Echinoderes rex n . sp . ( Kinorhyncha : Cyclorhagida ) , the largest Echinoderes species found so far

A new kinorhynch species, Echinoderes rex n. sp., is described from the Korea Strait. The new species is characterized by a pair of diminutive lateral terminal spines (19-23 μm) and a trunk length of 482-528 μm, making it the largest Echinoderes described so far. Unique for the new species is also the presence of a type of putative glandular cell outlets that have not been described previously. This paper presents light and scanning electron micrographs of the new structure. In addition, E. rex n. sp. is characterized by having a single middorsal spine on segment 4, lateroventral tubules on segment 5 and 8 and lateroventral acicular spines on segments 6 and 7. A pair of distinct and conspicuously large sieve plates, only described from two other species of the genus, is present on segment 9 in E. rex n. sp. A comparison is made with other species of Echinoderes and the similarities are discussed.


INTRODUCTION
Echinoderes Claparède, 1863 is with its 67 valid species the most diverse kinorhynch genus.The genus is characterized by the presence of 16 placids in the neck region (midventral placid usually the broadest), the first two segments always consisting of closed cuticular rings and the following nine segments consisting of one tergal and two sternal plates.Middorsal spines are present in some species, usually restricted to segments 4 to 8; lateral terminal spines are present in all species; and lateral terminal accessory spines are-if present in the species-restricted to females.A midterminal spine is never present in adults (Higgins, 1990;Sørensen and Pardos, 2008).
Among species of Echinoderes the sexes are usually distinguished by the presence of lateral terminal accessory spines in females only, and pairs of short penile spines in males.However, females of some species deviate from this pattern as they lack lateral terminal accessory spines.These species include Echinoderes coulli Higgins, 1977, E. isabelae G a Ordóñez et al.,  2007, E. capitatus Zelinka, 1928, E. maxwelli Omer-Cooper, 1957 and E. teretis Brown, 1985 (See Zelinka,  1928; Omer-Cooper, 1957; Higgins, 1977; Brown,  1985; Nebelsick, 1992; G a Ordóñez et al., 2007).All of these species, except E. coulli but including E. cantabricus Pardos et al., 1998, also share another feature that is unusual for the genus: the presence of only one middorsal spine located on segment 4.
Sieve plates in species of Echinoderes are oftenif observed-described as circular, small clusters of pores, often visible in scanning electron microscope only (Kristensen and Higgins, 1991;Pardos et al., 1998).Large and distinctive sieve plates consisting of an elongated pore field and a posteriorly located nonporous plate with one single pore centred was described for the first time from E. coulli (see Kristensen and Higgins, 1991).Similar sieve plates have only been observed in one other species, E. teretis (see Brown, 1985).
In the present study we describe a new species of Echinoderes, E. rex n. sp., from the western Pacific Ocean, based on light and scanning electron microscopical examinations.It is the largest species in the genus known so far, with an average trunk length of 505 µm.The common sexual dimorphism found in Echinoderes is not present in this new species because paired lateral terminal accessory spines are lacking in females.Presence of middorsal spines is restricted to segment 4 and-for the first time since the description of E. coulli and E. teretis-a pair of similar distinctive sieve plates is observed.(Kristensen and Higgins, 1984;Sørensen and Pardos, 2008) and extracted in the field through a 63 µm mesh sieve.The concentrated sample was fixed in 4% formalin mixed with sea water, and the meiofauna was subsequently extracted by flotation in Ludox ® (DuPont) HS 40 (Burgess, 2001).The kinorhynch specimens were sorted out from the mixed meiobenthos under a highmagnification LEICA MZ 8 stereomicroscope.Speci-Specimens for scanning electron microscopy (SEM) were dehydrated through a graded series of ethanol, trans-ferred to acetone and critical-point dried.The dried specimens were mounted on aluminum stubs, sputter coated and examined with a JEOL JSM-6335F field emission scanning electron microscope.Specimens for light microscopy (LM) were transferred to distilled water, dehydrated through a graded series of glycerin and mounted in Fluoromount G ® .The mounted specimens were examined and photographed using Nomarski differential interference contrast with an Olympus BX60 microscope equipped with an Olympus DP20 camera.Measurements were made with Cell^D software for analysis of light microscopical photos.

