A new species of Cephalorhyncha Adrianov, 1999 (Kinorhyncha: Cyclorhagida) from the Aegean Coast of Turkey

Kinorhynchs are marine, microscopic ecdysozoan animals that are found throughout the world’s ocean. Cephalorhyncha flosculosa sp. nov. is described from the Aegean Coast of Turkey. Samples were collected from intertidal zones at two localities. The new species is distinguished from its congeners by having flosculi in midventral positions on segment 3–8, and by differences in its general spine and sensory spot positions. Until now, species of Cephalorhyncha were only known from the Pacific Ocean, hence, this record of the genus at the Aegean Sea not only expands its geographic distribution to Turkey, but is likely to expand it throughout the Mediterranean Sea and much of southern Europe. The new species of Cephalorhyncha represents the fifth kinorhynch species recorded from Turkey, and increases also the number of known Cephalorhyncha species to four.

Effective management and conservation of biodiversity and ecosystems depends largely on the taxonomic determination of species composition. Unfortunately, insufficient taxonomic information intercepts our ability to make informed decisions about conservation and sustainable management of biodiversity and ecosystems. Turkey has numerous sandy beaches distributed along its coastline, but the marine meiofauna has been poorly explored despite the fact that the country has a vast coastline of about 8300 km. From a taxonomic perspective, mostly the Copepoda have been studied to a large extent [17,18]. Oppositely, the marine and meiobenthic Kinorhyncha have been very poorly explored. Băcescu [19] reported the first kinorhynch species, Pycnophyes communis Zelinka, 1908, from the Black Sea. Later, Sönmez et al. [20] reported Echinoderes gerardi Higgins, 1978 and a species resembling Echinoderes bispinosus Higgins, 1982 from the Aegean coast of Turkey, and Ürkmez et al. [21] found Echinoderes dujardinii Claparède, 1863 from subtidal sediments of Sinop Bay, Black Sea. In

Open Access
Helgoland Marine Research *Correspondence: nozlemkoroglu@gmail.com 1 Silifke Vocational School Aquaculture Program, Mersin University, 33940 Mersin, Turkey Full list of author information is available at the end of the article the present paper, a new species of Cephalorhyncha collected at two intertidal localities from the Aegean Coast of Turkey is described.

Methods
Samples were collected from two intertidal localities ( Fig. 1): the north coast of Kara Island, ca. 5 km southeast of Bodrum on the Turkish mainland (36°59′08″N, 27°27′13″E), and at Çalış on the beach of Fethiye (36°39′33″N, 29°06′35″E) using the Karaman-Chappius method [22]. This method consists of a funnel-shaped excavation into the sediment down to the groundwater level, allowing sea water to fill the bottom of the pit, and then collecting that water with a suitable cup and filtering it through plankton net. The filtered samples were fixed in 70% alcohol and kinorhynch specimens picked up using an Olympus SZ-16 stereomicroscope. Specimens for light microscopic examination were dehydrated through a graded glycerin series, and mounted in Fluoromount-G between two cover slips, and attached to a plastic H-S slide mount which enables from viewing from both sides. The specimens were examined with an Olympus BX51 compound microscope equipped with DIC optics. Photos were taken with an Olympus DP27 camera, and line art illustrations were made in Adobe Illustrator CS6. Specimens for scanning electron microscopy were dehydrated through a graded alcohol-acetone series, and were subsequently critical point dried, mounted on aluminium stubs, sputter coated with a platinum-palladium mix, and examined with a JEOL JSM-6335F Field emission scanning electron microscope.
Terminology follows Sørensen and Pardos [3] and Neuhaus [2]. Specimens used for morphological comparison include type material of Cephalorhyncha liticola (Natural History Museum of Denmark, Cat. Nos. KIN-205 to  and topotype specimens of C. asiatica, kept in the personal collection of MVS.

