Pteropods from the Kuril-Kamchatka Trench and the sea of Okhotsk (Euopisthobranchia; Gastropoda)

Journal Pre-proofs PTEROPODS FROM THE KURIL-KAMCHATKA TRENCH AND THE SEA OF OKHOTSK (EUOPISTHOBRANCHIA; GASTROPODA) P.C. Kohnert, A.F. Cerwenka, A. Brandt, M. Schrödl PII: DOI: Reference:

S0079-6611(19)30439-2 https://doi.org/10.1016/j.pocean.2019.102259 PROOCE 102259

To appear in:

Progress in Oceanography

Received Date: Revised Date: Accepted Date:

14 May 2019 15 December 2019 27 December 2019

Please cite this article as: Kohnert, P.C., Cerwenka, A.F., Brandt, A., Schrödl, M., PTEROPODS FROM THE KURIL-KAMCHATKA TRENCH AND THE SEA OF OKHOTSK (EUOPISTHOBRANCHIA; GASTROPODA), Progress in Oceanography (2019), doi: https://doi.org/10.1016/j.pocean.2019.102259

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier Ltd.

PTEROPODS FROM THE KURIL-KAMCHATKA TRENCH AND THE SEA OF OKHOTSK (EUOPISTHOBRANCHIA; GASTROPODA) KOHNERT, P. C.a,b*, CERWENKA, A. F.a., BRANDT, A.c, d & SCHRÖDL, M.a,b,e

a

SNSB-Bavarian State Collection of Zoology, Section Mollusca, Münchhausenstr. 21, 81247 Munich, Germany

b Department

Biologie II, Biozentrum, Ludwig-Maximilians-Universität, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany c Senckenberg

Research Institute and Natural History Museum, Department Marine Zoology, Senckenberganlage 25, 60325 Frankfurt, Germany d

Institute for Ecology, Evolution and Diversity, Goethe-University of Frankfurt, FB 15, Maxvon-Laue-Str. 13, 60439 Frankfurt am Main, Germany e Center

of Geobiology and Biodiversity Research, Ludwig-Maximilians-Universität, RichardWagner-Str. 10, 80333 Munich, Germany *Corresponding

author, Email: [email protected]

Abstract Pteropods are holopelagic marine snails and slugs that are of particular interest to science due to their role in in marine food webs, global carbon cycle and their potential sensitivity to ocean change. Due to their pelagic, often exclusively offshore occurrence, samples are difficult to obtain, resulting in a lack of knowledge about their physiology, ecology, anatomy, geographical ranges, phylogenetic relationships and reproductive biology. Despite a recent increase in interest surrounding pteropod taxonomy, many evolutionary uncertainties remain due to limited taxon sampling and inavailability of molecular vouchers, in particular for the lesser investigated groups Pseudothecosomata and Gymnosomata. The Northwest Pacific Ocean is one of the least investigated areas for pteropods and in the adjacent semi-enclosed Sea of Okhotsk basin, current knowledge is restricted to the epipelagic zone. We summarize results from plankton hauls (from up to 5900 m depth) conducted during the joint German/Russian SokhoBio and KuramBio II cruises to the Sea of Okhotsk and the Kuril-Kamchatka-Trench region. This study presents an integrative taxonomic overview of six pteropod species identified by detailed morphological methods, including serial semithin sectioning, µCT and SEM scanning supported by multimarker (COI, 28S, and H3) genetic barcoding. We found four species of Gymnosomata slugs (Clione limacina, Clione okhotensis, Notobranchaea grandis and Thliptodon sp.), three species of Euthecosomata snails (Limacina helicina and two genetically delimited Clio spp.) and one shelled pseudothecosome species that is probably new to science (Peracle n. sp.). Multilocus phylogenetic analyses support monophyly of major traditional groups such as Pteropoda, Thecosomata, Pseudothecosomata and Gymnosomata. Micro-CT scanning was applied for the first time on pteropod soft bodies, allowing direct comparison between detailed

anatomical peculiarities and molecular barcodes of the respective species. Furthermore, taxonomic positions, geographical ranges and potential dispersal barriers are discussed, with implications for future biodiversity comparisons. This study serves as a solid foundation for monitoring pteropods in a changing ocean.

Keywords: Pteropoda; Barcoding; Sea of Okhotsk; Kuril-Kamchatka-Trench; µCT; Anatomy

Introduction Pteropoda Cuvier, 1804 is a monophyletic clade of holopelagic euopisthobranch gastropods that possess paired parapodial appendages (so-called "wings") as an adaptation to their habitat. As they are found in almost all oceans and occur in large numbers, pteropods comprise a large part of the global zooplankton community, especially in polar and subpolar regions (Hunt et al., 2010). Pteropods play an important role as a food source for a variety of marine organisms (including fish, seabirds and whales), biogenic aragonite producers (Feely et al., 2004) and vectors of CaCO3 to the deep sea (Berner and Honjo, 1981). In traditional morphology-based pteropod phylogenies, there are two suborders: shelled Thecosomata (including Euthecosomata Meisenheimer, 1905 and Pseudothecosomata Meisenheimer, 1905) and (adult) shell-less Gymnosomata Blainville, 1824. Euthecosomata have extremely diverse shell forms, ranging from sinistrally spiraled to bilaterally symmetrical forms that can be straight or bulbous. Within the last decade, scientific interest in euthecosomes has increased, and the majority of phylogenetic and species delimitation analyses focus on them (Burridge et al., 2015; Burridge et al., 2016; Burridge et al., 2017; Corse et al., 2013; Gasca and Janssen, 2014; Hunt et al., 2010; Maas et al., 2013 Shimizu et al., 2017, Sromek, et al., 2015). Within Euthecosomata, the exceptionally thin aragonitic shells of the spiraled Limacinoidea Gray, 1840 were shown to be highly vulnerable to changing ocean chemistry (Bednaršek et al., 2016; Lischka et al., 2011, Lischka and Riebesell, 2012, Manno et al., 2017), particularly in high latitudes of the North Pacific (Comeau et al., 2012). In particular, the Limacina helicina species complex is used as indicator for sea water acidification and model organism for researching effects on calcifying organisms (e.g. Bednaršek et al., 2014). Problems in euthecosome taxonomy have arisen due to the destruction or loss of type-specimens and the degradation of their shells in museum collections. Within the Pseudothecosomata, the genus Peracle Forbes, 1844 have a sinistrally coiled calcareous shell, which is gradually replaced by a more or less developed pseudoconch in the family Cymbuliidae Gray, 1840 and completely absent in Desmopteridae Chun, 1889 (Van der Spoel, 1976). Gymnosomata have a very tiny, thimble-like shell in early larval stages that is discharged after a few days (Lalli and Conover, 1973; Lalli and Gilmer, 1989). Juvenile and adult individuals are consequently shell-less throughout their lives. In contrast to Euthecosomata and Pseudothecosomata, that feed by using a mucus net to trap other plankton organisms (Gilmer, 1972; Gilmer, 1974; Gilmer and Harbison, 1986; Lalli and Gilmer, 1989), Gymnosomata are voracious carnivore predators that feed exclusively on both aforementioned

