND DISCUSSION

The CO1 sequences of N. koreana were obtained from specimens collected from each of the following five locations in South Korea: Bogildo (34°08ʹ10ʺN, 126°33ʹ48ʺE), Cheongsando (34°10ʹ54ʺN, 126°33ʹ59ʺE), Mokpo (34°48ʹ 19ʺN, 126°21ʹ43ʺE), Masan (35°11ʹ11ʺN, 128°33ʹ53ʺE), and Sokcho (38°11ʹ50ʺN, 128°35ʹ00ʺE) using light traps according to the methods described by Song et al.(2013). The morphological identification, genomic DNA extraction, and PCR conditions were performed according to the methods described by Song and Min (2017). The sequences were aligned using Geneious 8.1.9 (Biomatters Ltd., Auckland, New Zealand). The CO1 sequences determined from this study and retrieved from GenBank were aligned using MEGA 10 (Kumar et al. 2018). Nucleotide sequence divergence was calculated using the Kimura two-parameter distances.

In our analysis, the maximum intra-species variation was 1.2% within Nebalia hessleri Martin, Vetter & Cash-Clark, 1996, while inter-species variation ranged from 9.0% (N. hessleri and Nebalia gerkenae Haney & Martin, 2000) to 34.8% (N. hessleri and N. pseudotroncosoi) (Table 1). This result is well agreed with the interspecific relationships among Nebalia species based on morphological characteristics (Song and Min, 2017). In conclusion, this study suggests that the CO1 sequences are very useful for the identification of Nebalia species, as confirmed by many other crustaceans.

Table 1. The minimum/maximum values of inter-species pairwise variation (%) between CO1 sequences from Nebalia species​​​​​​​