A peer-reviewed open-access journal ZooKeys | 112: 123—137 (2022) A Reet eee #ZooKeys https:/ / ZOO keys. pensoft.net Launched to accelerate biodiversity research A new provannid snail (Gastropoda, Abyssochrysoidea) discovered from Northwest Eifuku Volcano, Mariana Arc Chong Chen!', Hiromi Kayama Watanabe' | X-STAR, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa, 237-0061, Japan Corresponding author: Chong Chen (cchen@jamstec.go.jp) Academic editor: Martin Haase | Received 28 April 2022 | Accepted 9 June 2022 | Published 14 July 2022 https://z00 bank. org/2296D 13A-1207-4235-A785-A06CF1D7079C Citation: Chen C, Watanabe HK (2022) A new provannid snail (Gastropoda, Abyssochrysoidea) discovered from Northwest Eifuku Volcano, Mariana Arc. ZooKeys 1112: 123-137. https://doi.org/10.3897/zookeys. 1112.85950 Abstract Gastropods in the family Provannidae are characteristic members of deep-sea chemosynthesis-based com- munities. Recently, surveys of hydrothermal vents and hydrocarbon seeps in the western Pacific have revealed a high diversity of provannids, with new discoveries continuing to be made. Here, we report and describe a further new species, Provanna exquisita sp. nov., discovered from the Northwest Eifuku vol- cano on the Mariana Arc. This new species is distinguished from all other described Provanna species by its exaggerated sculpture characterised by two to three sharply raised, flange-like keels on the teleoconch whorls. ‘The status of P exquisita sp. nov. is also supported by a molecular phylogeny reconstruction using the mitochondrial cytochrome c oxidase subunit I (COI) gene, which suggested that it is most closely related to a clade of three species described from Okinawa Trough vents including P clathrata, P subglabra, and P fenestrata. Despite being one of the better-explored regions of the world in terms of hydrothermal vent biodiversity, new discoveries like P exquisita sp. nov. continue to remind us that we are nowhere near fully documenting the species diversity in these unique ecosystems—despite the species being threatened from imminent anthropogenic impacts such as deep-sea mining. Keywords Deep sea, hydrothermal vent, Mollusca, new species, Provannidae, Western Pacific Copyright Chong Chen & Hiromi Kayama Watanabe. This is an open access article distributed under the terms of the Creative Commons At- tribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 124 Chong Chen & Hiromi Kayama Watanabe / ZooKeys 1112: 123-137 (2022) Introduction Hydrothermal vent ecosystems in the deep sea host lush biological communities sus- tained by microbial chemosynthesis using hydrogen sulfide and other reducing sub- stances dissolved in the vent fluid. First discovered in 1977 on the Galapagos Rift (Corliss et al. 1979), over 300 active vents have been confirmed around the world, concentrated on mid-ocean ridges, volcanic arcs, and back-arc basins (Beaulieu and Szafranski 2020). The Izu-Ogasawara (Bonin)-Mariana (IBM) Arc in the western Pa- cific is home to over a dozen known active vent sites, typically located on submarine volcanoes (Watanabe et al. 2019). One of these is the Northwest Eifuku (NW Eifuku) Volcano, hydrothermal activity on which was discovered during the NOAA Ocean Exploration Program’s “Submarine Ring of Fire” (SROF) project that undertook sur- veys of a number of volcanoes on the Mariana Arc from 13.5°N to 22.5°N (Embley et al. 2007). The hydrothermally active sites at the summit of NW Eifuku discovered in March and April 2004 are notable for the presence of cold liquid carbon dioxide (CO,) discharge in addition to hot hydrothermal fluid venting from white smokers at the Champagne vent (Lupton et al. 2006). This CO, flux leads to an extreme habitat where dense animal colonies dominated by the bathymodioline mussel Bathymodiolus septemdierum Hashimoto & Okutani, 1994 are found, with pH as low as 5.36 (Limén and Juniper 2006; Tunnicliffe et al. 2009; Rossi and Tunnicliffe 2017). Gastropod molluscs are prevailing inhabitants of vent ecosystems (Warén and Bouchet 1993, 2001) and about two-thirds of gastropods found at vents occur in no other environments (Wolff 2005). Provannidae is a gastropod family found exclusively in chemosynthesis-based ecosystems (Chen et al. 2019; Linse et al. 2019), recently revealed to be paraphyletic due to genera in the closely related family