Molecular phylogeny of interstitial Polycopidae ostracods (Crustacea) and descriptions of a new genus and four new species

bs_bs_banner Zoological Journal of the Linnean Society, 2014, 172, 282 317. With 31 figures Molecular phylogeny of interstitial Polycopidae ostracods (Crustacea) and descriptions of a new genus and four new species HAYATO TANAKA 1 *, AKIRA TSUKAGOSHI 1 and IVANA KARANOVIC 2,3 1 Department of Environment and Energy System, Shizuoka University, Oya 836, Suruga-ku, Shizuoka 422-8529, Japan 2 Department of Life Science, Hanyang University, Seoul 133-791, South Korea 3 Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Tasmania 7001, Australia Received 13 February 2014; revised 20 May 2014; accepted for publication 27 May 2014 Family Polycopidae is one of the more abundant and diverse taxa occurring in marine interstitial environments. Most of the interstitial polycopids are so far known from Japan and belong to the genus Parapolycope Klie, 1936. In this paper we describe another four new species from Japan. A new genus, Kliecope gen. nov. is erected to include one new species Kliecope mihoensis sp. nov. and one new combination Kliecope oligohalina (Tanaka & Tsukagoshi, 2010) comb. nov. Although the morphology of Kliecope is similar to Parapolycope, the new genus has the following diagnostic characters: absence of an inward bulge on the antennular second podomere, presence of two setae bearing a sucker on the antennular third podomere, and absence of a dorsal seta on the basis of mandibula. Another three Parapolycope, Parapolycope setouchiensis sp. nov., Parapolycope subtidalis sp. nov., and Parapolycope miurensis sp. nov. are described as well. To test the phylogenetic relationship between the new genus and Parapolycope, we performed phylogenetic analyses based on the 14 18S rdna sequences of interstitial Polycopidae species, 12 of which were newly obtained from our material. The 18S gene proved to be suitable for phylogenetic analyses in polycopids with high intraspecific or intrageneric resolution. Here we present trees obtained with maximum likelihood, maximum parsimony, and neighbour-joining methods, and they support the divergence between Kliecope and Parapolycope with high bootstrap values.. doi: 10.1111/zoj.12176 ADDITIONAL KEYWORDS: Cladocopina integrative taxonomy Kliecope Parapolycope reproductive character displacement. INTRODUCTION The interstitial environment is a cryptic habitat characterized by a narrow, dark, and three-dimensional maze-like space. Since Nicholls (1935), these habitats have been intensively studied and are known to support high levels of biodiversity. Marine interstitial animals have some unique features not only in their morphology (such as miniaturization, elongated *Corresponding author. E-mail: cladocopina@gmail.com body, absence of eyes and pigmentation, and development of adhesion organs), but also in their ecology (salinity tolerance, desiccation resistance, geotaxis, negative photo taxis, laying small number of large eggs, and direct development) (Swedmark, 1964; Ito, 1985; Watanabe, Tsukagoshi & Higashi, 2008; Giere, 2009). Ostracods are a large group of bivalved crustaceans, living in every aquatic environment. Marine interstitial ostracods were first reported by Klie (1936), who described three new polycopid species from amphioxus sand and shell gravel in Helgoland, 282

MOLECULAR PHYLOGENY OF POLYCOPIDAE 283 Germany. Like other interstitial animals, these ostracods often show specialized morphological characters (Hartmann, 1973; Maddocks, 1976; Gottwald, 1983; Higashi & Tsukagoshi, 2012), e.g. they are much smaller than their surface-dwelling relatives (Danielopol & Bonaduce, 1990; Yamada & Tanaka, 2011) and have reduced or absent eyes (Kaji & Tsukagoshi, 2010). Reduction of body size often results in convergent evolution of many characters both of the carapace and soft parts, and therefore it is sometimes hard to reveal the true phylogenetic relationships amongst closely related taxa based on morphological characters alone (Gottwald, 1983). Molecular genetic methods are powerful tools for phylogenetic reconstruction. However, so far, molecular phylogenetic work focused on marine interstitial ostracods has only been carried out by Higashi et al. (2011), who used both morphology and a partial cytochrome c oxidase subunit I sequence to clarify that the size difference in the male copulatory organ between sympatric Microloxoconcha Hartmann, 1954, species is the result of an intrasexual dimorphism. Approximately 200 marine interstitial ostracods (two subclasses and 17 families) have been reported worldwide (Watanabe et al., 2008). Sixteen of these families and 37 genera belong to the subclass Podocopa, whereas in the subclass Myodocopa only one suborder Cladocopina and one family Polycopidae can be found in interstitial waters. The following nine genera of Polycopidae have been reported from this environment: Eupolycope Chavtur, 1981; Hexopolycope Chavtur, 1981; Micropolycope Chavtur, 1981; Orthopolycope Chavtur, 1981; Parapolycope Klie, 1936; Polycopetta Chavtur, 1981; Polycopiella Chavtur, 1981; Polycopinna Chavtur, 1981; Polycopissa Chavtur, 1981. Recently, Karanovic & Brandão (2012) provided for the first time a cladistic analysis of the Polycopidae based on 29 morphological characters scored for 91 Recent species (including 25 interstitial species). Karanovic & Tanaka (2013) also used morphological cladistic methods to study the phylogeny of 15 Parapolycope species. By contrast, molecular phylogenetic studies focused on Polycopidae have not been performed so far. Currently, GenBank contains only three sequences that belong to Polycopidae and have been used for inferring deeper phylogenies. Oakley & Cunningham (2002) used partial sequences of 28S rdna and 18S rdna of Polycope sp. to study the evolution and origin of the arthropod eye and Yamaguchi & Endo (2003) used an 18S rdna sequence of Polycope japonica Hiruta, 1983, to study the molecular phylogeny of the class Ostracoda. During our faunal survey of the Japanese coast, three new Parapolycope species were collected from the marine interstitial environment. The genus Parapolycope Klie, 1936, which is the most speciose of all interstitial ostracods, is characterized by the following morphological characters: antennula, antenna, and upper lip are sexually dimorphic; endopodite and exopodite of maxillula are very short; a small number of uropodal claws; and a long, flagellum-like male copulatory organ (Klie, 1936; Karanovic & Tanaka, 2013; Tanaka & Tsukagoshi, 2013a). We also found a species that seems to be very closely related to Parapolycope, but its unusual characters prompted us to describe a new genus to accommodate this and one previously described Parapolycope species. We tested the validity of this new genus by conducting a molecular phylogeny based on the 18S rdna sequence of 14 Polycopidae species. This genetic analysis is the first attempt to infer phylogenetic relationships amongst interstitial species of myodocope ostracods. MATERIAL AND METHODS COLLECTING AND TAXONOMIC METHODS Material was collected from various beaches in Japan (Fig. 1). Sand was washed five times in a bucket with fresh water, and the top layer of water was strained through a net (40-μm mesh size). The living specimens were picked up from the remaining deposits under a stereo-binocular microscope (SZH 10, OLYMPUS). Specimens were fixed in 8% formalin with neutral buffer (hexamethylenetetramine), and preserved in 80% ethanol at room temperature for description or 99.5% ethanol at 20 C for DNA extraction. 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 stereo-binocular microscope, and observed and drawn using a transmittedlight binocular microscope (BX 50, OLYMPUS) with a differential interference contrast system and a camera lucida. For scanning electron microscopy (SEM; JSM- 5600LV, JEOL) valves and soft parts were treated with the t-butyl alcohol freeze-drying method, and coated with osmium. The type series was deposited in the collection of the Shizuoka University Museum, identified by the registration numbers with the prefix SUM-CO. DNA EXTRACTION, PCR AMPLIFICATION, CLONING, AND SEQUENCING Taxa and collection information for the species used in molecular phylogenetic analyses are detailed in Table 1. Total DNA was extracted from 99.5% ethanolpreserved specimens using an E. Z. N. A. Insect DNA Isolation Kit (Omega Bio-tek) or a DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer s protocol. Partial 18S sequences were PCR amplified using the newly designed primer pair (P20F