Echinoderes
The terminology in the taxonomic account follows Pardos et al. (1998), Neuhaus and Higgins (2002), and Sørensen and Pardos (2008).For comparison, type specimens of Echinoderes coulli were loaned from the Smithsonian Institution: paratypic females type I, ranging from accession number USNM ; paratypic females type II, ranging from accession number USNM .Echinoderes maxwelli: holotypic male, accession number 1957:12:22:1 and allotypic female, accession number 1957:12:22:2 were loaned from the British Museum of Natural History.Furthermore, specimens of Echinoderes teretis were used for comparison.The specimens were collected in Candlagan Creek, 247 km south of Sydney, Australia, and were used in a previous study by Nicholas and Sørensen (2009)   on segment 10. Males with two lateral penile spines, females without lateral terminal accessory spines.Putative type II glandular cell outlets with conspicuous tuft of cuticular extensions present in various positions.Segment 9 with large, elongated sieve plate, located in a sublateral position, anterior to distinct pore.
Etymology.The species name "rex" is from Latin, meaning "king", and refers to the species' distinct and prominent appearance in light microscopy, as well as its considerable trunk length, making it the largest of all known species of Echinoderes.
Description.The adult specimen consists of a head, a neck and eleven trunk segments (Figs 1A,2A,3).Measurements and dimensions are given in Table 1.
A summary of sensory spot, spine and tubule positions is provided in Table 2. Scalid distribution could not be observed.
Segment 1 consists of one complete cuticular ring.The cuticle on this, and the following segments, appears to be relatively thick.Pachycycli are well-developed along the anterior margin of this and the second segment (Fig. 2A).Pairs of laterodorsal and midlateral rounded sensory spots are present near the anterior margin of the segment.A pair of ventrolateral rounded sensory spots is located halfway down the segment.These sensory spots, and all other found on this species, belong to type 1 and consist of a droplet-shaped field of several small, pointed cuticular papillae and two pores (Fig. 1B).A type I glandular cell outlet is present in the middorsal position.Cuticular hairs on this and all following segments emerge through slit-like perforation sites that are typical for bracteate hairs.However, an actual bractea is not present on the margin of the slit (see Fig. 1B-D for corresponding hairs on other segments).Perforation sites are densely scattered over the surface of the segment without any characteristic pattern.On this and the following nine segments, the posterior segment margin terminates into a pectinate fringe with slender, pointed and relatively long fringe tips.
Segment 2 consists of one complete cuticular ring.The cuticular structures found in the middorsal to sublateral positions on the tergal plate show sexual dimorphism.Females have a middorsal, droplet-shaped sensory spot and a pair of laterodorsal and midlateral droplet-shaped sensory spots.A pair of "modified type II glandular cell outlets" (Fig. 2F) is located subdorsally.These structures consist of a large opening surrounded by a tuft of cuticular extensions (Fig. 1D,E).Numerous minute cuticular papillae are located in a circle around the elongated fringes.The cuticle has fur-   Segment 4 possesses a short, soft middorsal acicular spine (12-17 µm) (Fig. 3A).A pair of droplet-shaped sensory spots is found in the laterodorsal position.Pairs of type I and modified type II glandular cell outlets are located in a subdorsal position.Furthermore, a pair of type I glandular cell outlets is found ventromedially and modified type II glandular cell outlets are located in the lateral accessory position.Perforation sites as on previous segments (Fig. 3A).These patches are found from segment 4 to 10. Segment 5 with one pair of short lateroventral tubules located close to the posterior margin of the segment (Fig. 3B).Paired droplet-shaped sensory spots are located in the subdorsal, midlateral and ventromedial positions.Furthermore pairs of type I glandular cell outlets are found subdorsally and ventromedially.
Segments 6 and 7 with one pair of short, lateroventral acicular spines (Fig. 3B).Sensory spots and type I glandular cell outlets as on segment 5. Lateral accessory modified type II glandular cell outlets are located on the posterior part of the segment.
Segment 8 with one pair of lateroventral tubules (Fig. 3B), one pair of droplet-shaped sensory spots and type I glandular cell outlets located in the subdorsal position (Fig. 3A).Type I glandular cell outlets furthermore located ventromedially.Paired modified type II glandular cell outlets present in the lateral accessory and midlateral positions.One additional middorsal modified type II glandular cell outlet was furthermore observed in a single specimen.
Segment 9 with one pair of subdorsal, midlateral and ventrolateral droplet-shaped sensory spots.In one specimen one pair of sensory spots was also found in the laterodorsal position.Type I glandular cell outlets located subdorsally and ventromedially.A pair of well-developed, large (15-21 µm) sieve-plate openings is located sublaterally (Fig. 3B).It consists of an elongated porous plate and a posterior pore situated in a nonporous, slightly prolapsed plate that overlaps with the outermost posterior part of the big porous plate (Figs.1C, 4A).
