Echinoderes pterus sp. n. showing a geographically and bathymetrically wide distribution pattern on seamounts and on the deep-sea floor in the Arctic Ocean, Atlantic Ocean, and the Mediterranean Sea (Kinorhyncha, Cyclorhagida)

Abstract Kinorhynchs rarely show a wide distribution pattern, due to their putatively low dispersal capabilities and/or limited sampling efforts. In this study, a new kinorhynch species is described, Echinoderes pterus sp. n., which shows a geographically and bathymetrically wide distribution, occurring on the Karasik Seamount and off the Svalbard Islands (Arctic Ocean), on the Sedlo Seamount (northeast Atlantic Ocean), and on the deep-sea floor off Crete and on the Anaximenes Seamount (Mediterranean Sea), at a depth range of 675–4,403 m. The new species is characterized by a combination of middorsal acicular spines on segments 4–8, laterodorsal tubes on segment 10, lateroventral tubes on segment 5, lateroventral acicular spines on segments 6–9, tufts of long hairs rising from slits in a laterodorsal position on segment 9, truncated tergal extensions on segment 11, and the absence of any type-2 gland cell outlet. The specimens belonging to the populations from the Arctic Ocean, the Sedlo Seamount, and the Mediterranean Sea show morphological variation in the thickness and length of the spines as well as in the presence/absence of ventromedial sensory spots on segment 7. The different populations are regarded as belonging to a single species because of their overlapping variable characters.


Introduction
The meiofauna, defined as the assemblage of microscopic benthic organisms passing through a 1 mm-sieve mesh and collected on a 40-63 µm-sieve mesh, is composed of various taxonomic groups, and occurs in diverse habitats including extreme environments such as polar regions, the deep sea, and seamounts (George 2013;De Broyer et al. 2014;Zeppilli et al. 2018). While meiobenthic organisms are generally thought to have a low dispersal ability because of their low mobility as well as their lack of a planktonic larval stage, some meiofaunal species can show a wide distribution pattern. This phenomenon is referred to the "meiofauna paradox" or "everything is everywhere hypothesis" (Giere 2009;Fontaneto 2011). Such wide distribution patterns have been explained or hypothesized by the stepping stone hypothesis (George 2013;Packmor and Riedl 2016), artificial dispersal (artificial invasion) (Herranz and Leander 2016;Pardos et al. 2016;Cvitković et al. 2017), or long range dispersal using currents and/ or drifting (Walters and Bell 1994;Neuhaus et al. 2014;Yamasaki et al. 2014). Some are even regarded as a pseudo-wide distribution via the detection of cryptic species (Jörger et al. 2012;Leasi et al. 2016).
Kinorhyncha is an ecdysozoan phylum which is exclusively composed of marine meiofaunal species. To date, more than 260 kinorhynchs species are known from around the world (Grzelak and Sørensen 2018a, b;Yamasaki et al. 2018). Many ecological studies on meiofauna from various regions and environments often report the presence of Kinorhyncha, but unfortunately provide only phylum-level identification (e.g., Grzelak and Kotwicki 2012;Nomaki et al. 2016;Riera et al. 2018). Most kinorhynch species have been recorded from a single or few localities within a limited region only, probably due to their low dispersal ability like other meiofaunal organisms, but most likely also because of limited sampling activities. So far, only few kinorhynch species have been recorded as geographically wide distributed species either from both shallow waters and the deep sea, e.g., Campyloderes cf. vanhoeffeni Zelinka, 1913, or from several shallow-water stations interrupted by the deep sea, e.g., Centroderes barbanigra Neuhaus et al., 2014, Echinoderes ohtsukai Yamasaki & Kajiraha, 2012, and Echinoderes tchefouensis Lou, 1934(Sørensen et al. 2012b, 2016Neuhaus et al. 2014;Herranz and Leander 2016).
In the present study, we describe a new kinorhynch species with a geographically and bathymetrically wide distribution, ranging from the Arctic Ocean to the Mediterranean Sea and from upper bathyal to lower abyssal depths. The interpopulational morphological variation of the new species is also discussed.

