Two new species of Jesogammarus from Japan ( Crustacea , Amphipoda , Anisogammaridae ) , with comments on the validity of the subgenera Jesogammarus and Annanogammarus

Two new species of anisogammaris amphipod, Jesogammarus (Jesogammarus) bousfieldi and J. (J.) uchiyamaryui, are described from mountain streams in Yamagata Prefecture and from brackish waters on Iki and Fukue Islands, Nagasaki Prefecture, Japan. Jesogammarus bousfieldi is morphologically almost similar to J. paucisetulosus Morino, 1984. However, J. bousfieldi is distinguished from J. paucisetulosus by the more number of marginal setae on the pleonites. Jesogammarus uchiyamaryui resembles J. ikiensis Tomikawa, 2015, but the former differs from the latter by two setae on the posterior margin of peduncular article 2 of antenna 1, short and straight accessory lobes of coxal gills on gnathopod 2 and pereopods 3–5, densely setose ventral margins of coxae of female gnathopods 1 and 2 and pereopod 3, and a shorter inner ramus of uropod 3. Phylogenetic analyses using nuclear 28S rRNA, mitochondrial cytochrome c oxidase subunit I, and the 16S rRNA markers showed the sister relationship between J. bousfieldi and J. paucisetulosus. However, the phylogenetic position of J. uchiyamaryui remains uncertain. Both new species were genetically highly diverged comparable to intraspecific divergence among other Jesogammarus species. The species diversity related to habitat and the subgeneric classification of Jesogammarus are briefly discussed.


Introduction
The amphipod genus Jesogammarus Bousfield, 1979 inhabits fresh and brackish waters of the Japanese archipelago, the Korean Peninsula, and the Chinese continent (Bousfield 1979, Morino 1984, 1985, 1986, 1993, Lee and Seo 1990, 1992, Tomikawa and Morino 2003, Tomikawa et al. 2003, Hou and Li 2004, 2005, Tomikawa 2015).Jesogammarus was erected by Bousfield (1979) as a monotypic genus with the type species Anisogammarus jesoensis Schellenberg, 1937.In the same article, Bousfield established monotypic Annanogammarus Bousfield, 1979, of which the type species was designated Gammarus annandalei Tattersall, 1922. Morino (1984) described a second species in Jesogammarus, J. paucisetulosus Morino, 1984 from Japan.Subsequently, Morino (1985) relegated Annanogammarus as a subgenus under Jesogammarus because the character combination of J. paucisetulosus and additional new species bridged the morphological gap between the two genera Jesogammarus and Annanogammarus.Morino concluded that Annanogammarus could be a distinct subgenus due to unequal coxal gill accessory lobes on gnathopod 2-pereopod 5 in length and simple robust setae on the palmar margin of female gnathopod 2. Molecular phylogenetic analyses of Jesogammarus recovered the monophyly of the subgenera Jesogammarus and Annanogammarus, respectively (Tomikawa et al. 2007, Tomikawa 2015).
The latest taxonomic study of the genus shows that Jesogammarus contains 18 species (Tomikawa 2015).The subgenus Annanogammarus consists of six freshwater species, and the subgenus Jesogammarus comprises 11 freshwater and one brackish-water species.
Two additional Jesogammarus species were collected during field surveys of fresh and brackish waters in Japan: one from mountain streams in Yamagata Prefecture and the other from brackish waters of Iki and Fukue Islands, All appendages of the examined specimens of the undescribed species were dissected in 70% 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) and the Zoological Collection of Kyoto University (KUZ).

PCR and DNA sequencing
The extraction of genomic DNA from appendage muscles of the Jesogammarus materials preserved in 99% ethanol followed Tomikawa et al. (2014).Primer sets Names of localities are shown in Table 1.
Table 1.Samples used for the phylogenetic analyses.The information on the vouchers or isolate numbers is accompanied by the collection localities and the INSDC accession numbers.Sequences marked with an asterisk were obtained for the first time in the present study.for the PCR and cycle sequencing reactions used in this study were as follows: for 28S rRNA (28S), 28SF and 28SR (Tomikawa et al. 2012); for cytochrome c oxidase subunit I (COI), Am-COI-H and Am-COI-T (Tomikawa 2015); for 16S rRNA (16S), 16STf (Macdonald III et al. 2005) and 16Sbr (Palumbi 1996; modified to correspond with "Fruit Fly").The PCR reactions and DNA sequencing were performed using the identical method mentioned in Tomikawa (2015).In total, 59 sequences from the unidentified Jesogammarus specimens and J. paucisetulosus were newly obtained in this study, and deposited with the International Nucleotide Sequence Database Collaboration (INSDC) through the DNA Data Bank of Japan (Table 1).

