Shallow water sea slugs (Gastropoda: Heterobranchia) from the northwestern coast of the Sea of Japan, north of Peter the Great Bay, Russia

The coast of northern Primorye region, north of Peter the Great Bay has been sparsely studied in regards to its molluscan fauna, with just a few works reviewing the distribution of local mollusks. This work presents a survey of the shallow water heterobranch sea slugs currently occurring around Kievka Bay to Oprichnik Bay, Russia. Thirty-nine species of sea slugs were found in this study and the new species Cadlina olgae sp. nov., described herein. Most (24) of the species occurring in the area have widespread ranges in the northern Pacific Ocean. The eight species are endemic for the Sea of Japan and adjacent part of the Sea of Okhotsk. Seven other occur also in northern Atlantic and Arctic waters. Thirteen found species are not known from Peter the Great Bay but known from adjacent northern Pacific waters. The finding of a previously undescribed species emphasizes the need of further surveys, particularly in subtidal and deeper waters, in order to improve the knowledge on this neglected fauna in Primorye.

The present study provides records of sea slugs found in shallow waters (above 30 m depth) between Kievka Bay (42.85 • N) and Oprichnik Bay (44,45 • N), Primorskiy Krai, Russia. The coast of this area consists of rocky formations with sparse sandy beaches and a quite narrow intertidal zone. Rocky platforms and boulder fields are common; however, some sheltered areas have open sandy beaches, usually exposed to strong surf (e.g., Rudnaya, Kievka Bays). The goal of this preliminary study is to contribute to the knowledge of the molluscan fauna in Russian Far East, particularly providing a tool useful for identification of live animals in the field.

MATERIALS AND METHODS
The material examined was collected during the summers of 2012-2016 in several locations between Kievka and Oprichnik Bays ( Fig. 1) of the northwestern Sea of Japan, Primorskiy Krai, Russia. All the collecting was made manually by SCUBA diving, mostly on rocky walls, platforms, and the pinnacles. Four specimens of Cadlina laevis collected in the White Sea Biological Station, Moscow University, White Sea, Russia were also examined. The specimens were deposited in the collections of the Museum of A.V. Zhirmunsky Institute of Marine Biology, Russian Academy of Sciences (MIMB) and Zoological Museum, Moscow State University (ZMMU).
Field study permits were not required for this study and none of the species studied herein are currently under legal protection. All the collected specimens were preserved in 95% ethanol. Photography was performed with a Nikon D300 or D810 cameras with a Nikkor 105/2.8G lens in appropriate Sea&Sea housings with Sea&Sea YS-D1 strobes when used underwater. All sizes given are living measurements, radular features were examined after carbon coating by field emission scanning electron microscope Zeiss Sigma using a ETSE detector at EHT 10 kV. Color plates were composed with Adobe Photoshop software and original colors of the images were not modified.
In order to characterize genetically and barcode the new species of Cadlina, DNA extraction was performed using DNEasy kit (Qiagen). Folmer's universal COI (Folmer et al., 1994), and 16S rRNA gene fragment primers (Palumbi, 1996) were used to amplify the region of interest for three specimens of Cadlina olgae sp.n. and two specimens in C. laevis. For two specimens of Limacina helicina, the COI fragment was amplified only. The master mix (for each sample) was prepared using 34.75 mL H 2 O, 5.00 mL PCR Buffer (Evrogen, Moscow, Russia), 5.00 mL 25 mM MgCl 2 , 1.00 mL 40 mM dNTPs, 1.00 mL 10 mM primer 1, 1.00 mL primer 2, 0.25 mL 5 mg/mL Taq, and 1.00 mL extracted DNA. Reaction conditions were an initial denaturation for 3 min at 95 C, 39 cycles of 1) denaturation for 45 sec at 94 • C, 2) annealing for 45 sec at 50 • C, and 3) elongation for 2 min at 72 • C, and a final elongation for 10 min at 72 • C. PCR products yielding bands of appropriate size (approximately 695 bp in COI, and 421 in 16S) were purified using the Montage PCR Cleanup Kit (Millipore). Cleaned PCR samples were quantified using a NanoDrop 3000 Spectrophotometer (Thermo Scientific). Sequencing was conducted by Sanger ddNTP termination method using BrightDye chemistry (Nimagen) and ABI 3500 Genetic Analyser (Applied Biosystems). The sequences were assembled and edited using BioEdit (Hall, 1999). BioEdit was also used to extract the consensus sequences The sequences used in this study are listed in the Table 1, most of acquired from GenBank sequences were obtained by Johnson (2010).
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:02814E3B-C41F-4AA7-80B9-D4DD2ED73FF2. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Retusa minima
Radula described and imaged in Chichvarkhin, Chichvarkhina & Chernyshev (2015). Ecology. Occurs at the depths of 16-20 m on rocky substrates in Senkina Shapka pinnacle. In Kievka Bay lives at the depth of 0.5-3 m on calcareous red algae. Feeding presumably on benthic bacteria or protists, reproduction unknown.
Distribution. Originally described from Kunashir Island, referred as Runcina elioti from the northern Hokkaido (Nakano, 2004). Likely distributed along the Sea of Japan coast between Amur river mouth and Peter the Great Bay, probably in the Korean peninsula (Chernyshev, 2006;Chichvarkhin, Chichvarkhina & Chernyshev, 2015