Materials and methods
Kinorhynchs were obtained from meiofauna samples collected from the central mount of the Karasik Seamount, Langseth Ridge in the Arctic Ocean (by the R/V Polarstern during the expedition PS101, Boetius and Purser (2017)), north of Svalbard in the Arctic Ocean (by the R/V Polarstern during the expedition PS92, Peeken (2016)), on the Sedlo Seamount in the Atlantic Ocean (by the R/V METEOR during the expedition M60/1, Christiansen and Wolff (2009)), in a deep-sea trench off Crete and on the adjacent deep-sea floor in the Mediterranean Sea (by the R/V METEOR during the expedition M71/2, Christiansen et al. (2015), and R/V Maria S. Merian during the expedition MSM14/1, Christiansen et al. (2012)), and on the Anaximenes Seamount in the Mediterranean Sea (by the R/V METEOR during the expedition M71/1, Denda and Christiansen (2011)) ( Fig. 1, Table 1). All sediment samples were fixed in 4-8% formaldehyde. Subsequently, the samples were washed with tap water on a 32-µm or a 40-µm mesh sieve in the laboratory, and the meiofauna was extracted from the sediment by centrifuging with a colloidal silica polymer (H.C. Stark, Levasil 200/40%, density 1.17 g/cm 3 ) and Kaolin, or with a colloidal silica polymer (Ludox TS50, density 1.4 g/cm 3 ). After extraction, the meiofauna was rinsed with tap water, sorted under a stereomicroscope, and subsequently preserved in 75% ethanol or 4% formaldehyde solution. Specimens collected during the expeditions PS92 and MSM14/1 were stained with Rose Bengal before sorting.
Specimens for light microscopy (LM) were dehydrated in glycerol and mounted as glycerol-paraffin slides on Cobb aluminum frames or mounted in Fluoromount G™ between two cover slips attached to a plastic H-S slide. LM specimens were observed with a Zeiss Axioskop 50 microscope, or with an Olympus BX51 microscope, and a Nikon E600 microscope. All microscopes were equipped with Nomarski differential interference contrast. A camera lucida equipped with a Zeiss Axioskop 50 microscope was used to make drafts for line art illustrations. Final line art illustrations were drawn with Adobe Illustrator CS6 based on the drafts. Measurements were made through a camera lucida or with Cell^D software. Specimens were photographed with a Zeiss AxioCam MRc5 or an Olympus DP27 camera.
Five specimens from the Karasik Seamount and 23 specimens from the Mediterranean deep sea were used for scanning electron microscopy (SEM) observation. The specimens were transferred from ethanol to distilled water through a graded series of ethanol, postfixed with OsO 4 in 0.05 M phosphate buffer (pH = 7.3) with 0.3 M sodium chloride and 0.05% sodium azide for 2.5 hours, dehydrated through a graded series of ethanol, critical-point dried with a BalTec CPD 030, mounted on aluminum stubs, sputter-coated with gold-palladium with a Polaron SC 7640, and observed with a Zeiss EVO LS 10 scanning electron microscope.
The terminology follows Neuhaus and Higgins (2002), Sørensen and Pardos (2008) and . All specimens, except those from Svalbard, have been deposited in the Museum für Naturkunde Berlin (= ZMB, former Zoological Museum Berlin), Germany, and catalogued in the collection "Vermes" in the "Generalkatalog  Tables 2 and 3 Diagnosis. Echinoderes with middorsal acicular spines on segments 4-8; laterodorsal tubes on segment 10; lateroventral tubes on segment 5; lateroventral acicular spines on segments 6-9; tufts of long hairs arising from slits in a laterodorsal position on segment 9; truncated tergal extensions on segment 11; without type-2 gland cell outlet. Etymology. The species name is derived from the Latinized Greek pterón (wing or feather), referring to the tufts of hairs on segment 9 which look like wings.
Material examined. Holotype: Adult male (ZMB 11608), collected at station 55 in the Mediterranean deep sea off Crete (Fig. 1A, E; Table 1), mounted as a glycerolparaffin slide on a Cobb aluminum frame.
Additional material for LM: all adults; seven males and 12 females, collected at station 152 on Karasik Seamount, mounted as glycerol-paraffin slides on Cobb aluminum frames (ZMB 11642-11660); one male and one female, collected at station 31 north of Svalbard, mounted in Fluoromount G (NHMD-202798 and NHMD-202799); one male and one female, collected at station 43 north of Svalbard, mounted in Fluoromount G (NHMD-202800 and NHMD-202801); one male, collected at station 717 on the Sedlo Seamount, mounted as a glycerol-paraffin slide on a glass slide (ZMB 11641) (Fig. 1A-D; Table 1).

