Two new species of Echinoderes (Kinorhyncha, Cyclorhagida), E. romanoi sp. n. and E. joyceae sp. n., from the Gulf of Mexico

Abstract Meiofauna sampling on the continental shelf of the northern Gulf of Mexico has been ongoing since 2007, on annual cruises in collaboration with the National Marine Fisheries Service laboratory in Pascagoula, Mississippi. This sampling has resulted in numerous new species of kinorhynchs from the shelf sediment, two of which are described in detail in this paper. Other species descriptions from this research effort include Echinoderes augustae, Echinoderes skipperae, and Echinoderes charlotteae. We now describe Echinoderes romanoi sp. n. and Echinoderes joyceae sp. n., which are unique in their spine, tube, and glandular cell outlet patterns.


Introduction
Diversity in the phylum Kinorhyncha has been underreported from the Gulf of Mexico, though recently new investigations are adding to our knowledge of the Gulf species. Currently there are few kinorhynchs identified to species from the Gulf, though their presence and abundance are well documented. The known species diversity in the Gulf of Mexico includes Echinoderes steineri (Chitwood, 1951), E. coulli Higgins 1977, E. remanei (Blake, 1930), Kinorhynchus langi (Higgins, 1964), Campyloderes cf. vanhoeffeni Zelinka, 1913, Centroderes barbanigra Neuhaus et al., 2014, Centroderes cf. drakei Neuhaus et al., 2014, E. skipperae Sørensen & Landers, 2014, E. augustae Sørensen & Landers, 2014, E. bookhouti Higgins, 1964and E. charlotteae Sørensen et al., 2016(Chitwood 1951, Harper et al. 1981, Shirley 2009, Neuhaus 2013, Neuhaus et al. 2014, Sørensen and Landers 2014, Fleeger et al. 2015, Sørensen et al. 2016). The last four Gulf records resulted from meiofauna surveys conducted by our labs in the Gulf in collaboration with the National Marine Fisheries Service (NMFS) lab in Pascagoula, Mississippi. This collaboration began in 2007 and continues currently, with sediment collection occurring on annual fall cruises. The first two reports from the kinorhynch analysis of this long term meiofauna study described three new species, E. augustae, E. skipperae, and E. charlotteae, and also provided a redescription of E. bookhouti (Sørensen andLanders 2014, Sørensen et al. 2016). This current contribution is the third in our series of new kinorhynch species discovered in the northern Gulf of Mexico, from continental shelf sediments, and describes Echinoderes romanoi sp. n. and E. joyceae sp. n. These new descriptions will be helpful for future taxonomic and morphological studies.

Materials and methods
Sediment was collected along the northern Gulf of Mexico continental shelf during several NOAA cruises from 2010 to 2015 in collaboration with the NMFS lab in Pascagoula, Mississippi, on NOAA ships Gordon Gunter, Pisces, and Oregon II. Sediment was collected in 2010-2012 using a Shipek® sediment grab, and in 2013-2015 using an Ocean Instruments® mini-multicorer. Specimens from the present study were obtained from 12 locations located along the northern Gulf of Mexico continental shelf ( Fig. 1, Table 1).
The samples were fixed immediately in 5-10% formalin on the cruise, and the meiofauna was subsequently extracted by Ludox centrifugation (Burgess 2001). After sorting the animals using a counting wheel, the kinorhynchs were stored in 70% isopropanol. They were processed for light microscopy by subjecting them to increasing concentrations of glycerin before mounting them in Fluoromount G® (Sørensen and Pardos 2008). They were examined and photographed using a Nikon E600 (Troy University) or an Olympus BX51 (University of Copenhagen) light microscope equipped with Nomarski interference contrast optics using digital cameras. Line art illustrations were based on mounted specimens that were drawn using Adobe Illustrator® CS6 or Adobe Photoshop Elements® software. Measurements were made with CellSens® software. All dimensions reported in the tables are based on LM measurements. All light microscopy type material is deposited at the Natural History Museum of Denmark (Copenhagen, Denmark). Specimens for scanning electron microscopy were observed at the Auburn University Research Instrumentation Facility (Auburn, Alabama). Specimens stored in 70% isopropanol were hydrated, post-fixed in OsO 4 vapor, then dehydrated to 100% ethanol through a graded series, critical point dried, mounted on aluminum stubs, and sputter-coated with gold. Specimens were photographed with a Zeiss EVO 50 SEM, using the backscatter and secondary electron detectors.   C Female, segments 10 to 11, dorsal view D Female, segments 10 to 11, ventral view. Abbreviations: gco1/2, glandular cell outlet type 1/2; ld, laterodorsal sensory spot; ltas, lateral terminal accessory spine; lts, lateral terminal spine; lvs, lateroventral spine; lvt, lateroventral tube; ml, midlateral sensory spot; pa, pachycyclus; pd, paradorsal sensory spot; pe, penile spine; pv, paraventral sensory spot; sd, subdorsal sensory spot; si, sieve plate; sl, sublateral sensory spot; vm, ventromedial sensory spot.