Distribution.
A common species known from California along North American and the Asian coast of Japan and Korea (Martynov & Korshunova, 2011 Identification. Color creamy white, size to 15 mm. Notum covered with bud-like (mushroom-like) tubercles.
Ecology. Feeds on a wide range of encrusting Bryozoans. In Senkina Shapka pinnacle feeds exclusively on different bush-like Bugula articulata. Occurs at the depth of 5-20 m. An ephemeral species that is abundant in May but totally disappeared in early autumn.
Distribution. Arctic and North Pacific species. Has been recently comfirmed from the Sea of Japan, far from its known distribution area (Chichvarkhin et al., 2016d).
Ecology. Occurs at 1-15 m depth under stones and on rocks. Feeds on encrusting bryozoans. Distribution. A common species known from California along North American and Asian coast to Peter the Great Bay. May occur in Japan and Korea (Martynov & Korshunova, 2011 Diagnosis. White semi-translucent oval shaped body with solid yellow dots, rather large yellow glands near the edge of mantle, and yellow rim formed by numerous tiny dots around notum. Oral tentacles short, triangular, folded at apex, rhinophores lamellar. Rachidian teeth with 2 bigger central and 4-6 smaller lateral denticles. Inner lateral teeth with equal number of denticles on both sides. Body size to 25 mm. Description. Body shape oval, rounded in juvenile specimens, lengths to 25 mm (14 mm in holotype, 11 mm in paratype) in fully extended living specimens (Figs. 4D and 4E). Body with uniformly white semi-translucent background, uniformly covered with small yellow dots on elevated tubercles. 4-10 larger yellow sub-epidermal glands along each side of mantle; edge of notum and foot covered with numerous tiny dots forming yellow rim, which looks solid without magnification (but less intense than in C. luteomarginata MacFarland, 1966). Notum moderately wide, wider than foot, contains no spicules. Rhinophores with 8-10 lamellae with few yellow dots on top. Oral tentacles very short, triangular, folded distally. Gills in holotype with five branchial leaves, with yellow pigment on tips. Radula (Fig. 4) of 55-60 rows, in 30th row 12.1.1.1.12. Rachidian tooth with two central larger central denticles and 2-3 smaller lateral denticles (Fig. 4A). First lateral teeth with bigger central denticle and four smaller denticles on both sides. The other lateral teeth are similar, with 4-5 outer denticles and no inner denticles (Fig. 4C).
Ampulla wide, long and convoluted in two folds. Prostate long, tubular with 1-2 loops, vas deferens very narrow with one loop, it expands in wider muscular ejaculatory portion. Penis narrow, bears an armature of very fine spines. Vagina wide and short, branched into a duct that connects seminal receptacle and uterine duct. Uterine duct is long, not shorter than bursa copulatrix. Seminal receptacle almost spherical, slightly smaller than oval bursa copulatrix. No vagina extension near the entrance into copulatory bursa Etymology. After my wife and colleague Olga Chichvarkhina.
Ecology. Occurs at various depths on rocky substrates, feeding unknown.
Distribution. Probably has wider distribution in the Sea of Japan.
Remarks. This species differs from Cadlina sp. (Martynov, 1999) with larger rachidians and fewer denticles in lateral teeth. Central denticles in the rachidian tooth of C. olgae are never split in 2-3 secondary denticles. The invalid (unpublished) species ''Cadlina potini' referred by Martynov (1999) is more similar to C. olgae but possesses 6 outer denticles in first lateral teeth (four in C. olgae), the other lateral teeth possess 15 lateral denticles (4-5 in C. olgae). Both these forms referred by Martynov, the radula possesses more rows with more teeth in each row. In C. laevis (Linnaeus, 1767), rachidian teeth possess up to six equal denticles (unequal in C. olgae) (Thompson & Brown, 1984). Examined specimens of C. laevis form the White Sea possess rachidian tooth with 2-4 poorly developed smooth denticles; first lateral tooth is crowned with three denticles on inner side and 5-7 denticles on the outer side (Figs. 4F and 4G), similar pattern is observed in C. sp.2 from Bering Sea (Fig. 4H) (four denticles on both sides in C. olgae). C. japonica Baba, 1937 clearly differs from C. olgae with: brownish pigment on the mantle, intense yellow pigmentation of gills, small hook-shaped rachidian tooth divided in two lobe-like denticles, and presence of small outermost lateral teeth (Baba, 1937b). C. luteomarginata MacFarland, 1966 differs from C. olgae with solid yellow rim around the mantle, more intense pigmentation on the tubercles, hook-shaped rachidian tooth with four small denticles, larger central denticle on all lateral teeth, and 7-8 very small denticles on all lateral teeth (Rudman, 2001;Johnson, 2001). Reproductive system is typical for Far Eastern C. laevis-group species described in Martynov (1999): it possesses rather polymorphic prostate and vas deferens containing one to five loops, thus they unlikely can be served as species-specific traits. Female reproductive system is similar to Martynov's (1999) ''C. potini'' (in C. olgae holotype is identical with Fig.  83 in this work) with no vaginal duct extension near bursa copulatrix entrance. I suppose, Martynov (1999) studied C. olgae but he mixed it with one or more species reporting radula/reproductive combinations, that do not fully coincide with my specimens. Thus thorough study of morphological variation in Cadlina needed to shed light onto the systematics of this genus in the northwestern Sea of Japan.
Molecular COI sequences suggest an evidence that Cadlina olgae is a member of cryptic species complex referred as C. laevis, which includes at least C. olgae, C. laevis, an undescribed species candidate from Bering Sea, and C. luteomarginata with at least two sister species (Fig. 5). Although the p-distance between these species is relatively low, lowered level of divergence is a characteristic for sibling species that descent during Pleistocene glaciations (Breslau, Valdés & Chichvarkhin, in press;Lindsay et al., 2016;Klienberger et al., 2016;Hallas, Simison & Gosliner, 2016). Likely, this phenomenon also occurs in amphiboreal species with direct development, e.g., Cadlina (Thompson, 1967) whose speciation took place during recent dispersal from a refugia.
The resulted number of species identified in ABGD analysis of COI and 16S. Using uncorrected distance matrices, the COI sequences showed a major barcode gap between a priori genetic distance thresholds of 0.01 and 0.036 in COI (0.01 and 0.013 in 16S). Using a value of P between this range (0.01 for both markers), the same 13 species were identified, and assignment of individuals to the species matched the NJ tree topology (Fig. 5). Importantly, however, the species identified are not polyphyletic. A series of species-specific diagnostic indels were found in the 16S after positon #240 (in C. laevis  (Table 2). These indels are likely a conservative trait in Cadlina species because C. luarna and C. rumia do not possess an insert as does C. olgae, while three species (C. japonica, C. luteomarginata, C. aff. luteomarginata) possess a four-base insert TTT(C)A, three others possess one Thymidine insert (C. flavomacualta, C. modesta, and C. sparsa), C. pellucida possesses a four-base TTTA insert, and C. sp.2 possesses an insert of seven bases TTTTAAA. I suppose this pattern has high phylogenetic weight, hence it is capable to adequately detect closely-related sibling species.  Identification. Small red-colored mollusk with elongate body. Body smooth: gill, tentacles or papillae on the notum are absent. Body size to 6 mm.
Ecology. Occurs at shallow depth of 0.1-2 m under rocks or on algae. Feeds on Spirorbidae tube worms.
Distribution. Known from Kievka and Peter the Great Bays in Russia, also from Japan (Martynov & Korshunova, 2011 Identification. Background body color varies bright white to light grey with orange pigment on the gills tips and papillae located on notum edge, darker orange colored tubercles scattered on notum. Body size to 15 cm.
Ecology. Occurs at 1-30 m depth, feeds on various bryozoans (Martynov, 1999). Distribution A common species known from California along North American and Asian coast to Japan and Korea (Martynov & Korshunova, 2011

