First Record of the Brackish Water Amphipod Jesogammarus (Jesogammarus) hinumensis (Amphipoda: Anisogammaridae) from Korea with DNA Barcode Analysis among Jesogammarus Species

Abstract

Jesogammarus (Jesogammarus) hinumensis Morino, 1993 was discovered firstly from a brackish water region in Jeju Island, Korea. To identification of the specimens we conducted both of morphological and molecular analyses. This species is characterized by having large eyes and a robust seta on the mandibular palp article 1. The morphology of this Korean specimens was well matched with the original description without variation. The mitochondrial cytochrome c oxidase subunit I(COI) sequences of the present specimens were also completely identical to the sequences of J. (J.) hinumensis collected from the type locality of the species. Thus, we concluded that the Jesogammarus species from Jeju Island is J.(J.) hinumensis, based on both morphological and molecular data.

INTRODUCTION

The anisogammarid genus Jesogammarus Bousfield, 1979 has been recorded from freshwater or brackish water in the Korean Peninsula, the Japanese Archipelago and the Chinese Continent, and currently comprises 20 species in two subgenera (Bousfield, 1979; Morino, 1984, 1985, 1986, 1993; Lee and Seo, 1990, 1992; Tomikawa and Morino, 2003; Tomikawa et al., 2003; Hou and Li, 2004, 2005; Tomikawa, 2015; Tomikawa et al., 2017). Two freshwater species, Jesogammarus(Jesogammarus) ilhoii Lee and Seo, 1992 and J. (Annanogammarus) koreaensis Lee and Seo, 1990 have been known from the Korean Peninsula (Lee and Seo, 1990, 1992). However, the species diversity of the genus in Korea is underestimated and many regions remain to be investigated.

Jesogammarus(J.) hinumensis Morino, 1993 was originally described from a brackish lake in Hinuma, Japan. Subsequently, this species has been recorded in various brackish water areas in Japan (Tomikawa, 2007). Jesogammarus (J.) hinumensis has been designated as an endangered species in Japan (Ariyama, 2012).

During field surveys of brackish water habitats in Jeju Island, the first author collected Jesogammarus specimens. Close examination of the external morphology and molecular analyses based on mitochondrial cytochrome c oxidase subunit I (COI) sequences revealed that these specimens are indubitably identical to J. (J.) hinumensis. In additional, for the future study using the DNA barcode of Jesogammarus in Korea, we provide revealed sequences by using two pairs of primers in the COI region. In this paper, we provide diagnosis and DNA barcode of this species collected from Jeju Island, Korea.

RESULTS AND DISCUSSION

Specimens were collected from a small brackish pond in Jeju Island (33°30.19ʹN, 126°53.51ʹE), Hado-ri, Jeju-si, Jeju-do, Korea using a fine-mesh hand-net on 11 Jun 2018 and 26 Apr 2019. Specimens were fixed and preserved in 99% ethanol. The Korean specimens completely agreed with the original description of J.(J.) hinumensis Morino, 1993. In addition to J.(J.) hinumensis, two species of Jesogammarus have robust setae on the mandibular palp article 1: J. (J.) fontanus Hou and Li, 2004 from a well in China and J. (J.) spinopalpus Morino, 1985 from freshwaters in Japan. Jesogammarus(J.) hinumensis differs from these two species by large eyes(vs. medium), the mandibular palp article 1 with one (vs. three) robust seta, and a few dorsal setae on pleonites 1-3 that are up to two (vs. more than six setae). The specimens examined in this study have been deposited in the collection of the Nakdonggang National Institute of Biological Resources, Korea (NNIBR22377-NNIBR22382).

The phylogenetic position of the newly identified Jesogammrus amphipod within the genus was estimated based on the sequences of the mitochondrial COI gene. DNA extraction and PCR reactions with COI primers Am-COI-H [CG (AG)GC(CGT)TA(CT)TT(CT)AC(CT)TC(ATC)GC(AC) ACTAT] and Am-COI-T [CGTCG(AGT)GG(CT)AT(ACG) CC(ACGT)CT(AGT)A(AG)(ATC)CCTA] were performed according to the methods described by Tomikawa (2015). Three sequences from the three Jesogammarus specimens were newly obtained in this study (NCBI Nos. MN068361, MN068362 and MN068363). Also, we provide sequences performed with universal primers LCO1490 and HCO2198 (Folmer et al., 1994) (NCBI Nos. MN068364, MN068365 and MN068366). Sequences of three specimens are completely identical from two COI regions. The sequences determined from primers Am-COI-H and Am-COI-T were used for the distance analysis(Tomikawa et al., 2007).

The sequences were aligned using Geneious 8.1.9 (Biomatters Ltd, Auckland, New Zealand). The COI gene sequences of other Jesogammarus species were obtained from GenBank. Sequence analyses were conducted using MEGA 7.0 (Kumar et al., 2016). Nucleotide sequence divergences were calculated using the Kimura two-parameter distance (Kimura, 1980). In our analysis, the sequences of the present specimens were completely identical to the sequence from J. (J.) hinumensis from the type locality of the species (Lake Hinuma, Japan), while the genetic distance between J. (J.) hinumensis and other Jesogammarus species are ranged from 20.5% to 30.2% (Table 1). Thus, we concluded that Jesogammarus species from Jeju Island is J.(J.) hinumensis based on both morphological and molecular data.

Table 1. Pairwise genetic distances among Jesogammarus species calculated using K2P model from COI gene sequences

Order Amphipoda Latreille, 1816

Family Anisogammaridae Bousfield, 1977

Genus Jesogammarus Bousfield, 1979

Jesogammarus(Jesogammarus) hinumensis Morino, 1993 (Fig. 1)

Jesogammarus (Jesogammarus) hinumensis Morino, 1993: 9, figs. 1-4; Tomikawa, 2007: 25.

Fig. 1. Jesogammarus (Jesogammarus) hinumensis Morino, 1993. Male 14.7 mm (NNIBR22377), Jeju Island, Korea. Habitus. Scale bar=5.0 mm.​​​​​​​

Diagnosis. Eyes large; pereonites without dorsal setae; dorsal margin of pleonites 1-3 each with 1-2 setae. Peduncles of antennae 1 and 2 with a few short setae on posterior margins. Mandible, palp article 1 with 1 robust seta (Fig. 2). Accessory lobes of coxal gills on gnathopod 2 and pereopods 3-5 well developed, both anterior and posterior lobes subequal in length or posterior lobe longer than anterior one. Female gnathopod 2, palmar margin of propodus with pectinate setae. Female pereopods densely setose. Uropod 3, inner ramus 0.15-0.27 times as long as outer ramus(Fig. 2); terminal article of outer ramus 0.1-0.2 times as long as proximal article. Telson length 1.0-1.3 times as long as wide (Fig. 2).

Fig. 2. Jesogammarus (Jesogammarus) hinumensis Morino, 1993. A, Mandible palp, lateral view; B, Mandible, medial view; C, Uropod 3, dorsal view; D, Telson, dorsal view. Scale bars: A-D=0.1 mm.​​​​​​​