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From wood to vent: first cocculinid associated with hydrothermal activity discovered in the

Article in Antarctic Science · April 2020 DOI: 10.1017/S095410202000022X

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Chong Chen Katrin Linse Japan Agency for Marine-Earth Science Technology British Antarctic Survey

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The user has requested enhancement of the downloaded file. Antarctic Science page 1 of 13 (2020) © Antarctic Science Ltd 2020 doi:10.1017/S095410202000022X From wood to vent: first cocculinid limpet associated with hydrothermal activity discovered in the Weddell Sea

CHONG CHEN 1 and KATRIN LINSE 2 1X-STAR, Japan Agency for Marine-Earth Science and Technology (JAMSTEC),2–15 Natsushima-cho, Yokosuka, Kanagawa Prefecture 237-0061, Japan 2British Antarctic Survey, High Cross, Cambridge CB3 0ET, UK [email protected] This article is registered in ZooBank under: urn:lsid:zoobank.org:pub:296FAB17-E989-4921-9E2D-2F8518B2D05F Cocculina enigmadonta n. sp. is registered in ZooBank under: urn:lsid:zoobank.org:act:0F15867C-6B73-40E3-B0C9-762F8CFE5730

Abstract: Lush 'oases' of life seen in chemosynthetic ecosystems such as hot vents and cold seeps represent rare, localized exceptions to the generally oligotrophic deep ocean floor. Organic falls, best known from sunken wood and whale carcasses, are additional sources of such oases. Kemp (59°42'S, 28°20'W) in the Weddell Sea exhibits active hydrothermal vents and a natural in close proximity, where an undescribed cocculinid limpet was found living in both types of chemosynthetic habitats. This represents the first member of the gastropod order Cocculinida discovered from hot vents, and also the first record from the . Here, we applied an integrative framework incorporating traditional dissection, electron microscopy, genetic sequencing and 3D anatomical reconstruction through synchrotron computed tomography in order to characterize this species. Together, our data revealed an unusual member of the Cocculina with a highly modified radula for feeding on bacterial film, described herein as Cocculina enigmadonta n. sp. Its phylogenetically derived position within the largely wood-inhabiting Cocculina indicates that it probably evolved from an ancestor adapted to living on sunken wood, providing a compelling case of the 'stepping stone' evolutionary trajectory from organic falls to seeps and vents.

Received 8 October 2019, accepted 7 March 2020

Key words: Cocculina, , Cocculiniformia, mollusc, new species, South Sandwich Arc

Introduction Among the , pectinodontids in the genus Pectinodonta typically have gut contents predominantly Deprived of sunlight and photosynthetic primary composed of microscopic pieces of wood, while the gut production, the deep ocean floor is generally oligotrophic contents of cocculinids also include large amounts of with the exception of -based ecosystems detritus and sediment, meaning that they probably feed driven by the Earth's own geochemistry, such as hot by grazing on the surface bacterial film (Marshall 1985). vents and cold seeps (Van Dover 2000). Microbial As organic falls decay, hydrogen sulphide is produced as chemosynthesis involves deriving energy from reducing a by-product, leading to a chemosynthetic ecosystem not compounds such as hydrogen sulphide, and it allows unlike vents or seeps (Smith & Baco 2003). As such, vents and seeps to host very high levels of organic falls have been hypothesized as evolutionary biomass comparable to those of coral reefs, with most 'stepping stones' towards endemism in vents and seeps species becoming specially adapted to these ecosystems (Smith et al. 2015), but with the exception of deep-sea and unable to live anywhere else (Van Dover 2000). bathymodioline mussels (Thubaut et al. 2013), little In addition, organic debris sinking from the sea surface evidence has been presented to show how such also generates localized, ephemeral 'oases' of nutrient transitions might have occurred. enrichment in the deep (Smith & Baco 2003). Primarily Recently,hydrothermal vents (Roterman et al. 2016) and comprising sunken wood and large mammal carcasses a natural whale fall (Amon et al. 2013) were discovered in typified by whales and collectively known as organic close proximity within Kemp Caldera (59°42'S, 28°20'W) falls, these habitats attract and are sources of food for in the Southern Ocean, < 100 km away from deeper numerous animals, including many typical foundation vents on segment E9 of the East Scotia Ridge (Rogers species such as the bone-eating zombie worm Osedax et al. 2012). Located just west of Kemp on the (Smith et al. 2015), the wood-boring bivalve Xylophaga volcanic South Sandwich Arc (Leat et al. 2016), Kemp and wood-ingesting limpets of the families Caldera is 900–1600 m in depth from sill to base with Pectinodontidae and Cocculinidae (Marshall 1985). white-smoker and diffuse-flow vents mainly concentrated