Abyssochrysidae becoming nested with genera considered to be provannids in phylogenetic reconstruc- tions (Johnson et al. 2010; Souza et al. 2020). The two families together form the superfamily Abyssochrysoidea (Souza et al. 2020). Currently, four genera from che- mosynthetic ecosystems, including the endosymbiotic Alviniconcha and Ifremeria, as well as the non-symbiotic Provanna and Desbruyeresia, are assigned to Provannidae, two genera including Abyssochrysos from non-chemosynthetic deep sea and Cordesia from organic falls are assigned to Abyssochrysidae, while Rubyspira from organic falls remains unassigned to either family (Souza et al. 2020). Familial affinities of the genera still remain in a state of flux. Provanna is the most species-rich abyssochrysoid genus, with 27 described species inhabiting hot vents, cold seeps, and organic falls between 450-5687 m deep around the globe (Sasaki et al. 2010; Linse et al. 2019). Recent explorations of vents and seeps in the western Pacific have revealed a high diversity of Provanna species (Sasaki et al. 2016; Chen et al. 2019; Ke et al. 2022). Here, we report a further previously unde- scribed species of Provanna with a striking sculpture, discovered from the hydrothermal vent field near the summit of NW Eifuku Volcano, providing a formal description and testing its relationship with other abyssochrysoid species using molecular phylogenetic reconstruction with the mitochondrial cytochrome c oxidase subunit I (COI) gene. New Provanna from NW Eifuku 125 Materials and methods Sample collection Provannid snails were collected from near the summit of NW Eifuku Volcano, Mari- ana Arc (Fig. 1) using the remotely-operated vehicle (ROV) JASON JIT on-board the R/V “Roger Revelle” cruise RR141 “Submarine Ring of Fire 2014 — Ironman” (chief scientist Craig Moyer and William Chadwick). Upon recovery on deck, snails were sorted from the biological material collected and preserved in 75% ethanol until fur- ther investigation in the laboratory. Jn situ images of NW Eifuku were taken using a video camera on ROV JASON I/ and supplemented by high-resolution photographs taken by a digital still camera of ROV ROPOS on-board the R/V “Thomas G. Thomp- son” cruise TN167 “Submarine Ring of Fire 2004” (chief scientist Robert W. Embley). Morphology Provannid snails were observed and dissected under an Olympus SZX7 dissecting mi- croscope and photographs were taken using a digital single reflex camera (Olympus OM-D E-M5 Mark III) mounted on the trinocular. For specimen photos, several photos taken at different focus levels were stacked automatically using Adobe Photo- shop 2022 software. Shell height (SH), shell width (SW), aperture height (AH), and aperture width (SW) were measured using digital Vernier callipers, with the values rounded up to the nearest 0.1 mm. In specimens with a damaged aperture, only SH and SW were taken. Electron microscopy The radula was dissected from the radula sac using fine tweezers and placed in 5% sodium hypochlorite solution to dissolve any remaining soft tissue, for about 5 min. The operculum was dissected and sulfide deposits on the surface were cleaned off using a soft brush. The radula and operculum were washed twice in Milli-Q water before mounting on aluminium stubs using carbon tape for scanning electron microscopy (SEM). A tabletop SEM (Hitachi TM3000) was used for observation and imaging of the radula and operculum. DNA extraction and sequencing Genomic DNA was extracted from a section of the provannid snail’s foot musculature using the QIAGEN DNeasy Blood and Tissue Kit (QIAGEN, Tokyo, Japan) follow- ing the manufacturer's standard instructions and then purified using GeneReleaser (BioVentures Inc., Marfreesboro, USA) also following the manufacturer's protocol. The quality of the extracted DNA was checked using a Thermo Scientific NanoDrop 2000 spectrophotometer. The Provanna-specific primer pair for the mitochondrial COI 126 Chong Chen & Hiromi Kayama Watanabe / ZooKeys 1112: 123-137 (2022) Figure |. Jn situ habitat of Provanna exquisita sp. nov. on the summit of Northwest Eifuku Volca- no. A overview of the habitat showing dominance by the deep-sea mussel Bathymodiolus septemdierum B close-up of the mixed provannid aggregation, arrowheads indicate examples of P exquisita sp. nov. Pho- tos taken by ROV ROPOS on dive #792 in Champagne