Molecular phylogenetic analyses
Thirty-two published sequences were obtained from the IN-SDC for use in molecular phylogenetic analyses (Table 1).
The phylogenetic positions of the unidentified species among Jesogammarus were estimated based on the gene fragments of 28S, COI, and 16S sequences.The align-ment of COI was trivial, as no indels were observed.The 28S and 16S sequences were aligned using MAFFT v. 7.305b L-INS-i (Katoh and Standley 2013).The lengths of 28S, COI and 16S sequences were 717, 362, and 420 bp, respectively.The concatenated sequences thus yielded 1,499 bp of alignment positions.One of each four completely identical sequence pairs (KUZ Z1803 and KUZ Z1804; KUZ Z1799 and KUZ Z1800; G1037 and G1038; and KUZ Z1805 and KUZ Z1806) was removed from the dataset using the "pgelimdupseq" command implemented in Phylogears v. 2.0.20142.0. .03.08 (Tanabe 2008)).
Phylogenetic relationships were estimated using maximum likelihood (ML) and Bayesian inference (BI).ML phylogenies were conducted using RAxML v. 8.2.8 (Stamatakis 2014) with GTRCAT, immediately after nonparametric bootstrapping (Felsenstein 1985) (BS) conducted with 1,000 replicates.The best-fit partitioning scheme for the ML analysis was identified with the Akaike information criterion (Akaike 1974) using PartitionFinder v. 1.1.1 (Lanfear et al. 2012) with the "all" algorithm: each of 28S, 1st to 3rd positions of COI and 16S were, respectively, treated as a separate partition.
Pereopod 3 (Fig. 4I): coxa with 7 marginal setae on ventral part, posteroproximal part with 2 setae; anterior and posterior margins of basis with long setae, anterodistal corner of basis with robust seta.
Pereopod 4 (Fig. 4J): coxa expanded with posterior concavity, bearing 2 setae on anterodistal corner and 6 setae on ventral margin; anterior and posterior margins of basis with long setae, anterodistal corner with robust seta.
Pereopod 6 (Fig. 5B): coxa bilobed, anterior lobe with anteroproximal setae, ventral margin of posterior lobe with 3 setae, posterodistal corner rounded with seta; posterior margin of basis weakly expanded with 12 setae, posterodistal corner with robust seta associated with small seta; anterior and posterior margins of merus to propodus with robust and slender setae.
Pereopod 7 (Fig. 5C): ventral margin of coxa weakly concave, bearing 2 setae on anterior part and 4 setae on posteroventral part; posterior margin of basis weakly expanded with 9 setae, posterodistal corner with robust seta associated with 2 small setae; anterior and posterior margins of merus to propodus with robust and slender setae.
Posterior margin of bases of pereopods 5-7 more expanded than in male (Fig. 7A-C).
Etymology.Jesogammarus bousfieldi was named in remembrance of the late Dr Edward Lloyd Bousfield, who enthusiastically guided and encouraged many Japanese amphipodologists, and sadly passed away on 7 September 2016.
Distribution and habitat.This species is known only from Yamagata Prefecture.The specimens were collected from small mountain streams.Ovigerous females were collected from November to April.
Pereopod 3 (Fig. 11I): coxa with 4 and 2 marginal setae on anterodistal and posterodistal parts, respectively; anterior and posterior margins of basis with long setae, anterodistal corner of basis without robust seta.
Pereopod 4 (Fig. 11J): coxa expanded with posterior concavity, bearing 2 setae on anterodistal corner and 4 setae on ventral margin; anterior and posterior margins of basis with long setae, anterodistal corner with robust seta.
Pereopod 5 (Fig. 12A): coxa bilobed, anterior lobe with 2 apical setae, ventral margin of posterior lobe with 2 setae, posterodistal corner not pointed with seta; posterior margin of basis weakly expanded, with 9 setae; anterior and posterior margins of merus to propodus with robust and slender setae.
Pereopod 6 (Fig. 12B): coxa bilobed, anterior lobe with apical seta, anterior margin with long setae, ventral margin of posterior lobe with 2 setae, posterodistal corner weakly pointed with seta; posterior margin of basis weakly expanded with 9 setae, posterodistal corner with robust seta; anterior and posterior margins of merus to propodus with robust and slender setae.
Pereopod 7 (Fig. 12C): ventral margin of coxa weakly concave, bearing 3 setae on anterior margin and 4 setae on posteroventral margin; posterior margin of basis    weakly expanded with 7 setae, posterodistal corner with 2 robust setae; anterior and posterior margins of merus to propodus with robust and slender setae.
Telson (Fig. 12P) almost as long as wide, cleft for 63% of length; each lobe with 2 or 3 robust setae and slender setae.