Dendronotus kamchaticus Ekimova et al., 2015 (Figs. 7A and 7F)
Dendronotus frondosus -Martynov, 2006;Ekimova et al., 2015;Martynov & Korshunova, 2011:152-155 (part.) Identification. Oral veil with 4-6 lip papillae and branched appendages. Primary stalks of veil appendages tall and slender, giving rise to numerous secondary branches with short tertiary branches. Rhinophoral sheath divide into 5-6 crown papillae that about same length. Lateral papillae (about one-third or one-half of sheath length) branches off sheath base and expanded with secondary branches. Rhinophores bear 14-20 lamellae. Background color is transparent white, with complex pattern of light, dark, and red-brown spots and stripes. On dorsal side spots and stripes merge and form characteristic striped pattern. Lateral sides of body devoid of stripes but covered with brown spots. Size to 25 mm.
Ecology. Occurs at 10-20 m depth on cnidarians. Distribution. Described from Kamchatka, recently found in Rudnaya and Peter the Great Bays. Probably possess wide distribution along Far Eastern shore (Ekimova et al., 2016). Identification. Body slim elongate laterally compressed with 4-10 pairs of branched papillae. Oral veil with 10-14 short lip papillae and 4-5 secondary branched appendages. Rhinophoral sheaths with long stalk and five crown appendages. Lateral papillae moderate in size with small secondary branches. Light to dark brown body with opaque golden groups of dots. Size to 20 mm.