Class
All paratypes are mounted in Fluoromount G® and deposited at the Natural History Museum of Denmark. Additional nontype material is listed in Table 1. See Figure 1 for localities and Table 1 for detailed station information.
Diagnosis. Echinoderes with middorsal spines on segments 4-8, and spines in lateroventral positions on segments 6-9. Tubes present in lateroventral position on segment 5. Glandular cell outlets type 2 present in subdorsal, laterodorsal, sublateral, and ventrolateral positions on segment 2, in midlateral position on segment 5, and in sublateral position on segment 8.
Description. Adults with head, neck and eleven trunk segments, ranging from 196-247 µm in trunk length (Figs 2-4). For complete overview of measures and dimensions, see Table 2. Distribution of cuticular structures, i.e., sensory spots, glandular cell outlets, spines and tubes, is summarized in Table 3.
The head (Fig. 3A, B, 4B) consists of a retractable mouth cone and an introvert. The mouth cone has nine outer oral styles. The introvert sectors are defined by 10 primary spinoscalids in ring 1. Each primary spinoscalid consists of a basal sheath with approximately 7 long extensions forming a fringed margin, and a distal end piece with a blunt tip. It was only possible to obtain information about the appearance and arrangement of scalids for introvert sectors 2, 3, and 4, which have the following characteristics: single central scalids of Rings 02 and 04, and paired scalids of Rings 03 and 05.
The neck (Figs 2A, B, 3A, B) has 16 placids, measuring 10 µm in length. The midventral placid is broadest, measuring 9 µm in width at its base, whereas all other are narrower, measuring 5-6 µm in width at their bases. The trichoscalid plates, each with a trichoscalid, are present in subdorsal, laterodorsal and ventromedial positions.
Segment 1 (Figs 2A, B, 3A-C, 4A) consists of a complete cuticular ring. Sensory spots are located anteriorly in subdorsal, laterodorsal, and ventromedial positions; sensory spots minute and rounded with two anterior cuticular hairs. Glandular cell outlets type 1 present middorsally, sublaterally and lateroventrally. Cuticular hairs sparse on dorsal and ventral surface. A line of cuticular hairs is located below the intersegmental joint line. Pectinate fringe of posterior segment margin with typical fringe tips.
Segment 2 (Figs 2A, B, 3A-D, 4A) consists of a complete cuticular ring. Pachycyclus of the anterior segment margin interrupted in middorsal and lateroventral positions. Sensory spots located in laterodorsal, midlateral, and paraventral positions; sensory spots on this and following segments minute and rounded. Glandular cell outlets type 2 located in subdorsal, laterodorsal, sublateral and ventrolateral positions. Glandular cell outlets type 1 located in paraventral positions. Secondary pectinate fringe present on this segment and on the following segments 3-10.
Segment 3 (Figs 2A, B, 3A-C), and remaining segments, consisting of one tergal and two sternal plates. Pachycyclus of the anterior segment margin interrupted middorsally, midventrally, and at the tergosternal junctions. Sensory spots present in subdorsal and sublateral positions. Cuticular hairs evenly distributed over tergal and sternal plates, between the pectinate fringe and intersegmental joint line, with a line of cuticular hairs below the joint line. Glandular cell outlets type 1 in paradorsal and paraventral positions.  