A new species of Jesogammarus from the Iki Island, Japan (Crustacea, Amphipoda, Anisogammaridae)

Abstract A new species of anisogammarid amphipod, Jesogammarus (Jesogammarus) ikiensis sp. n., is described from freshwaters in the Iki Island, Nagasaki Prefecture, Japan, based on results of morphological and molecular analyses. The new species is distinguished from all members of the genus by the combination of small number of setae on dorsal margins of pleonites 1–3, short and small number of setae on posterior margins of peduncular articles of antennae, mandibular article 1 without setae, well developed posterior lobes of accessory lobes of coxal gills on gnathopod 2 and pereopods 3–5, and pectinate setae on palmar margin of female gnathopod 2. A key to all the species of Jesogammarus is provided.


Introduction
The amphipod genus Jesogammarus Bousfield, 1979 has been recorded from fresh and brackish waters of the Japanese archipelago, the Korea peninsula, and the Chinese continent (Bousfield 1979;Morino 1984Morino , 1985Morino , 1986Morino , 1993Seo 1990, 1992;, Hou and Li 2004. To date, 17 species in two subgenera, Jesogammarus Bousfield, 1979 andAnnanogammarus Bousfield, 1979, have been recognized. In 2010, Mr. Y. Tohyama of Hiroshima University provided a few specimens of freshwater amphipod collected from the Iki Island, Nagasaki Prefecture, Japan. They proved to belong to a previously unknown species of Jesogammarus. The Iki Island is located between Kyushu and the Tsushima Island, and 14 km from east to west and 17 km from north to south (Fig. 1). During field surveys of freshwater amphipods in the Iki Island, made in 2010-2015, a significant number of specimens of this species have been accumulated. Close examination of the external morphology and molecular analyses based on mitochondrial DNA sequences revealed that the Iki species is distinct from its congeners, and it is described as a new species.

Morphological observation
All appendages of the examined specimens of Jesogammarus ikiensis sp. n. were dissected in 99% ethanol and mounted in gum-chloral medium on glass slides under a stereomicroscope (Olympus SZX7). Specimens were 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 specimens are deposited in the Tsukuba Collection Center of the National Museum of Nature and Science, Tokyo (NSMT).

DNA extraction, PCR amplification, and DNA sequencing
Total genomic DNA was extracted from pereopod musculature of each sequenced amphipod (Table 1) (Macdonald et al. 2005) and 16Sbr [CCGGTTT-GAACTCAGATCATGT] (Palumbi et al. 1991). PCR reactions containing 0.5 µl template solution, 2 mM MgCl 2 , 2.5 mM dNTP, 10 pmol of each primer, and 5U/ µl Taq polymerase (TaKaRa Ex Taq®) in 1X buffer provided by the manufacturer were performed in 10-µl volumes in an PC-320 thermal cycler (ASTEC). Amplification conditions were as follows: an initial denaturation for 7 min at 94 °C; 35 cycles of denaturation for 45 s at 94 °C, annealing for 1 min at 42-50 °C depending on samples, and extension for 1 min at 72 °C; and final extension for 7 min at 72 °C. Amplification products were purified by the silica method (Boom et al. 1990). All sequencing reactions were performed according to the manufacturer's instructions using the BigDye Terminater v3.1 Cycle Sequencing Reaction Kit (Applied Biosystems, Foster City, CA). Cycle sequencing conditions were 25 cycles of 10 s at 96 °C, 5 s at 50 °C, and 4 min at 60 °C. Sequencing reaction products were purified by ethanol precipitation. Labeled fragments were analyzed using an ABI 3130x Genetic Analyzer (Applied Biosystem). Sequences were obtained from both strands of the gene segments for verification using the same primers. The nucleotide sequences have been submitted to the DNA Databank of Japan (DDBJ) nucleotide-sequence database (linked to the EMBL and GenBank databases) ( Table 1).