vent, 21°29.25'N, 144°02.49'E, 1608 m deep. New Provanna from NW Eifuku 127 gene, Pg394L (5’°-CTGATTTTTCGGACATCCTG-3’) and Pg1253R (5’-TGTT- GAGGAAAGAAAGTAATATTAA-3’) were used for amplification via polymerase chain reaction (PCR) in a 20 ul reaction volume consisting of 1 yl template DNA, 1 ul of each primer, 10 pl of Premix Ex7Zaq HS DNA polymerase (TaKaRa, Shiga, Ja- pan) and 7 ul de-ionized sterilized water. A Veriti Thermal Cycler (Applied Biosystems) was used for PCR with the following protocol: 94 °C for 2 min followed by 30 cycles of (94 °C for 30 s, 45 °C for 30 s, 72 °C for 30 s), ending with 72 °C for 90 s. The successful PCR product was purified using ExoSAP-IT (Affymetrix) following stand- ard protocols and submitted to FASMAC Corporation (Kanagawa, Japan) for Sanger sequencing. Sequencing was done using the universal primer HCO2198 (Folmer et al. 1994) and the Provanna-specific Pg696R (5’-CAGGATGTCCGAAAAATCAG- 3’) in addition to Pg394L and Pg1253R. Molecular analyses Geneious Prime 2021.2.2 (https://www.geneious.com/) was used to align and manually correct the sequences obtained into a consensus sequence, deposited in GenBank under the accession number ON324570. This newly generated sequence of the NW Eifuku Provanna and a sequence of Cordesia atlantica Souza, Passos, Shimabukuro & Sumida, 2020 (Souza et al. 2020) was added to a 530-bp alignment with other abyssochrysoid COI sequences available on GenBank used in a previ- ous publication by Linse et al. (2019), using the MUSCLE alignment in Geneious. Four non-abyssochrysoid caenogastropods were used as outgroups, including two cerithiids, Cerithium zonatum (Wood, 1828) and Bittiolum varium (Pfeiffer, 1840), and two littorinids, Littorina littorea (Linnaeus, 1758) and Echinolittorina vidua (Gould, 1859). Phylogenetic reconstruction was conducted using Bayesian inference with Mr- Bayes v. 3.2 (Ronquist et al. 2012), using the nucleotide substation models HKY+I+G for the first and second codon positions and GTR+1+G for the third codon position selected by the Bayesian information criterion in PartitionFinder v. 2.1.1 (Lanfear et al. 2016). Markov chain Monte Carlo chains were run for 1 million generations with topologies being sampled every 100 generations. ‘The first 25% of trees were discarded as “burn-in” and convergence was checked using the software Tracer v. 1.7 (Rambaut et al. 2018). The Kimura-2-parameter (K2P) distance (Kimura 1980) between COI sequences of different Provanna species was calculated using the package MEGA X (Kumar et al. 2018) using the same alignment. Specimen repository Specimens examined in the present study were deposited in public museum collec- tions, including Museum national d’Histoire naturelle (MNHN), Paris, France and the National Museum of Nature and Science, Tsukuba (NSMT), Japan. 128 Chong Chen & Hiromi Kayama Watanabe / ZooKeys 1112: 123-137 (2022) Results Systematics Subclass Caenogastropoda Cox, 1960 Superfamily Abyssochrysoidea Tomlin, 1927 Family Provannidae Warén & Ponder, 1991 Provanna Dall, 1918 Type species. Provanna lomana (Dall, 1918). Provanna exquisita sp. nov. https://zoobank.org/57593 DBE-5809-41CC-B5A7-AA1A5726A1F7 Figs 2A—D, 3A—D Provanna aft. fenestrata—Giguére and Tunnicliffe 2021: supplementary table $2 Type locality. Hydrothermal vent near the summit of Northwest Eifuku Volcano (Lupton et al. 2006; Rossi 2016) on the Mariana Arc; 21°29.2567'N, 144°02.4813’E, 1606 m deep (“Golden Lips” site 40 m away from the Champagne vent). Habitat tem- perature 2.7 °C, sulfide level negligible. ROV JASON II dive #799, 2014/xii/14, R/V Roger Revelle cruise RR1413 “Submarine Ring of Fire 2014 — Ironman”. Type material. Holotype (Fig. 2A), MNHN-IM-2000-37945; SH 10.4 mm, SW 9.0 mm, AH 6.1 mm, AW 4.5 mm. Paratype #1 (Fig. 2B), NSMT-Mo 79360; SH 11.6 mm, SW 10.3 mm, AH 7.1 mm, AW 5.6 mm. Paratype #2 (Fig. 2C, Fig. 3A-D), MNHN-IM-2000-37946; SH 13.1 mm, SW 9.4 mm; aperture damaged, soft parts extracted and used for DNA barcoding and dissected for SEM. Paratype #3 (Fig. 2D), NSMT-Mo 79361; SH 9.4 mm, SW 7.2 mm; aperture broken. All type material were live collected from the type locality and preserved in 75% ethanol. Diagnosis. A large Provanna reaching over 13 mm in shell height (exceeds 15 mm if spire intact), teleoconch whorls with two or three sharply raised, flange-like spiral keels crossing