pteropod suborders. Gymnosomes possess characteristic manifold extendable pharyngeal organs that allow them to catch prey and ingest their entire soft bodies. For Pseudothecosomata and Gymnosomata, proper identification using phylogenetic approaches is lacking due to the limited availability of molecular vouchers, deformation of fixed specimens and lack of in situ observations. Both suborders are subsequently poorly studied scientifically and their morphological and genetic diversity, as well as their geographical ranges have rarely been explored. Although molecular phylogenetics has confirmed the monophyly of pteropods (Klussmann-Kolb and Dinapoli, 2006), interrelationships of the above-mentioned suborders are still unresolved (Burridge et al., 2017; Corse et al., 2013). Internal pteropod phylogeny thus is an active and promising field of research once suitable material from poorly studied areas like the Northwest Pacific becomes available for molecular studies. While modern species delimitation methods such as Automated Barcode Gap Discovery (ABGD) (Puillandre et al., 2012) rely solely on molecular data, predictions of a species’ adaptive abilities and potentials require soft body anatomical and physiological information. Much of the existing pteropod anatomical knowledge pre-dates the widespread use of molecular data (Meisenheimer, 1905; Tesch, 1913; Van der Spoel, 1967; Van der Spoel, 1976), therefore lacking genetic vouchers to accompany morphological descriptions. Microanatomical peculiarities of euthecosomes were recently reinvestigated using virtual 3D reconstruction based on serial semithin sections (Kubilius et al., 2014; Laibl et al., 2019), but to date do not include gymnosome or pseudothecosome species. The Northwest Pacific and adjacent semi-enclosed sea basins (such as the Sea of Okhotsk) are among the numerous ocean regions that lack a comprehensive synthesis of pteropod taxonomic identification (Janssen et al., 2019). Only two pteropod species were previously reported from the Sea of Okhotsk and the Kuril-Kamchatka-Trench (KKT) area by Sirenko (2013), the euthecosome Limacina helicina ochotensis Shkoldina, 1999 and the gymnosome Clione limacina (Phipps, 1774). Recently Yamazaki and Kuwahara (2017) described the new species Clione okhotensis from the southern Sea of Okhotsk. In this study, we give an integrative morphological and molecular overview of pteropod species obtained during the joint German-Russian SokhoBio and KuramBio II cruises to the Sea of Okhotsk and the adjacent KKT region. In the KKT region, plankton hauls from the meso- and partly bathypelagic water column revealed the presence of four previously unrecorded and one undescribed pteropod species. We used pteropod samples collected in the arctic and subarctic sector of the Atlantic Ocean and the White sea for comparison to KKT samples in our molecular analyses. Insert Fig. 1 here Material and Methods Pteropod specimens were collected during vertical hauls of up to 5900 m from 21 sampling localities (see Fig. 1). Information about sampling gear and parameters are summarized in Table 4 (supplementary material) and in the cruise report of KuramBio II (Brandt et al., 2016). Specimens were observed macro- and microscopically on board and identified morphologically. They were fixed in EtOH 96% (DNA extraction) and 4% formalin

(morphological vouchers). All obtained pteropods were transferred to the Bavarian State Collection of Zoology (ZSM Munich) for further study. DNA extraction, amplification and sequencing Genomic DNA was extracted either from tissue subsamples or whole animals, depending on their size. We followed the CTAB extraction protocol by Knebelsberger and Stöger (2012) combined with Nucleospin®Tissue DNA purification columns (Macherey-Nagel, Düren, Germany) for DNA recovery according to the manufacturer’s instructions. Amplification of mitochondrial Cytochrome c oxidase subunit 1 (COI), 28S ribosomal RNA and histone H3 gene was performed using a Biometra TProfessional thermocycler with primers and PCR protocols summarized in Table 2 (supplementary material). PCRs were performed in 25 µl volumes containing 23 µl of molecular water, 0.5 µl of respective primers (10 µmol) and 1 µl of extracted genomic DNA. PCR products were purified using the DNA Clean & ConcentratorTM-5 Kit (Zymo Research, Irvine, U.S.A.) and eluted with the provided buffer. Sequencing reactions were performed at the LMU Sequencing Center using ABI BigDye Terminator v3.1 and analyzed on an ABI 3730 48 capillary sequencer with 50 cm capillary length. A total number of 194 new sequences from 75 pteropod individuals were successfully amplified, including sequences from fifteen Limacina helicina and ten Clione limacina specimens from western Greenland, Svalbard and the White Sea. All sequences are publicly available in GenBank and accession numbers are summarized in Table 1 (supplementary material). Phylogenetic analysis and species delimitation Sequences were quality checked using the BLASTn function implemented in Geneious R8 and by translation into proteins for partial COI and H3 markers. Alignments were generated with the Mafft alignment (Katoh and Standley, 2013) Geneious R8 plugin. Resulting COI and H3 alignments were manually trimmed to a length of 498 and 335 bp, respectively. Original 28S alignment (1137 bp) was masked using the online version of Gblocks 0.91 (Castresana, 2000; Dereeper et al., 2008) set to the least stringent settings, resulting in a total 28S alignment length of 920 bp. RAxML analyses (Stamatakis, 2014) for single gene and concatenated trees (1753 bp) were calculated at the Cipres Science Gateway using the GTR GAMMA model with 1000 bootstrap replicates. Intra- and interspecific pairwise tree distance was calculated by means of the species delimitation plugin in Geneious R8. Automated Barcoding Gap Discovery (ABGD) species delimitation (Puillandre et al., 2012) was performed online on separated alignments of the respective clusters of sequences (www.abi.snv.jussieu.fr/public/abgd/abgdweb.html). Micro-computed tomography scanning (µCT): Seven specimens (five soft bodies and two shells) were scanned in a Phoenix Nanotom m cone beam CT scanner (GE Inspection Technologies, Germany). Specimens used for µCT scanning were fixed either in 4% aqueous formalin solution or 96% EtOH. Gymnosome and pseudothecosome specimens were incubated in PTA (1% phosphotungstic acid in aqueous solution), whereas we used IKI (1% iodine metal (I2) +2% potassium iodine (KI) in aqueous solution) staining (Metscher, 2009) for the Clio and Limacina specimens. Staining durations

and µCT parameters are summarized in Table 3 (supplementary material). Due to their fragility, limacinid shells were left unstained prior to scanning. All specimens were scanned in 96% EtOH and mounted in a plastic pipette-tip. Serial semithin sectioning: Two formalin-fixed specimens (Clione okhotensis and Peracle sp.) were embedded in Epon epoxy resin and serially sectioned to histological microscopy slices of 2 µm thickness following the procedure described in Neusser (2006), Kubilius et al. (2014) and Laibl et al. (2019). Results are not shown in figures but were used to verify µCT data. Scanning electron microscopy (SEM): For the scanning of radulae, the tissue of respective specimens was dissolved in a 10% aqueous potassium hydroxide solution for 12-24h. Radulae were dissected from tissue remnants, washed and dehydrated in 96% EtOH, mounted on a tungsten wire on top of a SEM stub and dried in a heating cabinet. Shells were taken directly from 96% EtOH-fixed samples and subsequently air-dried. All samples were coated with gold in a GaLa (Gabler Labor Instrumente Handels GmbH) sputter coater in an argon atmosphere for 240s. SEM images were taken using a LEO 1430 VP SEM (Zeiss, Germany) at 1-10 kV. Results Phylogenetic analysis, Fig. 2 Our multilocus phylogenetic analysis supports the monophyly of Pteropoda and the three traditional subgroups Euthecosomata, Pseudothecosomata and Gymnosomata. Euthecosomes present in the studied area were Limacina helicina (Phipps, 1774) and two morphologically similar species of the genus Clio Linnaeus, 1767. Sequences of Limacina helicina obtained from the cruise material cluster together with other L. helicina sequences from other arctic and subarctic localities used for comparison (from the Atlantic sector of the Arctic Ocean and the White sea). Clio sequences are different from existing Clio sequences in NCBI GenBank and show a well-supported terminal bifurcation in the tree caused by considerable differences in at least two markers. Sequences of pseudothecosome specimens (Peracle n. sp.) obtained during Sokhobio and Kurambio II cruises cluster together in a monophyletic group that is sister to Peracle reticulata (d’Orbigny, 1835), the only species of Peracle with more than one marker deposited in NCBI GenBank. Most gymnosome specimens from the cruise material were Clione elegantissima (Dall, 1871), and Clione okhotensis Yamazaki & Kumahara, 2017, both representing well-separated groups in the multilocus tree presented herein. Sequences from Clione limacina (Phipps, 1774) from the Atlantic sector of the Arctic and the White Sea cluster in a third group and form a well-supported clade together with C. elegantissima and C. okhotensis. For Notobranchaea grandis Pruvot-Fol, 1942 and Thliptodon sp., we were not able to generate molecular data, since only one specimen was collected for each species.

