Identification key to Nephtyidae (Annelida) of the Black Sea

Abstract Currently, nine species of Nephtyidae (Annelida) are known from the Black Sea. A new user-friendly identification key is presented with a brief description for each species based on type material and recently collected specimens from the Black Sea.

Almost all samples were first fixed in 10% formaldehyde and then transferred to 70% ethanol (24 specimens of M. longicornis were fixed directly in 70% ethanol). Specimens were stained with methylene blue (water solution) and examined using stereomicroscopy. Pharynx characters were studied on worms with a fully everted pharynx. Photographs were taken using a Carton DSZT70 stereomicroscope equipped with a MDC 320 Microscope Digital Camera. Line drawings were prepared by tracing stereomicroscope photographs in CorelDRAW. To examine the ultrastructure of chaetae, some chaetigers were dissected, critical point dried, coated with 25 nm Au-Pd and observed with a Camscan S-2 Cambridge Scanning Electron Microscope (SEM).
All the descriptions and drawings are original except for that of I. foretmontardoi (after Ravara et al. 2010). They were made without preparing slides as a cover glass deforms parapodial structure. All parapodia are shown in anterior view, unless otherwise stated. The terminology used in the key is given in Figures 1-4.

Remarks on the key
Nephtyids are rather similar in their morphology and often difficult to distinguish. The most used taxonomic characters to separate the species are: parapodial morphology, branchiae shape, number of branchiferous chaetigers, ornamentation of the chaetae (only visible under a compound microscope), and pharynx structure. The number of the most anterior chaetiger with developed postacicular lobes was also included in descriptions, as this is an important systematic character (Dnestrovskaya 2013). Not all characters are developed in juveniles, and it is not always possible to identify fragmented animals without specialized training.
All parapodia in Nephtyidae are biramous. Both noto-and neuropodia consist of acicular, pre-and postacicular lobes, and dorsal (notopodial) and ventral (neuropodial) cirri. In Nephtys and Micronephthys species, the acicular lobes are supported by one acicula and may be conical, rounded, or bilobed ( Fig. 1). In I. foretmontardoi, the anteriormost parapodia have up to five aciculae in the neuropodia and four in the notopodia. The number of aciculae decreases gradually towards the posterior end of the body. Single aciculae of posterior parapodia have curved tips. Smaller specimens of Inermonephtys have a lower number of aciculae per parapodium (after Ravara et al. 2010). Shape of parapodial lobes varies along the body, so the user should be sure of examining the parapodia from the chaetiger recommended in the key or key drawings. All morphological details of the parapodia can usually be seen under the stereomicroscope. Several undamaged parapodia from both sides of the worm should be examined.
The branchiae are inserted in the parapodia below the dorsal cirri and may be involute or recurved. They may be slender, digitiform, folia ceous, or rounded-fleshy. A small papilla may be present at the base of the branchiae under the notopodial cirrus. The shape and proportions of branchiae vary along the body, so they should be examined on the chaetigers that are recom mended in the key. The chaetiger on which the branchiae begin should be checked on both sides of the worm.
The prostomium is subquadrangular to subpentagonal (shape depends on whether the proboscis is everted or not). A pair of conical antennae is present in the anterior corners of the prostomium (absent in Inermonephtys). A pair of palps is inserted ventrolaterally. A pair of nuchal organs is located dorsolat erally on the posterior margin of the prostomium (Fig. 2).
The pharynx is a large eversible muscular proboscis, usually covered with soft papillae located in different areas that can be seen when pharynx is everted or dissected   ( Fig. 3). All pharyngeal papillae are absent in Inermonephtys. In Micronephthys and Nephtys the anterior margin of the pharynx is surrounded by 18-20 bifid terminal papillae separated dorsally and ventrally by gaps; each gap may bear a single conical papilla. The subterminal region has 14 to 22 longitudinal rows of conical to digitiform papillae decreasing in size towards the base of the pharynx. A single longer subterminal papilla may be present middorsally and midventrally. The proximal surface may be smooth or covered with small warts (flat outgrowths) or small papillae (conical or rounded) that slightly rise above the surface. Phar ynx dissection is not always necessary but may be useful to confirm identifications.
Examining several specimens rather than a single individual is strongly recommended for identification. Staining with methylene blue (but not methyl blue!) will significantly highlight morphological characters of all structures.

