A new species of the genus Nicippe from Japan (Crustacea, Amphipoda, Pardaliscidae)

Abstract A new species of the pardaliscid amphipod, Nicippe recticaudata, from off Cape Toi, Japan, is named and described. This is the first record of Nicippe Bruzelius, 1859 from the western Pacific coast of the Japanese archipelago. Additionally, nucleotide sequences of nuclear 28S ribosomal RNA and histone H3 as well as mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA from the holotype and paratypes were determined. The morphological characteristics and the COI distance values enforced the distinctiveness of N. recticaudata sp. n. among the known Nicippe species. Nicippe recticaudata sp. n. closely resembles N. tumida Bruzelius, 1859 in having a two-dentate posterior margin of usoromite 1. However, the former is distinguished from the latter by the posterior margin of merus of pereopod 4 with 5–6 setae, anterior margin of merus of pereopod 5 with 9–10 setae, and telson with straight inner margin, tapering proximally. A key to the species of Nicippe is provided.

Contrary to the cosmopolitan cryptic species complex N. tumida, the other three species had been recorded from the type localities: N. buchi was collected from lava tubes off Lanzarote, North Atlantic Ocean (Andres 1975); N. rogeri from the central Chatham Rise, off east New Zealand (Lörz and Schnabel 2015); and N. unidentata was recorded from the Palmer Archipelago on the Antarctic Peninsula (Barnard 1932;Biswas et al. 2009).
Recently, unidentified specimens belonging to Nicippe were obtained from off Cape Toi, Miyazaki Prefecture, Japan, at a depth of 265-367 m. This is the first record of the genus from the western Pacific coast of the Japanese archipelago. Following a detailed examination of the specimens and their genetic data, these amphipods are described as a new species herein.

Sample
The present specimens were collected from off the southern tip of Kyushu (St-12) during a research cruise of the T/S Toyoshio-Maru (Hiroshima University) to Kyushu and the Nansei Islands, southwestern Japan in 2006(Cruise # 2006. Specimens were collected with a sledge-net (mouth opening 145 cm × 15 cm, mesh opening 328 µm). The gear was towed along the bottom at a speed of 2 knots for 20 minutes. Samples were immediately fixed and preserved in 99% ethanol on-board ship. In the laboratory, specimens of Nicippe were sorted from amphipod samples under a stereomicroscope. For DNA extraction, muscle tissue was removed from the dorsal side of the pleon of each of three specimens.

Morphological observation
All appendages of the examined specimen were dissected in 70% ethanol and mounted in gum-chloral medium on glass slides under a stereomicroscope (Olympus SZ61). The specimens were examined using a light microscope (Olympus BH2) and illustrated with the aid of a camera lucida. The body length from the tip of the rostrum to the base of the telson was measured along the dorsal curvature to the nearest 0.1 mm. The specimens are deposited in the Tsukuba Collection Center of the National Museum of Nature and Science, Tokyo (NSMT) and the Zoological Collection of Kyoto University (KUZ).