Table 2. Measurements from light microscopy of Echinoderes romanoi sp. n. (in µm) from the Gulf of Mexico, including number of measured specimens (n) and standard deviation (SD). Abbreviations: (ac): acicular spine; LTAS: lateral terminal accessory spine; LTS: lateral terminal spine; LV: lateroventral; MD, middorsal; MSW-7: maximum sternal width, measured on segment 7 in this species; S: segment lengths; SW-10, standard width, always measured on segment 10; TL: trunk length. Segment 6 (Figs 2A, B, 3A, B, E, 4E) with middorsal and lateroventral acicular spines. Sensory spots present in paradorsal, subdorsal, midlateral, and ventromedial positions; ventromedial sensory spots slightly closer to midsternal junction than those on preceding segment. Pachycycli, glandular cell outlets type 1, and cuticular hairs as on preceding segment. Segment 7 (Figs 2A, B, 3A, B, E, 4C,E) with middorsal and lateroventral acicular spines. Sensory spots present in paradorsal, midlateral, and ventromedial positions; ventromedial sensory spots aligned with those on segment 5. Pachycycli, glandular cell outlets type 1, and cuticular hairs as on preceding segment. Segment 8 (Figs 2A, B, 3A, B, E, 4D, E) with middorsal and lateroventral acicular spines. Sensory spots present in paradorsal position. Glandular cell outlets type 2 in sublateral position. Pachycycli, glandular cell outlets type 1, and cuticular hairs as on preceding segment. Segment 9 ( Etymology. This species is named after the late Dr. Frank A. Romano III, Jacksonville State University, Alabama, for his contributions to the study of meiofauna and for his initiation of our ongoing meiofauna survey. Remarks. Echinoderes romanoi sp. n. is characterized by the presence of middorsal spines on segments 4 to 8, lateroventral tubes on segment 5, lateroventral spines on segments 6 to 9, and glandular cell outlets type 2 on segments 2 (4 pairs), 5, and 8. This combination of spines, tubes, and glandular cell outlets is unique among all species in the genus. The spine/tube arrangement is not unusual among congeners: 36 additional species share the presence of middorsal spines on segments 4 to 8 and lateroventral tubes/spines on segments 5 to 9, and out of these, ten also lack tubes on segment 2 as  Adrianov, 1999, E. stockmani Adrianov, 1999, E. obtuspinosus Sørensen et al. 2012, and E. bookhouti Higgins, 1964(Blake 1930, Higgins 1964a, 1964b, Higgins 1977, Higgins and Kristensen 1988, Adrianov and Malakhov 1999, Sørensen et al. 2012, 2016. Many of the descriptions of these ten species do not include glandular cell outlet type 2 information, though there are a variety of characteristics that distinguish E. romanoi n. sp. from each of the 10 other taxa.  Higgins (1964a), which has a trunk length of 282-358 µm. Echinoderes brevicaudatus has lateral dorsal tubes on segment 10, and short stubby lateral terminal spines, distinct from the new species. E. cernunnos has glandular cell outlets type 2 located similarly to E. romanoi on segments 2, 5 and 8, though E. cernunnos also has glandular cell outlets type 2 in the midlateral position on segment 7 and additionally has elongated spinous tergal extensions. Echinoderes koreanus has spines in the lateral dorsal positions on segments 7 and 8, and tubes in the laterodorsal position on segment 10, unlike the new species. Echinoderes stockmani is distinguished by having the lateral spines on segment 8 distinctly longer than those   on segment 9, unlike E. romanoi n. sp. Echinoderes obtuspinosus has glandular cell outlets type 2 similarly to E. romanoi on segments 2 and 8. However, E. obtuspinosus has glandular cell outlets type 2 in the subdorsal position on segment 4 and none on segment 5. Further, E. obtuspinosus has short stubby lateral terminal spines. Finally, E. bookhouti has lateral accessory spines on segment 8, and lacks glandular cell outlets type two on segment 5 and in the laterodorsal and sublateral position on segment 2. Description. Adults conspicuously small (183-209 µm in trunk length), with head, neck and eleven trunk segments (Figs 5-7). For complete overview of measures and dimensions, see Table 4. Distribution of cuticular structures, i.e., sensory spots, glandular cell outlets, spines and tubes, is summarized in Table 5.

