Two new species in the Echinoderes coulli group (Echinoderidae, Cyclorhagida, Kinorhyncha) from the Ryukyu Islands, Japan

Abstract Two new species belonging to the Echinoderes coulli group are described with their external morphologies and sequences of nuclear 18S rRNA and 28S rRNA genes, and mitochondrial COI gene. The first species, Echinoderes komatsui sp. n., is characterized by absence of acicular spines, and presence of lateroventral tubules on segments 5 and 8, laterodorsal tubules on segment 10, inverted triangle or wide oval shaped large sieve plates, lateral terminal accessory spines in female, and short tips of ventral pectinate fringe on segment 10. The second species, Echinoderes hwiizaa sp. n., is characterized by absence of acicular spines, and presence of lateroventral tubules on segments 5 and 7–9, midlateral tubules on segment 8, laterodorsal tubules on segment 10, large narrow oval shaped sieve plates on segment 9, and thick, short and blunt lateral terminal spines about 10–15% of trunk length. The diagnostic characters and key to species of E. coulli group are provided as well.


Introduction
Echinoderes is the most species-rich genus in the marine phylum Kinorhyncha. At present, 82 Echinoderes species have been reported worldwide, from the intertidal zone to abyssal depths and from polar to tropical regions (Sørensen and Pardos 2008, Herranz et al. 2013, Neuhaus 2013, Sørensen 2013, 2014. Only a few species have been reported from brackish waters and most of these belong to the Echinoderes coulli species group. The E. coulli group was proposed for the first time by Ostmann et al. (2012), as comprising species adapted to highly fluctuating estuarine habitats. Currently the group contains seven species (Echinoderes applicitus Ostmann et al., 2012;Echinoderes coulli Higgins, 1977; Echinoderes marthae Sørensen, 2014;Echinoderes maxwelli Omer-Cooper, 1957; Echinoderes ohtsukai Yamasaki & Kajihara, 2012; Echinoderes rex Lundbye et al., 2011;and Echinoderes teretis Brown, 1999) (Omer-Cooper 1957, Higgins 1977, Brown 1985, Adrianov and Malakhov 1999, Lundbye et al. 2011, Ostmann et al. 2012, Yamasaki and Kajihara 2012, Sørensen 2014. These species share the following morphological features: (1) middorsal spines are absent, or reduced to a short spine and occur only on segment 4; (2) lateroventral acicular spines are absent, or if present, very short and occur on segments 6 and 7; (3) lateral tubules are present at least on segments 5 and 8; (4) relatively large sieve plates consisting of a sieve area and a posterior pore are present; (5) lateral terminal accessory spines are absent in both sexes. The large sieve plates, which function in osmotic regulation, appear to be adaptive to estuarine habitats.
Among the seven species in the E. coulli group, only E. rex has not been reported from an estuarine environment, but instead inhabits subtidal marine waters (Lundbye et al. 2011). Two alternative hypotheses might explain the origin of adaptations to brackish water environments: (1) the adaptive characters arose only once, and E. rex has secondarily returned to a fully marine environment, or (2) adaptive characters arose several times independently in the E. coulli group (Ostmann et al. 2012, Yamasaki andKajihara 2012). No phylogenetic analysis has been performed to determine which hypothesis is correct yet.
In this paper we describe two new species of the E. coulli group collected from the Ryukyu Islands, southern Japan. In addition to the morphological descriptions, we include the sequences of three genes for each of these species. We also summarize the morphological diagnostic characters for the group and provide a key to species.

