A new species of Sunamphitoe Bate, 1857 (Crustacea: Amphipoda: Ampithoidae) from Hokkaido, Japan

Abstract A new species of ampithoid amphipod, Sunamphitoe gigantea sp. nov., is described from Katsurakoi, Hokkaido, Japan. The new species most closely resembles S. eoa, but can be distinguished from it by the shorter flagellum of antenna 1, the maxilla 1 inner plate that bears two slender setae, the short dactylus of male gnathopod 2, and the presence of a group of long setae on anterior margins of pereopods 3 and 4 bases. Nucleotide sequences of mitochondrial cytochrome c oxidase subunit I obtained from the type specimens also differentiated this new species from the congeners. Ontogenetic morphological changes of the male gnathopod 2 are briefly discussed.

An extremely large-bodied Sunamphitoe species (maximum length, more than 40 mm) was found on the surface of a kelp Saccharina longissima which was collected by the second author from Katsurakoi, south-east coast of Hokkaido, Japan. In this paper, we herein describe and illustrate the species as Sunamphitoe gigantea sp. nov. Additionally, nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) obtained from the type specimens were also provided for DNA barcoding. Phylogenetic analysis for COI sequences of Sunamphitoe species was also carried out.

Morphological observation
Body length was measured from the tip of rostrum along the dorsal margin to the posterior margin of telson (measurements were done on the curved body). Specimens were dissected under a binocular stereomicroscope, and appendages were fixed on slide mounts with Hoyer's medium. A part of pleopods were stored in 99% ethanol for subsequent DNA analysis.
Observations and line drawings were made by using a light microscope and a binocular stereomicroscope with aid of drawing tube. All the specimens examined in this study were deposited in the National Museum of Nature and Science, Tokyo (NSMT).

DNA extraction and COI sequencing
The following DNA extraction and COI sequencing were carried out at the Bioengineering Lab. Co., Ltd (Kanagawa, Japan). Genomic DNA was extracted from a part of pleopods of seven specimens (including the holotype and the allotype) by using Lysis buffer for PCR (TaKaRa). The target sequences of COI were amplified using the method of two-step tailed PCR procedure for library preparations. This two-step tailed PCR method consists of first-stage and second-stage PCRs. In the firststage PCR, gene specific amplification is performed. In the second-stage PCR, index-sequences (for sample identification) and adapter-sequences (for subsequent sequencing) are added. The first-stage and second-stage PCR primers are shown in Table 1. The first-stage PCR was carried out with two kinds of primer sets (see Table 1): for each, a total volume of 20.0 µl containing 1.0 µl of template DNA (of which the concentration was not standardized), 0.5 µl of each primer (each 10.0 µM), 10.0 µl of 2× Gflex PCR Buffer, 0.4 µl of Tks Gflex (TaKaRa) and 7.6 µl of deuterium depleted water (DDW). The first-stage PCR conditions were as follows: the initial denaturing step was set at 94°C for 1 min; followed by 35 cycles of 10 s at 98°C, 15 s at 52°C and 30 s at 68°C; the final elongation was set for 5 min at 68°C. The second stage of PCR was carried out in a total volume of 10.0 µl containing 2.0 µl of mixture of first-stage PCR products performed with two kinds of primer set, 1.0 µl of 10 × Ex Buffer, 0.8 µl of dNTPs (each 2.5 mM), 0.5 µl of each primer (each 10.0 µM), 0.1 µl of Ex Taq (5 U µl −1 ) (TaKaRa), and 5.1 µl of DDW. The programme of amplification for the second-stage PCR was 94°C for 2 min, 12 cycles (94°C for 30 s, 60°C for 30 s, 72°C for 30 s) and 72°C for 5 min. Concentration of the prepared amplicon libraries was evaluated using Synergy H1 (BioTek) and QuantiFluor dsDNA System (Promega). Quality of the library was evaluated using Fragment Analyzer and dsDNA 915 Reagent Kit (Advanced Analytical Technologies). The amplified products were then sequenced on an MiSeq (Illumina) 2 × 300 bp platform. After sequencing, the sequences with start regions which completely matched with the primer sequences were extracted, and then the primer sequences were removed by using the fastq_barcode_splitter in the Fastx Toolkit. Low-quality sequences (values <20) were removed. Short sequences (length <40 bp) and their paired sequences were discarded by using the Sickle Tools.
Target sequence of COI was divided into the first and the second half. For both first and second halves of target COI sequences, the processed sequences were merged by using the Paired-end merge script FLASH. The merged sequences having a high frequency were selected for both first and second half of COI sequences, and then these two sequences were combined into one total target sequence of COI by using CAP 3. All the obtained COI sequences were deposited into the International Nucleotide Sequence Database Collaboration (INSDC) through the DNA Data Bank of Japan.