Insert Fig. 2 here Limacina helicina (Phipps, 1774), Fig. 3 Specimens of Limacina helicina were present at every station during both cruises as veligers, juveniles and/or adults. Shells of adult specimens had a height of up to 3.64 mm and a diameter of 4.1 mm (H/D ratio 0.888), while shells of juvenile specimens had a height of 1.09 mm and a diameter of 1.17 mm (H/D ratio 0.932). First 3.5-4 whorls are elevated and unsculptured (Fig. 3 B, C, G-I), while remaining whorls are rather depressed, rapidly grow in diameter and show distinct fine radial riblets (Fig. 3 D, E, F). Twisted columella externally clearly visible in ventral part of shell, aperture wide and ear-shaped, umbilicus wide open. Soft body with anterior headfoot complex and posterior visceral sac covered by the shell. Dorsal mantle cavity with large mantle and anal gland, elongated osphradium, anus and nephroporus in anterior-most portion of the last whorl. Paired wings with small and pointed wing protrusions on the anterior edge. Three ventral foot lobes, the median of which bears the operculum (lost in many specimens). Balancer present. Head area situated between base of the wings. One pair of asymmetric and anatomically different (rhinophoral) tentacles. “Right” tentacle situated frontally on the head area and retractable into a sheath. Left tentacle on the left lateral side of the neck region with a rudimentary eye-like structure. Mouth opening with dark brown pigmentation. No jaws present. Radula formula [1-1-1]. Median unicuspidal tooth with broad, shovel-like base and numerous small denticules along both lateral edges. Lateral teeth long, unicuspidal and slightly curved, with denticulation only on the inner margin. Wide gizzard with four pointed and one blunt gizzard plate. Fifth plate in dorsal position, much smaller and slightly shifted to the posterior. Voluminous digestive gland with embedded intestine forming a large loop. Central nervous system (CNS) highly condensed with paired cerebropleural and pedal ganglia. Supraesophageal cerebral commissure extremely long. Subesophageal buccal ganglia completely fused. Visceral loop with two almost fused visceral ganglia. Peripheral osphradial ganglion present directly above the osphradium in the roof of the mantle cavity. Renopericardial complex situated in the posterior part of the body whorl comprising heart with auricle and ventricle, pericardium, small kidney and nephrostome, opening into the mantle cavity. Genital system with gonad situated in the uppermost whorls of the shell, gonoduct with conspicuous widening in the middle part and complex of nidamental glands within the right side of the visceral mass. Genital pore followed by external ciliated seminal groove connecting to cephalic copulatory organ retractable into the penial sheath. Opening of the latter close to the base of the right (median) tentacle. Eighteen specimens of different life stages and from different stations of SokhoBio and KuramBio II cruises were selected for amplification of genetic markers. Phylogenetic analysis of concatenated COI, 28S and H3 genes and additional ABGD species delimitation using COI only indicated that all sequences belong to a single species. Intraspecific distance is 0.002 for the concatenated dataset and the closest related species is Limacina retroversa (J. Fleming, 1823) with an interspecific distance of 0.142. Insert Fig. 3 here Clio cf. andreae/polita, Fig. 4

Clio specimens were exclusively obtained during the KuramBio II cruise and not present in any plankton tow conducted during the SokhoBio expedition. Fourteen specimens were obtained from stations 2, 3, 4, 5, 8, 10 and 11 within depth fractions of -3000 m to -250 m. Exceptions were present at station 5 (one specimen above 250 m) and station 11 (two specimens below 3000 m). Shells max. 10 mm, bilaterally symmetrical, elongated triangular and curved dorsally in the apical part. Protoconch bulbous, almost spherical. Soft body with anterior head-foot complex and posterior visceral sac covered by the shell. Specimen scanned in µCT immature, genital components such as gonad and cephalic copulatory apparatus only present as anlage. Mantle cavity with mantle gland, nephroporus and anus ventrally within anterior part of the shell. Osphradium and anal gland not detectable. Paired wings and single, median foot lobe with densely packed glandular cells along the anterior edge. Head area between wing-bases bears mouth opening and a pair of equally developed eyes. Radula not examined. Gizzard with four pointed and one blunt gizzard plate. Fifth plate in ventral position and much smaller. Intestinal loop strongly winding towards the anal opening on the anterior left part of the mantle cavity. Renopericardial complex left ventrolaterally within apical section of the shell/visceral mass, nephroporus opens to the most posterior section of the mantle cavity. CNS highly condensed with paired cerebropleural and pedal ganglia. Long supraesophageal cerebral commissure. Subesophageal buccal ganglia completely fused. Two almost completely fused ganglia on the visceral loop. Three specimens were sequenced, but COI data was only available for one specimen, making an ABGD species delimitation analysis impossible. Considerable uncorrected p-distances are however present in H3 (10%) and 28S (4,95%) alignments. Insert Fig. 4 here Peracle sp., Fig. 5 Peracle specimens were obtained during SokhoBio and KuramBio II cruises in varying quantities exclusively from stations in the Kuril area outside the Sea of Okhotsk basin. In KuramBio II specimens were mostly present in the depth fraction from -250 to 0 m, except for station 4 where several individuals were captured between 500-1000 m. Shells were completely destroyed/removed during sampling (for all specimens but one) and thus we are unable to show the full set of morphological shell characters for comparison with other known species. In a single specimen obtained with an almost intact shell, some details were observed and are described here. Sinistral shell extremely flimsy, depressed with approximately 2.5 whorls rapidly widening towards the broad aperture. Macroscopically no spiral sculpture, spiral striation, rostrum or other apertural prolongations visible. Distinct transversal growth striae (Fig. 5A), operculum absent. Bilateral wings fused to a swimming disc from which the typical proboscis projects ventrally. Moth opening with conspicuous, dark brown colored bordering (Fig 5B). Paired, well-developed jaws with 4 sharply edged ridges each. Radula formula [1-11] with approximately 12 transversal rows. SEM scans of the radula (Fig. 5 C, F) show pointed, shovel-like median teeth with bilateral denticulation. Lateral teeth hook shaped, with denticulation only along their inner margin. Salivary glands small. Eyes bilaterally equally developed and situated on short stalks in the head area closely above the base of the proboscis