Dendronotus dudkai Ekimova et al., 2016
Ecology. Occurs at 10-20 m depth on Obelia cnidarians. Distribution. This species has been detected just recently. It's confirmed distribution is two locations in Peter the Great Bay, and Rudnaya Bay, but may have wider distribution.
Remark. Recently, Dendronotus primorjensis Martynov, Korshunova and Sanamyan, 2015 has been described from Peter the Great Bay where at least three Dendronotus species occur. The description of the external morphology is quite brief and literally constitutes a redescription of D. kamchaticus because of the absence of white pigment agglomerations described for D. primorjensis is a characteristic of D. kamchaticus. However, described radula conforms to diagnosis of all species in the D. frondosus species complex. The illustrated holotype cannot be distinguished from D. kamchaticus, thus, D. primorjensis is probably a synonym of D. kamchaticus. The location of the type specimens of D. primorjensis is unknown: probably they do not exist because of their unavailability in referred collection, while the authors refuse providing them for examination. Also, the authors cannot provide or publish D. primorjensis nucleotide sequences that they refer as ''distinct from the other Dendronotus species.'' Therefore, we suggest considering D. primorjensis as nomen nudum or a synonym of a species of D. kamchaticus that is likely occurs at the type locality of D. primorjensis (Ekimova et al., 2016). Robilliard, 1972 (Fig. 7D) Robilliard, 1972:421-432 Identification. Semi-translucent pale yellow to intensive orange body and flattened papillae. White dots scattered across the body, the tips of papillae white. No white rim around foot. Size to 150 mm.

Dendronotus cf. albopunctatus
Ecology. Occurs on rocky substrates at various depths. Feeding unknown. Distribution. A common species known from California along North American and Asian coast to Japan and Korea (Martynov & Korshunova, 2011 Identification. Body white semi-translucent. Tiny white dots on oral tentacles, rhinophores, and on cerata below cnidosacs. Cerata with pinky-red appendages of digestive gland.
Ecology. Found on sunken rope colonized with Obelia hydroids. Distribution. Known from all Russian Pacific seas and Hokkaido shore in Japan (Martynov & Korshunova, 2011 Identification. Body white. Cerata brownish-red, never bright red in studied area. White solid stripe on oral tentacle and less solid pigmentation on the rhinophores. Cnidosacs smaller than in similar C. cf. nobilis. White stripe on tail. Body size to 35 mm.

Distribution.
A common species known from all Far Eastern seas of Russia, North America, Arctic and the northwestern Atlantic (Martynov & Korshunova, 2011;Behrens & Hermosillo, 2005 -Martynov, 2006;Martynov, 2013.  Identification. Body yellow to orange. Cerata, oral tentacles and proximal parts of the rhinophores white with blue pigmentation in basal part. Body size to 15 mm.

Triinchesia viridis
Identification. Body white. The rhinophores and oral tentacles are translucent, 2/3 proximal part of them is pigmented white. Cerata dusted with white pigment, with brownish-green digestive gland appendages. Cnidosac is distinctive, white under translucent cap. Body size to 15 mm.
Ecology. Found on algae covered with the hydroids. Distribution. Widely distributed in the northern Pacific and the northern Atlantic (Martynov & Korshunova, 2011 Identification. Body white semi-translucent. Rhinophores longer than oral tentacles lack pigmentation. Cerata pink with white dots and white cnidosacs. Body length to 30 mm. Ecology. Occurs at the depths of 2-20 m. Feeds on hydroids colonized hermit crabs' shells. Oviposits on the same shells and hydroid colonies. Distribution. Known from Vladimir Bay, Rudnaya Bay, and Kievka Bay (Chichvarkhin et al., 2016b). Presumably reported from Bering Sea (Martynov & Korshunova, 2011;Martynov, Sanamyan & Korshunova, 2015). Also known from the NE Pacific and Atlantic (Chichvarkhin et al., 2016b).
Remark. Cuthona hermithophila has been described from Kievka Bay recently. We have thoroughly investigated a population from there and few other populations. All of them are nearly indistinguishable from nominative C. nana (Chichvarkhin et al., 2016b).  Martynov, 1992(Figs. 11A-11G) Martynov, 1992