Discussion
Nephtys caeca, N. ciliata, N. longosetosa, and N. paradoxa were absent not only in our collection from the Black Sea, but also in the collections of other museums where the Black Sea's fauna traditionally was studied (ZIN; IMBR, Dr E. Lisitskaya pers. comm.; IMBU, Dr O. Bondarenko pers. comm.); they are absent in the keys by Vinogradov and Losovskaya (1968).
The mention of these species is based on identification by Rullier (1963) from the region near the Bosphorus. In later articles, Marinov (1977), Kiseleva (2004), Şahin and Çinar (2012), and Çinar et al. (2014) all referred to the same samples.
Perejaslavtseva (1891: 236) wrote: "Steamerships that constantly are coming from Constantinople and the Mediterranean, could bring to the Black Sea the specimens of the strait Bosphorus fauna", and recent researchers agree with her (Ivanov and Belokopytov 2011). However, I believe that the ranges of N. caeca, N. ciliata, N. longosetosa, and N. paradoxa are too widely circumscribed. These species are probably absent from the Black Sea fauna and some may even be absent from the Mediterranean; at least their presence in these faunas needs confirmation. Close investigation of some other species with wide distributions (including the Arctic Ocean and Mediterranean Sea) has shown that in reality they are species complexes. For example, Jirkov's (2018) recent revision of Thelepus cincinnatus (Fabricius, 1780) (Terebellidae) resulted in four different species: one from the deep Arctic, a second arcto-boreal, a third boreal-Mediterranean, and a fourth Mediterranean species.
The Nephtyidae of the Black Sea could be divided into two groups by the presence of different types of chaetae in the postacicular rows. The north-boreal species (N. caeca, N. ciliata, N. longosetosa, and N. paradoxa) have spinose chaetae, while south boreal-Lusitanian species (N. hombergii, and N. hystricis) have serrate chaetae with only single lateral rows of spines along one side of the chaeta (Dnestrovskaya and Jirkov 2011). Nephtys cirrosa has specific geniculate chaetae, M. longicornis has dentate chaetae and lyrate chaetae with unequal rami whereas I. foretmontardoi has lyrate chaetae with subequal rami (Ravara et al. 2010) (Fig. 4). Despite the shape of spines in postacicular chaetae only being visible under a compound microscope, they were added in descriptions as a supplementary character.
No key is complete and perfect. The key given below should be used with caution and confirmed with descriptions of the species concerned.

Remark
-C1 with distinct dorsal cirri; notopodial cirri in all chaetigers half the length of the branchiae or even shorter; in middle chaetigers postacicular lobes more than twice as long as acicular lobes; aciculae lobes of noto-and neuropodia without any external outgrowths; proximal region of pharynx covered with small warts; geniculate chaetae absent .. Body length up to 250 mm, up to 150 chaetigers (Rainer 1991). Parapodial preacicular lobes poorly developed rounded. Acicular lobe bilobed in anteriormost and middle regions of large worms. Postacicular lobes from C2 in neuropodia, C3 in notopodia, extending well beyond acicular lobes. Neuropodial postacicular lobes subequal in length to notopodial postacicular lobes or only slightly longer. Spinose chaetae in postacicular rows. Middorsal subterminal papilla of pharynx similar in size to largest subterminal papillae or absent; up to 6 subterminal papillae per row; proximal region covered with flattened warts. Amphiboreal and Lusitanian, reported from Black Sea near Bosphorus (Rullier 1963), but these records require confirmation; from the lower intertidal to nearly 1000 m (Rainer 1991), but according to our data it occurs at upper shelf depths.