284 H. TANAKA ET AL. Figure 1. A, map of Japan; B, type localities of Kliecope mihoensis gen. et sp. nov. and Parapolycope subtidalis sp. nov.; C, type locality of Parapolycope setouchiensis sp. nov.; D, type locality of Parapolycope miurensis sp. nov. Table 1. Taxa and collection information for the species used in the molecular phylogenetic analyses. GenBank accession numbers of 18S rdna sequences are shown Species Locality Sampling date Latitude Longitude Acc. no. K. mihoensis sp. nov. Orange Beach, Shizuoka, Japan 27/11/2008 35 01 15 N 138 30 58 E AB934911 K. oligohalina comb. nov. Kanogawa river mouth, Shizuoka, Japan* 8/4/2007 35 04 52 N 138 51 37 E AB934912 P. japonica Tsuga Beach, Wakayama, Japan 11/3/2008 33 31 16 N 135 50 52 E AB934913 P. spiralis Miho-masaki Beach, Shizuoka, Japan* 7/10/2007 35 01 13 N 138 31 13 E AB934914 P. digitolabrum Sotoura Beach, Shizuoka, Japan 27/11/2008 34 40 35 N 138 58 25 E AB934915 P. psittacina Orange Beach, Shizuoka, Japan* 27/11/2008 34 57 15 N 139 08 31 E AB934916 P. uncata Koajiro Beach, Shizuoka, Japan* 23/11/2007 34 57 15 N 139 08 31 E AB934917 P. setouchiensis sp. nov. Kitagi island Beach, Okayama, Japan* 11/10/2009 34 22 29 N 133 31 54 E AB934918 P. subtidalis sp. nov. Kiiohsima-sue Beach, Wakayama, Japan 8/5/2009 33 27 12 N 135 49 20 E AB934919 P. miurensis sp. nov. Maguchi Beach, Kanagawa, Japan* 17/4/2010 35 08 45 N 139 40 48 E AB934920 Pe. quadrispinata Hayakawa Beach, Kanagawa, Japan 22/11/2007 35 13 57 N 139 08 37 E AB934921 Pi. sp. Sesoko Beach, Okinawa, Japan 1/10/2008 26 38 60 N 127 51 22 E AB934922 Po. japonica Aburatsubo Inlet, Kanagawa, Japan No information 35 08.9 N 139 36.6 E AB076657 Po. sp. Leigh, New Zealand No information No information No information AF363310 Acc., accession; K., Kliecope gen. nov.; P., Parapolycope Klie, 1936; Pe., Polycopetta Chavtur, 1981; Pi., Polycopiella Chavtur, 1981; Po., Polycope Sars, 1866. Asterisks indicate type localities. Data from Yamaguchi & Endo (2003). Data from Oakley & Cunningham (2002).

MOLECULAR PHYLOGENY OF POLYCOPIDAE 285 5 -ACCTGGTTGATCCTGCCAGT-3, P1822R 5 - TGATCCTTCYGCAGGTTCAC-3 ). The reaction solution (0.25 μl TaKaRa Ex taq, 5 μl 10X Ex Taq buffer, 4 μl deoxyribonucleotide triphosphate mixture, 2 μl of each primer prepared in 10.0 μm, 5 μl template DNA, and 31.75 μl sterilized distilled water) was prepared. The PCR protocol consisted of an initial denaturation step at 94 C for 2 min, followed by 45 cycles of 20 s denaturation at 94 C, 30 s annealing at 54 C, extension at 72 C for 2 min, and a final extension at 72 C for 10 min. Quantity and length of the PCR products were checked by 1% Agarose S gel (Nippon Gene) electrophoresis, stained with ethidium bromide. The products were purified for the sequencing reaction with ChargeSwitch Pro PCR Clean-up Kit (Invitrogen), according to the manufacturer s protocol. Cloning was carried out by using the TOPO TA Cloning Kit for sequencing (Invitrogen). The resulting plasmids were purified using the Miniprep DNA Purification Kit (Takara Bio). DNA inserts were checked with the same method as for PCR products. Sequencing was carried out by either the Macrogen Japan sequencing service or the Dragon Genomics Center (TaKaRa Bio). The sequence primers were as follows: T3 5 -ATTAACCCT CACTAAAG-3, T75 -AATACGACTCACTATAG-3, and a specific primer for 18S rdna (18S-F2 5 -CCT GAGAAACGGCTRCCACAT-3 ) designed by Yamaguchi & Endo (2003). SEQUENCE ANALYSES AND PHYLOGENETIC RECONSTRUCTIONS A homology search of 18S rdna sequences was performed by BLAST (Altschul et al., 1990, 1997) for the DNA Data Bank of Japan (DDBJ, http:// www.ddbj.nig.ac.jp/). Two already existing 18S rdna sequences of Polycopidae species (Polycope sp. and Polycope japonica) were downloaded from GenBank. The sequences were aligned with MAFFT v. 7.127b (Katoh et al., 2002, 2005) using the E-INS-i algorithm. The ambiguous regions of the aligned sequences were detected by using GBLOCKS 0.91b (Castresana, 2000) and removed manually. The number of base differences per site from between sequences was calculated in MEGA5 (Tamura et al., 2011). Phylogenetic analyses were carried using three methods: maximum likelihood (ML) method in PhyML v. 3.0 (Guindon & Gascuel, 2003; Guindon et al., 2010), and neighbourjoining (NJ) and maximum parsimony (MP) methods in MEGA5 (Tamura et al., 2011). jmodeltest v. 2.1 (Darriba et al., 2012) was used to find the best-fit evolutionary model for the present data set under the corrected Akaike information criterion (AICc, Hurvich & Tsai, 1989) for the ML method. For the ML, NJ, and MP methods, bootstrap values (Felsenstein, 1985) were calculated with 1000 replications. RESULTS TAXONOMY SUBCLASS MYODOCOPA SARS, 1866 ORDER HALOCYPRIDA DANA, 1853 SUBORDER CLADOCOPINA SARS, 1866 FAMILY POLYCOPIDAE SARS, 1866 GENUS KLIECOPE GEN. NOV. Diagnosis Carapace elliptical and dorsal line straight in lateral view. Antennula four podomeres, in males third podomere with two setae carrying a sucker. Antennal exopodite nine podomeres; endopodite three podomeres, in males third podomere with one stout, hook-shaped claw extending backwards. Mandibular endopodite two podomeres, first podomere with one ventral seta, basis without dorsal seta. Endopodite and exopodite of maxillula strongly reduced. Endopodite of fifth limb indistinct and with two setae. Both uropodal lamellae with six claws in males and females. Upper lip showing sexual dimorphism. Male copulatory organ consisting of a hook-shaped, short tube. Type species Kliecope mihoensis sp. nov. Other species Kliecope oligohalina (Tanaka & Tsukagoshi, 2010) comb. nov. Etymology The new genus is named after Dr h.c. Walter Klie in honour of his contribution to the study of interstitial Ostracoda. Remarks This new genus is most similar to Parapolycope with the following distinguishing characters: absence of the inward bulge of antennular second podomere, presence of two setae bearing a sucker of antennular third podomere, and absence of a dorsal seta on mandibular basis. Kliecope can be easily distinguished from all other genera by a reduced exopodite of the maxillula. Although we have included only two species in this genus, it is very likely that the following four species also belong in it: Parapolycope serridentata (Hartmann, 1959), Parapolycope aidae (Hartmann, 1959), Parapolycope noodti (Hartmann, 1959), and Parapolycope minuta (Hartmann, 1974). All these species also lack an inward bulge of antennula, do not have seta on the basis of mandibula, and have six claws of both uropodal lamellae. However, it is not sure if they also have two setae with a sucker on the male antennula, which is one of the diagnostic features of Kliecope, because their descriptions are not complete. In the case of