1 2 CHONG CHEN AND KATRIN LINSE

Cocculinidae is a family of hermaphroditic, roughly symmetrical gastropods with limpet-like shells, currently placed in the superfamily (subclass Neomphaliones, order Cocculinida) (Bouchet et al. 2017). Previously known only from organic falls, primarily sunken woods (Marshall 1985, McLean & Harasewych 1995, Ardila & Harasewych 2005), 7 genera and over 40 species are currently recognized within the family (Zhang & Zhang 2018). Despite its near-global distribution, no records have been reported from the Southern Ocean. Cocculina, the nominal genus of Cocculinidae with over 20 species, is one of the most abundant invertebrates commonly associated with sunken wood, although two species have been described from cetacean carcasses, including Cocculina craigsmithi McLean 1992 from off California and Cocculina delphinicula Zhang & Zhang, 2018 from the East China Sea (McLean 1992, Zhang & Zhang 2018). Cocculinoidea was originally considered to form a monophyletic clade with Lepetelloidea, another superfamily of morphologically and ecologically similar limpets (Haszprunar 1987, 1988), but these superfamilies are now considered to be two separate, convergent radiations (Aktipis & Giribet 2012). Cocculinoids are more closely affiliated with neomphaloids, which are also restricted to chemosynthetic habitats but mainly radiated in hot vents (Warén & Bouchet 2001), while lepetelloids are placed within the subclass Vetigastropoda (Cunha & Giribet 2019). Fig. 1. Map of a. South Sandwich Arc showing the The Kemp Caldera cocculinid is the first member of the location of Kemp Caldera and b. the distribution of order Cocculinida from environments Cocculina enigmadonta n. sp. within Kemp Caldera (black and poses an opportunity to investigate a potential stars, hydrothermal vents; white star, natural whale fall). transition from organic falls to hot vents. It is also the first cocculinid from the Southern Ocean, although its locality around a central resurgent cone (Fig. 1). Although a subset is barely within the limit of the Southern Ocean (60°S), of taxa known from the East Scotia Ridge vents was present which is still debated. Here, we characterize the new at Kemp Caldera vents, some were absent (e.g. Kiwa yeti species morphologically and genetically, provide its formal crabs). In contrast, Kemp Caldera hosts large assemblages description and investigate its evolutionary history and of chemosymbiotic vesicomyid clams that are absent from adaptations to the hydrothermal vent environment. the East Scotia Ridge vents. Perhaps the most astonishing taxon presently known only from Kemp Caldera was an undescribed cocculinid limpet found in great abundance Materials and methods (Fig. 2) that represented a molluscan order that was Research cruises previously unknown from hot vents. The whale fall was also found near the resurgent cone in During expedition JC42 of RRS James Cook in 2010, Kemp Caldera, ∼250 m from the nearest hydrothermally cocculinid limpets were sampled using the suction active area (Amon et al. 2013). Of the species found on sampler or manipulator of the remotely operated vehicle the whale bones, which included typical whale fall taxa (ROV) Isis within Kemp Caldera on the volcanic South such as polychaete worms in the genera Osedax and Sandwich Arc from hydrothermal vents as well as from a Ophryotrocha (Amon et al. 2013), only two species were natural whale fall (Fig. 1) (Amon et al. 2013). In shared with the nearby vents: the vetigastropod limpet diffuse-flow areas of the hot vents, limpets were collected Lepetodrilus concentricus Linse, Roterman & Chen, from substrates including sulphide chimneys, sulphide 2019, known from both East Scotia Ridge and Kemp rubble, vesicomyid clam shells or whale bones (Fig. 2). Caldera vents (Linse et al. 2019), and an undescribed Upon recovery, specimens were preserved in pre-cooled cocculinid limpet (Amon et al. 2013). 96% ethanol. In 2019, expedition PS119 of RV HOT VENT COCCULINID LIMPET 3

Fig. 2. In situ photographs of Cocculina enigmadonta n. sp. in Kemp Caldera living on a. rock and b. vesicomyid clam shells at the hydrothermally active area near the resurgent cone, c. decaying whale vertebrae at a natural whale fall, d. sulphur crust at the hydrothermally active area on the caldera rim, e. co-occurrence of C. enigmadonta n. sp. with Lepetodrilus concentricus and the sea spider Sericosura sp. Arrowheads indicate positions of C. enigmadonta n. sp. individuals. 4 CHONG CHEN AND KATRIN LINSE