Phylogenetic analysis
A phylogenetic analysis was conducted by using MEGA 7.0 software (Kumar et al., 2016). In addition to the COI sequence of our materials, COI sequences of other Sunamphitoe spp. retrieved from INSDC were also used for the analysis ( Table 2). All sequences were aligned using Clustal W (Thompson et al., 1994). Nucleotide sequence divergences within and between species were calculated using Kimura 2-parameter distances. The maximum likelihood method was used to construct a tree. The strength of clade support was assessed with bootstrap resampling with 1000 replicates (Felsenstein, 1985). Ampithoe valida COI sequence (INSDC accession number: GU048489) was chosen as an outgroup.

DIAGNOSIS
Body very large, maximum length of more than 40 mm. Antenna 1 flagellum less than 2 times length of peduncle. Antenna 2 slender; flagellum without dense plumose setae on ventral margin. Mandibular palp present, with 3 articles. Maxilla 1 inner plate with 2 slender setae. Maxilla 2 inner plate narrow, outer plate broader than inner plate. Male gnathopod 2 enlarged; propodus, palm straight, not clearly defined, without distinct protrusion, with dense setae on posteroproximal corner; dactylus reaching to posterodistal angle of propodus, not beyond carpus. Female gnathopod 2 much smaller than that of male; propodus palm well defined, with spine. Pereopods 3 and 4 bases, anterior margin with group of long setae subproximally. Pereopods 5-7 not enlarged; basis, posterodistal lobe absent or indistinct, not reaching ischium; merus, carpus not expanded. Uropod 3 peduncle about 3 times as long as rami. Telson, posterior margin rounded, not acute.
Urosome. Uropod 1 ( Figure 6B) peduncle with dorsolateral row of spines on distal 0.6, dorsomedial row of spines on distal 0.7, ventrolateral row of slender setae on proximal 0.8, distal end with long ventral spur; outer ramus about 0.7 times as long as peduncle (distoventral spur excluded), inner ramus longer and more slender than outer ramus, both outer and inner rami bearing rows of spines on both lateral, mesial margins, lacking slender setae. Uropod 2 ( Figure 6C) peduncle with dorsolateral and dorsomedial rows of spines on distal half, without slender setae, distal end with ventral spur; outer ramus about 0.8 times as long as peduncle (distoventral spur excluded), inner ramus longer and more slender than outer ramus, both outer and inner rami bearing rows of spines on both lateral and mesial margins, lacking slender setae. Uropod 3 ( Figure 6D1) peduncle cylindrical, reaching beyond posterior margin on telson, with several tufts of slender setae laterally, medially, several spines dorsodistally, distolaterally, lateral half of distoventral margin slightly extended, concealing base of outer ramus, fringed with slender setae; outer ramus slightly tapering, about 0.4 times as long as peduncle, finely spinulate dorsolaterally to ventrolaterally ( Figure 6D2), with several short setae laterally, 2 recurved spines distally; inner ramus subquadrate, slightly shorter than outer ramus, with several small spines dorsodistally, dense slender setae ventrodistally. Telson ( Figure 6E), roundly trapezoid, wider than long, posterior margin roundly convex, both lateral margins with 3-5 slender setae, short pappose seta, small telsonic cusp, dorsal surface with 1-2 short simple seta(e), long simple seta on both left, right side.

VARIATIONS
Peduncular article 1 of antenna 1 bears 1 or 2 small spines distoventrally. Flagellar articles of antennae 1 and 2 increase numbers in large individuals. Coxae are sometimes notched on the ventral margin. Spines and setae on pereopods and uropods vary in numbers individually.
Male gnathopod 2 shows ontogenetic morphological change: in small males, palm well defined bearing a spine, and dactylus        Figure 8A, B), while in large males, posterior margin of propodus is straight and the palm is obscure without spine, and dactylus is elongate reaching proximal end of propodus ( Figure 8C).

COLOURATION IN LIFE
Body (Figure 9) generally yellowish (or sometimes greenish) light brown without mottling, without sexual dimorphism; eyes red; antennae 1, 2 flagellae white or whitish yellow, with dark brown band subdistally.

HABITAT
Found on the surface of fronds of a kelp Saccharina longissima inhabiting subtidal rocky shore.

