Setaphyes elenae sp. nov., a new species of mud dragon (Kinorhyncha: Allomalorhagida) from Skagerrak (north-eastern Atlantic Ocean)

. Meiofauna sampling in the proximity of Syd-Hällsö Island (Strömstad, Sweden) revealed a new species of Kinorhyncha from the Skagerrak. The species, Setaphyes elenae sp. nov., is distinguished from its congeners by the arrangement of the middorsal cuticular specializations (it has shortened, distally rounded middorsal processes on segments 1 and 9 and middorsal elevations throughout segments 2–8), as well as by the presence of paired laterodorsal setae on segments 3, 5, 7 and 9 and ventromedial setae on segments 3, 5 and 7 in both males and females. The finding of a new species from the northeastern Atlantic Ocean, provides new valuable information for the recently established genus in the Allomalorhagida.


Introduction
The phylum Kinorhyncha Reinhard, 1887 encompasses a group of meiobenthic, free-living invertebrates that mainly inhabit the upper centimetres of marine and estuarine sediments, although some species have been found living in hard substrata or associated with macroalgae and marine phanerogams (Higgins 1988). Kinorhynchs have been described from shallow to deep-sea waters across the worldwide oceans, but certain areas have received much more attention to the detriment of others due to the specialists sampling strategies (Sánchez et al. 2012;Neuhaus 2013;Sørensen et al. 2013;Cepeda et al. 2019).
In the context of global change and decreasing studies of taxonomy, especially those of small-sized taxa, there is a need to improve taxonomic information about meiofaunal organisms, even in geographic areas that are supposed to be relatively well-studied (Mora et al. 2011;Sørensen & Grzelak 2018). A recent sampling done in the proximities of Syd-Hällsö Island (Strömstad, Sweden) revealed a new species of Kinorhyncha from Skagerrak. Additionally, the species Echinoderes cf. eximus and Pycnophyes ancalagon Sørensen & Grzelak, 2018, the latter recently described from the Svalbard Archipelago (Arctic Ocean), are firstly reported for the boreal area.

Study site
Sampling was undertaken at a single locality near Syd-Hällsö Island (Strömstad, Sweden) during summer 2017, at the eastern-most limit of the North Sea: 58°56.846′ N, 11°4.896′ E (Fig. 1B). This area encompasses the Skagerrak Strait that connects the North Sea with the Kattegat sea region (Fig. 1A).
Skagerrak, with a surface area of about 32 000 km 2 , is the deepest area of the North Sea basin, with a maximum depth of 700 m (Weering et al. 1993;Rosenberg et al. 1996). Skagerrak is dominated by a deep-reaching flow of water from the central and northern North Sea, with a salinity of about 35 psu and to a lesser extent by a weaker inflow from the southern North Sea with a salinity of about 31-34 psu influenced by river inputs (Rosenberg et al. 1996). Additionally, the shallowest waters of Skagerrak are also subject of upwelling events that cause low-saline currents along the Swedish and Norwegian coastlines (Rodhe 1996).
Skagerrak forms a natural topographic sediment trap, receiving inputs from the entire north-western European drainage systems and the North Sea shoreline (Weering et al. 1993). Thus, Skagerrak has been evidenced as a major depository of fine-grained sediments in the North Sea, with 59% of particles with an average size less than 63 μm (Weering et al. 1987). Nevertheless, areas with coarse gravel, sandy coves and sandbars are also present in the area (Curini-Galletti et al. 2012).

Sampling and specimen preparation
Sampling was done during the summer of 2017 by dredging. The collected sample was taken at a depth of 55-65 m and mainly consisted of very fine, soft mud. Sediment was kept in large plastic boxes and stored at a constant temperature of 15ºC. Meiofaunal organisms were then extracted by MgCl 2decantation and live material was studied and sorted using stereo microscopes.
Kinorhynchs were preserved in 100% ethanol. For light microscopy (LM), unmounted specimens were dehydrated through a graded series of glycerine. After being kept in 100% glycerine for 24 h, kinorhynchs were mounted on glass slides with Fluoromount G ® sealed with Depex ® . Mounted specimens were studied with an Olympus © BX51-P microscope with differential interference contrast (DIC) optics equipped with an Olympus © DP-70 camera. Identification to genus level was done using the dichotomous keys provided by Sørensen & Pardos (2008) for cyclorhagids and the genus diagnoses provided by Sánchez et al. (2016) for allomalorhagids. For scanning electron microscopy (SEM), some unmounted kinorhynchs were sonically cleaned during 10-15 s and led to chemical point drying using a hexamethyldisilazane-ethanol series. Finally, specimens were coated with gold and mounted on aluminium stubs to be studied with a JSM ® 6335-F JEOL SEM at the ICTS Centro Nacional de Microscopía Electrónica (UCM, Spain). Type material was deposited at the Natural History Museum of Denmark (NHMD). Line drawings and image plates composition were done using Adobe ® Photoshop and Illustrator CC-2014 software.

Morphometric statistical analyses
Differences in selected morphometric measures (total trunk length, standard width and lateral terminal spines' length) of Setaphyes elenae sp. nov. and its congeners were tested. For this, we selected several specimens of S. dentatus (n = 18) and S. flaveolatus (n = 14), which are the most morphologically similar, and are also distributed through the north-eastern Atlantic Ocean. Specimens of S. dentatus and S. flaveolatus are from several Atlantic and Mediterranean locations surrounding the Iberian Peninsula, stored at the Meiofauna Collection of the UCM.
Normality and homoscedasticity of the variables were tested using the Saphiro-Wilk's test (together with visual methods of density and Q-Q plots) and the Bartlett's test, respectively. Tukey multiple comparison test and pairwise comparisons between group levels with corrections for multiple testing were used to determine which means amongst the set of means differ from the rest. A one-way analysis of variance (ANOVA) was used to test differences, but when heteroscedasticity was detected, a Welch's  (2020) ANOVA was performed instead. All the statistical analyses were run in R ver. 1.1.453 using the 'stats' basic package and the 'car' package ver. 3.0.5 (Fox et al. 2019).