Polarstern visited Kemp Caldera, with ROV MARUM subunit I (COI) gene using the universal primer pair QUEST dives to known hydrothermal areas near the LCO1490 and HCO2198 (Folmer et al. 1994). The PCR resurgent cone, as well as newly discovered venting areas conditions were as follows: 94°C for 2 min followed by on the inner caldera rim. 35 cycles of 94°C for 20 s, 50°C for 20 s and 72°C for 2 min, ending with 72°C for 7 min. Amplification was confirmed with 1% agarose gel electrophoresis using Morphology SYBR green. The PCR purification and DNA Morphological investigation and dissection were carried sequencing of forward and reverse strands were out under a Zeiss Stemi SV6 dissection microscope. performed at LGC Berlin, Germany. Alignment was Radulae were dissected and cleaned using half-strength carried out using Geneious R11 (https://www.geneious. commercial bleach, rinsed, air dried and mounted on com) and manually corrected. In downstream analyses, carbon tape for scanning electron microscopy (SEM). only those sequences with both good-quality matching The SEM imaging was undertaken using a Hitachi forward and reverse reads were used. TM3000 SEM (British Antarctic Survey, Cambridge, For phylogenetic reconstruction, sequences of described UK). Shell length (SL) and shell width (SW) were species in the order Cocculinida and its sister order measured using an eyepiece graticule in the Stemi SV6 Neomphalida in subclass Neomphaliones, as well as the for a total of 168 specimens with unbroken shells. morphologically similar order Lepetellida in subclass Soft parts of a specimen were visualized in hutch #3 of the Vetigastropoda, were downloaded from GenBank. Two beamline BL20B2 at the SPring-8 synchrotron facility distantly related caenogastropods, Murex pecten Lightfoot, (Hyogo, Japan) after rehydration for 48 h in Milli-Q water 1986 and Neptunea antiqua (Linnaeus, 1758) were selected and stained using 1% iodine solution for 24 h. Scans were as the outgroup. PartitionFinder 2 (Lanfear et al. 2016)was done using monochromatic X-rays at 25 keV using a used to select the most suitable evolutionary model using Hamamatsu Photonics K.K. charge-coupled device camera. the Bayesian information criterion. This selected the Samples were rotated through 180° yielding 1860 images general time-reversible γ-invariable mixture (GTR + I + R) with effective pixel sizes of 6.5 μm(Chenet al. 2018b, model for all codon positions in the 621 bp alignment used Sasaki et al. 2018). The resulting images were processed in in the final analyses. Reconstruction was carried out by Adobe Photoshop CC for contrast enhancement and then Bayesian inference using MrBayes 3.2 (Ronquist et al. imported into Amira v5.3.3 (FEI Visualization Sciences 2012). Metropolis-coupled Monte Carlo Markov chains Group). The images were aligned into a single stack and were run for two million generations, sampling topologies organs of interest were selected manually, their 3D surfaces once every 100 generations, with the first 25% discarded as rendered and their volumes calculated. The rendered burn-in to ensure that chains sampled a stationary position. surfaces were post-processed by smoothing to generate the TRACER v1.7 (Rambaut et al. 2018) was used to assess final tomographic model following previously published convergence, confirming split frequencies being < 0.01 methods (Ruthensteiner 2008). before the analysis terminated. Specimens were deposited in the invertebrate collections at New sequences generated from this study were the Natural History Museum, London (NHMUK), the deposited in GenBank under accession numbers University Museum of Zoology, University of Cambridge MN539277–MN539281. (UMZC), Muséum National d'Histoire Naturelle, Paris (MNHN) and the University Museum, University of Tokyo (UMUT). Results Systematics Genetics Order Cocculinida Thiele, 1909 Genomic DNAwas extracted using whole specimens with the DNeasy Tissue Extraction Kit following the Superfamily Cocculinoidea Dall, 1882 manufacturer's instructions (QIAGEN, Crawley, UK), Family Cocculinidae Dall, 1882 with quality checks carried out using a Nanodrop 2000 spectrophotometer. Polymerase chain reaction (PCR) Genus Cocculina Dall, 1882 with standard reagents in 25 μl total volume (2.5 μlof 10× buffer containing 15 mM Mg2+, 0.5 μlof10mM Cocculina enigmadonta n. sp. deoxynucleoside triphosphates, 2.5 μlofeach10μM (Fig. 3) primer, 0.125 μl od 2.5 U Taq DNA polymerase, 5 μlof – 'Q' solution, 10.875 μl double-distilled water and 1 μl Pyropelta sp. Amon et al. (2013) genomic DNA) was used to amplify the barcoding ZooBank registration LSID: urn:lsid:zoobank.org: fragment of the mitochondrial cytochrome c oxidase act:0F15867C-6B73-40E3-B0C9-762F8CFE5730 HOT VENT COCCULINID LIMPET 5

Fig. 3. Cocculina enigmadonta n. sp., representative type specimens in dorsal, ventral and lateral (left) views. a. Paratype #1 (NHMUK 20191096), b. paratype #4 (MNHN-IM-2014-7542), c. holotype (NHMUK 20191095), d. paratype #2 (UMZC 2019.47). Scale bars = 5 mm.