Molecular phylogenetic analyses
The nucleotide sequences were aligned using the multiple alignment algorithm in Clustal W (Thompson et al. 1994) with default setting (i.e., gap opening penalty = 15, gap extension penalty = 6.66, transition weight = 0.5). Phylogenetic relationships were reconstructed by the Neighbor-Joining method (NJ; Saitou and Nei 1987), the equally weighted maximum parsimony method (MP), and the maximum likelihood method (ML) with MEGA6 software (Tamura et al. 2013). There was no indel in COI sequences of the ingroup taxa. On the other hand, eight indels were found in 16S sequences of the ingroup taxa, which were treated as missing data in all analyses.
In the NJ analysis, the Kimura 2-parameter (K2P) model (Kimura 1980) of nucleotide substitution was used to estimate genetic distances. In the MP analysis, a tree was obtained using the Close-Neighbor-Interchange algorithm, in which the initial trees were obtained with the random addition of sequences (10 replicates). The ML analysis used the T92 + G + I model for COI and HKY + G for 16S and COI + 16S; this was selected as the best-fit model using the Bayesian information criterion (BIC) in MEGA6. To estimate statistical support for branching patterns, 1,000 bootstrap replications each (Felsenstein 1985) were performed for the NJ, MP, and ML analyses. As outgroup taxa, three anisogammarid species, Eogammarus kygi (Derzhavin, 1923), E. possjeticus (Tzvetkova, 1967), and Spasskogammarus spasskii (Bulycheva, 1952), were used (Table 1).  Table 1.