Table 2.
Measurements of adult Echinoderes pterus sp. n. Measurements are given in micrometers, except for the ratios, and are summed for all specimens and listed separately for each population. Columns N and SD indicate sample size and standard deviation, respectively. Abbreviations: (f ), length in females; ldt, length of laterodorsal tube; ltas, length of lateral terminal accessory spine; lts, length of lateral terminal spine; lvs, length of lateroventral spine; lvt, length of lateroventral tube; (m), length in males; mds, length of middorsal spine; msw, maximum sternal width; n.a., data not available; s, segment length; sw, standard width; tl, trunk length. Digits after abbreviation indicate segment number.      head (Fig. 3B), but located interior and posterior of inner oral styles in nature. Five thin and tube-like inner oral styles in ring 03, five thick spinose inner oral styles in ring 02, and ten spinose inner oral styles in ring 01. Two spinose structures present at basal part of ring -01 inner oral styles between sectors 2 and 3, 4 and 5, 7 and 8, and 9 and 10 (Figs 3C, 5). Each outer oral style consisting of rectangular basal part and triangular distal part, with basal part alternating in size between five larger ones in odd sectors and four smaller ones in even sectors (Figs 3B, C, 5). Each outer oral style with six long spinose processes bifurcated at their tips. One pair of additional short spinose processes originating slightly more anteriorly and laterally on either side of each outer oral style. Introvert composed of one ring of primary scalids, five rings of spinoscalids, and one ring of trichoscalids (Figs 3B, D, 5). Each primary spinoscalid consisting of basal sheath and distal end piece. Basal sheath with two layers of proximal fringes. End piece long, covered with minute hairs proximally, bluntly ending at distal tip. Each spinoscalid of rings 02-05 composed of basal sheath with fringed edge and distal long-spinose end piece. Spinoscalids in rings 02 and 03 longer than those in rings 04 and 05. Thin hair-like structures present at basal part of each spinoscalid. Trichoscalids arising from trichoscalid plates. Each trichoscalid covered with long hairs. Neck with 16 placids (Figs. 2A, B, 4B, C). Midventral placid broadest (Fig. 4C). Remaining placids similar in size. Two trichoscalid plates present ventrally and four dorsally, each associated with ventromedial, subdorsal, and laterodorsal placid, respectively (Fig. 4B, C).
Segment 1 consisting of complete cuticular ring. Sensory spots located in subdorsal and laterodorsal position (Figs 2A, 4D, 6A, B). Few hairs flanking each sensory spot. Two type-1 gland cell outlets present in tandem in middorsal and additional single pair in lateroventral position (Figs 2A, B, 4D, 6A, C). Posterior part of this and following ten segments with primary pectinate fringe (Figs 2A, B, 4D, 6A, C). Pectinate fringe teeth of primary pectinate fringe thin and long. Segment devoid of cuticular hairs except for hairs associated with sensory spots (Figs 3A, 6A, C).
Segment 2 with complete cuticular ring as segment 1. This and following eight segments with thick pachycyclus at anterior margin of each segment (Figs 2A, B, 4A, D-F). Pachycyclus interrupted middorsally in segments 3-9 as well as at tergosternal and midsternal junctions in segments 3-10. Cuticular hairs rising from perforation sites in anterior and central area of this and following eight segments (Fig. 6A); hairs long, rather thin and flexible, and tending to curl up (Figs 6D, 7C). Sensory spots present in middorsal, laterodorsal and ventromedial position (Figs 2A, B, 6A, C). Type-1 gland cell outlets present in middorsal and ventromedial position.
Segment 3 and following eight segments consisting of one tergal and two sternal plates ( Fig. 2A, B). No sensory spots present. Type-1 gland cell outlets situated in middorsal and ventromedial position.

Differential diagnosis
Echinoderes pterus sp. n. can be easily distinguished from all the other congeners by the presence of the tufts of hairs on segment 9. Such a structure has never been described for any other kinorhynch, and is thus a unique character for the new species. This is also the case for the conspicuously thick and long lateroventral spines on segment 9, although this character appears to be restricted to males in the Karasik Seamount, Svalbard, and the Sedlo Seamount populations.