Diagnosis: A large-sized Cocculina (upto15.2mminSL) Paratype #3 (UMZC 2019.48), SL 11.4 mm, SW with thick periostracum, irregular shell margins as adults 9.0 mm, same data as paratype #2. Paratype #4 (Fig. 3b, and a highly characteristic radula exhibiting finely serrated MNHN-IM-2014-7542), SL 14.1 mm, SW 10.8 mm, cusps on the inner lateral teeth and a single broad, blunt 'Great Wall', 59°41.597'S, 28°21.221'W, 1431 m deep, on cusp on the outermost lateral tooth. basalt rock, ROV Isis Dive #149, 2010/ii/9. Paratype Type material: Holotype (Fig. 3c, NHMUK 20191095), #5 (UMUT RM33193), SL 9.4 mm, SW 7.3 mm, SL 9.2 mm, SW 7.0 mm, live collected, fixed and stored in 'Winter Palace', 59°41.695'S, 28°20.982'W, 1434 m deep, 96% ethanol, 'North of Glacier', Kemp Caldera vent site, on sulphur chimney, ROV Isis Dive #149, 2010/ii/9. 59°41.701'S, 28°21.050'W, South Sandwich Arc, 1429 m Paratype lot #6, 11 specimens (NHMUK 20191097), deep, on clam shell, ROV Isis Dive #149, 2010/ii/9. same data as the holotype. Paratype lot #7, 31 specimens Paratype #1 (Fig. 3a, NHMUK 20191096), SL (UMZC 2019.49), same data as paratypes #2–3. 12.1 mm, SW 9.6 mm, same lot as the holotype. Paratype Paratype lot #8, 21 specimens (MNHN-IM-2014-7543), #2 (Fig. 3d, UMZC 2019.47), SL 4.1 mm, SW 3.0 mm, same data as paratype #4. Paratype lot #9, nine 'Seep Fields', 59°41.675'S, 28°21.081'W, 1431 m deep, on specimens (UMUT RM33194), same data as paratype sulphide precipitate, ROV Isis Dive #150, 2010/ii/9. #5. All specimens were live collected from within the 6 CHONG CHEN AND KATRIN LINSE

Kemp Caldera vent site on RRS James Cook expedition specimens (MNHN-IM-2014-7544), 'Clam Road', JC42 and fixed and stored in 96% ethanol. 59°42.024'S, 28°21.233'W, 1431 m deep, on basalt rock, Further materials measured (Fig. 4): five specimens ROV Isis Dive #151, 2010/ii/10. Fourteen specimens (NHMUK 20191098), 'NE Ash Mount Top', (UMUT RM33196), 'Tube Worm Field', 59°41.660'S, 59°41.670'S, 28°21.036'W, 1430 m deep, on basalt rock, 28°21.087'W, 1399 m deep, on basalt rock, ROV Isis Dive ROV Isis Dive #147, 2010/ii/6. Seventeen specimens #151, 2010/ii/10. Five specimens (MNHN-IM-2014- (NHMUK 20191099), 'Clam Road', 59°42.023'S, 7545), 'End of Glacier', 59°41.683'S, 28°21.075'W, 28°21.023'W, 1486 m deep, on vesicomyid clam, ROV Isis 1427 m deep, on sulphide precipitate, ROV Isis Dive Dive #149, 2010/ii/9. Seven specimens (NHMUK #151, 2010/ii/10. All specimens were live collected from 20191100), 'Glacier', 59°41.713'S, 28°21.069'W, 1320 m within the Kemp Caldera vent site on RRS James Cook deep, on sediment, ROV Isis Dive #149, 2010/ii/9. Ten expedition JC42 and fixed and stored in 96% ethanol. specimens (UMUT RM33195), from natural whale fall, Description: Shell (Fig. 3) large for cocculinids 59°41.600'S, 28°21.116'W, 1445 m deep, on whale bone, (maximum length 15.2 mm), white, extremely thin and ROV Isis Dive #149, 2010/ii/9. Two specimens (UMZC translucent. Protoconch unknown as apex lost to 2019.50), 'Ash Mount', 59°42.065'S, 28°21.237'W, 1462 m corrosion in all specimens available. Interior of apical deep, on basalt rock, ROV Isis Dive #149, 2010/ii/9. area secondarily thickened to compensate corrosion. Eight specimens (UMZC 2019.51), 'End of Glacier', Teleoconch lacking in sculpture except for fine 59°41.597'S, 28°21.211'W, 1431 m deep, on sulphide concentric growth lines. Apex directed posteriorly at precipitate, ROV Isis Dive #149, 2010/ii/9. Nine about 45° angle when preserved in adults (Fig. 3c). Shell margins in one plane in early growth stages (Fig. 3d), but strongly irregular in later stages (Fig. 3a & b). Lateral profile rather tall in early growth stages, rapidly becoming flattened in later stages, anterior slope slightly convex, posterior slope strongly concave. Dorsal outline oval, with irregular edges when adult. Thick, greenish, smooth periostracum present, often covered by additional layer of sulphide deposits. Radula (Fig. 5a & b) rhipidoglossate, formula n - (1 + 3) - r - (3 + 1) - n (n = numerous, r = rachidian), 61 countable rows in specimen figured. Rachidian plate rectangular, greatly broadened laterally, poorly developed, lacking true cusps, anterior edge concave, thickened. First lateral tooth with broad, flattened, straight shaft ending distally in one overhanging, triangular, pointed cusp finely serrated with 20–24 denticles. Second to third lateral teeth similar but progressively narrower. Outermost lateral tooth massive, well reinforced, with one broad, blunt cusp. Marginal teeth ∼50 on each side, diminishing in size outwards. Each marginal tooth narrow, elongate, ending distally in one cusp finely serrated into ∼20 denticles. External anatomy (Figs 5 & 6). Animal whitish in colour. Eyes lacking. Snout short. Cephalic tentacles (Fig. 6c, label 't') simple, conical, approximately symmetrical, protruding slightly beyond snout when preserved, but two to three times as long as snout when alive (Fig. 2a). Mouth ventrally situated, with cuticular epithelium lining typical of cocculinids, resembling coat of bristles or hairs (Fig. 5c), surrounded by thick, laterally broadened oral lappets (Fig. 6a, label 'ol'). Copulatory organ (Figs 5c, arrow & 6a, label 'co') situated at right side of foot, posterior of right oral Fig. 4. Size measurements of Cocculina enigmadonta n. sp., lappet. Mantle margin simple, lacking tentacles. Shell showing a. the relationship between shell length and shell muscle horseshoe shaped, encircling posterior two-thirds width and b. the size frequency distribution. of dorsal surface. One pair of simple posterior epipodial HOT VENT COCCULINID LIMPET 7