Systematics
Phylum Kinorhyncha Dujardin, 1851 [23] Class Cyclorhagida Zelinka, 1928 Order Echinorhagata Sørensen et al., 2015 FamilyEchinoderidae Zelinka, 1894 [24] Genus Cephalorhyncha Adrianov, 1999 Cephalorhyncha flosculosa sp. nov  Additional material from same date and locality as holotype includes two specimens mounted in Fluoromount-G and stored in the personal reference collection of the first author, and three specimens, mounted for SEM, and stored in the personal reference collection of the second author. In addition, three specimens were collected on June 29, 2011 from intertidal sand at Kara Island ( Fig. 1), 36°59′08″N, 27°27′13″E by Nuran Özlem Yıldız, mounted for SEM, and stored in the personal reference collection of the second author.

Diagnosis
Segment 1 forming complete ring; segment 2 with one tergal and one sternal plate, with complete tergosternal junctions, but only partially developed midsternal junction; segments 3-11 with one tergal and two sternal plates. Middorsal spines present on segments 4-8; ventrolateral tubes present on segments 2, lateroventral tubes on segment 5, lateroventral spines on segments 6-9, lateral accessory tubes on segment 8, and midlateral tubes on segment 10. Flosculum-shaped sensory spots present at posterior segment margins in midventral positions of segments 3-8.

Etymology
The species is named flosculosa (L.), meaning "having or bearing little flowers". The name was inspired by the flower-shaped sensory spots in the midventral positions of segments 3-8.

Description
Body consists of head, neck and trunk with 11 segments (Figs. 2a, b, 3a, 4a, b). Segment and spine lengths are summarized in Table 1. For spines, sensory spots and glandular cell outlets positions see Table 2.
Mouth cone with nine outer oral styles; outer oral styles alternate in length between longer and shorter ones (five long and four short), but all of them consist of two joined units (Figs. 4a, 7e). Exact arrangement of scalids could not be examined in any of the mounted specimens, since the introverts were either partly retracted or too dirty. Neck with 16 placids with conspicuously rounded anterolateral corners (Figs. 3c, d); midventral placids broader than remaining ones (Figs. 2b, 3b, d). The trunk is laterally compressed, which makes the specimens appear broad in lateral perspective, but rather narrow in dorsal or ventral view. This lateral compression also makes mounting for LM in a dorso-ventral orientation quite challenging.
Segment 1 consists of a closed ring (Figs. 2a, b, 3c, d, 4a). Glandular cell outlet type 1 present in middorsal (Figs. 2a, 6a) and lateroventral positions near anterior margin of segment (Figs. 2b, 5a). Sensory spots present in subdorsal and laterodorsal positions (Figs. 2a, 6a, 7a); sensory spots large, with a central pore, numerous short micropapillae, and a few considerably longer ones along the posterior margin. Cuticular hairs lightly scattered on the dorsal and lateral sides (Fig. 6a); ventral side completely devoid of hairs. Posterior segment margin terminates into a well-defined pectinate fringe with short fringe tips.
Segment 2 consists of one tergal and one sternal plate. Sternal plate partially differentiated into two plates by an epicuticular midsternal junction that, opposed to the midsternal junctions on the following segments, does not divide the ventral side into two articulating sternal plates (Figs. 2b, 3b, d). Tergosternal lines well-developed. Long tubes (23 µm from SEM) present in ventrolateral positions (Figs. 2b, 5a). Sensory spots present in middorsal, laterodorsal (Figs. 2a, 6a, 7a) and ventromedial (Figs. 2b, 5a) positions; sensory spots more droplet-shaped than those on the preceding segment, and without the extended marginal hairs. Cuticular hairs lightly scattered on the dorsal and lateral sides; ventral side with very few hairs only, near the tergosternal junctions; ventromedial and paraventral areas devoid of hairs but with longitudinal wrinkles in the cuticle. Posterior segment margin extend into midventral tip; pectinate fringe well-developed, with conspicuously long and flexible fringe tips, especially in sublateral-ventromedial positions; a special lanceolate or acuminate fringe tip is located at each tergosternal junction (Fig. 5a, b, d).
Segment 3 and following eight segments consisting of one tergal and two sternal plates. Segment with regular sensory spots in subdorsal positions (Figs. 2a, 6b), and glandular cell outlets type 1 in paraventral positions (Figs. 2b, 3f ). An unpaired flosculum-shaped sensory spot furthermore present midventrally (Figs. 2b, 5b, d, f ); the flosculum is small and cone-shaped, located between the posteroventral corners of the two sternal plates, at the very posterior margin of the soft cuticle that forms the articulation between the plates (Fig. 7f ). Cuticular hairs lightly scattered on the dorsal and lateral sides; sternal plates with 3-4 lines of cuticular hairs, anteriorly on segment, in the ventrolateral and ventromedial areas; paraventral areas without hairs, but with hair-like cuticular extensions, not emerging through perforation sites. Dorsal, lateral and ventral parts of posterior segment margins with conspicuously long and flexible fringe tips, being even longer than on segment 2 (Fig. 3f ); acuminate fringe tips present at each tergosternal junction (Fig. 5b).