(Fig. 5D). Dorsal pallial cavity with extensive mantle gland and well-developed, folded gill with five fringes (Fig 5E). In total nine specimens were sequenced, four of them from SokhoBio station 10 and five from KuramBio II stations 3, 4, 5, 6 and 8. All specimens reached full gene coverage except for one individual missing COI. In our phylogenetic analysis, they form a monophyletic clade with an intraspecific distance of 0.016 and an interspecific distance of 0.291 to the closest species Peracle reticulata (d’Orbigny, 1835), resulting in an intra-/interspecific ratio of 0.05. Insert Fig. 5 here Clione elegantissima (Dall, 1871), Fig. 6 A-E Specimens of Clione elegantissima were obtained from stations 6, 8, 9 and 10 (SokhoBio) and stations 3, 4, 5, 6, 10 and 11 (KuramBio II). Most individuals were fully mature with a maximum body length of 18 mm. Buccal cones and posterior tip of the visceral sac of distinct red color. Head with pronounced bilateral labial tentacles situated laterally to the mouth opening (Fig. 6A). Short rhinophoral stalks bearing rudimentary eyes in the dorsal neck region. Head cavity bears six buccal cones, paired hook sacs (approx. 12-15 hooks/sac), radula (Fig. 6 B, D, E), central nervous system and the retracted cephalic copulatory apparatus. Radula with >30 transversal rows of 10-11 lateral teeth and one median tooth plate [11-1-11]. Lateral teeth long, slender, spiny and increasingly curved towards the sagittal plane. Median tooth plate broad and shovel-shaped, with small median indentation and eight denticules on each side of the anterior margin. CNS with paired and well separated cerebral, pedal and pleural ganglia and two discernable visceral ganglia on the visceral loop. Peripheral ganglia present in the form of paired buccal, rhinophoral and optic ganglia, all connected directly to the cerebral ganglion. Osphradium with distinct underlying ganglion on visceral sac shortly behind right wing. Parapodial wings and complex of three ventral foot lobes, including paired lateral and single median foot lobe. Lateral foot lobes anteriorly merged, median foot lobe slender and distally pointed. Visceral sac cylindrical and slender, pointed towards the posterior tail, which contains numerous unicellular glands (Fig. 6 C). Distinct red bordering of the anal field. Main visceral organs comprise voluminous stomach with adjacent intestine, circulatory and excretory system and the major part of the genital system. Narrow intestine branches off the stomach in its middle right ventrolateral section and runs towards the anus situated directly under the osphradium. Heart situated ventrolaterally to the right. Auricle and ventricle not distinguishable. Pericardium voluminous. Pericardial cavity connected to kidney via ciliated nephrostome and adjacent nephridial duct. Kidney voluminous, extends within the right lateral portion of the visceral sac and opens via narrow nephroporus next to the anus. Visceral genital components comprise dorsal gonad, long and narrow proximal gonoduct, well developed complex of genital accessory glands and short distal gonoduct opening via the genital pore situated right laterally above the posterior margin of the wing. DNA amplification of five specimens from SokhoBio and KuramBio II cruises was successful, all sequences clustered in a monophyletic clade corresponding to C. elegantissima (Fig. 2). Intraspecific distance was 0.005 and interspecific distance 0.035 to the closest species C. okhotensis, resulting in a ratio of 0.14. Insert Fig. 6 here

Clione okhotensis Yamazaki and Kumahara, 2017, Fig. 6 F-I Clione okhotensis was the second most common species and present at all stations except for station 4 (SokhoBio) and stations 4, 5, 6, 8, 9 and 11 (KuramBio II). Within the latter cruise, it was always found in the -250 to 0 m fraction, except for station 5 where a single specimen was obtained from lower than -500 m. Developmental stages comprised larvae without wings and juvenile specimens with a maximum body length of 4.5 mm, no fully adult specimens were observed. Specimens translucent with bright yellow to orange color of inner organs and three ciliated bands on the head, at two- thirds of the visceral sac and around the tail (Fig. 6 G, H). Numerous subepidermal droplets form collar in the anterior portion of the visceral sac, in which the head-foot complex can be completely retracted when the animal is disturbed (FIG. 6 F). Head with paired labial tentacles adjacent to the mouth opening and paired, very short rhinophoral tentacles in the dorsal neck region which bear rudimentary eyes. Head cavity with six buccal cones, paired hook sacs (approx. 30 hooks/sac), radula, paired salivary glands, CNS and cephalic copulatory apparatus. CNS with paired cerebral, pedal, and pleural ganglia and two visceral ganglia on the visceral loop. Ganglia well separated with relatively long connectives. Paired buccal, optical and rhinophoral ganglia independently connected to the cerebral ganglia. Optical and rhinophoral ganglia in close proximity to each other, situated directly underneath the rhinophoral tentacles. Bulbous osphradium with underlying ganglion shortly behind the right wing on the visceral sac and connected to the right visceral ganglion. Cephalic copulatory organ retracted, provided with sucker and long, slender accessory copulatory organ. Visceral sac contains posterior parts of the digestive system, poorly developed genital components and the circulatory and excretory system. Stomach voluminous and lined by glandular digestive epithelium (Fig. 6 I). Intestine arises from middle section of stomach and runs along ventral side of the visceral cavity towards the anus situated ventrally of the osphradium on the right lateral side of the anterior visceral sac. Heart not clearly identifiable. Voluminous pericardium present in left ventrolateral portion between median and posterior ciliated bands. Short renopericardial duct with well developed, densely ciliated nephrostome. Kidney constitutes elongated tube that runs along intestine to open via a tiny nephroporus next to the anus. Tip of visceral sac with tightly packed unicellular, subepidermal glands. DNA was extracted from a total of nine specimens. Amplification success was low for the COI marker (two sequences), but 28S and H3 data is available for all specimens. Sequences cluster in a monophyletic clade with an intraspecific distance of 0.003. The nearest species is C. limacina with an interspecific distance of 0.056. Intra-/Interspecific distance ratio is 0.05. Notobranchaea grandis Pruvot-Fol, 1942, Fig. 7 A single specimen of Notobranchaea grandis was captured during the KuramBio II expedition at station 1 in the mesopelagic depth fraction from -1000 to -500 m. Specimen with shiny white color of head and visceral sac and conspicuous dark brown coloration of labial tentacles and anterior wing edges (Fig 7A). No ciliated rings present. Lateral foot lobes merged anteriorly forming a V-like structure. Very short median foot tubercle situated centrally between lateral foot lobes (Fig. 7 A, B). Epidermis of visceral sac contains numerous clear droplets. µCT scanning revealed four buccal cones, spiny jaws, paired hook

sacs (approx. 10-15 hooks /sac) and a radula within the head area. Radula was unfortunately lost during preparation for SEM scanning. Circumesophageal CNS with paired, well separated cerebral, pedal, and pleural ganglia and two visceral ganglia on the visceral loop. Very short rhinophoral tentacles bear rudimentary eyes dorsally in the neck area. Each rhinophoral tentacle with two distinct underlying peripheral ganglia. Dark brown labial tentacles retracted into the head after capture. Cephalic copulatory organ with long and slender accessory part and wellpronounced sucker in the ventral posterior part of the head at the level of lateral foot lobes. Voluminous stomach filling large parts of the visceral body cavity. Short intestine leading to papillate anus positioned mid-ventrally on the visceral sac, slightly shifted to the right side. Genital system well developed, comprises gonad, proximal gonoduct, complex of genital accessory glands and short distal gonoduct, the latter opens to the general genital opening shortly above the left wing. Well-developed heart with auricle and thick muscular ventricle positioned laterally to the left within the posterior part of the visceral sac. Voluminous kidney situated laterally to the left and follows the intestine to open to the exterior via narrow nephroporus next to the anus. Three longitudinal crests with haemolymph sinuses present in the tail area which form a triradiate gill. Repeated attempts to generate molecular data after µCT staining/scanning procedures were unsuccessful. Insert Fig. 7 here Thliptodon sp., Fig. 8 Single specimen captured at station 1 within the mesopelagic depth fraction from -500 to -250 m. Size 14 mm, badly damaged but major body characteristics identifiable. Individual mostly transparent with light brown stomach and creamy white gonad visible through the body wall. Head proportion enlarged to approx. one-third of the total body length, triangular wings consequently origin from a more posterior position compared with other gymnosome species. Tail rounded with a small ciliary band. Well-developed genital components, conspicuous dark brown spot within the complex of genital accessory glands. Tissue subsampling heavily damaged specimen and morphological investigation of remnants is pending. DNA extraction from tissue subsample was unsuccessful. Insert Fig. 8 here Discussion Limacina helicina Limacina helicina was by far the most abundant species sampled during both cruises. Rarely present under a depth of -250m, our data supports its exclusively epipelagic occurrence. Limacina helicina has a long and confusing taxonomic history. Originally, it was thought to have a bipolar distribution with high variability in shell morphological characters (such as height to diameter (H/D) ratio, elevation of whorls and presence of radial riblets). Since