Materials and methods
Sediment samples were taken at two stations by hand at low tide (Fig. 1). Station 1 is an intertidal flat with a mangrove area in Oura Bay, Okinawa Island, Japan (26°33.35'N, 128°2.57'E); samples were collected on 26 May 2013 and 8 July 2013. Station 2 is an intertidal flat in Kabira Bay, Ishigaki Island, Japan (24°27.58'N, 124°8.57'E); samples were collected on 23 June 2013. All samples consisted of mud mixed with sand, without black sulfide sediments.
Kinorhynchs were extracted from the samples by using the bubbling and blot method (Higgins 1988, Sørensen andPardos 2008). Extracted animals including kinorhynchs were washed with fresh water and preserved in 99% EtOH. In the laboratory, kinorhynch specimens were sorted under a stereomicroscope. Some specimens were used for DNA extraction. The others were observed by light microscopy (LM) or scanning electron microscopy (SEM). Total genomic DNA was extracted from selected single individuals with a DNeasy Tissue Kit (Qiagen, Tokyo), following the protocol of Yamasaki et al. (2013). After DNA extraction, the exoskeleton of each specimen was mounted for LM as described below. Parts of the nuclear 18S and 28S rRNA genes and the mitochondrial COI gene were amplified by PCR using primer sets 18S-F1/18S-R9 for 18S; 28S-01/28Sr, 28Sf/28S-3KR, and 28S-2KF/28jj-3' for 28S; and LCO1490/HCO2198 for COI (see Table 1 for primer references and sequences). PCR conditions for 18S and 28S were 95 °C for 1 min; 35 cycles of 95 °C for 30 sec, 45 °C for 1 min 30 sec, and 72 °C for 3 min; and 72 °C for 7 min. Conditions for COI were 95 °C for 1 min; 35 cycles of 95 °C for 30 sec, 45 °C for 1 min 30 sec, and 72 °C for 90 sec; and 72 °C for 7 min. All nucleotide sequences were determined by direct sequencing with a BigDye Terminator Kit ver. 3.1 (Life Technologies, Co., USA) and a 3730 DNA Analyzer (Life Technologies, Co., USA). Sequence fragments were assembled by using MEGA 5 (Tamura et al. 2011). After assembly, sequences were deposited in GenBank under accession numbers AB899164-AB899171.  Folmer et al. (1994) Specimens for LM were transferred into dehydrated glycerin to replace the ethanol with glycerin and were then mounted in Fluoromount G® between two cover slips attached to a plastic H-S slide. They were observed, sketched, and photographed with an Olympus BX51 microscope equipped with a Nikon DS-Fi1c camera and a drawing tube. Line illustrations were drawn in Adobe Illustrator CS5, based on scanned camera lucida drawings of mounted specimens. Measurements were made with a Nikon DS-L3 camera control unit.
Specimens for SEM were immersed in 100% butanol for several minutes, freeze dried, mounted on aluminum stubs, sputter-coated with gold-palladium, and observed with a JEOL JSM-6060LV scanning electron microscope at 15 kV accelerating voltage.

Order
Paratypes: Three adult females and two adult males RUMF-ZK-00003-00007); two exoskeletons (RUMF-ZK-00008-00009) from DNA-extracted specimens (one adult female and one adult male); all collected at same locality as the holotype; all mounted in Fluoromount G®. Paratype RUMF-ZK-00003 was collected on 26 May 2013, and the others on 8 July 2013.
Additional material: Six specimens for SEM (one adult female, three adult males, and two adults gender undetermined), collected at same locality as holotype on 8 July 2013, mounted on aluminum stubs.
Diagnosis. Echinoderes without acicular spines; lateroventral tubules present on segments 5 and 8, laterodorsal tubules on segment 10, and large, sieve plates on segment 9 with an inverted triangular or oval shape; females with lateral terminal accessory spines. Pectinate fringe of the sternal plate on segment 10 with short tips.
Head consists of retractable mouth cone and introvert (Figs 4E,5). Mouth cone with inner oral styles and nine outer oral styles. Exact number and arrangement of inner oral styles not examined. Each outer oral style consists of rectangular basal part and triangular distal part (Fig. 4B). Basal parts of outer oral styles alternating in size: five large in odd sectors of introvert, and four small in even sectors (Figs 4B, 5). Intro- Table 2. Measurements for adult Echinoderes komatsui sp. n. (in micrometers). Columns N and SD show sample size and standard deviation, respectively. Abbreviations: (f ), female condition of sexually dimorphic character; LD, length of laterodorsal tubule; LTAS, length of lateral terminal accessory spine; LTS, length of lateral terminal spine; LV, length of lateroventral tubule; (m), male condition of sexually dimorphic character; MSW, maximum sternal width; S, segment length; SW, standard width; TL, trunk length. vert composed of seven rings of spinoscalids and one ring of trichoscalids (Figs 4E, 5). Ring 01 includes ten primary spinoscalids each with basal sheath and smooth long end piece (Fig. 4E). Each basal sheath with three overlapping fringes. Proximal fringe extends into three flat projections, like a trident, covering next fringe. Middle fringe with two lateral projections overlapping end piece. Distal fringe with five threads projecting between two projections of middle fringe. End piece of primary spinoscalids is longest unit. Rings 02 and 04 with 10 spinoscalids; rings 03 and 05 with 20 spinoscalids. Spinoscalids of rings 02-05 equal length. Rings 06 and 07 not examined in detail, but ring 06 with at least seven relatively short spinoscalids, and ring 07 with nine leaf-like scalids (Fig. 5). Six trichoscalids attached with trichoscalid plate in sectors 2, 4, 5, 7, 8, and 10. Neck with 16 placids (Figs 2A, B, 4D, 5). Midventral placid broadest (ca. 18 μm at basal width and ca. 10 μm at tip width). Remaining placids similar in size, but differ alternately in tip width (ca. 12 μm at basal width and ca. 5-6 μm at tip width) (Fig. 4D).