Table 1 Measurements from light microscopy of the four type specimens of C. flosculosa sp. nov (in µm)
-Missing data; digits in the character column indicate segment numbers A/S absent structure, MD middorsal spine, LTAS lateral terminal accessory spine, LTS lateral terminal spine, LV lateroventral spine, ML midlateral spine, MSW-5 maximum sternal width, measured on segment 5 in this species, MSW-8 maximum sternal width, measured on segment 8 in this species, S segment lengths, SW-10 standard width, always measured on segment 10, TL trunk length, VL ventrolateral tube Flosculum-shaped sensory spot not present. Cuticular hairs and posterior segment margin as on preceding segment (Fig. 3g). Segment 10 with strong and rigid midlateral acicular spine (Figs. 2b, c, d, 3h, 6e, 7d). Regular sensory spots present in subdorsal positions (Figs. 2a, c, 6e, 7d), and glandular cell outlets type 1 in ventromedial positions (Figs. 2b, d, 5e). Cuticular hairs on dorsal and lateral sides as on preceding segments; ventral side only with few, marginal hair-like cuticular extensions. Pectinate fringe of posterior segment margin with rather short fringe tips, differing considerably from those on preceding segments (Fig. 3h), and acuminate fringe tips at tergosternal junctions absent. Segment 11 with long lateral terminal spines (Fig. 6f ); females in addition with lateral terminal accessory spines (Fig. 2a), males with three pairs of penile spines (Figs. 2c,  d, 3h). Sensory spots present in subdorsal (Figs. 2a, c, 6e) and ventromedial positions near the posterior margin of the segment (Figs. 2b, d, 5e), and glandular cell outlets type 1 present in subdorsal (Figs. 2a, 6f ), ventrolateral and ventromedial (Fig. 2d). Tergal plate covered by short cuticular hair-like structures, and extending into short pointed tergal extensions; sternal plates without cuticular hairs, but with short, densely set hair-like cuticular extensions near margins of rounded sternal extensions.

Differential diagnosis
The composition of segment 2, consisting of one tergal and one sternal plate, with the sternal plate being partially divided by an incomplete midsternal junction, clearly places the new species in the echinoderid genus Cephalorhyncha [13,14,25]. Currently, the genus accommodates three species: C. asiatica (Adrianov, 1989), C. nybakkeni (Higgins, 1986), and C. liticola . Cephalorhyncha flosculosa sp. nov. can be differentiated from its three congeners by its midventral flosculi on segments 3-8.
Cephalorhyncha flosculosa sp. nov. is most easily distinguished from C. liticola by its midventral flosculi, and other minor differences in distributions of sensory spots. For example, C. flosculosa sp. nov. has paradorsal sensory spots on segment 9 and no paradorsal sensory spots on segment 10, whereas C. liticola shows the opposite, with paradorsal sensory spots on segment 10 only. C. flosculosa sp. nov. also has subdorsal sensory spots on segments 9 and 10; these are missing in C. liticola.
Cephalorhyncha flosculosa sp. nov. clearly shows the closest resemblance with C. liticola. Especially the strongly laterally compressed trunk and the exceptionally long fringe tips from the posterior segment margins are highly characteristic for both species (compare Figs. 1, 3 in the present contribution with Figures 10 and 11 in Sørensen [16]). They also have their minute sizes in common. With trunk lengths of 139-209 µm in C. flosculosa sp. and 127-210 µm in C. liticola, they are indeed the two smallest kinorhynch species.
The new species is distinguished from C. nybakkeni by the presence of lateral accessory tubes on segment 8. Such tubes are absent in C. nybakkeni (Higgins, 1986). C. asiatica (Adrianov, 1989) is distinguished from C. flosculosa sp. nov. by the presence of strong midlateral spines on segment 10; C. asiatica has short, laterodorsal tubes on this segment [12,13]. Unpublished SEM images of C. asiatica show that this species also tends to be laterally compressed, but to a lesser degree than C. flosculosa sp. nov. and C. liticola, and that C. asiatica also shows conspicuously long fringe tips from the posterior segment margins, but only from the sternal plates. SEM images of C. nybakkeni are not available, but LM images shown by Neuhaus and Blasche [14; Figure 11f-m] suggest that long fringe tips Although it may seem as a rather vague character, the conspicuously long fringe tips, at least from the sternal plates, could potentially represent an additional diagnostic trait for the genus.