Fig. 5. Scanning electron micrographs of Cocculina enigmadonta n. sp. showing a. a section of the radula ribbon with b. a close-up of the lateral teeth, c. anteroventral view of the animal with white arrowhead indicating the copulatory organ and d. posterior view with arrowheads indicating the single pair of epipodial tentacles. Scale bars, a. and b. = 100 μm, c. and d. = 500 μm.

tentacles present (Figs 5d, arrows & 6a, label 'et'). in C-shape anterodorsally to enter stomach (Fig. 7d). Operculum absent. Pallial cavity shallow, occupying Stomach very large, curving in reversed C-shape approximately anterior third of body length. Single, posteroventrally (Fig. 7, label 'st'). Intestine emerging triangular pseudoplicate gill (Fig. 6a & d, label 'g') posteriorly to left from posterior end of stomach, situated on pallial mantle roof at right side, dorsal of quickly recurving anteriorly to make 3.5 loops before right oral lappet, with ∼10–12 vestigial leaflets without exiting anteriorly to left side, then abruptly turning right sensory bursicles. Osphradium situated left of into a straight rectum. Anus situated at right side of pseudoplicate gill, without visible zonation. Posterior mantle roof in pallial cavity, posterior to pseudoplicate part of mantle roof occupied by pericardium on left and gill. Digestive gland (Figs 6b & 7a, label 'dg') expansive, rather large kidney on right (Fig. 6b, labels 'pc' and 'k', one part filling dorsal aspect of posterior viscera, while respectively). Hypobranchial gland lacking. another part fills space between intestinal loops. Internal anatomy (Fig. 7). Radular sac approximately Intestine contents composed of whitish organic matter half as long as body length, posterior end of radular sac mixed with sulphide particles. Animal hermaphroditic, bifurcated with lateral protrusions. Radular sac twisted with a single hermaphroditic gland (Fig. 6b, label 'hg') 180° anteroventrally in a C-shape at posterior end. situated at posterior end of animal ventral to digestive Single, lateral pair of massive radular cartilages present, gland, divided into distinct regions for producing sperm bending outwards posteriorly inside laterally expanding or egg. Testis part (Fig. 7, label 'te') of hermaphroditic buccal mass (Fig. 7c, label 'rc'). Oesophagus exiting gland dorsal to ovary part (Fig. 7, label 'o'), usually posteriorly to left from buccal mass, gradually recurving testis part smaller than ovary part. Size of 8 CHONG CHEN AND KATRIN LINSE

Fig. 6. External anatomyof Cocculina enigmadonta n.sp., a. ventral view with shellremoved,b. dorsal view with shell removed, c. dorsal aspect of the head after removing the mantle, d. ventral (left) and dorsal (right) enlargements of the pseudoplicate gill. co = copulatory organ, dg = digestivegland, et = epipodial tentacle, f = foot, g = pseudoplicate gill, gd = gonoduct, hg = hermaphroditic gland, k = kidney, m = mantle edge, mo = mouth, ol = oral lappet, pc = pericardium, st = stomach, t = cephalic tentacle. Scale bars: a.–c. = 1 mm, d. = 0.5 mm. hermaphroditic gland, especially ovary part, greatly Etymology:'Enigma' (Latin), 'riddle' or 'enigma'; variable among individuals of different ripeness. '-donta, odon' (Greek), teeth. In reference to its radular Gonoduct emerges from right side of hermaphroditic morphology, unusual for the genus. gland, running along shell muscle anteriorly. Distribution: Only known from Kemp Caldera, so far it Dimensions: Specimens range between 2.1 and 15.2 mm has been collected from hydrothermally influenced areas in SL and between 1.1 and 11.9 mm in SW (Fig. 4), and a natural whale fall, both near the central resurgent typically ∼10 mm in length and ∼8 mm in width. cone (Fig. 1b). Recent survey by RV Polarstern confirmed The relationship between SL and SW was found to its presence in newly discovered hydrothermally be generally constant across the size range available influenced areas near the northern part of the inner for study. caldera rim. In vent habitats, C. enigmadonta n. sp. was HOT VENT COCCULINID LIMPET 9