Character
Segment 1 consists of complete cuticular ring with thick pachycyclus at anterior margin. Bracteate cuticular hairs densely cover entire dorsal side, and posterior area of ventral side ( Fig. 2A, B). Rounded subdorsal and laterodorsal sensory spots located close to anterior margin of the segment ( Fig. 2A). Rounded ventrolateral sensory spots located central between anterior and posterior segment margins (Figs 2B, 4G). Type 1 glandular cell outlets situated anteriorly in middorsal and lateroventral positions ( Fig. 2A, B). Posterior part of the segment with pectinate fringe showing longer fringe tips laterally ( Fig. 2A, B). Segment 2 with complete cuticular ring, like segment 1 ( Fig. 2A, B). This and following eight segments with thick pachicycli at anterior margins. Bracteate cuticular hairs densely cover whole area. One oval sensory spot in middorsal position, two pairs in laterodorsal position, and one pair in ventrolateral position (Figs 2A, B, 4F, G). All sensory spots central in position. Two pairs of type 2 glandular cell outlets in both subdorsal and laterodorsal positions (Figs 2A, B, 4C, F). Pair of type 2 glandular cell outlets in lateroventral position. All type 2 glandular cell outlets of segment 2 and following six segments situated centrally of the segment. In LM observation, type 2 glandular cell outlets show funnel shaped structure, whereas in SEM observation, they . Echinoderes komatsui sp. n., scanning electron micrographs. A Entire animal, lateral view B outer oral styles, lateral view C close up of laterodorsal type 2 glandular cell outlet on segment 2 D neck, overview E partly retracted head, overview F segments 2-3, lateral view G segments 1-3, ventral view. Complete circles indicate type 2 glandular cell outlets; dashed circles indicate sensory spots. Abbreviations: mvp, midventral placid; oos, outer oral style; pss, primary spinoscalid; sp, spinoscalid followed by ring number.
show single small pore in slightly protruded cuticular surface (Fig. 4C). Posterior margin of the segment ends as pectinate fringe showing longer tips than tips of preceding segment (Figs 2A, B, 4F).
Segment 3 and following eight segments consist of one tergal and two sternal plates ( Fig. 2A, B). This and following seven segments entirely covered with bracteate cuticular hairs except for anterior area (Figs 2A, B, 3B, C). Paired sensory spots in subdorsal and midlateral positions (Figs 2A, B, 4F). Type 1 glandular cell outlets of segment 3 and following seven segments situated at anterior part of segment in ventromedial position (Fig. 2B).Pair of type 2 glandular cell outlets in subdorsal and midlateral positions ( Fig.  2A, B). Pectinate fringe on segment 3 and five following segments as on segment 2. Segment 4 without sensory spots. Type 2 glandular cell outlets in subdorsal, laterodorsal, and lateroventral positions ( Fig. 2A, B).
Segment 9 with two pairs of subdorsal sensory spots and one pair of laterodorsal and ventrolateral sensory spots (Figs 2A, B, 3C, 6B). Pair of sieve plates with wide sieve area and single posterior pore situated in midlateral position. Sieve area variable in shape, forming inverted triangle in some specimens and broad oval in others (Figs 2A, B, 3C, 6B). Tips of pectinate fringe slightly shorter in length than on preceding segment.
Segment 10 with thin laterodorsal tubules (Figs 2A, 6C). Length of laterodorsal tubules in males about twice as long as those in females. Paired subdorsal and ventrolateral sensory spots situated close to posterior margin of segment (Figs 2, 3D, 6C, D). Posterior middorsal margin elongated, extending to segment 11 in some specimens (Figs 2A, 6C), but truncate in other specimens. Posterior margin ends as pectinate fringe with short tips, except in ventrolateral area, which without pectination.
Etymology. The species is named after Dr H. Komatsu (National Museum of Nature and Science, Tokyo, Japan), a taxonomist of brachyuran crabs and the first person to find Echinoderes komatsui sp. n.
Paratypes: Two adult females and two adult males (RUMF-ZK-00012-00015); three exoskeletons (RUMF-ZK-00016-00018) from specimens used for DNA extraction (one adult female and two adult males); all collected at the same locality as the holotype; all mounted in Fluoromount G®.
Other material: six specimens for SEM (four adult females, one adult male, and one adult gender undetermined), collected at the same locality as the holotype, mounted on aluminum stubs.

