A new species of Hangangbathynella (Crustacea, Bathynellacea, Parabathynellidae) from South Korea

Abstract A new parabathynellid bathynellacean species, Hangangbathynella mihoensissp. nov., was found in the groundwater of the Geumgang River in South Korea. This is the first report of Hangangbathynella from a tributary of the Geumgang River. All previously-reported species were found in the Hangang River and the origins of the two rivers are distinct from each other. The new species can be distinguished from its congeners by the two-segmented mandibular palp and the absence of epipods on thoracopod II. In this study, we provide a description of the new species and an identification table for the genus Hangangbathynella. In addition, we obtained partial sequences of the mitochondrial cytochrome c oxidase subunit I gene for DNA barcoding.


Introduction
Bathynellacea Chappuis, 1915 is an order of groundwater crustaceans, known to have high levels of endemism owing to their limited dispersal capacity (Camacho et al. 2012). The group exclusively inhabits fresh and brackish subterranean waters and is distributed throughout all continents except Antarctica (Camacho et al. 2012). They have no eyes, simplified appendages, and a reduced number of segments in the antennule, antenna, mouthpart structures and thoracopods. As opposed to other adult malacostracans, the appendages of adult Bathynellacea are similar to those of their larvae (Schminke 1981).
The taxonomic study of Korean bathynellaceans was begun by Morimoto (1970), who described seven species through a speleological survey: two species belonging to Parabathynellidae and five belonging to Bathynellidae. To date, 30 species of the family Parabathynellidae, belonging to five genera, have been described from South Korea (Morimoto 1970;Cho et al. 2008;Park and Cho 2008, 2013, 2015a, b, 2016Schminke 2011;Park and Eun 2012;Shin 2014): 17 species of Allobathynella Morimoto & Miura, 1957, two of Eobathynella Birstein & Ljovuschkin, 1964, four of Nipponbathynella Schminke, 1973, three of Arisubathynella Park & Eun, 2012, and four of Hangangbathynella Park & Cho, 2013 As suggested by the generic name, the genus Hangangbathynella was first discovered and established at Hangang River in South Korea by Park and Cho (2013) and has been recorded, to date, only in the tributary of the Hangang River.
Hangangbathynella specimens were first collected during field surveys of groundwater habitats of the tributary of the Geumgang River in the Korean Peninsula. Based on the morphological examination of these specimens, we herein describe and illustrate them as a new species. In addition, we obtained mitochondrial cytochrome c oxidase subunit I (COI) gene sequence data for molecular barcoding and provide an identification table to species of the genus Hangangbathynella.