Fig. 7. Internal anatomy of Cocculina enigmadonta n. sp. shown through 3D reconstruction, displaying a. dorsal view and b. lateral (left) view showing all reconstructed organs, c. radula apparatus within the buccal mass and d. ventral view showing the alimentary tract. Anterior of the animal is indicated by an arrowhead for each part of the figure. a = anus, bu = buccal mass, dg = digestive gland, g = pseudoplicate gill, i = intestine, mo = mouth, o = ovary part of the hermaphroditic gland, oe = oesophagus, r = radula, rc = radula cartilage, re = rectum, st = stomach, te = testis part of the hermaphroditic gland. Length of the animal is 6.14 mm.

found on basalt rock, sediment rubble, vesicomyid clam cocculinid species. Furthermore, it is the only cocculinid shells and precipitated sediments, and it lives in close known to occur in a hydrothermal vent environment and proximity to vesicomyid clams and actinostylid also the only cocculinid presently known from the anemones. It is often associated with an abundance of the Southern Ocean. In an earlier study (Amon et al. 2013), vetigastropod limpet Lepetodrilus concentricus and sea it was erroneously reported as 'Pyropelta sp.'. spiders in the genus Sericosura (Fig. 2e). Whether the lateral ridges of the wide rachidian tooth in Remarks: The radular characters of C. enigmadonta cocculinids represent vestigial first lateral teeth or not n. sp. are highly modified from the standard cocculinid remains unsettled. This view was originally proposed by radula and unlike any other Cocculina described to date Marshall (1985), with some authors (e.g. Zhang & in having finely serrated cusps on the inner lateral teeth Zhang 2018) accepting it and others (e.g. McLean & and a unicuspid outermost lateral tooth. Nevertheless, Harasewych 1995) continuing to consider there to be its external and internal anatomy agree well with only four lateral teeth. The outermost, massive, lateral Cocculina and indicate that it should be placed in this tooth has sometimes been termed the pluricuspid tooth genus. Its radula, combined with a thick periostracum (McLean & Harasewych 1995). Here, we tentatively and highly irregular shell margins, makes this species treat the vestigial ridge as part of the rachidian tooth impossible to confuse with any other described and suggest there to be four lateral teeth on either side, 10 CHONG CHEN AND KATRIN LINSE