With respect to other characters, the spine/tube pattern of E. pterus sp. n., i.e., with middorsal acicular spines on segments 4-8, laterodorsal tubes on segment 10, lateroventral tubes on segment 5, and lateroventral acicular spines on segments 6-9, but without any other spine and tube is not shared with any of 109 congeners.
The head morphology of E. pterus sp. n. seems to be shared with only a few species of Kinorhyncha. In the new species, the ring -02 and -03 inner oral styles occur in odd and even sectors, respectively. Such an arrangement is known for Dracoderes abei Higgins & Shirayama, 1990(see Sørensen et al. 2012a, whereas the position of these styles seems to be reversed in all cyclorhagid species for which the arrangement of the inner oral styles is known, i.e., Antygomonas caeciliae Dal Zotto, 2015, Antygomonas incomitata Nebelsick, 1990, Antygomonas oreas Bauer-Nebelsick, 1996, Antygomonas paulae Sørensen, 2007, Cateria gerlachi Higgins, 1968, Ce. barbanigra, Centroderes bonnyae Neuhaus et al., 2014, Centroderes drakei Neuhaus et al., 2014, Centroderes readae Neuhaus et al., 2014, Cephalorhyncha liticola Sørensen, 2008, Semnoderes armiger Zelinka, 1928 Tubulideres seminoli Sørensen et al., 2007, Triodontoderes anulap Sørensen & Rho, 2009(see Bauer-Nebelsick 1996Sørensen 2007Sørensen , 2008Sørensen et al. 2007Sørensen and Rho 2009;Dal Zotto 2015;Neuhaus et al. 2014;Neuhaus and Kegel 2015). However in Cat. gerlachi, only a single specimen mounted for light microscopy had its inner oral styles everted enough to be recognizable, and the mouth cone was separated from the specimen (Neuhaus and Yamasaki, unpubl. obs.). Neuhaus and Kegel (2015, fig. 3) illustrated ring -02 and -03 inner oral styles in the position they assumed to be correct. This raises the question how accurate identification of the position was in other species by these and other authors. Since the exact arrangements and the shapes of inner oral styles have been infrequently observed in Echinoderes, it is not possible to conclude whether those in E. pterus sp. n. are unique among the genus or not. We hope that further observations of head structures in other species of Echinoderes will allow a comprehensive comparison of this character in the future.

Geographically and bathymetrically wide distribution in Kinorhyncha
Echinoderes pterus sp. n. shows a geographically and bathymetrically wide distribution, from near the North Pole to the eastern Mediterranean Sea through the northeast Atlantic Ocean, and from 675 m to 4,403 m depth (Fig. 1, Table 1). For other kinorhynchs, such a geographically and bathymetrically wide distribution is only known for Cam. vanhoeffeni, Centroderes spinosus (Reinhard, 1881), and S. armiger. The former one was reported worldwide at a depth ranging from 0-5,118 m from several localities in the Atlantic Ocean, Pacific Ocean, Indian Ocean, and the Antarctic Sea . The latter two were found in the Mediterranean Sea, Black Sea, northeastern Atlantic Ocean, and North Sea at depths ranging from 14 m to 444 m (Ce. spinosus) and from 15 m to 444 m (S. armiger) .
There are few other kinorhynchs which have been reported to show either a geographically or a bathymetrically wide distribution. Species with a geographically wide distribution are e.g., Ce. barbanigra found in the Gulf of Mexico, the Caribbean Sea, Bermuda, and the Dominican Republic at a depth ranging from 2 m to 57.5 m, E. ohtsukai found on both the eastern and western coasts of the Pacific Ocean in the intertidal zone, and E. tchefouensis found in the East China Sea, South China Sea, Celebes Sea, Singapore Strait, and Mariana Islands at a depth ranging from 0 m to 140 m (Sørensen et al. 2012b(Sørensen et al. , 2016Yamasaki and Kajihara 2012;Neuhaus et al. 2014;Herranz and Leander 2016). Species from a bathymetrically wide range are e.g., Echinoderes arlis Higgins, 1966, Echinoderes drogoni Grzelak & Sørensen, 2018, Echinoderes eximus Higgins & Kristensen, 1988, Echinoderes peterseni Higgins & Kristensen, 1988, and Echinoderes rhaegali Grzelak & Sørensen, 2018, all

A single or multiple species?