Echinoderes joyceae
The head (Fig. 7A-C) consists of a retractable mouth cone and an introvert. Mouth cone with nine outer oral styles that alternate in length between slightly shorter and slightly longer ones. No outer oral styles present anterior to introvert sector 6. A fringe with three long spikes is located at the base of each outer oral style. It was only possible to obtain complete information about appearance and arrangement of scalids for introvert sectors 8 and 9. Sector 8: single central scalids of Rings 02 and 04, and paired scalds of Rings 03 and 05. No scalids present posterior to Ring 05, except for a single trichoscalid that attaches through a trichoscalid plate. Sector 9: single central scalids of Rings 02, 04, and 06, and paired scalds of Rings 03 and 05. No trichoscalids present.

Figure 7.
Scanning electron micrographs showing overviews and details in head and trunk morphology of female Echinoderes joyceae sp. n. A Lateral overview B Mouth cone and introvert sector 8 C Head and segments 1 to 2, lateral view D Laterodorsal parts of segments 1 to 6 E Trunk segments 1 to 10, lateral view F Laterodorsal parts of segments 4 to 7 G Detail showing sublateral parts of segments 7 to 9 H Detail showing sub-and laterodorsal parts of segment 10 and left tergal extension of segment 11 I Segments 7 to 11, laterodorsal and caudal view. Abbreviations: gco2, glandular cell outlet type 2; ld, laterodorsal sensory spot; ldt, laterodorsal tube; ltas, lateral terminal accessory spine; lvs, lateroventral spine; lvt, lateroventral tube; mds, middorsal spine; ml, midlateral sensory spot; oos, outer oral style; pd, paradorsal sensory spot; psp, primary spinoscalid; sd, subdorsal sensory spot; sec 8, introvert sector 8; si, sieve plate; sp2-5, spinoscalids of rings 2 to 5; tr, trichoscalid; vlt, ventrolateral tube. spines on segments 5 to 9, and laterodorsal tubes on segment 10. This combination of spines and tubes is not unusual among congeners, and is shared with five other species: E. bermudensis Higgins 1982, E. kristenseni Higgins 1985E. abbreviatus Higgins 1983E. hispanicus Pardos et al., 1998and E. intermedius Sørensen, 2006. (Higgins 1982, 1983, 1985Pardos et al. 1998, Sørensen 2006. These species are distinguished from E. joyceae using a number of characteristics. All five of these species have tergal extensions on segment 11 distinct from E. joyceae. Additionally, the new species has a distinctive distribution of glandular cell outlets type 2, with locations in the subdorsal position on segment 2. Amongst the abovementioned species this is only found in E. kristenseni, which also has glandular cell outlets type 2 in lateroventral positions of segment 2, not present in Echinoderes joyceae sp. n. The most unique character combination in Echinoderes joyceae sp. n. though, is the presence of glandular cell outlets type 2 in midlateral positions of segment 6 and in sublateral positions of segment 8. This combination is not found in any other species of Echinoderes. Furthermore, Echinoderes joyceae sp. n. is characteristic by its minute size. With a trunk length ranging from 183 to 209 µm, Echinoderes joyceae sp. n. is among the smallest known Echinoderes.

Discussion
This study describes E. romanoi sp. n. and E. joyceae sp. n., and reports the known distribution of the two new species along the northern Gulf of Mexico continental shelf. In common with the previous shelf species reported so far during this long term meiofauna sampling is the broad distribution of the taxa. Echinoderes romanoi sp. n. and E. joyceae sp. n. are distributed across wide regions of the United States' Gulf shelf, with the location of E. romanoi extending from central Louisiana to western Florida, and with E. joyceae sp. n. extending from Texas to Florida. Similarly, E. augustae, E. skipperae, E. charlotteae, and E. bookhouti (Sørensen andLanders 2014, Sørensen et al. 2016), all reported during this survey from the northern Gulf shelf, have broad distributions either extending across half of the U.S. Gulf shelf or across the entire Gulf. As more samples are processed during our survey, more locations for all of these species will be determined. Their distribution will likely cover the entire shelf from Florida to Mexico, given the trend observed so far. Despite the broad distribution of these Echinoderes species across the Gulf, it is interesting that they have not been observed in coastal marshes. In a recent study on the effects of the Deepwater Horizon oil spill from 2010 in Barataria Bay, Louisiana (Fleeger et al. 2015), all identified kinorhynchs from a subsample of 100 animals were identified as E. coulli, with no offshore species present (identifications in the Fleeger et al. 2015 paper were made by M.V. Sørensen). Echinoderes coulli is an estuarine species, which has not been observed in our offshore samples. Our sampling on the continental shelf has consistently sampled sediment from high salinity waters, yielding species that may not tolerate estuarine conditions. Sampling along the U.S. shoreline in high and low salinity waters are needed to determine if the shelf species are found in the intertidal zone or marshes.