transitions appeared to be fluent, an extensive system of subspecies and “formae” was created. Van der Spoel (1967) described formae as “groups of individuals developed by variation of species or subspecies and differing from other individuals by the mere fact that the variation has gone in a distinct direction by influences of the environment”. From the southern hemisphere, L. h. antarctica Woodward, 1854 and L. h. rangii, (d’Orbigny, 1835) have been described, while in the northern hemisphere the L. helicina complex contained three formae: L. h. helicina (Phipps, 1774), L. h. acuta (Spoel, 1967) and L. h. pacifica Dall, 1871. Shkoldina (1999) added L. h. ochotensis from the Sea of Okhotsk. A first genetic approach by Hunt et al. (2010) revealed a >30% difference between southern and northern specimens in phylogenetic analysis using a single gene (COI), consequently identifying Limacina antarctica as separate species. Subsequent works focused on northern Limacina helicina specimens (Abyzova et al., 2018; Chichvarkhin, 2016; Shimizu et al., 2017; Sromek et al., 2015), but found a much lower genetic diversity in the COI sequences than in southern specimens. This disparity resulted in a rejection of hypotheses that northern formae might also constitute genetically well-delimited species. Our ABGD analysis and phylogenetic reconstruction based on a three-marker set convincingly shows that Limacina helicina specimens obtained from SokhoBio and KuramBio II cruises belong to the same species as specimens from the Atlantic sector of the Arctic. This observation suggests genetic connectivity of the arctic population. Even specimens collected off California are reported to belong to the same species (Janssen et al., 2019) and thus would constitute their southernmost distribution. In addition, anatomical data of the soft-body obtained by means of µCT scanning fully corresponds with a Limacina helicina specimen from western Greenland recently investigated by Laibl et al. (2019). Interestingly, Shimizu et al. (2017) proved that the H/D ratio as well as aperture shapes and growth patterns differed significantly between early and late developmental stages, which is consistent with our observations on juvenile and adult shells. The operculum was only present in juvenile forms, which is an observation frequently described for Limacina helicina and indicates the tendency to lose the operculum with increasing growth/size of the individual. Clio cf. andreae/polita Our phylogenetic analyses of the Clio specimens revealed two well-separated clades. While one clade is represented by two specimens from a depth lower than -3000m, the other clade is represented by a singleton obtained from an alleged juvenile specimen captured within the epipelagic fraction (-250-0 m). An ABGD species delimitation could not be conducted due to the missing COI sequences of two of the three genetically investigated individuals. However, the relatively large p-distances of 28S and H3 (4.95 % and 10 %, respectively) strongly point towards the existence of morphologically at least semi-cryptic, but genetically well-separated species. Unfortunately, the formalin fixation of the CT-scanned specimen damages DNA, prohibiting assignment to any of the above-mentioned genetic clades. Morphologically, the sequenced specimens were determined as either Clio andreae (Boas, 1886) or Clio polita Pelseneer, 1888. Both species are reported to inhabit bathypelagic depth as adults, while juveniles were emphasized to live near the surface in the upper 200 m (Van der Spoel, 1976). Janssen et al. (2019) addressed the taxonomically controversial synonymy of C. andreae and C. polita suggested by Van der Spoel (1976). They argue for retention of both species, due to a weak but distinct longitudinal and transversal ornament in C. andreae, that is absent in C. polita.

The revision of images taken during the sorting of plankton samples, however, did not allow the detection of such minor indicative morphological shell features. The taxonomic affiliation of specimens from the KKT area thus remains questionable since type localities of C. andreae and C. polita are in the Atlantic Ocean. Additionally, holo- and syntypes of the respective species are either in bad condition or lost (Janssen et al., 2019). Thus, a comprehensive recollection of material is indispensable for a further detailed integrative taxonomic investigation and the verification of putatively different bathymetrical preferences of genetically recovered species. This is the first record of Clio cf. andreae/polita from the KKT area and to our knowledge the westernmost distribution in the NW Pacific Ocean. Peracle sp. Molecular data together with a comprehensive investigation of the soft body via µCT scanning, serial sectioning and SEM scanning of the radula revealed the investigated specimens to belong to the genus Peracle. Morphological peculiarities of the latter genus are a sinistrally coiled, calcareous shell, pallial cavity with pallial gland and folded gill, the foot forming a wing-shaped swimming disc with a proboscis arising from the mouth opening, well-developed jaws and symmetrical tentacles with eyes (Van der Spoel, 1976). The species described herein, however, differs from most of the other eight to date accepted Peracle species (WoRMS, 2019) by rather depressed whorls without sculpture or ornamentation, the absence of a rostrum with columellar membrane and the missing operculum. Since the shells of specimens obtained during our cruises were mostly broken, it is possible that rostral structures and operculum (if previously present) were damaged and/or lost during net collection. Interestingly, a very similar observation was made for Peracle valdiviae (Meisenheimer, 1905), originally described as Procymbulia valdiviae, Meisenheimer, 1905. Because no specimen could be sampled with a completely intact shell, the original description contained no specifications on shell morphology. The anatomical investigation of specimens showed characters that were interpreted as linking the pseudothecosome genera Peracle and Cymbulia, consequently causing Meisenheimer to erect the new genus Procymbulia. After subsequent reinvestigations of Tesch (1948), who managed to reconstruct the shell from broken remnants of a resampled specimen he identified as P. valdiviae, the genus Procymbulia was incorporated to Peracle. However, Gilmer (1990) argued for the resurrection of Procymbulia in his description of Procymbulia philiporum based on a single specimen observed by the Johnson Sea-link submersible at a depth of 902 m that was brought to the surface using a protective closed 7.5 l acrylic cylinder. Images taken of the living specimen clearly showed that the shell was enclosed in a large gelatinous pseudoconch, which could not be removed without the destruction of the shell (Gilmer, 1990: figs. 1-8). The depicted shell has a very high resemblance to the apical shell part of the Peracle sp. observed in this study. To verify the presence of potential apomorphic characters that would justify the resurrection of the genus Procymbulia as proposed by Gilmer (1990), further detailed microanatomical studies are needed. Janssen et al. (2019) list two other Peracle species that are known from the Pacific Ocean: Peracle diversa (Monterosanto, 1875) and Peracle reticulata (d’Orbigny, 1835). Both species differ from the specimens studied herein by highly elevated whorls with a reticulated pattern. In contrast to the limited documented sampling stations, most Peracle species are suspected to have a cosmopolitan distribution (Van der Spoel, 1976). Molecular vouchers for Peracle species are