Etymology.The specific name honors Mr Ryu Uchiyama (nature photographer), who provided many photos of living amphipods throughout KT's amphipodological study.
Distribution and habitat.This species is known from Iki and Fukue Islands, Nagasaki Prefecture.The specimens were collected from river mouths subject to tidal action.An ovigerous female was collected in March.
The freshwater J. bousfieldi described from Yamagata Prefecture has been treated as a population of J. paucisetulosus, which was thought to be widely distributed in Ibaraki, Yamagata and Niigata Prefectures (Tomikawa 2007, Tomikawa andMorino 2012).The latter species was originally described from a spring brooklet in Mito, Ibaraki Prefecture, Japan (Morino 1984).The J. bousfieldi specimens were clearly distinguished from those of J. paucisetulosus collected from Ibaraki Prefecture by the number of marginal setae on dorsal margins of pleonites 1-3.Additionally, our molecular phylogenetic analy- ses showed that J. bousfieldi was highly diverged from the true J. paucistulosus although they possessed sister-species relationships.Their genetic divergence could be comparable to the detected intraspecific divergences among other Jesogammarus species.Thus, we concluded that J. bousfieldi could be regarded as a distinct new species.Unfortunately, we could not examine specimens of J. paucisetulosus from Niigata Prefecture.A further taxonomic study is necessary to determine the taxonomic position of the population living there.
Contrary to the highly diverged freshwater Jesogammarus species, the number of known species indigenous to brackish habitats is limited: only J. hinumensis has been recorded from brackish lakes and estuaries in Japan (Morino 1993).Therefore, Jesogammarus uchiyamaryui is the second brackish-water species in this genus.This species is morphologically similar to the freshwater J. ikiensis.Although both species are distributed on Iki Island (Tomikawa 2015), they were not collected from the same localities or at the same time on the island (Tomikawa, personal observation).The habitat preferences of these two species seem to be completely different.Moreover, the phylogeny showed that J. uchiyamaryui was not a sister species of J. ikiensis, indicating that they did not share the last common ancestor.Because species diversity of the brackish Jesogammarus remains far from investigated, it is highly possible that additional undescribed species may be found in brackish-water environments within the distributional range of the genus Jesogammarus.
The morphological characteristics and the phylogenetic positions of the two new species raises a question about the validity of the two subgenera, Jesogammarus and Annanogammarus, under the genus Jesogammarus.These two subgenera are characterized only by two morphological characters: 1) accessory lobes of coxal gills of gnathopod 2-pereopod 5 subequal in length or posterior accessory lobe longer than anterior accessory lobe in the subgenus Jesogammarus vs. unequal in length, posterior accessory lobe often rudimentary in the subgenus Annanogammarus; and 2) palmar margin of propodus of female gnathopod 2 with pectinate robust setae in the subgenus Jesogammarus vs. without pectinate robust setae in the subgenus Annanogammarus.Jesogammarus bousfieldi and J. uchiyamaryui bear these subgeneric diagnostic characteristics that are identified with the subgenus Jesogammarus.Contrary to the monophyly of Annanogammarus implicated by previous phylogenetic studies (Tomikawa et al. 2007, To-mikawa 2015), the phylogenies obtained here failed to recover the monophyly of the subgenus Jesogammarus.Although the precise phylogenetic position of J. uchiyamaryui remains uncertain, the findings highlight that the subgeneric classification under the genus Jesogammarus may be abrogated along with synonymizing Annanogammarus with Jesogammarus.Nevertheless, a future systematic study is essential to reveal whether Annanogammarus should be treated as a subjective junior synonym of Jesogammarus.

Figure 1 .
Figure 1.Map showing the collection localities of the specimens examined in this study.The closed squares indicate the localities of J. bousfieldi sp.n.The closed diamonds denote the localities of J. uchiyamaryui sp.n.The closed circles indicate the localities of the referred materials used for the phylogenetic analyses.The symbols in red denote the type locality of each of the new species.Names of localities are shown in Table1.

Figure 15 .
Figure 15.Bayesian inference tree for 1,499 bp of nuclear 28S rRNA and mitochondrial COI and 16S rRNA markers.Numbers on nodes represent bootstrap values for maximum likelihood and Bayesian posterior probabilities.