The morphological comparison between populations of E. pterus sp. n. reveals that the new species shows an inter-population variation (Fig. 10, Table 2). The most obvious difference is found between males of the Arctic populations (Karasik Seamount + Svalbard) + the Sedlo Seamount population with lateroventral acicular spines on segment 9 being conspicuously thicker and longer than the preceding spines, as opposed to those of the Mediterranean populations (Mediterranean deep sea + Anaximenes Seamount), which have lateroventral spines on segment 9 of similar thickness to the other spines and only slightly longer than the preceding ones (compare Fig. 8B and Fig. 8C; see Fig. 10 for measurements). Such a large difference is not found between females of these populations. In addition, the length of the remaining spines is slightly longer in the Arctic populations than in the Mediterranean populations (Fig. 10). The population on the Sedlo Seamount, although it is represented by a single specimen in this study, shows similarities in spine length to the Arctic populations in the middorsal acicular spines on segments 4, 5, 8 and the lateroventral acicular spines on segments 6, 7, 9, whereas it shares a similar spine length with the Mediterranean populations in the other spines. The ventromedial sensory spots on segment 7 reveal variation insofar as they are absent in the Arctic and Sedlo Seamount populations but present in the Mediterranean populations.
Considering the geographically and bathymetrically wide distribution of E. pterus sp. n., the presence of inter-population variation in morphological characters, as well as the potentially low-distribution ability of kinorhynchs, it should be considered whether E. pterus sp. n. represents one or multiple species. In the case of the other geographically and bathymetrically wide distribution kinorhynchs, intra-and inter-populational variation of several morphological characters, e.g., body length, arrangement of gland cell outlets, and sensory spots, has been detected in Cam. vanhoeffeni. However, it was still regarded as a single species due to the overlapping characters between/within populations and the absence of the type material . Variation in the occurrence of sensory spots within one species has also been reported for several other kinorhynchs, e.g., Cat. gerlachi, Cateria styx Higgins, 1968, Ce. spinosus, Ce. barbanigra, and Ce. readae (Neuhaus et al. , 2014Neuhaus and Kegel 2015).
Echinoderes pterus sp. n. may on the one hand represent two species, e.g., one species in the Arctic Ocean and on the Sedlo Seamount and the second species in the Mediterranean, or it may even belong to three species, i.e., one in the Arctic Ocean, another on the Sedlo Seamount, and the third in the Mediterranean, with only a few morphological differences. However, there is the possibility that the different populations belong to the same species with the observed morphological variations, which gradually change from the Arctic Ocean via the Sedlo Seamount to the Mediterranean populations or vice versa. Although we cannot reject these possibilities, we currently regard all populations as a single species. Further investigations of the species, for instance the sampling and observation of populations in intermediate localities and/or molecular phylogeographic studies, should provide more information about the population connectivity of the species and support one of the two hypotheses.
Whichever hypothesis is correct, all populations in this study are undoubtedly closely related to each other. They have expanded their habitat range with or without speciation, however, their distribution process is open to question: did they distribute from the Arctic Ocean via the Atlantic Ocean to the Mediterranean, from the Mediterranean via the Atlantic Ocean to the Arctic Ocean, or from the Atlantic Ocean to both the Arctic Ocean and the Mediterranean? Indeed the species represents interesting material for studying the "meiofauna paradox" or the "everything is everywhere hypothesis". We cannot provide a strongly-supported answer based on our current data. Further data about the species distribution range and population connectivity would also enable us to approach the question in future studies. specimens collected during the expedition M71/2 by R/V METEOR, Kristine Kämpf (Museum für Naturkunde Berlin) for assisting in the preparation of LM specimens, Anke Sänger (Museum für Naturkunde Berlin) for the technical support at the SEM, and Dr Jason Dunlop (Museum für Naturkunde Berlin) for English editing. This study was supported by a grant from the Deutsche Forschungsgemeinschaft DFG to KHG (GE 1086/20-1) and to BN (NE 931/6-1) and by the SYNTHESYS Projects (DK-TAF-5319 and DK-TAF-6523) to KG, which were financed by European Community Research Infrastructure Action under FP7 (http://www.synthesys.info/). The study was completed also thanks to funding provided by the National Science Centre, Poland (grant no. 2016/20/S/NZ8/00432 and 2015/19/B/NZ8/03945). Material collected during R/V Polarstern TRANSSIZ cruise (ARK XXIX/1; PS92) was carried out under grant number AWI_PS92_00 and organized by Arctic in Rapid Transition (ART).