Diagnosis
Setaphyes with shortened, distally rounded middorsal processes on segments 1 and 9, and middorsal elevations on segments 2-9, superficially covered by tufts of elongated, thick hairs whose tips sometimes surpass the posterior margin of segment. Unpaired setae in paradorsal position on segments 1-9. Laterodorsal setae on segments 3, 5, 7 and 9; paralateral setae absent. Lateroventral setae on segments 2-10. Ventromedial setae on segments 3, 5 and 7. Paired, small, dot-shaped intracuticular structures (maybe outlets of glandular cells) present in several positions throughout the trunk, with a specific arrangement that differs from males to females. Males with paired, sexually dimorphic ventromedial tubes on segment 2, and females with paired, sexually dimorphic ventrolateral setae on segment 2. Lateral terminal spines present, relatively short, slender. Segment 11 retractable into segment 10.

Etymology
The species is dedicated to Ms Elena González, sister of the second author.

Material examined
Holotype ATLANTIC OCEAN • ♀ adult (mounted in Fluoromount G ® ); near Syd-Hällsö Island, Skagerrak Additional non-type material ATLANTIC OCEAN • 8 specs (four mounted for LM and four mounted for SEM); same collection data as for holotype; Meiofauna Collection UCM.

Description
See Table 1 for measurements and dimensions, Table 2 for summary of cuticular elevation, process, seta, tube, nephridiopore and sensory spot locations, and Table 3 for summary of intracuticular, dot-shaped structure locations.
Head with retractable mouth cone and introvert. The collected specimens were not suitable for head examinations, hence data on number and arrangement of scalids and oral styles are not available.
Neck with four dorsal and two ventral sclerotized placids ( Fig. 2A-B, D). Dorsal placids rectangular, with a slightly convex anterior margin; mesial ones broader than lateral ones (Fig. 2B). Ventral placids morphologically similar to dorsal ones but much more elongated, getting thinner towards the lateral sides ( Fig. 2A, D).
Segment 5 similar to segment 3 in the arrangement of the cuticular elevation, setae and sensory spots (Figs 2A-B, 4F, H).
Segment 6 similar to segment 4 in the arrangement of the cuticular elevation, setae and sensory spots ( Fig. 2A-B).
Segment 7 similar to segments 3 and 5 in the arrangement of the cuticular elevation, setae and sensory spots ( Fig. 2A-B).

Discussion
Setaphyes elenae sp. nov. clearly agrees with the main diagnostic characters of the genus, including the absence of ventrolateral setae on segment 5, the presence of unpaired paradorsal setae on segments 2-9 and paired lateroventral setae on segments 2-10, ball-and-socket joints only conspicuous at segments 2-3 (reduced on posterior segments), and the presence of scattered, dot-shaped, intracuticular structures (sometimes called as cuticular scars) at both tergal and sternal plates (Sánchez et al. 2016). However, it can easily be distinguished from the remaining species of the genus by the unique arrangement of setae and middorsal cuticular specializations.
species of the genus also possess laterodorsal setae also even segments. In addition, S. elenae sp. nov. is characterized by bearing shortened, distally rounded middorsal processes on segments 1 and 9 exclusively, while both S. dentatus and S. flaveolatus have middorsal processes on segments 1 and 7-9. The pattern of ventromedial setae also differs between S. dentatus (on segments 4-5 and 7-9, females furthermore on segment 3), S. flaveolatus (on segments 5 and 7-8, females furthermore on segment 3) and S. elenae sp. nov. (on segments 3, 5 and 7). Sánchez et al., 2018, S. dentatus, S. elenae sp. nov. and S. flaveolatus are also characterized by having patterns of cuticular ornamentation that can be used to discriminate congeners (Sánchez et al. 2018). Longitudinal, parallel, fold-like cuticular thickenings are present on segment 10 in S. dentatus and S. cimarensis, which are absent in S. elenae sp. nov. and S. flaveolatus. Moreover, S. flaveolatus has a continuous, reticule-like ornamentation only present in the middle region of the tergal plate, whereas S. dentatus and S. elenae sp. nov. possess a similar ornamentation extended throughout the anterior margin of the plate, and S. cimarensis is characterized by having small, rounded, isolated depressions near the anterior margin of the plate with a net-like structure (Sánchez et al. 2018).

Setaphyes cimarensis
The only statistically significant differences were found in the total trunk length (TL) between Setaphyes elenae sp. nov. and S. flaveolatus (p = 0.00042), so we can conclude that these two morphologically similar species may be also distinguished by the total trunk length (Fig. 5A). Oppositely, S. dentatus and S. elenae sp. nov. overlap, and statistically significant differences were not found (p = 0.51153) (Fig. 5A). Setaphyes flaveolatus is the smallest of the tested species, with a total trunk length of 500-600 μm, followed by S. dentatus and S. elenae sp. nov. with a total trunk length of 600-800 μm (Fig. 5A). The remaining analysed morphometric measurements were not significantly different between S. elenae sp. nov. and the aforementioned congeners ( Fig. 5B-C).