ECOLOGICAL NOTE
Specimens of the new species were found occupying a nest which was constructed by the apical part of kelp blade being rolled up ( Figure 10) as reported in some other congeners (e.g. Cerda et al., 2010). The following four patterns were observed as inhabitant(s) of single nest: (1) single male only, (2) single female only, (3) single female with many juveniles and (4) a pair of male and ovigerous female.
Male gnathopod 2 shows ontogenetic morphological changes (see VARIATIONS). Furthermore, it was found that a male and an ovigerous female coexisted in a nest, though the male gnathopod 2 was not fully developed (NSMT-Cr 26735 and 26736; NSMT-Cr 26738 and 26739). This suggests that male sexual maturity may occur prior to morphological maturity of their gnathopod 2. Males were smaller than coexisting ovigerous females in these two cases.

GENETIC ANALYSIS
In total, 643-658 bp of COI sequences were obtained from seven specimens including the holotype and the allotype (INSDC accession numbers, LC472973-LC472979 for finally obtained COI sequences, DRA008087 for raw fastq data). During the alignment procedure, all positions containing gaps or missing data were eliminated, and then, a total of 506 bp was used for the analysis. Intra-specific divergence of COI sequences within the new species was less than 2%, while inter-specific divergences between the new species and congeners were greater than 14% (Table 3).
In the phylogenetic tree of the COI sequences (Figure 11), all the sequences of S. gigantea sp. nov. formed a monophyletic clade that was supported by 100% bootstrap value. The sister clade of S. gigantea sp. nov. comprised of S. eoa and S. aorangi, though it was supported by a low bootstrap value. Species-level clade was supported by a high bootstrap value for each Sunamphitoe species, indicating the usefulness of COI sequences for DNA barcoding. However, all the higher-level clades (higher than species level) were supported by low bootstrap values, indicating that using only COI sequences may be insufficient for determining the phylogenetic relationship among Sunamphitoe species and other kinds of sequencing may be necessary for further validation.

ETYMOLOGY
The species name 'gigantea' is derived from their large body size.

REMARKS
Among congeners, Sunamphitoe gigantea sp. nov. most resembles S. eoa (von der Brüggen, 1907) in the conspicuously large body (maximum body length is 42.6 mm in S. gigantea sp. nov. and

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Masafumi Kodama et al. S. baegryeongensis (after Kim & Kim, 1988;Kim et al., 2012) S. eoa from NW Pacific (after von der Brüggen, 1907;Gurjanova, 1938;Tzvetkova, 1967;Kim et al., 2012) S. eoa from NE Pacific (after Barnard, 1954) Maximum body length 42.6 mm 9 mm 38 mm in Russia 20.5 mm in Korea Length of peduncle of uropod 3 About 2.5-3.0 times longer than rami About 2 times longer than rami About 2.5-3.0 times longer than rami About 2 times longer than rami 38 mm in S. eoa according to Tzvetkova, 1967), well developed mandibular palp, slender antenna 2, undefined male gnathopod 2 palm with the straight margin, unenlarged pereopods 5-7. However, the new species can be distinguished from S. eoa by the following points: (1) antenna 1 flagellum is much shorter than that of S. eoa; (2) maxilla 1 inner plate bears 2 slender setae in the new species, while that of S. eoa bears 3 setae; (3) gnathopod 2 dactylus of large male just reaches to posteroproximal end of propodus, whereas that of S. eoa reaches beyond carpus; (4) pereopods 3 and 4 bases bear a group of long setae on the sub-proximal area of anterior margin, while those of S. eoa lack long setae on its anterior margin. Moreover, S. gigantea sp. nov. and S. eoa also differ genetically in COI (17.2-18.0%) greater than the threshold distances (3.5-4%) proposed for amphipod species discrimination (Witt et al., 2006;Rock et al., 2007;Hou et al., 2009). Therefore, we concluded that S. gigantea sp. nov. undoubtedly represents a novel species. The morphological comparisons among the new species and closely related species were summarized in Table 4. Immature males of this new species are also close to Barnard's (1954) description of S. eoa from Oregon (note: Conlan & Bousfield (1982) suggested that Barnard's (1954) material might be another species rather than S. eoa). However, immature males of the new species differ from Barnard's material in the following characters: (1) gnathopod 2 palm is well defined with a small spine; (2) gnathopod 2 propodus lacks a large process on posterodistal corner; (3) pereopod 3 basis has a group of long slender setae on anterior margin; (4) distoventral spur on uropod 2 peduncle is longer; and (5) uropod 3 peduncle is longer.