A new species of Microcharon from marine interstitial waters, Shizuoka, Japan (Isopoda, Lepidocharontidae)

Abstract A new species of Microcharon Karaman, 1934 (Asellota: Lepidocharontidae) is described from Miho-Uchihama beach, Shizuoka, Japan. Microcharon tanakai sp. n. differs from its congeners by having nine simple, five penicillate setae on antennal article 6; one simple distal seta on article 1 of the mandibular palp and having the apical lobe of male pleopod 1 convex, rounded, armed with seven setae. A key to Asian species of the genus and 16S rRNA of the new species are provided.


Introduction
Members of the genus Microcharon Karaman, 1934, are free-living interstitial isopods. Being highly adapted to the narrow spaces of the interstitial environment, the species possesses an elongated body without visual organs or pigmentation (Coineau 1994(Coineau , 2000Wilson and Wägele 1994). When Karaman (1934) described Microcharon, it was included in the family Microparasellidae Karaman, 1934. However, following a recent revision (Galassi et al. 2016), the genus is currently a member of the family Lepidocharontidae. Besides Microcharon, Lepidocharontidae also includes Lepidocharon Galassi & Bruce, 2016 and Janinella Albuquerque, Boulanouar & Coineau, 2014. The most prominent diagnostic character of Microcharon is the shapes of pereonites 1-7. Pereonites are cylindrical and each is rectangularly shaped in dorsal view whereas, Lepidocharon and Janinella have trapezoidal pereonites (Galassi et al. 2016).
The genus Microcharon is one of the best studied groups of the family Lepidocharontidae and 69 species have been described from all over the world (Boyko et al. 2008). The majority of species are known from Europe, especially from the Mediterranean region (Coineau 1994;Boyko et al. 2008;Galassi et al. 2016). On the other hand, only the following three Asian species have been recorded so far: M. halophilus Birstein & Ljovuschkin, 1965;M. kirghisicus Jankowskaya, 1964; and M. raffaellae Pesce, 1979. They are known from Turkmenistan, Kyrgyzstan, and Iran respectively (Coineau 1994;Boyko et al. 2008;Galassi et al. 2016). Diversity of Microcharon in East Asia remains particularly unknown. Only Shimomura et al. (2006) briefly noted a single species of Microcharon collected together with an ingolfiellidean amphipod from marine interstitial of Okinawa archipelago.
During a survey of the interstitial fauna of Miho-Uchihama beach (Shizuoka, Japan), a small number of psammolittoral isopods were collected together with other marine interstitial fauna such as, harpacticoid copepods, nematodes and ostracods. The isopod specimens had a typical Microcharon body plan, but a unique combination of morphological characters which lends support to the establishment of the new species described herein. Beside its description an identification key to the four Asian species of this genus currently recorded. In addition, a partial sequence of 16S rRNA gene was obtained and this may be useful for the future phylogenetic study studies of Microcharon and the family Lepidocharontidae.

Specimen collection and identification
Seven female and two male specimens were collected from coarse sand from Miho-Uchihama beach, Shizuoka City, Shizuoka Prefecture, Japan on 7 February 2015 ( Fig. 1). Coarse sand samples were washed five times in a bucket with fresh water. The top layer of water was strained through nets of 40 µm mesh size and material was immediately preserved in 99% ethanol. Sorting from sediment sample and dissection of specimens were done under an Olympus SZX 12 dissecting microscope. Dissected appendages were mounted onto glass slides in lactophenol. Line drawings were prepared using a Zeiss Axioskop 50 compound microscope equipped with a camera lucida. All studied material was deposited at the invertebrate collection of the National Institute of Biological Resources (NIBR), Korea. Two female and one male were transferred to isoamyl acetate for 20 minutes and dried in a critical-point dryer Hitachi E-1010. Dried specimens were mounted onto a SEM stub and coated with gold using a sputter coater to a thickness of 15-30 nm. Coated specimens were examined and photographed with a Hitachi S-3400 scanning electron microscope at Chungang University (Seoul). Measurements were done following the method of Riehl and Brandt (2010). All measurements were taken from the dorsal view of line drawings using the distance measurement tools of Adobe Acrobat Professional. The ratios of appendages were given in distal to proximal order, excluding setae. The body ratios were given in anteromedial to posteromedial point order excluding appendages. Whole body length was measured from biggest female specimen. Terminology is largely based on Galassi et al. (2016).