286 H. TANAKA ET AL. Parapolycope oligohalina, which is here included in the new genus, the presence of the two suckers on the male antennula was confirmed with SEM. KLIECOPE MIHOENSIS SP. NOV. (FIGS 2 8) Type series Holotype: adult male (SUM-CO-2178), Right valve length 151 μm and height 92 μm, left valve length 145 μm and height 87 μm, soft parts mounted on slide and valves preserved in a cardboard cell slide, Paratypes: 12 adult males (SUM-CO-2179 2190) and eight adult females (SUM-CO-2191 2198). Type locality The holotype specimen was collected from the Mihomasaki Beach, Shizuoka City, Shizuoka Prefecture, the Pacific coast of central Japan, 35 01 15 N, 138 30 58 E (Fig. 1B), on 4 July 2013; interstitial environment at 50 cm below the sand surface at 6 m inland from the low tide shoreline. The substrate consisted mainly of granular gravel. Diagnosis Carapace elliptical and dorsal line straight in lateral view. Carapace surface covered with a number of small, shallow pits. Serration along anteroventral margin with 16 sharp processes in right valve. Anteroventral region of marginal infold widened. Description of adult male Carapace (Figs 2, 3A E, 4). Right valve length 146 156 μm and height 91 98 μm, left valve length 141 147 μm and height 86 94 μm (Table 2). Carapace elliptical and dorsal line straight in lateral view. Carapace anterior half surface covered with a number of shallow pits (Fig. 3A, B). A number of thin serrations on anteroventral margin of left valve. Serration along anterior margin with 16 sharp processes on right valve (Fig. 2). Adductor muscle scars round and consisting of three closely spaced scars (Fig. 2). Marginal infold of each valve developed along anterior to mid-ventral margins (Figs 3C, D, 4B, G). Along inner margin of right valve, bar and groove on mid-dorsal to midventral (Fig. 4A, B), one socket (part of hinge structure) developed at dorsal end (Fig. 4C), posterior bar on mid-dorsal to posterior end (Fig. 4D), posterior element of hinge structure (Fig. 4E), and ridge on posterior end to mid-ventral (Fig. 4F). Along inner margin of left valve, bar and groove on anterodorsal to dorsal end (Fig. 4H), one knob (part of hinge structure) developed at dorsal end (Fig. 4I), bar on mid-dorsal to posterior end (Fig. 4J), posterior element of hinge structure (Fig. 4K), and bar along posteroventral margin (Fig. 4L). Bellonci organ. Absent. Upper lip (Figs 5A, 6A). Dorsal round and ventrally straight, and distal part elongated semicircular dorsally in lateral view. Antennula (Figs 5B, 6B D). Uniramous, four articulated podomeres. First podomere rectangular, with tufts of setulae on dorsal margin, lateral surface. Second podomere almost same length as first podomere, with one annulated seta at dorsoproximal end, two tufts of setulae on dorsal margin (Fig. 6B). Third podomere about three-quarters as long as first podomere, with one short seta at dorsodistal end and four ventrodistal setae consisting of one seta with large disc-shaped sucker, one annulated seta with small disc-shaped sucker, one seta with comb-like setulae, and one simple seta (Fig. 6C, D). Fourth podomere small, with three medium and two long annulated setae. Antenna (Figs 5C, D, 6E). Typically biramous, with exopodite and endopodite consisting of nine and three podomeres, respectively. Basis rectangular, tapered distally. Exopodite: first podomere about half as long as basis. Podomere length decreasing in size from second to eighth, each podomere with one long annulated seta; ninth (distal-most) podomere very small, with one long annulated seta with short process, one medium seta, and one short seta at distal end. Endopodite: first podomere about half as long as basis. Second podomere two-thirds as long as first podomere, with three short setae along dorsal margin, and six annulated setae at Figure 2. Kliecope mihoensis gen. et sp. nov., male holotype (SUM-CO-2178). A, internal lateral view of left valve; B, internal lateral view of right valve. Scale bar = 50 μm.

MOLECULAR PHYLOGENY OF POLYCOPIDAE 287 Figure 3. Scanning electron micrographs of valves of Kliecope mihoensis gen. et sp. nov. A, B, male paratype (SUM- CO-2179); C, D, male paratype (SUM-CO-2180); E, male paratype (SUM-CO-2181); F, G, female paratype (SUM-CO- 2193); H, I, female paratype (SUM-CO-2194); J, female paratype (SUM-CO-2195). A, external lateral view of right valve (RV); B, external lateral view of left valve (LV); C, internal lateral view of LV; D, internal lateral view of RV; E, dorsal view of carapace; F, external lateral view of RV; G, external lateral view of LV; H, internal lateral view of LV; I, internal lateral view of RV; J, dorsal view of carapace. Scale bar = 50 μm. distal consisting of two long, one long with some spines, two medium, one short. Third podomere one-quarter as long as first podomere, with one slender, hookshaped claw extending backward (Fig. 6E), one long annulated seta at ventral margin, and one long annulated, one medium annulated, and one short setae at distal end. Mandibula (Fig. 5E). Coxal endite with four sharp teeth. Basis with three plumose setae on ventral margin. Exopodite absent. Endopodite consisting of two podomeres. First podomere with one plumose seta on ventral margin near proximal end and two long, setulous annulated setae at dorsodistal end. Second podomere very small, bearing one medially plumose and one stout setulous annulated seta. Maxillula (Fig. 5F, F, F ). Precoxa (Fig. 5F ) with eight plumose setae of different lengths. Coxa (Fig. 5F ) with two short plumose setae on lateral surface near ventroproximal margin, and three long plumose setae on ventral margin. Basis with one medium and one long plumose setae on ventral margin. First podomere of endopodite with one short seta dorsodistally, and two long annulated setae at ventroproximal margin. Second podomere, with two long annulated setae, and two long, stout setulous setae with few bilateral spines. Exopodite consisting of two podomeres. First podomere with one tuft of setulae on dorsal margin. Second podomere with one very long setae and five long setae on ventral margin, and one tuft of setulae on dorsal margin.

288 H. TANAKA ET AL. Figure 4. Scanning electron micrographs of valves of Kliecope mihoensis gen. et sp. nov., internal lateral view, male paratype (SUM-CO-2180). A F, right valve; G L, left valve. A, anterodorsal bar and groove; B, anterior part of marginal infold; C, socket at dorsal end of hinge structure; D, posterodorsal bar of hinge structure; E, posterior element of hinge structure; F, posteroventral ridge; G, anterior part of marginal infold; H, anterodorsal bar and groove; I, knob at dorsal end of hinge structure; J, posterodorsal bar of hinge structure; K, posterior element of hinge structure; L, posteroventral bar. Fifth limb (Fig. 5G). Coxa bearing epipodite with ten long plumose setae, and two short setulous setae on dorsodistal margin. Basis with two plumose setae along dorsal margin, one slender setulous seta on ventral margin. Endopodite with two long plumose setae. Exopodite one stout, short setulous seta. Uropod (Figs 6F, 7A). Both lamellae with six stout claws and one tuft of setulae anteroventral end. Male copulatory organ (Figs 6F, 7A). Arising from outer surface of body on left side of terminal trunk segment as a hook-shaped, slender tube. Description of adult female Mandibula, maxillula, and fifth limb similar to those of adult male.

MOLECULAR PHYLOGENY OF POLYCOPIDAE 289 Figure 5. Kliecope mihoensis gen. et sp. nov. A E, G, male holotype (SUM-CO-2178); F, male paratype (SUM-CO- 2180). A, left lateral view of upper lip; B, antennula; C, antenna (A2) except exopodite; D, exopodite of A2; E, mandibula; F, maxillula (Mxl); F, precoxa of Mxl; F, coxa of Mxl; G, fifth limb. Abbreviations: ba, basis; cx, coxa; en, endopodite; ep, epipodite; ex, exopodite; pc, precoxa. Scale bar = 20 μm.