Flosculi
Van der Land [26] described flosculi from the priapulid Tubiluchus corallicola as "small organs in the shape of a flower". Since then, various types and shapes of flosculi have been reported from species of Priapulida and Loricifera [27][28][29][30]. In kinorhynchs, Adrianov [12] considers the mushroom-shaped sensory structures (type 3 sensory spots sensu Nebelsick [31] to be a kind of flosculi. These flosculi are typically found on the most posterior segments, and appear to be more numerous in juvenile specimens [2,32,33].
The flosculi which is reported from C. flosculosa sp. nov. differ from the typical type 3 sensory spots. Whereas type 3 sensory spots are mushroom-shaped, with a relatively long stalk equipped with short, terminal micropapillae, the flosculi in C. flosculosa sp. nov. have a shorter stalk, and the terminal micropapillae are longer and pointier. Nebelsick [31] already stressed that the homology of flosculi across the scalidophoran groups still remained to be examined, and likewise, we find it premature to suggest that the flosculi of C. flosculosa sp. nov. are homologous with type 3 sensory spots found on posterior segments of allomalorhagid and kentrorhagid kinorhynchs. What is interesting though, is that the flosculi of C. flosculosa sp. nov. are rather unusual structures, in regard to their shape as well as their position. This kind of flosculi has to our knowledge not been shown from any kinorhynch before, which in itself makes them unusual, and also their position is completely unique. Whereas all kinorhynchs typically have cuticular structures distributed around their trunk segments-from middorsal to paraventral position-it is not very common to possess unpaired, midventral structures, and we are not aware of any corresponding examples of midventral structures on the trunk segments.

Habitat preferences and zoogeography
Cephalorhyncha flosculosa sp. nov. was collected from the intertidal zone and from a sandy beach with gravel at 40-70 cm sediment depth. This special habitat preference is shared with two of its congeners: C. nybakkeni was described from the intertidal zone of a Californian beach [15] and C. liticola is intertidal as well, from a Hawaiian beach [16]. In all three cases, specimens could be found in the sand down to 70 cm depth. This suggests that at least some species of Cephalorhyncha share a certain preference for this kind of habitats. However, C. asiatica is described from subtidal mud [13], hence the habitat choice does not characterize the genus as such.
Zoogeographically, the finding of C. flosculosa sp. nov. in the Aegean Sea also attracts special attention, as it represents the first recording of the genus outside the Pacific Ocean. The finding of a representative of Cephalorhyncha in the Mediterranean Sea, basically as far away from the Pacific Ocean as possible, suggests that species of the genus could be found in any other World ocean as well. This confirms a trend that appears to be general for many kinorhynch genera, namely that their species may have a rather patchy distribution, but still on the generic level show an almost global distribution. Similar examples are the genus Dracoderes, which since its discovery appeared to be an East Asian genus [13,34], but has more recently been found in samples from Spain [35]; Paracentrophyes was originally described from the Atlantic Ocean [36,37], but later was recorded from the West Pacific also [38]; and Fissuroderes that was known from the Pacific only [14], until it showed up in the West Atlantic [39]. Other kinorhynch genera are still known from only single localities or restricted areas, but we find it very likely that they over time will be recorded from other regions of the world as well, and that most, if not all, kinorhynch genera will turn out to have a nearly cosmopolitan distribution. The number of taxonomists working on obscure taxa such as kinorhynchs has always been rather low which explains our limited knowledge about them.