nevertheless rare and exist for only three species (16 sequences of 3 markers) and are publicly available on NCBI Genbank (www.ncbi.nlm.nih.gov/genbank). Eleven sequences refer to Peracle reticulata (d’Orbigny, 1835), four to Peracle bispinosa Pelseneer, 1888, and one to Peracle valdiviae (Meisenheimer 1905). Clione elegantissima Our multimarker phylogenetic analysis and ABGD delimitation fully corroborate the results of Yamazaki and Kuwahara (2017), who revealed Clione limacina and Clione elegantissima to constitute separate species with an allopatric occurrence in the North Atlantic and North Pacific, respectively. Both species were mentioned to differ morphologically in body size, as well as in size and number of hooks. Sexually mature C. elegantissima individuals investigated in this study reached a size of 18 mm but may not represent fully grown individuals. Clione limacina, on the other hand, is known as the largest gymnosome, reaching sizes of 85 mm in the North Atlantic (Conover and Lalli, 1974). From our µCT data, a smaller number of hooks per sac were found for C. elegantissima (<15) than for C. limacina (up to 30). Based on SEM scans of the median radula plate, we add a third distinguishing character for species delimitation. While there is a distinct central cusp in C. limacina (Lalli & Gilmer, 1989: fig. 55b), C. elegantissima has a concave indentation in the same position (Fig. 6E). In contrast to Chichvarkhin (2016), who reported a total disappearance of Clione limacina by the end of May in the northern Sea of Japan, we found them to be at least rarely present in August 2015 in the Sea of Okhotsk and in August/September in the Kuril-Kamchatka region. Clione okhotensis Yamazaki and Kumahara, 2017 Diagnostic morphological/anatomical characters of Clione okhotensis are the bright orange-red color of the visceral mass, its small size (max. 8 mm), very short buccal cones, three ciliated rings retained in the adult stage, large orange-red visceral mass and no space between head tentacles and mouth (Yamazaki & Kumahara, 2017). This study complements the original description with details on the nervous, genital, circulatory and excretory systems which will be discussed in a separate paper showing microanatomical peculiarities (Kohnert, in prep.). Historically, C. okhotensis has likely been misinterpreted as juvenile Clione limacina, due to the retention of paedomorph characters in the adult stage and the high resemblance of both species. Paedomorphic characters such as the retention of three ciliated rings in sexually mature individuals are also known from Paedoclione doliiformis Danforth, 1907, which occurs in the NW Atlantic and has a maximum body length of 2.5 mm (Lalli and Conover, 1973). The major morphological difference between the genera Clione and Paedoclione is the reduced number of buccal cones (six in Clione and four in Paedoclione). The driving force in the evolution of P. doliiformis may have been a specialization in predation on smaller thecosome species such as Limacina retroversa (Fleming 1823) and juvenile L. helicina. This differs from C. okhotensis, that feeds on adult individuals of L. helicina and shows no reduction in buccal cones (Yamazaki et al. 2017), rendering the role of paedomorphosis in gymnosome evolution and speciation enigmatic. C. okhotensis was found throughout all stations sampled, which further expands its reported distribution to all semi-enclosed West Pacific sea basins (Sea of Japan and Sea of Okhotsk) and the adjacent Kuril-Kamchatka area. How far C. okhotensis projects into the NW

Pacific remains to date unclear. The sympatric occurrence of Clione elegantissima and Clione okhotensis, however is not limited to the Sea of Okhotsk but also extends to the pacific side of the KKT area. Notobranchaea grandis Morphological data based on µCT scanning corroborates our first identification of the specimen as Notobranchaea grandis. The latter is the only species within the genus that shows the characteristic dark-brown coloration of labial tentacles and anterior wing edges (Van der Spoel, 1976). According to Lalli and Gilmer (1989), specimens within the genus Notobranchaea Pelseneer, 1886 possess either four buccal cones or none, radula and jaw and paired hook sacs with 9-20 hooks per sac. This description also matches with our specimen, which clearly possesses four buccal cones and hook sacs with 10-15 hooks per sac in the anterior head cavity. Although there are direct observations of feeding and/or stomach analyses of several gymnosomes (Clionidae and Pneumodermatidae, as well as Cliopsis and Hydromyles species (Lalli and Gilmer, 1989), nothing is known about prey preferences of Notobranchaea. The genus Notobranchaea, together with Clione Pallas, 1774, Paraclione (Tesch, 1903), Paedoclione Danforth, 1907, Thalassopterus Kwietniewski, 1910 and Fowlerina Pelseneer, 1906, belongs to the family Clionidae, which is distinguished from other gymnosomes by the presence of buccal cones (Van der Spoel, 1976). Of these genera, three species (Clione limacina, Paedoclione doliiformis and Paraclione sp.) have been observed to feed on coiled shelled euthecosome species (Lalli & Gilmer, 1989: table 23). We therefore suggest that buccal cones constitute organs specialized for grasping onto spirally shaped shells. Because the only coiled shell pteropod found sympatrically was the mesopelagic Peracle sp., we hypothesize that the latter constitutes the main prey of Notobranchaea. This hypothesis would imply a coevolutionary history of Clione/Notobranchaea species together with spiral-shelled Limacina and Peracle species, respectively. Thliptodon sp. The single specimen obtained during the KuramBio II cruise was clearly identifiable to the genus Thliptodon due to the barrel-shaped body, extremely enlarged head (compared to other gymnosomes) and triangular wings attached to the body at one-third of the body length or even more posterior. Thliptodon currently contains four accepted species (WoRMS, 2019), all of which are suspected to have a mesopelagic and mostly cosmopolitan distribution. Reported morphological interspecific differences are restricted to minor radula characteristics. Since a thorough investigation of the remnants of the individual obtained during the KuramBio II expedition is still pending and DNA extraction was unsuccessful, we are not able to assign it to any morphological or molecular voucher. The nearest geographical report is a brief description from Tokioka (1950) who described the species Thliptodon akatukai based on gross external morphological observations of a single specimen from off Seto, Japan. However, the author determined only minor differences in wing shape compared with T. gegenbauri Boas, 1886, which led to the current status as taxon inquirendum (WoRMS, 2019). Thliptodon diaphanus (Meisenheimer, 1902) is also reported from Japanese waters and might correspond to our specimen. Genetic sequences of undetermined Thliptodon spp. are available from only one

Atlantic and two East Pacific specimens, the latter forming separated clades in a multimarker RAxML tree presented by Burridge et al. (2017). The existing morphological and molecular data on Thliptodon species is too limited for proper species identification/delimitation. A thorough reinvestigation of the genus including integrative taxonomical approaches is needed and will depend on a geographically wide range resampling in mesopelagic depths. Conclusions We showed that µCT scanning is a suitable tool to generate easily accessible morphological data for pteropod gastropods. It allows exact measurements such as height, diameter, width and thickness of shells, but also an exploration of all major organ systems in high resolution and the determination of individual sexual stages. Furthermore, digital scans are easy to share with the scientific community and not subject to negative preservation effects such as shell degradation or destruction frequently observed in pteropod type specimens from museum collections. Our detailed descriptions thus are a reliable basis for future comparisons. Together with molecular multilocus barcoding, this integrative approach has the power to improve taxonomic research on pteropods, particularly of interest for the proposed “indicator species” within the genus Limacina. The herein presented dataset, including four first records and one species possibly new to science, contributes to the Sea of Okhotsk and the KKT as some of the best investigated areas regarding pteropod diversity. However, it also clearly demonstrates how limited and insufficient our knowledge of pteropod diversity and distribution still is, in particular regarding mesopelagic species. A thorough inventory of present pteropod diversity from all ocean areas is in our view inevitable for future monitoring and conservation efforts in times of rapidly changing ocean chemistry. Acknowledgements The material was collected and sorted within the framework of several large international projects. KuramBio II and SokhoBio projects were financially supported by the PTJ (German Ministry for Science and Education) grant 03G0857A, KuramBio I BMBF grant 03G0223A, as well as KuramBio II BMBF grant 03G0250A to Prof. Dr. Angelika Brandt, University of Hamburg, now Senckenberg Research Institute and natural History Museum, Frankfurt, Germany. We thank the crews of the RVs Sonne and Akademik M.A. Lavrentyev for their help on board and all student helpers and technicians for support and help with sorting of the extensive expedition material. We would also like to thank Marina Malyutina (Institute of Marine Biology Far-Eastern Branch of Russian Academy of Sciences) for the possibility to participate in SokhoBio and Angelika Brandt (Senckenberg Frankfurt) for the possibility to join theKuramBio II expedition. Christina Egger (ZSM Munich) is thanked for taking SEM images. Silke Lischka (Geomar Kiel) and Alexander Martynov (Lomonosov Moscow State University) are deeply appreciated for sampling Clione and Limacina specimens at Svalbard and the White Sea, respectively. We are very grateful to Katie Wolcott, Arie Janssen (Naturalis Biodiversity

Center, Leiden), Bastian Brenzinger (ZSM Munich) and two anonymous reviewers who provided very helpful comments on the manuscript. This is KuramBio publication # 64. Funding Amplification and sequencing of genes was partly financed by a research grant from the Malacological Society of London and by the German Research Foundation (DFG Schwerpunktprogramm Antarktis und arktische Polargebiete, SCHR667/16-1). Competing interest statement: The authors do not have a competing interest to declare. Declarations of interest: none