Materials and methods
Hangangbathynella specimens were collected from the interstitial groundwater of sandbanks near the Miho Stream (tributary of the Geumgang River) in South Korea ( Fig. 1). The groundwater was pumped using a core (Lee and Park 2016) and filtered using a 50 μm fine-mesh net. The specimens were immediately fixed in 95% ethanol and dissected in glycerol under a stereomicroscope (SZX12, Olympus, Japan). Dissected appendages were mounted using Eukitt Quick-hardening mounting medium (Sigma-Aldrich, St. Louis, MO, USA) for permanent slides. Observations and drawings were performed using an optical microscope at 1000× magnification (DM2500, Leica, Germany). Specimens for scanning electron microscopy (SEM) were dehydrated in serial ethanol solutions, transferred into hexamethyldisilazane (Sigma-Aldrich, St. Louis, MO, USA), covered with platinum, and observed using a Hitachi SEM model S-4300SE (Hitachi, Japan). The materials were deposited in the collection of the National Institute of Biological Resources (NIBR), Incheon, South Korea.
Genomic DNA was extracted using the LaboPass Tissue Genomic DNA Isolation Kit Mini (Cosmo GENETECH, Seoul, South Korea) according to the manufacturer's instructions. Partial COI sequences were obtained using the primers Bathy_F1 (5'-ACWAAYCAYAAAGATATYGGRAC-3') and Bathy_R1 (5'-CC-CCCTCGAGCTTGTACAGCTCGTCCATGC-3'). Polymerase chain reaction amplification was conducted under the following conditions: pre-denaturation at 94 °C for 2 min, followed by denaturation at 95 °C for 20 s, annealing at 48 °C for 20 s and extension at 72 °C for 1 min for 40 cycles, with a final extension at 72 °C for 5 min. Diagnosis of the genus. Body elongated and cylindrical. Antennule seven-segmented. Antenna seven-segmented. Incisor process of mandible with four teeth, a triangular proximal tooth, and molar process with more than six spines, with distal spine furcated distally. Maxilla four-segmented without medial seta on second segment. Exopod of thoracopods I-VII two-segmented. First endopodal segment of thoracopods I-VII with one tiny seta on outer distal margin. Male thoracopod VIII oval with massive protopod; epipod present in the form of gourd-shaped process reaching penial region; basis approximately half the size of protopod, without basial setae; endopod small, with two apical setae; exopod with two distal protuberances. Female thoracopod VIII as a one-segmented small process with tiny teeth and two distal setae. Pleopod absent. Uropod: protopod sympod with a row of homonomous spines; endopod with a spur distally, with two distal setae and two plumose setae on dorsal surface; exopod with a basi-ventral seta. Pleotelson with one lateral seta on each side. Anal operculum protruded. Furcal rami with four to six spines, and one long plumose seta, and one simple seta. Type material. Holotype: male, dissected on 9 slides (NIBRIV0000879484). Allotype: female, dissected on 12 slides (NIBRIV0000879485). Paratypes: 9 females (NIBRIV0000879486, NIBRIV0000879487, NIBRIV0000881724, and NI-BRIV0000881737-881742) and 6 males (NIBRIV0000881731-881736).
Description of adult male (Figs 2-5). Body length 1.13 mm ( Fig. 2A), approximately 10 times as long as wide. Head as long as anterior three thoracic segments combined.
Antennule (Fig. 2B) seven-segmented, first segment with one small seta on inner distal margin, two simple dorsal setae of different sizes, three plumose setae on outer margin; second segment with two simple setae on inner distal margin, one group of four plumose setae on outer margin; third segment with one simple dorsal and one ventral seta on inner distal margin, and with two simple setae of different sizes and one tiny seta on outer margin; inner flagellum of third segment with three simple setae; fourth segment with one stub seta and one plumose seta on dorsal margin, two stub setae and two plumose setae on outer distal apophysis, which is slightly protruded; fifth segment with one dorsal simple seta, distally with two setae on inner distal margin; sixth segment with three simple setae on inner distal margin, two aesthetascs and one simple seta dorsally, one aesthetasc on outer margin; seventh segment with three subterminal aesthetascs and four distal simple setae.
Mandible (Fig. 2E) with incisor process of four teeth; tooth of ventral edge triangular; spine row consisting of seven spines; palp two-segmented with one apical seta not exceeding incisor process in length.
First pleopod absent ( Fig. 2A). Uropod (Fig. 5A) with load-shaped sympod bearing eight spines of similar size on inner margin; endopod 28% as long as sympod length, with one large distal spur, two barbed setae and two plumose setae on dorsal surface; exopod longer than endopod, 50% as long as protopod, with one outer seta, two terminal setae and one inner medial seta; inner setae strong, longer and thicker than outer terminal seta. Pleotelson (Fig. 5B) with one lateral seta on each side. Anal operculum (Fig. 5B) protruded. Furcal rami (Fig. 5B) slightly longer than wide, with four spines and two setae, longer one plumose and shorter one barbed.
Description of adult female. The female differs from the male in thoracopod VIII. Thoracopod VIII (Fig. 6E) as a gourd-shaped protuberance with two distal setae and two tiny spines.
Antenna (Fig. 6A) seven-segmented, two proximal segments without setae; third segment with one simple seta on inner edge; fourth segment with one simple seta on inner edge and one simple seta on outer distal margin; fifth segment without setae; sixth segment with one simple and one plumose seta on outer distal margin; distal segment with three simple setae and one plumose seta. Labrum (Fig. 6B) with 14 teeth. Mandible (Fig. 6C) with incisor process of four teeth, tooth of ventral edge triangular, spine row consisting of seven spines. Maxillule (Fig. 6D) two-segmented.
Etymology. The species name is derived from the stream (Miho, a tributary of the Geumgang River) in which the species was collected. Molecular analysis. A total of 606 base pairs of COI sequences (GenBank accession numbers: MW429327, MW429328, and MW624440) were obtained from three individuals of Hangangbathynella mihoensis sp. nov. (NIBRIV0000879486, NIBRIV0000879487, and NIBRIV0000881724). The intra-specific distance of the COI sequences ranged from 0.5% to 1.0%. Although the phylogenetic relationships of Hangangbathynella could not be confirmed owing to the lack of molecular data on Korean bathynellaceans, this result suggests that COI will be useful in distinguishing Korean bathynellacean species in the future.

Discussion
The new species belongs to the genus Hangangbathynella, and species of this genus have been recorded only in the Hangang River to date (Park and Cho 2013). However, the new species was first found in the Miho Stream, which is a tributary of the Geumgang River (Fig. 1).
Hangangbathynella mihoensis sp. nov. most closely resembles H. taechooni in the following characteristics: 1) the antennule's 6 th segment bears three simple setae on inner distal margin, 2) the antenna's 4 th segment bears two setae, and 3) the antenna's 6 th segment bears one simple and one plumose seta. However, the new species differs from H. taechooni by the following features (features of H. taechooni in parentheses):