COI genetic diversity calculation
To calculate genetic diversity between the present specimens and the other Nicippe sample, one COI sequence (CMBIA134-11.COI-5P) of the amphipod identified as N. tumida was obtained from BOLD (Ratnasingham and Hebert 2007). The individual was collected from the eastern coast of the Pacific Ocean (Palos Verdes Peninsula, California, U.S.A.).
The COI sequences were manually aligned, because no indels were observed. Pairwise comparisons of uncorrected p-distances for three COI sequences obtained in this study (658 bp) and that obtained from BOLD (651 bp) were calculated using MEGA7.0.16 (Kumar et al. 2016). All missing positions were eliminated for each sequence pair. Diagnosis. Dorsal margin of urosomite 1 with 2 pointed teeth; posterior margin of merus of pereopod 4 with 5-6 setae; anterior margin of merus of pereopod 5 with 9-10 setae; telson with straight inner margin, tapering proximally.
Mouthparts. Upper lip ( Fig. 2D) with shallowly concave ventral margin, lobes symmetric. Mandibles (Fig. 2E-G): slightly asymmetric, incisor margins broad, straight, anterodosal corner rounded, anteroventral corner with a strong tooth; left lacinia mobilis ( Fig. 2F) broad, about 0.8 × length of incisor, multi-dentate; right incisor ( Fig. 2G) with 2 teeth on proximal to anterodorsal corner; right lacinia absent; accessory setal row of left and right mandibles each with 2 robust setae, and a proximal tuft of seta; molar absent; mandibular palp 3-articulate with length ratio of 1.0 : 3.6 : 2.7, article 2 with 12 setae, article 3 with 5 posterolateral and 3 apical setae. Lower lip (Fig. 2H) with broad outer lobes, inner lobes coalesced. Maxilla 1 (Fig. 2I) with inner and outer plate and palp; inner plate small with apical seta; outer plate subrectangular with 7 spine-teeth and 1 stout plumose seta, the lateral one strongest and longest; left and right palps symmetric, palp 2-articulate, article 1 lacking marginal setae, article 2 expanded distally, with 8 robust and 8 slender setae on its apical margin. Maxilla 2 ( Fig. 2J) with moderately slender inner and outer plates; inner plate bearing row of plumose setae on apical to medial margin; outer plate slightly longer than inner plate with apical plumose setae. Maxilliped ( Fig. 2K) with inner and outer plates and palp; inner plate not reaching base of palp, with long plumose seta and short simple seta apically; outer plate narrowly rounded, reaching base of article 2 of palp, with setae along apical to medial margin; palp raptorial, 4-articulate, long, article 2 longest with inner marginal rows of setae, article 3 covered with 4 clusters of setae, article 4 slender with serrate inner margin.
Gnathopod 1 (Fig. 3A): coxa ovate with seta on anterodistal corner; basis long, expanded distally, anterior margin straight, posterior margin arched; ischium short, triangular, subequal in length to merus; carpus with short rounded lobe ventrally with long setae; propodus oval, about as wide as carpus, palm straight with long setae; dactylus slender, slightly curved, inner margin smooth with tooth near the base.
Telson (Fig. 5D) length 2.0 × width, cleft for 88% of length in V-shape with straight inner margins of incision, each lobe with 4 setae laterally; apex of each lobe incised, lateral part of apex slightly longer than medial part, with small robust seta, lobes slightly tapering distally.
Coloration. Color in life unknown; faded in preservative (Fig. 1). Etymology. The specific name is a compound adjective derived from the Latin words rectus, and caudatus referring to the fact that the inner margin of the telson of this species is straight, a diagnostic character of the species.
The obtained three COI sequences (LC214958-LC214960) were completely identical to each other. Based on the 651 bp aligned sequences, the COI uncorrected pdistance between N. recticaudata and the eastern North Pacific "N. tumida" was 17.1%.  Remarks. Although the present specimens showed two characteristics in the degree of callynophore of male antenna 1 and the length of peduncular article 5 of male antenna 2, the obtained genetic data revealed that these morphological variants (specimens with strongly developed callynophore and long peduncular article 5 of antenna 2, e.g., KUZ Z1807 vs specimens with weakly developed callynophore and short peduncular article 5 of antenna 2, e.g., NSMT-Cr 25456) shared completely identical COI sequences, and thus they were identified as the same species. The character states of the well developed callynophore of antenna 1 and the elongate peduncular article 5 of antenna 2 were observed only in males of N. recticaudata. Males with these characteristics might be regarded as fully mature individuals.
According to the conventional classification of Nicippe species, this species would be identified as N. tumida based on the possession of the two-dentate posterior margin of urosomite 1. However, Nicippe recticaudata clearly differs from the "true" N. tumida defined by Bruzelius (1859) and Sars (1895) in the following three characteristics (character states of N. tumida in parentheses): posterior margin of merus of pereopods 4 with 5-6 setae (more than 10); anterior margin of merus of pereopod 5 with 9-10 setae (more than 20); and telson with straight inner margin, tapering proximally (sinuous inner margin, weakly expanding proximally). Moreover, the calculated COI genetic distance (17.1%) between N. recticaudata specimens and the N. tumida sample from California (Northeastern Pacific) revealed that the new species is genetically diverged from the Californian population of N. tumida; 3.5-4% COI distances have been proposed as the threshold for amphipod species discrimination (Witt et al. 2006;Rock et al. 2007;Hou et al. 2009). The N. tumida individuals inhabiting Californian waters were once reported to possess the morphological characteristics resembling those of N. recticaudata (Barnard 1959). Contrary to their morphological similarities, therefore, the present COI data highlighted that the population from Northwestern Pacific (N. recticaudata), and that inhabiting Northeastern Pacific ("N. tumida" in Californian waters) are different species. In summary, the present tumida-like Japanese population of Nicippe is considered as distinctive, and thus was described as a new species of the genus.
A Nicippe specimen identified as N. tumida was recorded from the East China Sea (Ren 2012). However, its description as well as figures clearly shows the diagnostic characteristics of N. recticaudata, and thus the Chinese sample in Ren (2012) definitely belongs to the present new species. As well, N. tumida has been recorded from around the Japanese Archipelago, e.g., from Sea of Japan and Sea of Okhotsk (Gurjanova 1951;Bulycheva 1957). Because these previous records lacked the detailed descriptions related to the diagnostic characters of N. recticaudata, their identities remain unclear. A worldwide systematic revision is essential to elucidate the cryptic species diversity in N. tumida.