References Abyzova, G.A., Nikitin, M.A., Popova, O.V., Pasternak, A.F., 2018. Genetic population structure of the pelagic mollusk Limacina helicina in the Kara Sea. PeerJ Preprints, 6, e27016v27011. Bednarsek, N., Feely, R.A., Reum, J.C., Peterson, B., Menkel, J., Alin, S.R., Hales, B., 2014. Limacina helicina shell dissolution as an indicator of declining habitat suitability owing to ocean acidification in the California Current Ecosystem. Proc Biol Sci, 281, 20140123. Bednaršek, N., Harvey, C.J., Kaplan, I.C., Feely, R.A., Možina, J., 2016. Pteropods on the edge: Cumulative effects of ocean acidification, warming, and deoxygenation. Progress in Oceanography, 145, 1-24. Berner, R.A., Honjo, S., 1981. Pelagic sedimentation of aragonite: its geochemical significance. Science, 211, 940-942. Burridge, A.K., Goetze, E., Raes, N., Huisman, J., Peijnenburg, K.T.C.A., 2015. Global biogeography and evolution of Cuvierina pteropods. Bmc Evolutionary Biology, 15, 39. Burridge, A.K., Hörnlein, C., Janssen, A.W., Hughes, M., Bush, S.L., Marlétaz, F., Gasca, R., Pierrot-Bults, A.C., Michel, E., Todd, J.A., Young, J.R., Osborn, K.J., Menken, S.B.J., Peijnenburg, K.T.C.A., 2017. Time-calibrated molecular phylogeny of pteropods. PLOS One, 12, e0177325. Burridge, A.K., Janssen, A.W., Peijnenburg, K.T.C.A., 2016. Revision of the genus Cuvierina Boas, 1886 based on integrative taxonomic data, including the description of a new species from the Pacific Ocean (Gastropoda, Thecosomata). ZooKeys 619, 1-12. Brandt. A and Shipboard scientific party (2016) RV Sonne SO-250 Cruise Report / Fahrtbericht Tomakomai - Yokohama (Japan) 1608-26092016. 2016 SO-250 KuramBio II (Kuril Kamchatka Biodiversity Studies). 174 pp. Castresana, J., 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution, 17, 540-552.

Chichvarkhin, A., 2016. Shallow water sea slugs (Gastropoda: Heterobranchia) from the northwestern coast of the Sea of Japan, north of Peter the Great Bay, Russia. PeerJ, 4, e2774. Comeau, S., Gattuso, J.-P., Nisumaa, A.-M., Orr, J., 2012. Impact of aragonite saturation state changes on migratory pteropods. Proceedings of the Royal Society B: Biological Sciences, 279, 732-738. Conover, R.J., Lalli, C.M., 1974. Feeding and growth in Clione limacina (Phipps), a pteropod mollusk. 2. Assimilation, metabolism, and growth efficiency. Journal of Experimental Marine Biology and Ecology, 16, 131-154. Corse E, R.J., Cuoc C, Pech N, Perez Y, et al., 2013. Phylogenetic analysis of Thecosomata Blainville, 1824 (Holoplanktonic Opisthobranchia) using morphological and molecular data. PLOS One, 8, e59439. Dereeper, A., Chevenet, F., Blanc, G., Dufayard, J.-F., Claverie, J.-M., Lescot, M., Audic, S., Buffet, S., Guindon, S., Guignon, V., Lefort, V., Gascuel, O., 2008. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Research, 36, 465-469. Feely, R.A., Sabine, C.L., Lee, K., Berelson, W., Kleypas, J., Fabry, V.J., Millero, F.J., 2004. Impact of anthropogenic CO2 on the CaCO3 system in the oceans. Science, 305, 362-366. Gasca, R., Janssen, A.W., 2014. Taxonomic review, molecular data and key to the species of Creseidae from the Atlantic Ocean. Journal of Molluscan Studies, 80, 35-42. Gilmer, R.W., 1972. Free-floating mucus webs: A novel feeding adaptation for the open ocean. Science, 176, 1239-1240. Gilmer, R.W., 1974. Some aspects of feeding in thecosomatous pteropod mollusks. Journal of Experimental Marine Biology and Ecology, 15, 127-144. Gilmer, R.W., 1990. Procymbulia philiporum new species, with a discussion of the genus Procymbulia Meisenheimer, 1905 (Gastropoda: Thecosomata). The Nautilus, 104, 111-119. Gilmer, R.W., Harbison, G.R., 1986. Morphology and field behavior of pteropod molluscs: feeding methods in the families Cavoliniidae, Limacinidae and Peraclididae (Gastropoda: Thecosomata). Marine Biology, 91, 47-57. Hunt, B., Strugnell, J., Bednarsek, N., Linse, K., Nelson, R.J., Pakhomov, E., Seibel, B., Steinke, D., Würzberg, L., 2010. Poles Apart: The “bipolar” pteropod species Limacina helicina is genetically distinct between the Arctic and Antarctic Oceans. PLOS One, 5, e9835. Janssen, A.W., Bush, S.L., Bednarsek, N., 2019. The shelled pteropods of the northeast Pacific Ocean (Mollusca: Heterobranchia, Pteropoda). Zoosymposia, 13, 305-346. Katoh, K., Standley, D.M., 2013. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution, 30, 772-780. Klussmann-Kolb, A., Dinapoli, A., 2006. Systematic position of the pelagic Thecosomata and Gymnosomata within Opisthobranchia (Mollusca, Gastropoda) - revival of the Pteropoda. Journal of Zoological Systematics and Evolutionary Research, 44, 118-129.

Knebelsberger, T., Stöger, I., 2012. DNA Extraction, Preservation, and Amplification. In J.W. Kress, L.D. Erickson (Eds.), DNA Barcodes: Methods and Protocols (pp. 311-338). Totowa, NJ: Humana Press. Kubilius, R.A., Kohnert, P., Brenzinger, B., Schrödl, M., 2014. 3D-microanatomy of the straight-shelled pteropod Creseis clava (Gastropoda: Heterobranchia: Euthecosomata). Journal of Molluscan Studies, 80, 585-603. Laibl, C.F., Schrödl, M., Kohnert, P.C., 2019. 3D-microanatomy of a keystone planktonic species, the northern polar pteropod Limacina helicina helicina (Gastropoda: Heterobranchia). Journal of Molluscan Studies, eyy063. Lalli, C.M., Conover, R.J., 1973. Reproduction and development of Paedoclione doliiformis, and a comparison with Clione limacina (Opisthobranchia: Gymnosomata). Marine Biology, 19, 13-22. Lalli, C.M., Gilmer, R.W., 1989. Pelagic snails: the biology of holoplanktonic gastropod mollusks: Stanford University Press. Lischka, S., Büdenbender, J., Boxhammer, T., Riebesell, U., 2011. Impact of ocean acidification and elevated temperatures on early juveniles of the polar shelled pteropod Limacina helicina: mortality, shell degradation, and shell growth. Biogeosciences, 8, 919. Lischka, S., Riebesell, U., 2012. Synergistic effects of ocean acidification and warming on overwintering pteropods in the Arctic. Global Change Biology, 18, 3517-3528. Maas, A.E., Blanco-Bercial, L., Lawson, G.L., 2013. Reexamination of the Species Assignment of Diacavolinia Pteropods Using DNA Barcoding. PLOS One, 8, e53889. Manno, C., Bednaršek, N., Tarling, G.A., Peck, V.L., Comeau, S., Adhikari, D., Bakker, D.C., Bauerfeind, E., Bergan, A.J., Berning, M.I., 2017. Shelled pteropods in peril: Assessing vulnerability in a high CO 2 ocean. Earth-Science Reviews. Meisenheimer, J., 1905. Pteropoda. Wissenschaftliche Ergebnisse der deutschen TiefseeExpedition auf dem Dampfer "Valdivia" 1898-1899 Vol. 9 (pp. 1-314). Neusser, T.P., Heß, M., Haszprunar,G. and Schrödl, M., 2006. Computer-based threedimensional reconstruction of the anatomy of Microhedyle remanei (Marcus, 1953), an interstitial acochlidian gastropod from Bermuda. Journal of Morphology, 267, 231-247. Puillandre, N., Lambert, A., Brouillet, S., Achaz, G., 2012. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology, 21, 1864-1877. Shimizu, K., Kimoto, K., Noshita, K., Wakita, M., Fujiki, T., Sasaki, T., 2017. Phylogeography of the pelagic snail Limacina helicina (Gastropoda: Thecosomata) in the subarctic western North Pacific. Journal of Molluscan Studies, 84, 30-37. Shkoldina, L., 1999. On the systematics of the pteropod mollusk Limacina helicina from the Sea of Okhotsk. Russian Journal of Marine Biology, 25, 330-336.

