A new species of the rare genus Priscomilitaris from the Seto Inland Sea, Japan (Crustacea, Amphipoda, Priscomilitaridae)

Abstract A new species of the priscomilitarid amphipod, Priscomilitaris heike, from the Seto Inland Sea, Japan, is named and described. This new species is the third species of Priscomilitaridae and the second species of Priscomilitaris. Additionally, nucleotide sequences of nuclear 28S rRNA and histone H3 as well as mitochondrial cytochrome c oxidase subunit I from its holotype were determined. Priscomilitaris heike sp. n. is distinguished from its congener, Priscomilitaris tenuis Hirayama, 1988, by having deep antennal sinus, long flagellar article 1 of antennae 1 and 2, long mandibular palp article 2, 10 robust setae on outer ramus of maxilla 1, and rounded epimeral plates. A key to the species of Proscomilitaridae is provided.


Introduction
Priscomilitaridae Hirayama, 1988 is a small family of amphipods comprising two monotypic genera Priscomilitaris Hirayama, 1988 and Paraphotis Ren, 1997 from coastal waters in Japan and China (Hirayama 1988;Ren 1997;Myers and Lowry 2003). Priscomilitaris was erected by Hirayama (1988) along with a new species P. tenuis Hirayama, 1988 from Ariake Sea, Japan. There has been no record of this genus since its original description, and thus several areas await intensive taxonomic surveys.
The Seto Inland Sea is a vast inland sea separating Honshu, Shikoku, and Kyushu. More than 90 species of amphipods were recorded from the sea (Nagata 1965;Ariyama 1996Ariyama , 2015Ariyama , 2016. During field surveys of marine crustaceans in the Seto Inland Sea made by HT, an undescribed species of Priscomilitaris was collected. In this paper, we describe and illustrate this undescribed species, and provide a key to species of Priscomilitaridae. Additionally, we provide nucleotide sequences obtained from the undescribed Priscomilitaris species for future molecular systematic studies.

Sample
The present specimen was collected with a dredge (mouth 40 cm wide, 15 cm high, mesh size 5 mm) at 14 m depth off Abashima Island, Takehara City, Hiroshima Prefecture, Seto Inland Sea, Japan (34°19'30.6"N, 132°56'31.9"E: Fig. 1). The specimen was preserved in 80% ethanol. For DNA extraction, dorsal side muscle was removed from inside pleon of the specimen, and was transferred into absolute ethanol.

Morphological observation
All appendages of the examined specimen were dissected in 80% ethanol and mounted in gum-chloral medium on glass slides under a stereomicroscope (Olympus SZX7). The specimen was examined using a light microscope (Nikon Eclipse Ni) 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 nomenclature of the setal patterns on the mandibular palp follows Stock (1974). The unique holotype has been deposited in the Tsukuba Collection Center of the National Museum of Nature and Science, Tokyo (NSMT).

PCR and DNA sequencing
The extraction of genomic DNA from pleon muscle followed Tomikawa et al. (2014). Primer sets for the PCR and cycle sequencing (CS) reactions used in this study were as follows: for 28S rRNA (28S), 28F and 28R (PCR and CS) (Hou et al. 2007) with 28SF and 28SR (CS) (Tomikawa et al. 2012) as internal primers; for histone H3 (H3), H3aF and H3bR (PCR and CS) (Colgan et al. 1998); for cytochrome c oxidase subunit I (COI), jgLCO1490 and jgHCO2198 (Geller et al. 2013), respectively, with M13F and M13R tails (Messing 1983), used for PCR, and then M13F and M13R used as primers for CS, followed the method outlined in Raupach et al. (2015). The PCR reactions and DNA sequencing were performed using the modified method mentioned in Nakano (2012). The PCR reactions were performed using a T100 Thermal Cycler (Bio-Rad) using an Ex Taq Polymerase Kit (Takara Bio Inc.) for 28S plus H3, and Taq Polymerase Kit (Takara Bio Inc.) for COI. The PCR mixtures were heated to 94°C for 5 min, followed by 35 cycles at 94°C (10 s each), 50°C for 28S and H3 or 49°C for COI (20 s each), and 72°C (1 min 24 s for 28S, 24 s for H3, 42 s for COI), and a final extension at 72°C for 6 min. The sequencing mixtures were heated to 96°C for 2 min, followed by 40 cycles at 96°C (10 s each), 50°C (5 s each) and 60°C (1 min for 28S, and 42 s for H3 and COI). The obtained sequences were edited using DNA BASER (Heracle Biosoft S.R.L.). These DNA sequences were deposited with the International Nucleotide Sequence Database Collaboration (INSDC) through the DNA Data Bank of Japan (DDBJ).

Family Priscomilitaridae Hirayama, 1988
Remarks. This family name was subsequently used as Priscomilitariidae by Myers and Lowry (2003). The generic name of its type species, Priscomilitaris, ends in a Latin word, militaris (genitive militaris, stem militar-). Therefore, the stem of this family name should be Priscomilitar-according to the Art 29.3. of the Code (ICZN 1999). The original spelling by Hirayama (1988) is thus obviously correct. Because Myers and Lowry (2003) did not provide a statement for any demonstrably intentional change of the spelling Priscomilitaridae, the spelling Priscomilitariidae is an incorrect subsequent spelling according to the Art 33.3. of the Code. This incorrect spelling is used in the influential web sources, e.g. WoRMS (Horton and Lowry 2015). The incorrect spelling of Priscomilitaridae on those web registries should be emended to avoid additional erroneous citations of the spelling of this family name.
Antenna 2 (Fig. 3B): length 0.9 times as long as antenna 1; length ratio of peduncular articles 3-5 1.0 : 1.7 : 1.7; article 3 quadrate with 2 single setae and a pair of setae on posterior margin; article 4, anterior margin with 2 short setae and one long seta, posterior margin with 4 single setae and pair of setae; article 5 with 2 short setae on anterior margin, and 4 pairs and 2 clusters of setae on posterior margin; flagellum 5-articulate, article 1 long, length 2.5 times as long as article 2, article 5 minute; calceoli absent.
Uropod 1 (Fig. 5C): extending beyond uropod 2; peduncle long, length 1.6 times as long as inner ramus, dorsolateral margin with robust seta and numerus minute setae; inner ramus length 1.1 times as long as outer ramus, inner and outer margins with minute robust setae, apical part with robust seta; inner margin of outer ramus with minute robust setae, outer submargin with seta, apical part with robust seta. Uropod 2 (Fig. 5D): extending beyond uropod 3; peduncle almost as long as inner ramus, distal part of dorsolateral margin with minute robust setae; inner ramus slightly longer than outer ramus, inner distal and outer margins with minute robust setae; outer ramus with minute robust setae on outer and inner distal margins. Uropod 3 (Fig. 5E): extending beyond telson, uniramous; peduncle short, with facial seta and minute robust setae along with distal margin; ramus long, length 2.4 times as long as peduncle, 1-articulate with terminal robust seta. Telson (Fig. 5F): entire, fleshy, length 0.6 times width, with 2 clusters of 6 setae on distal submargin. Female unknown.
Distribution. This species is known only from the type locality. Etymology. After 'Heike' (literally 'House of Taira') that controlled the Seto Inland Sea, the Chugoku region, the Shikoku region as well as the Kyushu region during the Heian Period. The specific name is a Japanese word, not a Latin or Latinized one.