DNA extraction and amplification
Two females from the type locality sample were identified without dissection under an Olympus SZX 12 dissecting microscope. Before amplification, specimens were transferred into distilled water for 20 minutes to remove ethanol and then macerated with a small glass rod. Whole specimens were used to isolate genomic DNA with the aid of the LaboPassTM Kit (COSMO Co. Ltd., Korea) following the manufacturer's protocols. 16S rRNA was amplified with polymerase chain reaction (PCR) using PCR premix (BIONEER.Co) in TaKaRa PCR thermal cycler (TaKaRa Bio Inc., Otsu, Shiga, Japan). The primers used were 16sar-L (5`-CWAAYCATAAAGAYATTGGNAC-3`) and 16sar-H (5`-ACTTCAGERTGNCCAAARAAYCA-3`) (Palumbi et al. 1991).
The amplification protocol consisted of the initial denaturation at 94°C for 2 min and 35 cycles each consisting of denaturation at 94°C for 50 sec, annealing at 50°C for 50 sec, extension at 72°C for 1 min 20 sec; the final extension was at 72°C for 7 min. Successful amplifications were confirmed by electrophoresis on 1% agarose gel. The PCR products were purified for sequencing reactions, using the Labopass PCR Purification Kit (COSMO Co. Ltd., Korea) following the guidelines provided with the kit. DNA was sequenced on an ABI automatic capillary sequencer (Macrogen, Seoul, Korea) using the same set of primers. All obtained sequences were visualized using Finch TV version1.4.0 (http://www.geospiza.com/Products/finchtv.shtml). Each sequence was checked for the quality of signal and sites with possible low resolution, and corrected by comparing forward and reverse strands.
Material examined. Holotype: adult female, (NIBRIV0000787789) completely dissected and mounted in lactophenol on four slides; paratype 1 female (NI-BRIV0000787790) dissected on three slides, paratype 2 female (NIBRIV0000787791) dissected on one slide; adult male pleotelson dissected on three slides; 2 females and 1 male used for SEM.
Diagnosis. Antennular article 6 smallest, with one aesthetasc, one penicillate, three simple setae distally; article 1 of mandibular palp with one distal simple seta; lateral lobe of maxillula with eleven robust setae; distal apex of male pleopod 1 convex, round, with three apical, four subapical setae; protopod of male pleopod 2.9 times longer than wide; protopod of uropod 3.1 times longer than wide, with fifteen setae.
Description of the female holotype. Body ( Fig. 2A): elongate, slender in whole appearance, total length, 1.95 mm, measured from anteromedial point of cephalon to posteromedial point of pleotelson, body approximately 8.5 times longer than wide, maximum body width in pereonite 3, 0.92 times of maximum width of pleotelson; color of preserved specimens transparent, whole surface of body with many ornamentation looking like lines.
Cephalon (Fig. 2B): 1.12 times longer than wide and 0.12 times of whole body; anterior margin with weak rostrum; lateral margins straight, with four pairs of simple setae, three pairs of simple setae along dorsomedial surface.
Pereon ( Fig. 2A, C): 0.68 times of whole body length, medial margin of tergite convex, pereonites with lateral margin, straight, pereonite 1, 0.62 times longer than wide, four simple setae along lateral margin, two setae on dorsal surface; pereonite 2, 1.12 times longer than wide, with four pairs of setae along lateral margin, two setae on anteromedial edge; pereonite 3, 1.06 times longer than wide, with four pairs of setae along lateral margin, two pairs of setae along dorsal surface; pereonite 4, 1.02 times longer than wide, with nine setae on both lateral and dorsal surface; pereonite 5, 0.99 times longer than wide, with six setae on both lateral and dorsal margin; pereonite 6, 1.13 times longer than wide, with eight simple setae on dorsal margin; pereonite 7, 1.02 times longer than wide, with two setae on both anterolateral corner and three pairs of setae along dorsal surface.
Pleotelson (Fig. 2D): 1.33 times longer than wide, wider than preceding pereonites, with several setae, becoming slightly narrow from basal part to distal end, weak cleft on middle of posterior rim.
Antenna (Fig. 3A, B): six podomeres, twelve flagellar articles; article 1 globular in dorsal view, with one lateral simple seta; article 2 semicircular, naked; article 3, 1.8 times longer than wide, with one distomedial simple seta, scale thick, robust, with two simple setae laterally (Fig. 8A); article 4, as long as wide, with two simple setae distolaterally; article 5, 3.02 times longer than wide, with three distoventral simple setae, one simple, one penicillate setae distodorsally; article 6, longest, 5.3 times longer than wide, with two simple, five penicillate setae on lateral margin, seven simple setae on distal margin; flagellar articles from 2 to 8 subequal in length, armature of each articles as follows: article 1 with two simple setae distally, article 2 