Segment 10 with a pair of short, but stout and slightly hook-shaped laterodorsal tubules that extend from the posteriormost part of the segment.In SEM they appear to extend from the intersegmental joint between segment 10 and 11, but LM reveals that they originate from segment 10.A pair of droplet-shaped sensory spots is present ventrolaterally.Six out of the nine examined animals furthermore have a pair of droplet-shaped sensory spots located subdorsally.Type I glandular cell outlets are located subdorsally and ventromedially.One additional middorsal type I glandular cell outlet was furthermore observed in a single specimen.The posterior margins of the sternal plates are rounded ventromedially.This feature is more distinct in females (Figs.3B, D, 5A,C).
Segment 11 with a pair of subdorsal droplet-shaped sensory spots in some specimens, but these are lacking in others.The lateral terminal spines (LTS) are remarkably short (Figs.2A, 3, 5A-C) ranging from 20 to 24 µm.Lateral terminal accessory spines are lacking in both sexes (Fig. 3).In females the gonopores are vis-ible ventrolaterally as two distinct protuberances (Fig. 5A).Males with two pairs of short and inconspicuous penile spines in a lateral accessory position and with longer lateral terminal spines than found in females (Fig. 5B,D).One of the penile spines is tubule-shaped and fringed (Fig. 5C,D).The tergal plates terminate into two, paired tergal extensions.The tergal extension forms a pair of short but stout spinous projections (Fig. 3).The posterior margin in between the tergal extensions is densely fringed and more or less straight in males, but more rounded in females (Fig. 5A,C).

Notes on diagnostic features
Echinoderes rex n. sp. is easily recognized by its diminutive lateral terminal spines (19-23 µm) (Figs 1A, 2A, 3A,B) and the length of its body (482-528 µm), making it the largest Echinoderes species described so far.It can also be distinguished by its spine/tubule composition that includes a single middorsal spine on segment 4, lateroventral tubules on segment 5 and 8 and lateroventral acicular spines on segments 6 and 7.The characteristic fringed cuticular structures, referred to as modified type II glandular cell outlets, are unique for the species and have not, to our knowledge, been reported previously from any kinorhynch species.Furthermore, the species is characterized by its large and distinctive sieve plates and the lack of lateral terminal accessory spines in females.The latter feature is shared only with six other known species of Echinoderes, E. capitatus, E. coulli, E. isabelae, E. maxwelli and E. teretis (see Zelinka, 1928; Omer-Cooper, 1957; Higgins, 1977; Brown, 1985; Nebelsick, 1992; G a Ordóñez  et al., 2007) in addition to a yet undescribed species of Echinoderes from Spain (Herranz and Pardos, pers. com.).
Of the six other species that lack lateral terminal accessory spines in the females, E. teretis is the species that most resembles E. rex n. sp. in having only one middorsal spine on segment 4, lateroventral tubules on segment 5 and 8 and lateroventral spines on segment 6 and 7. Echinoderes teretis differs from E. rex n. sp. by its remarkably smaller trunk length (207-264 µm) and overall trunk shape with conspicuously broad segments 4 to 6 (Fig. 2C).
Echinoderes rex n. sp. has only two pairs of penile spines, both located in a lateral position, whereas E. teretis possesses two pairs of lateral and one pair of dorsal penile spines.The length of the lateral terminal spines is furthermore considerably longer in E. teretis, being 106-141 µm (46-65% of trunk length) in this species, as opposed to only 20-24 µm (4% of trunk length) in E. rex n. sp.