Fig. 8. Bayesian phylogenetic tree constructed using 621 bp of the mitochondrial COI gene showing the systematic position of Cocculina enigmadonta n. sp. within genus Cocculina, family Cocculinidae. GenBank accession numbers are shown in parentheses. including the pluricuspid tooth. In any case, the outermost Discussion lateral teeth of C. enigmadonta n. sp. are completely smooth and not pluricuspid. Cocculina enigmadonta n. sp. is the only member of the order Cocculinida known from hydrothermal vents, and the fact that it also lives on whale bones means that it is Molecular phylogeny also the first cocculinid known to be capable of Bayesian phylogenetic reconstruction using 621 bp of the inhabiting two different types of chemosynthetic mitochondrial COI gene (Fig. 8) recovered C. enigmadonta ecosystems. Integrative evidence from external anatomy, n. sp. nested within a fully supported (Bayesian posterior internal anatomy and molecular phylogeny together probability (BPP) = 1) genus Cocculina represented by two demonstrated that C. enigmadonta n. sp. is a member of other described species, confirming its placement in this the diverse genus Cocculina (Haszprunar 1987, Ardila & genus. Cocculina enigmadonta n. sp. was recovered sister to Harasewych 2005). Furthermore, the phylogenetic Cocculina messingi McLean & Harasewych, 1995, and this reconstruction presented herein (Fig. 8) indicated that it pair were in turn sister to Cocculina subcompressa is a more derived member of the family compared to Schepman, 1908. The five individuals of C. enigmadonta typical wood fall inhabitants such as C. messingi and n. sp. sequenced emerged as a well-supported lineage C. subcompressa, having a highly unusual radula for the (BPP = 0.92) among the sequences sampled. The order group. This suggests that C. enigmadonta n. sp. probably Cocculinida, represented by a total of four species in evolved from an ancestor living on sunken wood that two cocculinid genera, was recovered with strong support subsequently adapted to the hydrothermal vent (BPP = 1) as a monophyletic clade sister to Neomphalida. environment, in line with the evolutionary 'stepping Within Neomphalida, a strongly supported Peltospiridae stone' hypothesis (Smith et al. 2015). As C. enigmadonta (BPP = 0.99) represented by four species was recovered n. sp. is also found on whale falls, cetacean carcasses sister to Melanodrymiidae represented by Leptogyra infalata may have provided an intermediate stepping stone Warén & Bouchet, 1993. The subclass Neomphaliones between wood and vent, as has been suggested for containing these two orders was found to be genetically bathymodioline mussels (Thubaut et al. 2013). The distinct (BPP = 1) from morphologically similar isopod Jaera tyleri Linse et al., 2014, from the same vetigastropod limpets in the order Lepetellida. whale fall in Kemp Caldera, also shows shifts in habitat HOT VENT COCCULINID LIMPET 11 and food source, as it belongs to a normally shallow-water, The shell of C. enigmadonta n. sp. is usually highly herbivorous genus (Linse et al. 2014). However, corroded and often covered by sulphide deposits, a phylogenetic reconstruction did not place it in a derived condition common in vent molluscs and attributable to position within the genus (Linse et al. 2014). the acidic conditions of the vent fluid (Warén & Bouchet Unlike bathymodioline mussels in which the scenario of 1993). The periostracum of C. enigmadonta n. sp. is stepwise evolution from sunken wood to hot vent is thickened compared to many other Cocculina species, associated with the acquisition of chemoautotrophic which have thin periostracum (McLean & Harasewych endosymbiotic bacteria (Smith et al. 2015), 1995). Although a thin periostracal layer is common in C. enigmadonta n. sp. clearly feeds by ingesting external the family, and even abyssal cocculinids in the genus food by radular activity and does not rely on symbionts Fedikovella lack a thick periostracal layer (Leal & for nutrition. Stomach contents of C. enigmadonta n. sp. Harasewych 1999), a number of wood-dwelling are composed of whitish organic matter mixed with cocculinids do have a well-developed periostracal layer occasional mineral particles. In gastropods that graze on (e.g. Coccocrater agassizii (Dall, 1908); Haszprunar bacterial mats in hydrothermal vent environments, such 1987). This is perhaps a response to a reducing mixtures often comprise bacterial matter mixed with the environment on wood falls. Thickening of the sulphide mineral particles that make up the vent periostracum in C. enigmadonta n. sp. is probably a chimney (Fretter 1989, Warén & Bouchet 1993). This is similar adaptation in order to limit shell corrosion, a in sharp contrast to typical cocculinids that ingest small feature also shared by many vent-endemic gastropods pieces of wood, which are the main components of their (Warén & Bouchet 1993). Cocculina craigsmithi,a intestine and stomach contents (Haszprunar 1987). In species that lives on decaying whale carcasses, also the case of the cocculinids, the stepwise evolutionary possesses a thickened periostracum, as well as a corroded transition from living on sunken wood (and whale apex (McLean 1992), indicating that cocculinids are bones) to hot vents probably became advantageous by capable of evolving a thick periostracum in response to simply allowing them to become gradually adapted to sulphidic reducing environments. sulphidic environments, which typically exhibit more Similarly to other cocculinids (Haszprunar 1987), environmental stress, such as higher concentrations of C. enigmadonta n. sp. is a hermaphrodite, with distinct hydrogen sulphide and heavy metals. areas of the hermaphroditic gland serving the functions The radula of C. enigmadonta n. sp., although still of testis and ovary (Fig. 7c). The individuals available to retaining the same formula, is highly modified from the study differed greatly in the ripeness of their gonads, standard cocculinid radula (Marshall 1985), in that the especially the ovary parts. Sexually mature individuals lateral teeth have become broadened and flattened and with a greatly expanded ovary containing hundreds of have lost well-defined major cusps. Instead, the inner white eggs were visually distinguishable, as the posterior lateral teeth now have rake-like, finely denticulate cusps ends of their bodies were greatly inflated and appeared resembling radula of grazing trochoid vetigastropods, to be much taller (compare Fig. 3b with Fig. 3a). and the outermost lateral tooth has only a single cusp Interestingly, these mature individuals were collected in that is not well differentiated from the shaft. Gastropods large quantities in a single sampling event. Figure 2a are capable of modifying their radula to suit various shows an aggregation of such sexually mature food sources over evolutionary timescales (Hickman individuals, which were collected in 2010 on basaltic 1983), and some species are known to modify their rocks. In aggregations, a stacking behaviour was radula to suit each food source (e.g. Padilla 1998). We observed, in which several individuals stacked on top of interpret the modifications seen in the radula of each other to form 'limpet towers' (Fig. 2a). This is C. enigmadonta n. sp. as probably arising from reminiscent of 'mating stacks' documented for the ecological factors in that the radula now exploits a Antarctic true limpet Nacella concinna (Strebel, 1908), different food source from other cocculinids. The which spawn in temporary stacks to increase fertilization flattened, unicuspid teeth of C. enigmadonta n. sp. with success (Picken & Allan 1983) or more permanent an increased number of marginal teeth are more adapted stacking in calyptreid slipper limpets, which are for sweeping the surfaces (Hickman 1983) of vent protandric hermaphrodites (Cledón et al. 2016). As habitats for the loosely attached bacterial film than the cocculinids exhibit internal fertilization (Haszprunar typical cocculinid radula. The fact that the anatomical 1987, 1988), stacking may represent a behaviour to features of C. enigmadonta n. sp., particularly the facilitate copulation, as has been speculated for the alimentary tract, remain typical for genus Cocculina vent-endemic gonochoristic peltospirid snails in (Marshall 1985, Haszprunar 1987, 1988, Sasaki 1998)is the genus Gigantopelta, which are also internal perhaps unexpected considering that a shift in food fertilizers (Chen et al. 2018a). Stacking has also been source often results in changes in the digestive anatomy observed in the vetigastropod vent limpet Lepetodrilus (Warén & Bouchet 1993, Chen et al. 2018b). fucensis, which live in extreme densities as high as 12 CHONG CHEN AND KATRIN LINSE