Description. Adult with head, neck and eleven trunk segments
Head consists of retractable mouth cone and introvert (Figs 9A, B, 10). Mouth cone with inner oral styles and nine outer oral styles. Exact number and arrangement of inner oral styles not observed. Each outer oral style composed of rectangular basal part and triangular distal part. Basal parts of outer oral styles alternate in size: five large in odd sectors of introvert, and four small in even sectors (Fig. 9A). Posterior to basal part of each outer oral style, two spinose hairs project anteriorly, covering outer oral style (Fig. 9A). Introvert composed of seven rings of scalids and one ring of tri-  (Figs 9B, 10). Ring 01 includes ten primary spinoscalids with basal sheath and long, smooth end piece (Fig. 9B). Each basal sheath with three fringes. Proximal fringe extends into three long projections, like a trident, covering next fringe. Middle basal fringe with two lateral projections, overlapping end piece. Distal fringe with five to seven threads projecting between two projections of middle fringe. End piece of primary spinoscalids is longest unit. Rings 02 and 04 with 10 spinoscalids, and rings 03 and 05 with 20 spinoscalids. Spinoscalids of rings 02-05 similar in length. Rings 06 and 07 could not be examined in detail, but at least seven relatively short spinoscalids present in ring 06, and 13 leaf-like scalids in ring 07. Six trichoscalids present each attached with trichoscalid plate in sectors 2, 4, 5, 7, 8, and 10. Neck with 16 placids (Figs 7A, B, 8B, 10). Midventral placid broadest (ca. 17 μm at basal width and ca. 11 μm at tip width); remaining placids with similar size (ca. 11 μm at basal width and ca. 5 μm at tip width).

Position MD PD
Segment 4 with pair of laterodorsal sensory spots and paired subdorsal type 2 glandular cell outlets (Fig. 7A, B). Pectinate fringes as on segment 2.

Discussion
The Echinoderes coulli group previously accommodated seven species: E. coulli, E. applicitus, E. marthae, E. maxwelli, E. ohtsukai, E. rex, and E. teretis (Omer-Cooper 1957, Higgins 1977, Brown 1985, Lundbye et al. 2011, Ostmann et al. 2012, Yamasaki and Kajihara 2012. These species share the following characters: (1) absence of middorsal acicular spines, or presence of a single spine on segment 4; (2) absence of lateroventral acicular spines, or presence of very short ones only on segments 6 and 7; (3) lateral tubules on at least segments 5 and 8; (4) relatively large sieve plates consisting of a sieve area and a posterior pore; (5) lateral terminal accessory spines lacking in both sexes. In addition, all species except for E. rex were reported from intertidal brackish habitats, such as intertidal flats or mangrove areas, where other echinoderid species are rarely found. These morphological and ecological similarities have been viewed as an evidence to consider the E. coulli group a monophyletic group.
Both their morphological characters and habitats suggest that E. komatsui sp. n. and E. hwiizaa sp. n. are closely related to the seven species which are known as members of the E. coulli group, and the two new species also belong to the group. Two new species both lack spines on segments 1-10 completely, have lateroventral tubules on segments 5 and 8 (additionally on segments 7 and 9, and midlateral tubules on segment 8 in E. hwiizaa sp. n.), and have relatively large sieve plates. In addition, E. komatsui sp. n. was collected in a mangrove area and E. hwiizaa sp. n. on an intertidal flat, both areas of variable and often reduced salinity.
Echinoderes komatsui sp. n. and E. hwiizaa sp. n. differ from other species in the E. coulli group in one particular feature, namely the lack of lateral terminal accessory spines in females. The former two species possess lateral terminal accessory spines in females, however, these spines are very short and seem to be poorly developed. One possibility is that the previous seven species included in the E. coulli group are more closely related to one another rather than to any of the two new species. However, the relationships within the group have never been examined with a cladistic analysis. Since the relationships within the group are still open to question, the future phylogenetic studies using more abundant morphological data and/or molecular markers are needed.
Below follows a revised diagnosis of the E. coulli group and a dichotomous key to species in the group, modified from Sørensen (2014).

Diagnosis of Echinoderes coulli group
Echinoderes without acicular middorsal spines, or with a very short spine only on segment 4; lateral spines very short on segments 6 and 7, or absent; midlateral, sublateral, lateral accessory, or lateroventral tubules on segments 5 and 8; sieve plates relatively large, consisting of oval or inverted-triangular sieve area and single posterior pore; lateral terminal accessory spines poorly developed or completely absent in females. Lateroventral tubules absent on segments 7 and 9; lateral terminal spines thin, pointed, and measuring more than 20% of trunk length ....E. marthae