Echinoderes maxwelli (Fig. 2D) has the same overall body shape as E. rex n. sp.although the trunk length differs (328 µm in E. maxwelli vs. 482-528 µm in E. rex n. sp.).Based on the original description of E. maxwelli (see Omer-Cooper, 1957) and the corrections and additional notes by Higgins (1977), the two species resemble each other in having one pair of tubules lateroventrally on segments 5 and 8, and males with only two pairs of penile spines, although penile spines in E. maxwelli are longer (1/6 the length of the lateral terminal spines) than those in E. rex n. sp.The lengths of the lateral terminal spines also differ considerably, being 184-212 µm (56-66% of the trunk length) in E. maxwelli.Type material was loaned from British Museum of Natural History, and based on our examinations we found the following additional similarity: E. maxwelli has a pair of diminutive spines in the lateroventral positions of segment 6 and 7 (Fig. 4E).Furthermore, we found characters that in LM could be interpreted as the same kind of modified type II glandular cell outlets (Fig. 2E) as observed in E. rex n. sp.However, examination with SEM would be required to confirm this.An inconspicuous, short, flexible structure that could be interpreted as a spine was found middorsally on segment 4 (Fig. 4F) but to ensure this, further examination is required.Opposite to E. rex n. sp., E. maxwelli has no perforation sites on the ventral side of the 1 st segment.The sieve-plate is remarkably large and triangular in shape extending from the lateroventral side to a more laterodorsal position (Fig. 4D).Echinoderes capitatus and E. rex n. sp.share the presence of a middorsal spine on segment 4 only, and the presence of paired ventrolateral tubules on segments 5 and 8.However, they differ regarding their distribution patterns and numbers of the tubules.Whereas E. rex n. sp.possesses tubules on segments 5 and 8, in the lateroventral position on both segments, E. capitatus has numerous tubules distributed in subdorsal to ventromedial positions between segments 2 to 9 (Nebelsick, 1992).Furthermore, the total body length of E. capitatus is 270-290 µm and the lateral terminal spines are long (64-88 µm) and medially curved.
Echinoderes isabelae and E. rex n. sp.share the presence of a single middorsal spine on segment 4, paired lateroventral tubules on segment 5 and 8 and paired spines in the same positions on segment 6 and 7.The two species can be distinguished by several characters: Firstly, the trunk length of E. isabelae is 223-240 µm and its body shape is bulbous with the 1 st and 2 nd segment being the broadest (G a Ordóñez et al.,  2007).Echinoderes isabelae also has pairs of several paired tubules on segment 2, a pair of sublateral tubules on segment 7, and paired lateroventral spines on segments 6 to 9. Its lateral terminal spines are considerably longer than in E. rex n. sp., being 87-94 µm, and males have three pairs of penile spines, as opposed to only two in E. rex n. sp.
Echinoderes rex n. sp. and the previously mentioned yet undescribed species from Spain, Echinoderes nov.sp., share the presence of a single middorsal spine on segment 4 only, the lateroventral pair of tubules on segment 5 and the paired spines in the same position on segments 6 and 7.However, the undescribed species differs by its overall body shape and by being considerably shorter (270 µm) than E. rex n. sp.Echinoderes nov.sp. also has pairs of tubules in a subdorsal, laterodorsal, lateral accessory and ventrolateral position on segment 2 and pairs of tubules in a subdorsal and lateral accessory position on segment 8, together with a pair of lateroventral acicular spines on the same segment.The three pairs of penile spines are distinct and long (28µm) and the lateral terminal spines are 97 µm.
Of the 6 species that lack a lateral terminal accessory spine in the females, E. coulli (Fig. 2B) is the one that deviates the most from E. rex n. sp.Their most conspicuous similarity, apart from the lack of lateral terminal accessory spines in females, is the small, lateroventral tubules on segment 5 and 8 and the short lateral terminal spines, being 5.6% of the trunk length in the E. coulli female type II and 3.8-4.8% in E. rex n. sp.Taxonomic significant differences between them include the trunk length (348-364 µm compared to the 482-528 µm in E. rex n. sp.) and the complete absence of middorsal spines in E. coulli.Furthermore, E. coulli has three pairs of penile spines and characteristic thin, lanceolate extensions of cuticle flares at the base of the gonoporeal area (Higgins, 1977).Such flares are not present in E. rex n. sp., and the species has only two pairs of penile spines Among the six species that are characterized by the absence of lateral terminal accessory spines in females, three species share another distinctive character: the very well developed sieve-plates on segment 9, as found in E. rex n. sp.(Fig. 4A).The species that share this feature are E. rex n. sp., E. coulli (Fig. 4B) and E. teretis (Fig. 4C).In E. rex (Fig. 4A) the elongated porous plate is narrower than in the two other species, and the posterior pore that is situated in a nonporous, slightly prolapsed plate is situated somewhat more posterior than in E. coulli and E. teretis.In E. coulli (Fig. 4B) the pore is located anteriorly and closer to the posterior end of the porous plate and in E. teretis (Fig. 4C) the pore is situated in the centre of the nonporous plate.The size and appearance of the elongated porous plate in E. coulli and E. teretis are similar.
