Description and scanning electron microscopic observation of a new species of the genus Polycopetta (Crustacea, Ostracoda, Cladocopina) from an interstitial habitat in Japan

Abstract A new species of the genus Polycopetta Chavtur, 1981, Polycopetta quadrispinata sp. n. is described from the interstitial environment of Mihomasaki Beach in Japan. These observations showed some morphological peculiarities of Polycopetta quadrispinata sp. n. compared with its congeners; Polycopetta monneroni Chavtur, 1979, Polycopetta curva Chavtur, 1979, Polycopetta bransfieldensis (Hartmann, 1987), and Polycopetta pax Kornicker and Harrison-Nelson, 2005. Three characteristics are described for the first time: (1) a seta with serrated tip on the male antennula, (2) the endopodite of the fifth limb consisting of two podomeres, (3) the long spermatozoa in the male posterior body. More detailed observations of the type species are needed in order to update the generic diagnosis.


introduction
The genus Polycopetta, belonging to the Suborder Cladocopina, was originally proposed by Chavtur (1979), but the name of this genus could not be used because the type species had not been designated. Subsequently, Chavtur (1981) assigned the type species and gave a diagnosis for the genus, after which Polycopetta Chavtur, 1981 was accepted as a valid name. The generic diagnosis of Polycopetta based on Chavtur (1981) is as follows: Carapace oval and concave anteriorly; frontal organ consists of one seta split at distal half; third podomere of male antennula with two ventral setae; male antennal endopodite bearing dorsal outgrowth and hook-like protrusion, and terminal podomere in both sexes with ventral protuberance; basis of fifth limb with one internal and three external setae, and exopodite with four terminal setae; outgrowth between the furcal lamellae rounded and armed with spines; male left furcal lamella with six claws.
Thus far four species have been described in this genus: the type species Polycopetta monneroni Chavtur, 1979, P. curva Chavtur, 1979 (Hartmann, 1987) and P. pax Kornicker & Harrison-Nelson, 2005. Three species are found in the sediment of the seafloor at depths of 60 to 265m, except for P. pax. This species has been reported from a Riftia pachyptila (giant tube worm) aggregation at a depth of 2500m.
During the faunal survey along the Pacific coast in Japan, a species of Polycopetta was found in interstitial habitats for the first time. In the present paper, the authors describe this new species, including observations of the detailed structure of the carapace and appendages, obtained by using a scanning electron microscope (SEM).

Materials and methods
Sand material was collected from the Mihomasaki beach, Shizuoka City, Shizuoka Prefecture, Japan ( Fig. 1) at 40 cm below the shoreline sand surface, at low tide. The samples were washed five times in a bucket with fresh water, and the top layer of water was strained through nets of 40 μm mesh size. The living specimens were picked out from the remaining deposits under a stereo-binocular microscope (SZH 10, OLYMPUS). The observed specimens were fixed in 8% formalin with neutral buffer (hexamethylenetetramine), and preserved in 80% ethanol at room temperature. The soft parts and valves were dissected with fine needles and mounted in Neo-Shigaral (Shiga Konchu Fukyusha, Tokyo, Japan), or glycerine, on glass slides under a stereobinocular microscope, and then observed and sketched using a transmitted-light binocular microscope (BX 50, OLYMPUS) with a differential interference contrast system and a camera Lucida. The valves and soft parts, treated with the t-butyl alcohol freeze-drying method, were also coated with osmium and observed by SEM (JSM-5600LV, JEOL).
The type series was deposited in the collection of the Shizuoka University Museum, identified by registration numbers with prefix SUM-CO. Type locality. The holotype specimen was collected from Mihomasaki beach, Shizuoka City, Shizuoka Prefecture, along the Pacific coast of central Japan, 35°01'13"N, 138°31'20"E ( Fig. 1B); in an interstitial environment at 40 cm below the shoreline sand surface. The substrate consisted mainly of clastic very coarse sand (median grain size is about 1.5 mm).
Diagnosis. Carapace oval and anteriorly concave in lateral view. Carapace peripheral surface covered with shallow pits, except on posterodorsal area, and with scale-like sculptures in anterior area. Anterior end of both valves with one conspicuous spine. Posteroventral margin of right valve with four conspicuous spines in both sexes. In each valve, 73 simple pores, 23 pore systems involving a circular depression and two    Frontal organ (Figs 7A, 8B). Spinous seta divided at mid-length. Distal half with long and proximal half with short setae, respectively (Fig. 8B).
Antennula (Figs 7B, 8C). Uniramous, four articulated podomeres. First podomere rectangular in shape and tapering distally, with setulae on dorsal margin, lateral surface and at ventrodistal end, respectively. Second podomere about four-fifths as long as first podomere, with one annulated setulous seta at dorsoproximal end, one simple seta on ventrodistal end (Figs 7B, 8C), and setulae on dorsal margin, lateral surface, ventral middle margin and at ventrodistal end, respectively. Third podomere about one-fifth as long as first podomere, with one short simple seta at dorsodistal end and one seta   with serrations at ventrodistal end (Figs 7B, 8C). Fourth podomere small, with four long setulous annulated setae.
Antenna (Figs 7D, D', 8D). Typically biramous, with exopodite and endopodite consisting of nine and three podomeres, respectively. Basis triangular and tapering distally. Exopodite: first podomere about one-third as long as basis; podomere lengths decreasing in size from second to eighth, each podomere with one long plumose annulated seta, respectively; ninth (distal-most) podomere very small, with one long annulated, one medium annulated and one short bare setae at distal end. Endopodite (Figs 7D, D', 8D): first podomere about two-thirds as long as first podomere of exopodite; second podomere half as long as first podomere, with one setulous seta along dorsal margin, one clavate process at proximal middle end (Fig. 7D') and five setae at distal end consisting of three long annulated, one medium annulated and one short annulated setulous. Third podomere one-fifth as long as first podomere, with one dorsal outgrowth (Figs 7D', 8D), and two long spinous annulated, one long annulated and one short setulous annulated setae at distal end.
Upper lip (Fig. 10A). Semicircular in lateral view, with fine setae on surface (Fig. 13A). Mandibula (Fig. 10B). Coxal endite with four teeth. Basis with four plumose annulated setae on ventral margin, and one plumose annulated seta at mid-lateral surface. Exopodite pear-shaped, distal end jagged, with thin setae, and one simple seta. Endopodite consisting of two podomeres. First podomere with three annulated plumose setae on ventral margin and two annulated long setulous setae at dorsodistal end. Second podomere very small, bearing two plumose setae at distal end. Maxillula (Fig. 10C, C', C"). Precoxa (Fig. 10C') with seven annulated plumose setae and one stout setulous seta on ventral side. Coxa (Fig. 10C") with two short and two medium plumose setae on lateral surface near ventroproximal margin, two short and two medium plumose setae on lateral surface of ventral middle margin. Basis rectangular, dorsally-convex in lateral view, with one medium and one long  plumose setae on ventral margin, and setulae along ventral margin. First podomere of endopodite with one long plumose seta at ventrodistal end. Second podomere three-fourths as long as first podomere, with two long and one medium annulated setulous setae on ventrodistal area, one short annulated and one medium setulous annulated seta at dorsodistal end. Third podomere small, with 4 long annulated setulous setae. Exopodite with four tufts along dorsal margin, and nine annulated setae at distal end.
Description of adult female. Mandibula, maxillula, fifth limbs, and upper lip similar to those of adult male. Carapace ( Fig. 2F-J). Carapace length and height larger than adult males. Antennula (Fig. 7C). Uniramus, four articulated podomeres. First podomere similar to that of adult male. Second podomere about four-fifths as long as first podomere, with one annulated setulous seta at dorsoproximal end, and setulae on dorsal margin, lateral surface, ventral middle margin and at ventrodistal end, respectively. Third podomere about one-fifth as long as first podomere, with one short simple seta at dorsodistal end. Fourth podomere small, with five long setulous annulated setae. Antenna (Fig. 7E). Only second and third podomeres of endopodite different from those of adult male. Endopodite consisting of three podomeres. Second podomere half as long as first podomere, with one setulous seta along dorsal margin and five annulated setae at distal end. Third podomere one-fifth as long as first podomere with four annulated setae at distal end. Furca (Fig. 12B). Each lamella with seven claws.
Dimensions. See Table 1.   Occurrence. So far known only from type locality. Etymology. Specific name quadrispinata, an adjective derived from the Latin prefix quadri-(four) and Latin adjective spinatus (spiny), referring to the four spines on the posteroventral margin of the right valve in both sexes.