with four simple setae distally, article 3 with three simple setae distally, article 4 with three simple setae distally, article 5 with four simple setae distally, article 6 with six simple setae distally, article 7 with three simple setae distally, article 8 with four simple setae distally, article 9 with four simple setae distally, article 10 with four simple setae distally, article 11 with three simple setae distally, article 12 with four simple setae distally Mandible (Figs 3E-G, 8B): body robust, curved inwardly; pars incisiva of both mandibles with four cusps; right mandible ( Fig. 3F), lacinia mobilis, with five cusps, tapering proximally three pinnate, three simple setae located below lacinia mobilis; pars molaris of both mandibles with one simple, one pinnate distal setae, slightly longer than pars molaris, almost same length as seta on palp; left mandible ( Fig. 3E), pars incisiva with four cusps, lacinia mobilis missing, with three pinnate and one naked robust setae located below pars incisiva; palp ( Fig. 3G) with three articles, inserted on cuticular projection; article 1 with one simple seta distally (Fig. 8B), article 2 longest, with two pinnate and several hair-like setae on distal margin, article 3 curved outwardly, with five pinnate and with several hair-like setae on lateral margin.
Maxillula (Fig. 4D): lateral lobe with eleven strong robust setae on distal edge, some denticulate, some setulose, two spinular rows along outer margin; mesial lobe nearly half as long and wide as outer endite, with three short simple and several hairlike setae on distal end. Maxilla (Fig. 4C): lateral rami slightly shorter than median one, with four pectinate setae on distal end respectively; mesial ramus coalescent with basis, much thicker than others with eight strong setae distally and one of them pectinate.
Maxilliped (Fig. 3C): epipodite wide, apically pointed, distal tip reaching distal part of first palp article; basis 2.1 times longer than wide; palp with 5 articles; article 1, 0.5 times longer than wide, with two simple setae on both distal corners; article 2, 1.7 times longer than article 1, with two simple setae on distomedial corner; article 3 tapering distally, with three simple setae along medial margin, one seta on distolateral corner; article 4 curved inwardly, with four simple setae distally; article 5 with five simple setae, 2 claws distally; endite 2.4 times longer than wide, with five apical pinnate setae, numerous spinules on distomedial corner.
Female operculum (Fig. 6C): 1.2 times longer than wide, with no ornamentation on dorsal surface, with four setae on distal margin. Uropods (Fig. 7E): protopod robust, slightly longer than pleotelson, length 3.1 times longer than wide, with thirteen simple and two robust setae proximomedially; endopod 0.5 times longer than protopod, 1.7 times longer than exopod, with nine simple and four penicillate setae; exopod 0.6 times longer than endopod, with four simple setae distally.
Pleopod 1 (Figs 7A, 9I-L, 10M): elongate, total length almost reaching posterior margin of pleotelson, composed of two coalescent halves, proximal part enlarged, gradually tapering at its distal part, approximately 1.9 times longer than maximum wide (measured at widest section of proximal part); separated in half by medial sperm tube running from triangular opening on proximal part of medial groove, distolateral edge of hyaline lamella straight, without ornamentations; parallel to lateral margin of pleopod, in lateral view distal end of lobes convex, rounded at distal apex, each with three simple setae distally, four subapical setae on ventral margin.
Etymology. The species is named in honor of the collector, Dr. Hayato Tanaka, to express our appreciation for his support in this study.

DNA amplification
Partial 16S rRNA sequence was obtained only from a single female. The final length of trimmed sequence was 493 base pairs (GenBank accession number KY498031) and BLAST (Altschul et al. 1990) analysis of the GenBank data base revealed that the obtained sequence was isopod in origin and was not contaminated. Chelator rugosus (Gen-Bank accession number KJ578668.1, Desmosomatidae) was the most similar sequence to M. tanakai resulted by Megablast optimization with the 74% identity, 1e-36 E-value, 92% of query cover, 165 of total and max score. On the other hand, when Blastn optimization was chosen, Betamorpha fusiformis (GenBank accession number EF116503.1, Munnopsidae) was the most similar sequence to that of our new species with 73% identity, 4e-67 E-value, 100% of query cover, and 266 of total and max score.
Foundation of Korea (NRF) and National Institute of Biological Resource (NIBR) funded by the Ministry of Environment (MOE), Republic of Korea. We would like to express our great appreciation to Dr. Nicole Coineau for her kind help and invaluable comments for the improvement of this paper.