290 H. TANAKA ET AL. Figure 6. Scanning electron micrographs of male soft parts of Kliecope mihoensis gen. et sp. nov. A, paratype (SUM- CO-2182); B, E paratype (SUM-CO-2183); C, paratype (SUM-CO-2184); D, F, paratype (SUM-CO-2185). A, left lateral view of upper lip; B, right lateral view of second and third podomeres of antennula (A1); C, left lateral view of third and four podomeres of A1; D, right lateral view of setae with sucker of A1; E, right lateral view of endopodite and hookshaped claw of antenna; F, left lateral view of posterior trunk segment, uropod, and copulatory organ. Arrows and arrowheads indicate small and large disc-shaped suckers, respectively. Abbreviation: en, endopodite. Carapace (Fig. 3F J). Right valve length 158 169 μm and height 93 103 μm, left valve length 149 162 μm and height 88 100 μm (Table 2). Upper lip (Fig. 8A). Semicircular in lateral view. Antennula (Fig. 8B). First podomere rectangular, with tufts of setulae on dorsal margin, lateral surface. Second podomere almost same length as first podomere, with one annulated setulous seta at dorsoproximal end, two tufts of setulae on dorsal margin. Third podomere

MOLECULAR PHYLOGENY OF POLYCOPIDAE 291 Figure 7. Kliecope mihoensis gen. et sp. nov., left lateral view of posterior trunk segment, uropod, and copulatory organ. A, male holotype (SUM-CO-2178); B, female paratype (SUM-CO-2192). Abbreviation: fs, female spermatheca. Scale bar=20μm. Figure 8. Kliecope mihoensis gen. et sp. nov., female paratype (SUM-CO-2191). A, left lateral view of upper lip; B, antennula; C, antenna except part of exopodite. Abbreviations: ba, basis; en, endopodite; ex, exopodite. Scale bar = 20 μm. about three-quarters as long as first podomere, with one short seta at dorsodistal end and one ventrodistal seta. Fourth podomere small, with five annulated setae. Antenna (Fig. 8C). Only second and third podomeres of endopodite differing from those of adult male. Second podomere half as long as first podomere, with one short setulous seta along dorsal margin, and five

292 H. TANAKA ET AL. Table 2. Dimension of valves of Kliecope mihoensis sp. nov. from the type locality Length (μm) Height (μm) Mean Observed range N Mean Observed range N Male RV 150 146 151 7 93 90 98 7 LV 145 141 147 6 89 86 94 6 Female RV 164 158 169 6 98 93 103 6 LV 156 149 162 6 93 88 100 5 LV, left valve; RV, right valve. annulated setae at distal consisting of two long, one long with some spines, two medium. Third podomere one-quarter as long as first podomere, with one long annulated seta at ventral margin, and one long annulated, one medium annulated and one short seta at distal end. Uropod (Fig. 7B). Both lamellae with six stout claws and one row of setae on anteroventral lateral surface. Female copulatory organ (Fig. 7B). Female spermatheca cylindrical in lateral view. Dimensions See Table 2. Distribution Shichigahama [Miyagi Prefecture (Pref.) 38 17 08 N, 141 03 80 E], Toyooka Beach (Chiba Pref. 35 03 04 N, 139 49 55 E), Hiratsuka (Kanagawa Pref. 35 18 48 N, 139 22 01 E), Kouzu (Kanagawa Pref. 35 16 40 N, 139 12 34 E), Orange Beach (Shizuoka Pref. 34 58 37 N, 139 05 44 E), Ohura Beach (Shizuoka Pref. 34 40 05 N, 138 56 28 E), Miho-masaki Beach (Shizuoka Pref. type locality, 35 01 15 N, 138 30 58 E), Awaji-tsuna (Hyogo Pref. 34 26 21 N, 134 54 41 E), Nushima (Hyogo Pref. 34 10 04 N, 134 48 52 E), Izari (Tokushima Pref. 33 48 07 N, 134 40 08 E), Ukitsu (Kochi Pref. 33 17 18 N, 134 08 42 E), Kashiwajima (Kochi Pref. 32 46 02 N, 132 37 38 E); Aira-fukuyama (Kagoshima Pref. 31 41 00 N, 130 48 41 E). All specimens were collected by Hayato Tanaka from interstitial water in Japan. Etymology This species is named after the type locality, Mihomasaki Beach. Remarks The morphology of Kliecope mihoensis sp. nov. is similar to Parapolycope aidae (Hartmann, 1959) from Los Cobanos in El Salvador, Parapolycope noodti (Hartmann, 1959) from Cristóbal in Panama, and Parapolycope minuta (Hartmann, 1974) from Xai-Xai in Mozambique. The males of Pa. minuta possess cross-shaped (kreuzförmig) seta on the antennula, and the other two species unusually do not have sexually dimorphic antennula. In addition, Kliecope mihoensis has two plumose setae on the basis of fifth limb and Pa. minuta has three. KLIECOPE OLIGOHALINA (TANAKA &TSUKAGOSHI, 2010) COMB. NOV. (FIG. 9) Synonymy Parapolycope oligohalina Tanaka & Tsukagoshi, 2010: Tanaka & Tsukagoshi, 2010: pp. 39 47, 56, figs 2 7; Karanovic & Tanaka, 2013: p. 26, 29, 32 34, fig. 12. Material examined Holotype: adult male (SUM-CO-1765), Paratypes: seven adult males (SUM-CO-1766 1769, 1775 1777), deposited in the Shizuoka University Museum, Japan. Additional specimens: two adult males (SUM-CO-2199, 2200) collected from Miho-masaki Beach. Diagnosis Carapace slightly elliptical and dorsal line straight in lateral view. Carapace surface covered with puncta of varying sizes; coarse in mid-dorsal to dorsal areas, fine in anteromedian, mid-anterior, and posterior areas. Serration along anteroventral margin: 11 sharp processes increasing in size toward ventral in right valve. Redescription of adult male We agree with the description of Tanaka & Tsukagoshi (2010) except for the characters of the antennula and exopodite of antenna. Antennula (Fig. 9A). Uniramous, four articulated podomeres. First podomere rectangular, with two and one tufts of setulae on dorsal margin and lateral surface, respectively. Second podomere almost same length as first podomere, with one setulous seta at dorsoproximal end, setulae on dorsal margin. Third podomere about two-thirds as long as second podomere, with one short seta at dorsodistal end and four ventrodistal setae

MOLECULAR PHYLOGENY OF POLYCOPIDAE 293 Figure 9. Scanning electron micrographs of male soft parts of Kliecope oligohalina comb. nov. A, right lateral view of distal part of antennula, male paratype (SUM-CO-2199); B, left lateral view of antenna, male paratype (SUM-CO-2200). Arrows and arrowheads indicate small and large disc-shaped suckers, respectively. Abbreviations: en, endopodite; ex, exopodite. Figure 10. Parapolycope setouchiensis sp. nov., male holotype (SUM-CO-2201). A, internal lateral view of left valve; B, internal lateral view of right valve. Scale bar = 100 μm. consisting of one seta with large, disc-shaped sucker, one seta with small, disc-shaped sucker, one stout seta with setulae, and one simple seta. Fourth podomere small, with two medium and two long annulated setae. Antenna (Fig. 9B). Typically biramous, with exopodite and endopodite consisting of nine and three podomeres, respectively. Basis rectangular tapered distally. Exopodite: first podomere about one-third as long as basis. Podomere length decreasing in size from second to eighth, each podomere with one long annulated seta; ninth (distal-most) podomere very small, with one long annulated seta, one medium seta, and one short seta at distal end. Distribution Kliecope oligohalina comb. nov. is known from the following localities: the mouth of Kano River (Shizuoka Pref. type locality, 35 04 52 N, 138 51 33 E), the mouth of Sagami River (Kanagawa Pref. 35 19 34 N, 139 21 59 E), the mouth of Nishina River (Shizuoka Pref. 34 46 00 N, 138 46 32 E), the mouth of Fuji River (Shizuoka Pref. 35 06 57 N, 138 38 25 E), the mouth of Okitsu River (Shizuoka Pref. 35 03 24 N, 138 32 05 E), and Miho-masaki Beach (Shizuoka Pref. 35 01 15 N, 138 30 58 E). All specimens were collected by Hayato Tanaka from interstitial water in Japan. Remarks Careful examination of the type specimens and additional material confirmed that Parapolycope oligohalina has two suckers on the male antennula, which prompted us to transfer it to the new genus. This species also has all of the other characters of Kliecope (see above). GENUS PARAPOLYCOPE KLIE, 1936 PARAPOLYCOPE SETOUCHIENSIS SP. NOV. (FIGS 10 16) Type series Holotype: adult male (SUM-CO-2201), right valve length 294 μm and height 220 μm, left valve length 288 μm and height 217 μm, soft parts mounted on slide and valves preserved in a cardboard cell slide, Paratypes: eight adult males (SUM-CO-2202 2209) and nine adult females (SUM-CO-2210 2218).