300 000 individuals per m2, although in this case access Author contributions to food particles has been hypothesized as the purpose CC and KL conceived and designed the study. KL joined for stacking behaviour (Bates et al. 2005). research expeditions and collected the specimens. Both authors collected, analysed and interpreted morphological Conclusion data by microscopy. CC carried out synchrotron scanning, 3D reconstruction and interpretation of the internal Through an integrative taxonomy framework anatomy. KL carried out morphometric measurements incorporating traditional dissection and observation, and generated the map used herein; CC prepared the fi electron microscopy, genetic sequencing and 3D other gures. KL collected genetic data, which were anatomical reconstruction using synchrotron computed analysed by CC. CC drafted the original manuscript, tomography scanning, we characterized an unusual which was revised and improved by KL; both authors fi cocculinid limpet discovered in hydrothermal vent and gave nal approval for submission and publication. whale fall habitats in Kemp Caldera, Southern Ocean. Phylogenetically nested in a derived position among Conflict of interest the typically wood-dwelling genus Cocculina, C. enigmadonta n. sp. was found to have a highly The authors declare that they have no conflicts of interest. modified radula suited for feeding on bacterial film instead of wood, but surprisingly retaining other anatomical features typical of the genus. Our results Financial support suggest that C. enigmadonta n. sp. probably evolved from The ChEsSO consortium and its expedition JC42 were an ancestor adapted to living on sunken wood like vast funded by the UK NERC Consortium Grant majority of Cocculina species. Most taxa we know today NE/DO1249X/1. PS119 was funded by BMBF and that are capable of living in hydrothermal vents are MARUM. Fieldwork in the East was relatively recently derived in the late Mesozoic or undertaken under the permit S3-3/2009 (JC42) issued by Cenozoic (Little & Vrijenhoek 2003, Vrijenhoek 2013). the Foreign and Commonwealth Office, London to The discovery of C. enigmadonta n. sp., the first species of Section 3 of the Antarctic Act 1994 and permit RAP its order found to have colonized hot vents and the first 2018/064 (PS119) issued by the and South representative from the Southern Ocean, sheds light on a Sandwich Government. KL's participation in PS119 was compelling case study of the 'stepping stone' evolutionary supported by a NERC national capability grant trajectory from organic falls to seeps and vents. (NC-science) to the British Antarctic Survey. CC was supported by a Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (18K06401). Acknowledgements The funders had no role in the study design, data The authors would like to thank the masters and crews of collection and analysis, decision to publish or preparation RRS James Cook and RV Polarstern on board of the manuscript. expeditions JC42 and PS119 for their professionalism and support, as well as the logistic and shipboard Details of data deposit support of the pilots and technical team of NMF and MARUM with their ROVs Isis and QUEST. We are very Data generated and/or analysed during the current study are grateful to Paul Tyler for leading the ChEsSO available in the NCBI GenBank repository, with accession consortium, Alex D. Rogers and Gerhard Bohrmann as numbers MN539277–MN539283. Specimens used in the principal scientists of cruises JC42 and PS119, Veerle present study are deposited in the following museums: Huvenne for the base map used herein, Elaine NHMUK (20191095–20191100), MNHN (MNHN-IM- Fitzcharles for PCR amplifications, Yukiko Nagai for 2014-7542–MNHN-IM-2014-7545), UMZC (2019.47– microscopy and Pete Bucktrout for macrophotography. 2019.51) and UMUT (RM33193–RM33196). Experiments at SPring-8 were performed under the approval of the SPring-8 Proposal Review Committee under the proposal number 2017A1720 and were References technically supported by Kentaro Uesugi and Masato AKTIPIS,S.W.&GIRIBET, G. 2012. Testing relationships among the Hoshino. Takenori Sasaki is thanked for enabling access vetigastropod taxa: a molecular approach. Journal of Molluscan to SPring-8 and for helping with sample scanning. We Studies, 78, 10.1093/mollus/eyr023. AMON, D.J., GLOVER, A.G., WIKLUND, H., MARSH, L., LINSE, K., thank Bruce Marshall, José H. Leal and two anonymous ROGERS, A.D., et al. 2013. The discovery of a natural whale fall in reviewers for comments and edits that improved earlier the Antarctic deep sea. Deep Sea Research II, 92, 10.1016/ versions of this paper. j.dsr2.2013.01.028. HOT VENT COCCULINID LIMPET 13

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