Another species of Echinoderes that resembles E. rex n. sp. is Echinoderes cantabricus Pardos et al., 1998, in having the characteristic, single, middorsal spine on segment 4. E. cantabricus resembles E. rex n. sp. in its overall body shape, though it is slightly smaller (328-408 µm).It has lateroventral tubules on segments 5 and 8 and spines in the same position on segments 6 and 7.The two species differ in the amount of tubules, E. cantabricus having a midlateral pair on segment 1 and four pairs, subdorsally, laterodorsally, sublaterally and ventrolaterally on segment 2, and the males having an additional subdorsal pair on segment 10.In addition to these differences, the sieve plate in E. cantabricus is smaller and more rounded.Females have lateral terminal accessory spines, and the two sexes show dimorphism regarding their lengths.Males of E. cantabricus have three pairs of penile spines, as opposed to only two in E. rex n. sp.
The internal relationships of Echinoderes are far from clarified.A clarification would require a prober phylogenetic analysis, and include reexamination of most of the not recently described species.However, with the description of E. rex n. sp.we see indications that may suggest potential monophyletic clades of species within the genus.For instance, the similarities discussed above could indicate a closer relationship between the species E. rex n. sp., E. capitatus, E. coulli, E. isabelae, E. maxwelli, E. teretis and the yet undescribed species of Echinoderes from Spain, which all are characterized by the absence of lateral terminal accessory spines in both sexes.Lateral terminal accessory spines in females is a sexually dimorphic character trait that is found in most species of the family Echinoderidae, including species of the genera Fissuroderes (see Neuhaus and Blasche, 2006), Polacanthoderes (see Sørensen, 2008) and Cephalorhyncha (see Adrianov and Malakhov, 1999).This would suggest that the presence of lateral terminal accessory spines is synapomorphic for all echinoderid species; the absence of these spines in the seven particular species mentioned above would thus be due to secondary reduction, and hence could be considered synapomorphic for these species.Within this potential clade, the presence of an enlarged sieve plate on segment 9 could indicate a closer relationship between E. rex n. sp., E. coulli and E. teretis.These, and other hypotheses about the interrelationships between species of Echinoderes, will hopefully be tested in a formal, phylogenetic analysis in the near future.
rex n. sp. was detected during sampling of meiobenthic animals under the KORDI (Korea Ocean Research and Development Institute) cruise of the research vessel RV Onnuri within the framework of the research programme POSEIDON (Pacific Ocean Study on Environmental and Interactions between Deep Ocean and National seas).Sediment samples were taken with a Smith McIntyre Grab from the subtidal zone of the Korea Strait.Specimens of Echinoderes rex n. sp. were collected at one station (34˚34′43″N, 127°45′52″E) at 13 m depth on May 26, 2008.The sediment consisted of mud with tiny shell particles.Meiofauna was freshwater shocked

Fig. 1 .
Fig. 1. -Scanning electron micrographs of Echinoderes rex n. sp.A, ventrolateral view; B, detail from segment 7 showing droplet shaped sensory spot as it appears in the ventrolateral positions; C, detail from segment 9 showing sieve plate consisting of an elongated porous plate and a single pore posteriorly; D, detail from midlateral position on segment 8 showing modified glandular cell outlet type II; E, detail from ventrolateral position on segment 2 showing modified glandular cell outlet type II; F, detail from segment 5 showing lateroventral tubule; G, detail from segment 6 showing lateroventral acicular spine.Abbreviations: ac, acicular spines; cf, cuticular furrows; cp, cuticular papillae; ef, elongated fringes; op, opening; po, pore; si, sieve plate; tu, tubule.

Table 2
. -Summary of nature and location of sensory spots, glandular cell outlets and spines arranged by series in Echinoderes rex n. sp.from the Korea Strait.Abbreviations: LA: lateral accessory; LD: laterodorsal; LV: lateroventral; MD: middorsal; ML: midlateral; PD: paradorsal; SD: subdorsal; SL: sublateral; VL: ventrolateral; VM: ventromedial; ac, acicular spine; f, female condition of sexual dimorphic character; gco1, glandular cell outlet type I; gco2, modified glandular cell outlet type II; lts, lateral terminal spine; m, male condition of sexual dimorphic character; si, sieve plate; ss1, sensory spot type 1 (for some sensory spots it was not possible to determine their types); tu, tubule; (!) only observed in one individual; (!!) observed in some, but not all of the examined animals.