294 H. TANAKA ET AL. Figure 11. Scanning electron micrographs of valves of Parapolycope setouchiensis sp. nov. A, B, male paratype (SUM- CO-2202); C, D, male paratype (SUM-CO-2203); E, male paratype (SUM-CO-2204); F, G, female paratype (SUM-CO- 2211); H, I, female paratype (SUM-CO-2212); J, female paratype (SUM-CO-2213). A, external lateral view of right valve (RV); B, external lateral view of left valve (LV); C, internal lateral view of LV; D, internal lateral view of RV; E, dorsal view of carapace; F, external lateral view of RV; G, external lateral view of LV; H, internal lateral view of LV; I, internal lateral view of RV; J, dorsal view of carapace. Scale bar = 50 μm. Type locality The holotype specimen was collected from Kitagi island Beach, Kasaoka City, Okayama Prefecture, the Seto inland sea of Japan, 34 22 29 N, 133 31 54 E (Fig. 1C), on 11 October 2009; interstitial environment at 20 cm below the sand surface at 5 m inland from the low tide shoreline. The substrate consisted mainly of very coarse, decomposed granite sand. Diagnosis Carapace oval in lateral view. Carapace surface covered with a number of shallow pits. Serration along anterior margin with 24 and 22 sharp processes on right valve and left valve, respectively, a dorsal-most process of right valve prominently large. Uropodal projection of male long, with numerous prominent spines or barbs. Three claws on each uropodal lamella in female.

MOLECULAR PHYLOGENY OF POLYCOPIDAE 295 Figure 12. Scanning electron micrographs of valves of Parapolycope setouchiensis sp. nov., internal lateral view, male paratype (SUM-CO-2203). A F, right valve; G L, left valve. A, anterodorsal bar and groove; B, anterior part of marginal infold; C, socket at dorsal end of hinge structure; D, posterodorsal bar of hinge structure; E, posterior element of hinge structure; F, ventral part of marginal infold; G, anterior part of marginal infold; H, anterodorsal bar and groove; I, knob at dorsal end of hinge structure; J, posterodorsal bar of hinge structure; K, posteroventral bar; L, ventral part of marginal infold. Male copulatory organ consisting of extending helical tube. Description of adult male Carapace (Figs 10, 11A E, 12). Right valve length 285 297 μm and height 208 220 μm, left valve length 280 292 μm and height 211 218 μm (Table 3). Carapace oval in lateral view. Carapace surface covered with a number of shallow pits (Fig. 11A, B). Serration along anterior margin with 24 and 22 sharp processes on right valve and left valve, respectively (Fig. 10), a dorsal-most process of right valve prominently large. Adductor

296 H. TANAKA ET AL. Figure 13. Parapolycope setouchiensis sp. nov. A, B, D, F, male holotype (SUM-CO-2201); C, E, male paratype (SUM- CO-2202). A, right lateral view of upper lip; B, antennula (A1; arrowhead indicates inward bulge); B, seta with comblike setulae of A1; C, antenna (A2) except exopodite; C, exopodite of A2; D, mandibula; E, maxillula (Mxl); E, precoxa of Mxl; E, coxa of Mxl; F, fifth limb (L5); F, one of the epipodites of L5. Abbreviations: ba, basis; cx, coxa; en, endopodite; ep, epipodite; ex, exopodite; pc, precoxa. Scale bar = 50 μm.

MOLECULAR PHYLOGENY OF POLYCOPIDAE 297 Figure 14. Scanning electron micrographs of male soft parts of Parapolycope setouchiensis sp. nov. A, B, male paratype (SUM-CO-2205); C, D, male paratype (SUM-CO-2206). A, right lateral view of upper lip; B, right lateral view of broad chitinous hook of upper lip; C, right lateral view of posterior trunk segment, uropod, and uropodal projection; D, right lateral view of uropodal projection with numerous prominent spines. muscle scars oval and consisting of three closely spaced scars (Fig. 10). Marginal infold of each valve developed along anterior to posteroventral margins (Figs 11C, D, 12B, F, G, L). Along inner margin of right valve, bar and groove on anterodorsal to dorsal end (Fig. 12A), one socket (part of hinge structure) developed at dorsal end (Fig. 12C), posterior bar on mid-dorsal to posterior end (Fig. 12D), bar and groove on posterior to midposteroventral end (Fig. 12E). Along inner margin of left valve, bar and groove on anterodorsal to dorsal end (Fig. 12H), one knob (part of hinge structure) developed at dorsal end (Fig. 12I), bar on mid-dorsal to posterior end (Fig. 12J), and bar along posteroventral margin (Fig. 12K). Bellonci organ. Absent. Upper lip (Figs 13A, 14A, B). Broad chitinous hook and a chain of cone-shaped setae on right lateral side. Distal part with anteroventral protrusion, tapering to wrinkled, and papilla-shaped termination in lateral view. Antennula (Fig. 13B, B ). Uniramous, four articulated podomeres. First podomere quadrate, with setulae on dorsal margin, lateral surface and ventrodistal end. Second podomere about 1.5 times as long as first podomere, proximal half of dorsal cuticle thick and with an inward bulge, with one annulated setulous seta at dorsoproximal end, one cluster of setae on middle of dorsal margin, and three tufts of setulae on lateral surface. Third podomere about two-thirds as long as first podomere, with one short, simple seta at dorsodistal end and five ventrodistal setae consisting of one seta with large disc-shaped sucker, one seta curving at tip armed with five falciform spines and minute setulae, one seta with comb-like setulae, and two simple setae. Fourth podomere small, with five long annulated setae. Antenna (Fig. 13C, C ). Typically biramous, with exopodite and endopodite consisting of nine and three podomeres, respectively. Basis triangular tapered distally. Exopodite: first podomere half as long as

298 H. TANAKA ET AL. Figure 15. Parapolycope setouchiensis sp. nov. A, male holotype (SUM-CO-2201), left lateral view of posterior trunk segment, uropod, and copulatory organ; B, female paratype (SUM-CO-2210), right lateral view of posterior trunk segment, uropod, and copulatory organ. Abbreviations: fs, female spermatheca; urp, uropodal projection. Scale bar = 50 μm. basis. Podomere length decreasing in size from second to eighth, each podomere with one long annulated seta; ninth (distal-most) podomere very small, with one long annulated seta at proximal, one medium annulated seta with short process and one short seta at distal end. Endopodite: first podomere half as long as basis. Second podomere two-thirds as long as first podomere, with three setae along dorsal margin consisting of one medium annulated and two short, and six annulated setae at distal consisting of three long, one long with some filaments, one medium, one short. Third podomere one-quarter as long as first podomere, with one stout, hook-shaped claw extending backward, one long annulated seta at ventral margin, and one long and one medium annulated seta at distal end. Mandibula (Fig. 13D). Coxal endite with four sharp teeth. Basis with three plumose setae on ventral margin and one annulated setulous seta on dorsal margin. Endopodite consisting of two podomeres. First podomere with two annulated plumose setae on ventral margin near proximal end and two annulated plumose long setae at dorsodistal end. Second podomere very small with setulae on dorsal margin, bearing one medially plumose and one claw-like, setulous annulated seta. Maxillula (Fig. 13E, E, E ). Precoxa (Fig. 13E ) with ten annulated plumose setae of different lengths. Coxa (Fig. 13E ) with one short and one medium plumose setae on lateral surface near ventroproximal margin, one short plumose setae on lateral surface near ventral middle margin, three medium plumose setae on ventral margin, and two tufts of setulae on dorsal margin. Basis with one short, one medium annulated, and two long plumose setae on ventral margin, and setulae along ventral and dorsal margins. First podomere of endopodite with one annulated setulous seta dorsodistally, and one long annulated and one long plumose seta at ventroproximal margin. Second podomere with two long, annulated setulous setae and two long, stout setulous setae with few bilateral spines. Exopodite consisting of two podomeres. First podomere with setulae along dorsal margin. Second podomere with