Remarks
Monophyly of the subgenera Jesogammarus and Annanogammarus were supported in COI, 16S, and COI + 16S trees (Figs 2, 3). Jesogammarus ikiensis sp. n. from Iki Island was included in the clade of the subgenus Jesogammarus. However, phylogenetic position of J. ikiensis was not clearly resolved in the phylogenetic trees based on the COI and 16S rRNA genes due to low bootstrap values. Jesogammarus ikiensis differs from the all Japanese congeners by large genetic distances (18.6-25.8% for COI and 12.7-18.7% for 16S) (Table 2), which were larger than intraspecific distances among many species of Jesogammarus. In addition, J. ikiensis was morphologically distinguished from its congeners. Thus, it can be concluded that J. ikiensis from Iki Island as a distinct new species and is described below.
Mouthparts. Upper lip (= labrum) ( Fig. 5D) with rounded distal margin, bearing fine setae. Lower lip (= labium) ( Fig. 5E) with broad outer lobes, inner lobes indistinct. Mandibles ( Fig. 5F-H) with left and right incisors six-and four-dentate, respectively, left lacinia mobilis five-dentate, right one bifid, bearing many teeth; molar process triturative, with plumose seta; accessory setal rows of left and right mandibles each with seven blade-like setae; left palp three-articulate with length ratio of 1.0 : 3.8 : 3.8, palp article 1 bare, article 2 with 28 setae, article 3 with two clusters and one pair of A-setae, one pair of B-setae, and many C-, D-, and E-setae, article 3 of right palp with three clusters of A-seta and one B-seta. Maxilla 1 (Fig. 5I) with inner and outer plates and palp; medial margin and apical submargin of inner plate with 31 plumose setae; outer plate subrectangular, with 11 serrate teeth apically (Fig. 5J); right palp twoarticulate, much longer than outer plate, article 1 lacking marginal setae, article 2 with seven robust and six slender setae on its apical margin, outer margin with three setae, left palp lacking setae on outer margin of article 2. Maxilla 2 ( Fig. 5K) with oblique inner row of 23 plumose setae on inner plate; outer plate slightly longer than inner plate. Maxilliped (Fig. 6A) with inner and outer plates and palp; inner plate ( Fig. 6C) with six robust setae along apical and inner margins; outer plate ( Fig. 6B) with plumose setae on apical margin and robust setae on inner margin; palp four-articulate, article 2 with inner marginal and submarginal rows of setae, article 3 with facial setae, article 4 slightly curved inward, with slender nail.
Gnathopod 2 (= pereopod 2) (Fig. 6F): coxa with seven marginal and one submarginal setae on ventral part, posteroproximal part with two setae; anterior and posterior margins of basis with long setae; carpus length 1.7 × width, anterior margin with cluster of setae and single seta; propodus almost as long as carpus and 1.5 × width of propodus, anterior margin with two clusters of setae, palmar margin (Fig. 6G) oblique, weakly convex, with 12 peg-spines (= robust setae) and one serrate seta; dactylus (Fig.  6G) as long as palmar margin, with posterior accessory blade longer than nail.
Pereopod 3 (Fig. 7A, B): coxa with seven marginal setae on ventral part, posterioproximal part with two setae; anterior and posterior margins of basis with long setae, anterio-distal corner of basis with robust seta.
Pereopod 4 (Fig. 7C, D): coxa expanded with posterior concavity, bearing one seta on anterodistal corner and five setae on posterodistal margin; anterior and posterior margins of basis with long setae, anterodistal corner with robust seta.
Pereopod 5 (Fig. 7F, G): coxa bilobed, anterior lobe with apical seta, ventral margin of posterior lobe with three setae; posterior margin of basis weakly expanded, with ten setae; anterior and posterior margins of merus to propodus with robust and slender setae.
Pereopod 6 (Fig. 8A, B): coxa bilobed, anterior lobe with apical seta and anterioproximal setae, ventral margin of posterior lobe with three setae; posterior margin of basis weakly expanded, with 18 setae; anterior and posterior margins of merus to propodus with robust and slender setae.
Pereopod 7 (Fig. 7D, E): ventral margin of coxa weakly concave, bearing slender setae on anterior part and three setae on posteroventral part; posterior margin of basis weakly expanded, with 20 setae; anterior and posterior margins of merus to propodus with robust and slender setae.
Uropods. Uropod 1 (Fig. 8J): peduncle with robust seta on basofacial part, inner and outer margins each with three robust setae, inner proximal part with three short setae; inner ramus length 0.8 × peduncle, inner margin with three robust setae and outer margin with robust seta and minute seta; outer ramus length 0.9 × inner ramus, inner and outer margins each with two and three robust setae. Uropod 2 (Fig. 8K): peduncle with three robust setae on inner and outer margins, respectively; inner ramus length 0.9 × peduncle, its inner and outer margins with two robust setae, respectively; outer ramus length 0.8 × inner ramus, its outer margin with robust seta. Uropod 3 (Fig. 9A): peduncle length 0.3 × outer ramus; inner ramus length 0.25 × outer ramus (both proximal and terminal articles), with two robust setae on inner margin; outer ramus two-articulate, inner margin of proximal article with five plumose setae, and several robust setae and simple setae, outer margin with robust setae and simple setae, terminal article length 0.2 × proximal article, with short setae apically.
Telson (Fig. 9B) length 1.1 × width, cleft for 59% of length in V-shape; each lobe with one lateral and one apical robust seta.   Gnathopod 1 (Fig. 10A): carpus length 1.7 × width, with cluster of setae and single seta on anterior margin; propodus almost as long as carpus and 1.5 × width of propodus, bearing two clusters and one pair of setae on anterior margin; palmar margin ( Fig.  10B) with seven robust setae and two pectinate setae.
Gnathopod 2 (Fig. 10C): carpus length 2.2 × width, with one cluster, one pair, and one single seta on anterior margin; propodus length 0.9 and 2.0 × carpus and width of propodus, respectively, bearing one cluster and one pair of setae on anterior margin; palmar margin ( Fig. 10D) with two robust and 10 pectinate setae.
Posterior margin of bases of pereopods 5-7 more expanded than in male ( Fig.  10F-H).
Variations. The number of setae and/or setal bundles on posterior margin of peduncular articles of antennae is variable: antenna 1, two or three on article 1, three or four on article 2, one or two on article 3; antenna 2, two to four on article 4, three to Figure 8. Jesogammarus (Jesogammarus) ikiensis sp. n., holotype, male, 13.1 mm, NSMT-Cr 24107, Ishida, Iki, Nagasaki Prefecture, Japan. A coxa-merus of pereopod 6, lateral view B carpus-dactylus of pereopod 6, lateral view C coxal gill of pereopod 6, lateral view D coxa-merus of pereopod 6, lateral view E carpus-dactylus of pereopod 6, lateral view F coxal gill of pereopod 7, lateral view G pleopod 1, medial view, distal parts of rami omitted H retinacula on peduncle of pleopod 1, medial view I bifid plumose seta (clothes-pin seta) on inner basal margin of inner ramus of pleopod 1, medial view J uropod 1, dorsal view K uropod 2, dorsal view. five on article 5. Most specimens have a pair of setae on dorsal margins of pleonites 1-3 but several specimens have three setae. The length ratio of inner ramus of uropod 3 to outer ramus ranged from 0.2 to 0.3 in both sexes. The number of plumose setae on inner margin of outer ramus of uropod 3 varied from two to eight in males and one to three in females. Ovigerous females have 58 to 175 eggs.