Sirenko, B.I., 2013. Check-list of species of free-living invertebrates of the Russian Far Eastern seas. St. Petersburg, Russia: Russian Academy of Sciences, Zoological Institute. Sromek, L., Lasota, R., Wolowicz, M., 2015. Impact of glaciations on genetic diversity of pelagic mollusks: Antarctic Limacina antarctica and Arctic Limacina helicina. Marine Ecology Progress Series, 525, 143-152. Stamatakis, A., 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30, 1312-1313. Tesch, J.J., 1913. Pteropoda / bearbeitet von dr. Johan Jacob Tesch. Mit 108 Abbildungen. Berlin: R. Friedländer und Sohn. Tesch, J.J., 1948. The thecosomatous pteropods. II. The Indo-Pacific. Dana Reports, Vol. 30 (pp. 1-45). Tokioka, T., 1950. Droplets from the plankton net. Publications of the Seto marine biological laboratory, 1, 151-157. Van der Spoel, S., 1967. Euthecosomata: A Group with Remarkable Developmental Stages. (Gastropoda, Pteropoda). Gorinchem: J. Noorduijn. Van der Spoel, S., 1976. Pseudothecosomata, Gymnosomata and Heteropoda (Gastropoda). Utrecht: Bohn, Scheltema & Holkema. WoRMS Editorial Board (2019). World Register of Marine Species. Available from http://www.marinespecies.org at VLIZ. Accessed 2019-05-11. doi:10.14284/170 Yamazaki, T., Kuwahara, T., 2017. A new species of Clione distinguished from sympatric C. limacina (Gastropoda: Gymnosomata) in the southern Okhotsk Sea, Japan, with remarks on the taxonomy of the genus. Journal of Molluscan Studies, 83, 19-26.

Competing interest statement: The authors do not have a competing interest to declare. Declarations of interest: none

Fig. 1 Map of sampling sites and species occurrence. A sampling stations during the SokhoBio (yellow) and KuramBio II (red) cruises to the Sea of Okhotsk and the Kurile-KamchatkaTrench (KKT) area. Dotted square indicates map section depicted in B-G. B Occurrence of Limacina helicina. C Occurrence of Clio spp. D Occurrence of Peracle n. sp. E Occurrence of

Clione elegantissima. F Occurrence of Clione okhotensis. G Occurrence of Notobranchaea grandis and Thliptodon sp.

Fig. 2 Maximum Likelihood tree based on a concatenated dataset of Cytochrome Oxidase I (COI), ribosomal 28S and Histone 3 (H3) genes calculated with 1000 bootstrap replicates. Bootstrap-support values >50% are shown at nodes, black squares near branch terminals indicate >99% bootstrap support. Newly generated sequences are coded according to sampling locality.

Fig. 3 Shell morphology of Limacina helicina ochotensis. A Living specimen of Limacina helicina ochotensis B SEM scan of subadult shell. C Detailed SEM scan of protoconch area (shell B). D-F CT scans of adult shell. D Apertural view. E Apical view. F Umbilical view. G-I CT scans of subadult shell. G Apertural view. H Apical view. I Umbilical view. Scale bars: A = 2mm; B = 250µm; C =50µm; D, E, F = 1mm; G, H, I = 250µm.

Fig. 4 Morphology of Clio sp. A Living specimen dorsal view. Arrows indicate bilaterally welldeveloped eyes. B CT volume rendering showing anterior visceral organs and central nervous system. C Volume rendering with indicated general body organization, left lateral view. D Photograph of living specimen, ventral view. Arrows indicate statocysts. Abbreviations: a anus, cns central nervous system, dgl digestive gland, gi gizzard, go gonad, he heart, hf head-foot complex, int intestine, mc mantle cavity, mfl median foot-lobe, mgl mantle gland, vs visceral sac, wi wings. Scale bars: A, B, D = 1mm; C = 2mm.

Fig. 5 Morphology of Peracle n. sp. A Photograph of specimen shortly after capture. Arrow indicates remnants of shell apex. B Photograph of soft body without shell, dorsal view. C SEM scan of radula showing details of lateral and median teeth. D CT scan of soft body stained with PTA, dorsal view. Arrows indicate short eye stalks. E CT scan of soft body showing mantle cavity organs, posterior view. F SEM scan of complete radula. Abbreviations: dgl digestive gland, gi gills, go gonad, lt lateral teeth, mgl mantle gland, mo mouth opening, mt median teeth, pr proboscis, sd swimming disc, sr shell remnants. Scale bars: A = 1mm; C, F = 50µm; D, E = 500µm.

Fig. 6 Morphology of Clione elegantissima (A-E) and Clione okhotensis (F-I). A Photograph of living specimen. B SEM scam of complete radula. C CT scan of complete specimen showing inner organs. D SEM scan of lateral teeth. E SEM scan of median teeth. F Clione okhotensis specimen, living, ventral view. G Volume rendering based on CT data, ventral view. H Volume rendering based on CT data, right lateral view I Sagittal section based on CT data. Abbreviations: a anus, acr anterior ciliated ring, bc buccal cones, cg cerebral ganglion, go gonad, hf headfoot, hs hook sacs, lm longitudinal muscle bundles, lfl lateral foot lobes, lrt lateral radula teeth, lt labial tentacles, mcr median ciliated ring, mfl median foot lobe, mrt median radula tooth, od “oily” droplets, osp osphradium, pcr posterior ciliated ring, ph

pharynx, sto stomach, vs visceral sac, wi wings. Scale bars: A, C = 5mm; B = 100µm; D, E = 20µm F, G, H, I = 1mm.

Fig. 7 Morphology of Notobranchaea grandis. A Photograph of living specimen. Arrow indicates median foot tubercule. B CT scan of fixed specimen, ventral view. Orientations of following CT sections indicated by white lines. C CT scan, sagittal section. D CT cross-section of head area. E CT cross-section of anterior visceral sac. F CT cross-section of posterior visceral organs. Abbreviations: a anus, bc buccal cones, cg cerebral ganglion, es esophagus, ey eyes, fl lateral footlobes, gag genital accessory glands, go gonad, lt labial tentacles, ns nephrostome, pg pedal ganglion, sto stomach, ve ventricle, vg visceral ganglion, wi wings. Scale bars A, B, C = 1mm; D, E, F = 500µm.

Fig. 8 External Morphology of Thliptodon sp., left ventrolateral view. Arrow indicates specimen of cercozoan Phaeodaria Haeckel, 1879 stuck in the epidermis of Thliptodon. Abbreviations: gag genital accessory glands, go gonad, he head, hf head-foot, sto stomach, vs visceral sac, wi wing. Scale bar = 500µm.

Highlights    

µCT-scanning is a suitable tool for exploring pteropod anatomy Clione limacina (Phipps, 1774) and Clione elegantissima Dall, 1871 are separate species Panarctic genetic connectivity is present in Limacina helicina (Phipps, 1774) More pteropod species than previously known are present in the Northwest Pacific