MOLECULAR PHYLOGENY OF POLYCOPIDAE 299 Figure 16. Parapolycope setouchiensis sp. nov., female paratype (SUM-CO-2210). A, right lateral view of upper lip; B, antennula; C, antenna except part of exopodite. Arrowhead indicates inward bulge. Abbreviations: ba, basis; en, endopodite; ex, exopodite. Scale bar = 50 μm. Table 3. Dimension of valves of Parapolycope setouchiensis sp. nov. from the type locality Length (μm) one very long, stout setulous seta, four long and three medium annulated setae on ventral margin, and one tuft of setulae on dorsal margin. Fifth limb (Fig. 13F, F ). Coxa bearing epipodite with 12 long plumose setae and two short setulous setae on dorsodistal margin. Basis with three plumose setae along dorsal margin and one slender, annulated setulous seta on ventral margin. Endopodite with one short, plumose annulated, one medium annulated, and two long plumose setae. Exopodite with one stout setulous seta. Uropod (Figs 14B, C, 15A). Left lamella with three stout claws and three rows of setae on anteroventral surface. Right lamella with four stout claws and ventrodistally situated uropodal projection, which is long, flexible, and covered with numerous prominent spines. Height (μm) Mean Observed range N Mean Observed range N Male RV 292 285 297 6 216 208 220 6 LV 286 282 292 6 216 211 218 6 Female RV 309 301 319 6 227 218 234 6 LV 307 294 321 6 226 219 234 6 LV, left valve; RV, right valve. Male copulatory organ (Fig. 15A). Arising from outer surface of body on left side of terminal trunk segment as a long stretched helically tube. Description of adult female Mandibula, maxillula, and fifth limb similar to those of adult male. Carapace (Fig. 11F J). Right valve length 301 319 μm and height 218 234 μm, left valve length 294 321 μm and height 219 234 μm (Table 3). Upper lip (Fig. 16A). Semicircular in lateral view. A number of setae on lateral surface. Antennula (Fig. 16B). Uniramous, four articulated podomeres. First podomere quadrate, with two tufts of setulae on dorsal margin. Second podomere about 1.5 times as long as first podomere, proximal half of

300 H. TANAKA ET AL. dorsal cuticle robust and with inward bulge, with one annulated setulous seta at dorsoproximal end, one cluster of setae on middle of dorsal margin, and two tufts setulae on lateral surface. Third podomere about two-thirds as long as first podomere, with one short simple seta at dorsodistal end, two short simple setae on ventrodistal end, and setulae along dorsal margin. Fourth podomere small, with five long annulated setae. Antenna (Fig. 16C). Only second and third podomeres of endopodite differing from those of adult male. Second podomere, with one annulated setulous seta on dorsodistal end and six annulated setae at distal consisting of three long, one long with some filaments, one medium, one short. Third podomere one-quarter as long as first podomere, with one long annulated seta at ventral margin, and one long and one medium annulated seta at distal end. Uropod (Fig. 15B). Each lamella with three claws. Right lamella with three rows of setae on anteroventral surface. Female copulatory organ (Fig. 15B). Female spermatheca double twisted tube in lateral view. Dimensions See Table 3. Distribution Tai Beach (Kyoto Pref. 35 34 53 N, 135 14 09 E), Komatsubara (Kagawa Pref. 34 15 10 N, 134 22 33 E), Aji (Kagawa Pref. 34 23 48 N, 134 07 44 E), Kitagi island Beach (Okayama Pref. type locality, 34 22 29 N, 133 31 54 E), Meotoura (Miyazaki Pref. 31 29 36 N, 131 23 04 E), Injo Beach (Kagoshima Pref. 30 33 27 N, 131 01 32 E). All specimens were collected by Hayato Tanaka from interstitial pore water in Japan. Etymology This species is named after the type locality, the Seto inland sea of Japan. Remarks Appendage morphology of Pa. setouchiensis sp. nov. is similar to that of Parapolycope spiralis Tanaka & Tsukagoshi, 2010. However, the new species can be easily distinguished from Pa. spiralis by the characteristics of the male uropodal projection. Namely, males of the new species bear a number of prominent spines on the uropodal projection, whereas they are absent in Pa. spiralis. PARAPOLYCOPE SUBTIDALIS SP. NOV. (FIGS 17 23) Type series Holotype: adult male (SUM-CO-2219), right valve length 228 μm and height 159 μm, left valve length 221 μm and height 156 μm, soft parts mounted on slide and valves preserved in a cardboard cell slide, paratypes: ten adult males (SUM-CO-2220 2229) and six adult females (SUM-CO-2230 2235). Type locality The holotype specimen was collected from Mihouchihama Beach, Shizuoka City, Shizuoka Prefecture, the Pacific coast of central Japan, 35 00 51 N, 138 31 07 E (Fig. 1B), on 31 July 2012; interstitial environment at 20 cm below the sand surface at 2 m seaward from the low tide shoreline. The substrate consisted mainly of very coarse sand with gravels. Diagnosis Carapace elliptical in lateral view. Carapace surface covered with a number of shallow pits. Serration along anterior margin with 21 and 22 sharp processes on right valve and left valve, respectively. Anterior part of male upper lip with blunt anterodorsal projection and long, tapering spine that curves around the anterior and ventral margins. Basis of fifth limb bearing two dorsal plumose setae. Uropodal projection of Figure 17. Parapolycope subtidalis sp. nov., male holotype (SUM-CO-2219). A, internal lateral view of left valve; B, internal lateral view of right valve. Scale bar = 100 μm.

MOLECULAR PHYLOGENY OF POLYCOPIDAE 301 Figure 18. Scanning electron micrographs of valves of Parapolycope subtidalis sp. nov. A, B, male paratype (SUM- CO-2221); C, D, male paratype (SUM-CO-2222); E, male paratype (SUM-CO-2223); F, G, female paratype (SUM-CO- 2232); H, I, female paratype (SUM-CO-2230); J, female paratype (SUM-CO-2233). A, external lateral view of right valve (RV); B, external lateral view of left valve (LV); C, internal lateral view of LV; D, internal lateral view of RV; E, dorsal view of carapace; F, external lateral view of RV; G, external lateral view of LV; H, internal lateral view of LV; I, internal lateral view of RV; J, dorsal view of carapace. Scale bar = 50 μm. male short, with numerous small spines or barbs. Male copulatory organ consisting of a wavy tube. Description of adult male Carapace (Figs 17, 18A E, 19). Right valve length 221 228 μm and height 148 159 μm, left valve length 210 221 μm and height 148 156 μm (Table 4). Carapace elliptical in lateral view. Carapace surface covered with a number of shallow pits (Fig. 18A, B). Serration along anterior margin with 21 and 22 sharp processes on right valve and left valve, respectively (Fig. 17). Adductor muscle scars round and consisting of three closely spaced scars (Fig. 17). Marginal infold of each valve developed along anterior to posteroventral margins (Figs 18C, D, 19B, G, H, N). Along inner margin of right valve, bar and groove on anterodorsal to dorsal end (Fig. 18A), one socket (part of hinge structure) developed at dorsal end (Fig. 18C), posterior bar on mid-dorsal to posterior end (Fig. 18D, E), bar and groove on posterior end to mid-posteroventral end (Fig. 18F). Along inner margin of left valve, bar and groove on anterodorsal to dorsal end (Fig. 18I), one knob (part of hinge structure)

302 H. TANAKA ET AL. Figure 19. Scanning electron micrographs of valves of Parapolycope subtidalis sp. nov., internal lateral view, male paratype (SUM-CO-2222). A G, right valve; H N, left valve. A, anterodorsal bar and groove; B, anterior part of marginal infold; C, socket at dorsal end of hinge structure; D, posterodorsal bar of hinge structure; E, posterior element of hinge structure; F, posteroventral bar and groove; G ventral part of marginal infold; H, anterior part of marginal infold; I, anterodorsal bar and groove; J, knob at dorsal end of hinge structure; K, posterodorsal bar of hinge structure; L, posterior element of hinge structure; M, posteroventral bar; N, ventral part of marginal infold. developed at dorsal end (Fig. 18J), bar on mid-dorsal to posterior end (Fig. 18K, L), and bar along posteroventral margin (Fig. 18M). Bellonci organ. Absent. Upper lip (Figs 20A, 21A). Anterior part with blunt anterodorsal projection and long, tapering spine that curves around the anterior and ventral margins.

MOLECULAR PHYLOGENY OF POLYCOPIDAE 303 Figure 20. Parapolycope subtidalis sp. nov. A E, male holotype (SUM-CO-2219); F, male paratype (SUM-CO-2220). A, left lateral view of upper lip; B, antennula, arrowhead indicates inward bulge; C, antenna (A2); C, endopodite of A2; D, mandibula; E, maxillula (Mxl); E, precoxa of Mxl; E, coxa of Mxl; F, fifth limb. Abbreviations: ba, basis; cx, coxa; en, endopodite; ep, epipodite; ex, exopodite; pc, precoxa. Scale bar = 50 μm.