Discussion
Existing species of Polycopetta are known from only a few specimens in seafloor sediment and deep sea tube worm aggregations. This study is the first report of a species of Polycopetta from the interstitial environment. Because 21 specimens were obtained, the authors could observe the details of their morphologies.
Four species of Polycopetta have been described: P. monneroni, P. curva, P. bransfieldensis, P. pax. P.a quadrispinata sp. n. and P. curva are similar to each other; i.e. both species have scale-like sculpture on the anterior carapace surface ( Fig. 2A, B), one clavate process at proximal middle end of second podomere of male antennal endo-podite (Fig. 8D), and four teeth as coxal endites of mandibula (Fig. 10B). They are distinguished by the number of spines at posteroventral margin of right valve, four for P. quadrispinata (Fig. 5A) and one for P. curva, respectively. This new species is distinguishable from each of the other three species by the number of coxal teeth (endites) of the mandibula (two in P. monneroni vs four (Fig. 10B) in P. quadrispinata the carapace surface ornamentation (absent in P. bransfieldensis vs scale-like sculpture and pits ( Fig. 2A, B) in P. quadrispinata and the number of adductor muscle scars (six in P. pax vs three (Fig. 5E) in P. quadrispinata This new species also differs from all previously described species by details of the chaetotaxy of the antennula, antenna, maxillula and fifth limb (see Table 2).
Our observation shows some morphological peculiarities of Polycopetta quadrispinata sp. n. when compared with its congeners. First, the third podomere of male antennula bears one seta with serrations at the ventrodistal end (Figs 7B, 8C). This seta has not been identified in the other species. Since this seta is only found in the male, itmust be related to sexual activity; however the function of this seta is unknown at the present time. Second, the endopodite of the fifth limb consists of two podomeres (Fig. 11B). Kornicker and Harrison-Nelson (2005) stated that the podomere number is only one in P. pax. Third, the long spermatozoa (Figs 12A, 13D) are described in Polycopetta for the first time. The males have been known for three species (P. monneroni, P. curva and P. bransfieldensis), but there is no information about their spermatozoa. In the family Polycopidae the sperm length of Eupolycope dispar (Müller, 1894) and Polycope cancellea Hartmann, 1954 have been reported (Hartmann 1955;1968). The length of the former species is 45 μm (carapace length is 300 μm), the latter is 15 μm (carapace length is 500 μm). The sperm length (750 μm) of the new species is extreme for this family. These characters are likely to be present in other incompletely described species. In future, more detailed observation of all of these species may be needed, in order to update the generic diagnosis.