304 H. TANAKA ET AL. Figure 21. Scanning electron micrographs of male soft parts of Parapolycope subtidalis sp. nov. A, C, D, male paratype (SUM-CO-2224); B, male paratype (SUM-CO-2225). A, left lateral view of upper lip; B, left lateral view of fifth limb; C, left lateral view of posterior trunk segment, uropod, and uropodal projection; D, left lateral view of uropodal projection with numerous small spines. Antennula (Fig. 20B). Uniramous, four articulated podomeres. First podomere rectangular, with tufts of setulae on dorsal margin, lateral surface and ventrodistal end. Second podomere same length as first podomere, proximal two-thirds of dorsal cuticle thick and with inward bulge, with one annulated setulous seta at dorsoproximal end, one cluster of setae on distal third of dorsal margin, setulae on dorsoproximal and dorsodistal margins, and one tuft of setulae on lateral surface. Third podomere about four-fifths as long as first podomere, with one short annulated seta at dorsodistal end and five ventrodistal setae consisting of one with large discshaped sucker, one with minute setulae, one with comblike setulae, and two simple setae. Fourth podomere small, with five long annulated setae. Antenna (Fig. 20C, C ). Typically biramous, with exopodite and endopodite consisting of nine and three podomeres, respectively. Basis triangular, tapered distally. Exopodite: first podomere one-third as long as basis. Second podomere one-quarter as long as first podomere; podomere length decreasing in size from third to eighth, each podomere with one long annulated seta; ninth (distal-most) podomere very small, with one long annulated seta at proximal, one medium annulated seta

MOLECULAR PHYLOGENY OF POLYCOPIDAE 305 Figure 22. Parapolycope subtidalis sp. nov. A, male holotype (SUM-CO-2219), right lateral view of posterior trunk segment, uropod, and copulatory organ; B, female paratype (SUM-CO-2231), left lateral view of posterior trunk segment, uropod, and copulatory organ. Abbreviations: fs, female spermatheca; urp, uropodal projection. Scale bar = 50 μm. Figure 23. Parapolycope subtidalis sp. nov., female paratype (SUM-CO-2231). A, left lateral view of upper lip; B, antennula, arrowhead indicates inward bulge; C, antenna except part of exopodite. Abbreviations: ba, basis; en, endopodite; ex, exopodite. Scale bar = 50 μm. with short process and setulae, and one short seta at distal end. Endopodite (Fig. 20C ): first podomere about one-third as long as basis. Second podomere twothirds as long as first podomere, with three setae along dorsal margin consisting of one medium and two short, and six annulated setae at distal consisting of three long, one long with some filaments, one medium, and one short. Third podomere one-quarter as long as first podomere, with one stout, hook-shaped claw extending backward, one long annulated seta at ventral margin, and two long annulated and one short setae at distal end. Mandibula (Fig. 20D). Coxal endite with four sharp teeth. Basis with three plumose annulated setae on ventral margin and one plumose seta on dorsal margin. Endopodite consisting of two podomeres. First podomere

306 H. TANAKA ET AL. Table 4. Dimension of valves of Parapolycope subtidalis sp. nov. from the type locality Length (μm) Height (μm) Mean Observed range N Mean Observed range N Male RV 223 217 228 8 153 148 159 8 LV 210 210 221 8 152 148 156 8 Female RV 239 233 245 5 162 154 168 5 LV 231 226 237 5 160 153 166 5 LV, left valve; RV, right valve. with one plumose seta on ventral margin near proximal end and two plumose long setae at dorsodistal end. Second podomere very small with setulae on dorsal margin, bearing one medially plumose and one clawlike setulous setae. Maxillula (Fig. 20E, E, E ). Precoxa (Fig. 20E ) with seven plumose setae of different lengths. Coxa (Fig. 20E ) with one short and one medium plumose setae on lateral surface near ventroproximal margin, one medium and three long plumose setae on ventral margin. Basis with two medium and one long plumose setae on ventral margin, and setulae along dorsal margin. First podomere of endopodite with one plumose seta dorsodistally, and two long annulated setae at ventroproximal margin. Second podomere, with one long annulated and one long setulous setae, and two long, stout setulous setae with few bilateral spines. Exopodite consisting of two podomeres. First podomere with setulae along dorsal margin. Second podomere with one very long, stout setulous seta, three long and two medium setulous setae, one long annulated seta on ventral margin, and one tuft of setulae on dorsal margin. Fifth limb (Fig. 20F). Coxa bearing epipodite with 12 long plumose setae, and two short setulous setae on dorsodistal margin. Basis with two plumose setae along dorsal margin, one slender setulous seta on ventral margin. Endopodite with one medium and one long plumose seta. Exopodite one stout setulous seta. Uropod (Figs 21C, D, 22A). Left lamella with three stout claws, one short proximal spine dorsally, and two rows of setae on anteroventral surface. Right lamella with four stout claws, one short proximal spine dorsally, and ventrodistally situated, short, straight uropodal projection with numerous short spines or barbs. Male copulatory organ (Fig. 22A). Arising from outer surface of body on left side of terminal trunk segment as a long, wavy tube. Description of adult female Mandibula, maxillula, and fifth limb similar to those of adult male. Carapace (Fig. 18F J). Right valve length 233 245 μm and height 154 168 μm, left valve length 226 237 μm and height 153 166 μm (Table 4). Upper lip (Fig. 23A). Semicircular in lateral view, with tapering conical, anterodorsal projection similar to that of male. A number of setae on ventral lateral surface. Antennula (Fig. 23B). Uniramous, four articulated podomeres. First podomere rectangular, with tufts of setulae on dorsal margin, lateral surface, and ventrodistal end. Second podomere same length as first podomere, proximal two-thirds of dorsal cuticle thick and with inward bulge, with one annulated setulous seta at dorsoproximal end, one cluster of setae on distal third of dorsal margin, setulae on dorsoproximal and dorsodistal margins. Third podomere about twothirds as long as first podomere, with three short simple setae at distal end. Fourth podomere small, with five long annulated setae. Antenna (Fig. 23C). Only second and third podomeres of endopodite differing from those of adult male. Second podomere, with one seta on dorsodistal end and six annulated setae at distal consisting of four long, one medium, one short. Third podomere one-quarter as long as first podomere, one long annulated seta at ventral margin, and two long annulated and one short setae at distal end. Uropod (Fig. 22B). Each lamella with four claws and one short proximal spine dorsally. Both lamella with three spines and one rows of setae on anteroventral surface. Female copulatory organ (Fig. 22B). Female spermatheca bearing villus-like structure in lateral view. Dimensions See Table 4. Distribution Miho-uchihama Beach (Shizuoka Pref. type locality 35 00 51 N, 138 31 07 E), Kiiohsima-sue (Wakayama Pref. 33 27 12 N, 135 49 20 E). All specimens were

MOLECULAR PHYLOGENY OF POLYCOPIDAE 307 collected by Hayato Tanaka from interstitial pore water in Japan. Etymology Subtidal, referring to the habitat of this species. Remarks The carapace and appendages of Pa. subtidalis sp. nov. are similar to those of Parapolycope psittacina Tanaka & Tsukagoshi, 2013a. The most notable difference between the two species is in the morphology of the male upper lip, which in Pa. subtidalis bears a slender projection, which is absent in Pa. psittacina. The chaetotaxy of the mandibula and fifth limb of the two species are also different. PARAPOLYCOPE MIURENSIS SP. NOV. (FIGS 24 30) Type series Holotype: adult male (SUM-CO-2236), right valve length 280 μm and height 207 μm, left valve length 273 μm and height 205 μm, soft parts mounted on slide and valves preserved in a cardboard cell slide, Paratypes: ten adult males (SUM-CO-2237 2246) and eight adult females (SUM-CO-2247 2254). Type locality The holotype specimen was collected from the sandy beach (Maguchi Beach) near the Maguchi fishing port, Miura City, Kanagawa Prefecture, on the Pacific coast of central Japan, 35 08 45 N, 139 40 48 E (Fig. 1D), on 25 May 2009; interstitial environment at 30 cm below the sand surface at low tide shoreline. The substrate consisted mainly of coarse sand with shell fragments. Diagnosis Carapace nearly elliptical in lateral view. Carapace surface covered with a number of shallow pits. Serration along anterior margin with 25 and 23 sharp processes on right valve and left valve, respectively. Anterodorsal part of male upper lip with slender, tapering spine curving around anterior to ventral margin. Basis of fifth limb bearing three dorsal plumose setae. Uropodal projection of male short, straight, covered with numerous small spinules or barbs. Male copulatory organ consisting of a wavy tube. Description of adult male Carapace (Figs 24, 25A E, 26). Right valve length 253 280 μm and height 188 207 μm, left valve length 244 273 μm and height 185 205 μm (Table 5). Carapace nearly elliptical in lateral view. Carapace surface covered with a number of shallow pits (Fig. 25A, B). Serration along anterior margin with 25 and 23 sharp processes on right valve and left valve, respectively (Fig. 24). Adductor muscle scars round and consisting of three closely spaced scars (Fig. 25A, B). Marginal infold of each valve developed along anterior to posteroventral margins (Figs 25C, D, 26B, G, H, N). Along inner margin of right valve, bar and groove on anterodorsal to dorsal end (Fig. 26A), one socket (part of hinge structure) developed at dorsal end (Fig. 26C), posterior bar and groove on mid-dorsal to posterior end (Fig. 26D, E), bar and groove on posterior to mid-posteroventral end (Fig. 26F). Along inner margin of left valve, bar on anterodorsal to dorsal end (Fig. 26I), one knob (part of hinge structure) developed at dorsal end (Fig. 26J), bar on middorsal to posterior end (Fig. 26K, L), and bar along posteroventral margin (Fig. 26M). Bellonci organ. Absent. Upper lip (Figs 27A, 28A, B). Distal part with slender spine extending from anterior to ventral margins. Antennula (Fig. 27B). Uniramous, four articulated podomeres. First podomere quadrate, with tufts of setulae on dorsal margin, lateral surface. Second podomere almost same length as first podomere, proximal two-thirds of dorsal cuticle thick and with inward Figure 24. Parapolycope miurensis sp. nov., male holotype (SUM-CO-2236). A, internal lateral view of left valve; B, internal lateral view of right valve. Scale bar = 100 μm.

308 H. TANAKA ET AL. Figure 25. Scanning electron micrographs of valves of Parapolycope miurensis sp. nov. A, B, male paratype (SUM-CO-2237); C, D, male paratype (SUM-CO-2238); E, male paratype (SUM-CO-2239); F I, female paratype (SUM- CO-2247); J, female paratype (SUM-CO-2248). A, external lateral view of right valve (RV); B, external lateral view of left valve (LV); C, internal lateral view of LV; D, internal lateral view of RV; E, dorsal view of carapace; F, external lateral view of RV; G, external lateral view of LV; H, internal lateral view of LV; I, internal lateral view of RV; J, dorsal view of carapace. Scale bar = 50 μm. bulge, with one annulated setulous seta at dorsoproximal end, one cluster of setae on distal third of dorsal margin, setulae on dorsoproximal and dorsodistal margins, and two tufts of setulae on lateral surface. Third podomere about three-quarters as long as first podomere, with one short seta at dorsodistal end and five ventrodistal setae consisting of one with large, disc-shaped sucker, one with minute setulae, one with comb-like setulae, and two annulated setae. Fourth podomere small, with five long annulated setae. Antenna (Fig. 27C, C ). Typically biramous, with exopodite and endopodite consisting of nine and three podomeres, respectively. Basis rectangular, tapered distally. Exopodite: first podomere one-third as long as basis. Second podomere one-fifth as long as first podomere; podomere length decreasing in size from third to eighth, each podomere with one long annulated seta; ninth (distal-most) podomere very small, with one long annulated seta at proximal, one medium annulated seta with short process and setulae, and one short annulated

MOLECULAR PHYLOGENY OF POLYCOPIDAE 309 Figure 26. Scanning electron micrographs of valves of Parapolycope miurensis sp. nov., internal lateral view, male paratype (SUM-CO-2238). A G, right valve; H N, left valve. A, anterodorsal bar and groove; B, anterior part of marginal infold; C, socket at dorsal end of hinge structure; D, posterodorsal bar and groove of hinge structure; E, posterior element of hinge structure; F, posteroventral bar and groove; G ventral part of marginal infold; H, anterior part of marginal infold; I, anterodorsal bar; J, knob at dorsal end of hinge structure; K, posterodorsal bar of hinge structure; L, posterior element of hinge structure; M, posteroventral bar; N, ventral part of marginal infold. seta at distal end. Endopodite (Fig. 27C ): first podomere about one-third as long as basis. Second podomere twothirds as long as first podomere, with three setae along dorsal margin consisting of one medium and two short, and five annulated setae at distal consisting of two long and three medium. Third podomere one-quarter as long as first podomere, with one stout, hook-shaped claw extending backward, one long annulated seta at ventral margin, and two long and one short annulated setae at distal end. Mandibula (Fig. 27D). Coxal endite with four sharp teeth. Basis with three plumose annulated setae on ventral margin and one plumose seta on dorsal margin. Endopodite consisting of two podomeres. First podomere with one plumose seta on ventral margin near proximal end and two long plumose annulated

310 H. TANAKA ET AL. Figure 27. Parapolycope miurensis sp. nov. A F, male holotype (SUM-CO-2236). A, left lateral view of upper lip; B, antennula, arrowhead indicates inward bulge; C, antenna (A2); C, endopodite of A2; D, mandibula; E, maxillula (Mxl); E, precoxa of Mxl; E, coxa of Mxl; F, fifth limb. Abbreviations: ba, basis; cx, coxa; en, endopodite; ep, epipodite; ex, exopodite; pc, precoxa. Scale bar = 50 μm. setae at dorsodistal end. Second podomere very small with setulae on dorsal margin, bearing one medially plumose and one claw-like setulous seta. Maxillula (Fig. 27E, E,E ). Precoxa (Fig. 27E ) with eight plumose setae of different lengths. Coxa (Fig. 27E ) with one short and one medium plumose seta on lateral surface near ventroproximal margin and ventral middle margin, respectively, and one medium and one long plumose seta on ventral margin. Basis with one medium and two long plumose setae on ventral margin, and setulae along dorsal margin. First podomere of endopodite with one setulous seta dorsodistally, and two long

MOLECULAR PHYLOGENY OF POLYCOPIDAE 311 Figure 28. Scanning electron micrographs of male soft parts of Parapolycope miurensis sp. nov. A, male paratype (SUM-CO-2240); B, male paratype (SUM-CO-2241); C, D, male paratype (SUM-CO-2242). A, left lateral view of upper lip; B, right lateral view of upper lip; C, right lateral view of uropod and uropodal projection; D, right lateral view of uropodal projection with numerous small spines. annulated setae at ventroproximal margin. Second podomere, with two long annulated setulous setae, and two long, stout setulous setae with few bilateral spines. Exopodite consisting of two podomeres. First podomere with setulae along dorsal margin. Second podomere with one very long, stout setulous seta, one long setulous setae, two long annulated setae, three long setae on ventral margin, and one tuft of setulae on dorsal margin. Fifth limb (Fig. 27F). Coxa bearing epipodite with 12 long plumose setae, and two short setulous setae on dorsodistal margin. Basis with three plumose setae along dorsal margin and one slender setulous seta on ventral margin. Endopodite with two long plumose setae. Exopodite with one stout setulous seta. Uropod (Figs 28A, B, 29A). Left lamella with three stout claws, one short proximal spine dorsally, and two rows of setae on anteroventral surface. Right lamella with four stout claws, one short proximal spine dorsally, and one ventrodistally situated, medium uropodal projection with a number of short spines. Male copulatory organ (Fig. 29A). Arising from outer surface of body on left side of terminal trunk segment as a long, slender, curved tube. Description of adult female Mandibula, maxillula, and fifth limb similar to those of adult male. Carapace (Fig. 25F J). Right valve length 282 303 μm and height 206 220 μm, left valve length 276 293 μm and height 207 217 μm (Table 5). Upper lip (Fig. 30A). Semicircular in lateral view with very small dorsal protrusion. A number of setae on ventral lateral surface. Antennula (Fig. 30B). Uniramous, four articulated podomeres. First podomere quadrate, with tufts of setulae on dorsal margin, lateral surface. Second