with weaker axial ribs to form a regularly latticed sculpture. Description. Shell (Fig. 2). Teleoconch thin and fragile, translucent, thickened where ribs or keels occur. Whorls highly convex, inflated for its genus. Suture dis- tinct, well defined, impressed. Spiral sculpture of 2 or 3 very strong, sharply raised, approximately equally-spaced blade or flange-like keels, positioned at shoulder, mid- whorl, just above suture. Some individuals lack shoulder keel; other 2 always present. Three additional weaker spiral ribs present anterior to suture. Axial sculpture of 14-18 regularly spaced, raised ribs running from suture to suture, approximately equal in strength. Together two directions of ribs intersect to form regular reticulate sculpture of regular rectangles. Nodes drawn out on spiral keels where intersection with axial New Provanna from NW Eifuku 129 ribs occur, resulting in undulated edges, on shoulder keel these develop into short spines. Aperture semicircular, taller than wide. Columellar variable from straight to sigmoidal. Siphonal notch distinct, shallow. Apex decollate, heavily corroded, leaving only 1.5—2.5 whorls of teleoconch whorls. Incompletely corroded periostracum pre- sent around apex, darkened in colouration. Secondary plug-like calcareous secretion present at apex, preventing exposure of visceral mass. Growth lines indistinct. Proto- conch unknown, as all specimens examined had corroded spire. Periostracum thick, golden brown. Operculum (Fig. 3D) present. Paucispiral, oval, bluntly pointed. Nucleus eccen- tric, 3.5 whorls. Yellowish-brown in colour, thin, semitransparent. Radula (Fig. 3A—C) taenioglossate, formula 2 + 1 + 1 + 1 + 2. Central tooth solid, with single triangular, bluntly pointed, overhanging main cusp. Solid lateral support ridges present on both sides of central supporting ridge. Shaft with 2 sharp protru- sions at anterior edge of lateral support ridge. Lateral teeth solid, inner edge sigmoidal. Laterals with 5 cusps, main cusp triangular, bluntly pointed. One moderately strong Figure 2. Provanna exquisita sp. nov., type specimens A=D holotype (MNHN-IM-2000-37945), shell height 10.4 mm E=H paratype #1 (NSMT-Mo 79360), shell height 11.6 mm I, J paratype #2 (MNHN- IM-2000-37946), shell height 13.1 mm K, L paratype #3 (NSMT-Mo 79361), shell height 9.4 mm. Scale bar: 1 cm, applies to all parts of the figure. 130 Chong Chen & Hiromi Kayama Watanabe / ZooKeys 1112: 123-137 (2022) inner cusp inside main cusp, 3 weaker cusps outside main cusp. Sharply raised protru- sion present on shafts of laterals below main cusp. Marginal teeth flat, broad, trun- cated distally. Distal tip rake-like, finely serrated into c. 22—24 denticles, outermost strongest. Further c. 10-12 minor denticles present on outermost cutting edge, below strongest cusp. Gross external anatomy examined to limited extent with 2 brittle, ethanol-pre- served specimens, revealing no notable deviations from published accounts for its ge- nus (Warén and Ponder 1991; Chen et al. 2019). Animal occupied approximately 1.5—2 whorls. Head with flattened snout, 1 pair of equally sized, tapering cephalic tentacles present; eyes lacking. Penis, neck furrow, epipodial tentacles lacking. Pallial edge smooth. Gill monopectinate, typical for its genus, not hypertrophied. Apex of visceral mass occupied by gonad, posterior of digestive gland. Distribution. So far, it is only known from a hydrothermal vent field on the sum- mit of Northwest Eifuku Volcano, Mariana Arc. In addition to the Golden Lips site where specimens were collected, it has also been visually confirmed from the Cham- pagne site 40 m away (Fig. 1B). Etymology. Exquisita (Latin, feminine adjective in the nominative singular), meaning “inquiring” or “exquisite”. Remarks. ‘The striking shell sculpture of Provanna exquisita sp. nov., especially the prominent sharply raised spiral keels, is unique among described Provanna species. The species with the closest morphology is Provanna fenestrata Chen, Watanabe & Sasaki, Figure 3. Provanna exquisita sp. nov., scanning electron micrographs A radula overview B close-up of central and lateral teeth C close-up of marginal cusps D operculum. Scale bars: 100 um (A); 20 pm (B); 50 um (C); 1 mm (D). New Provanna from NW Eifuku 131 2019 described from Okinawa Trough vents and also recently reported from a meth- ane seep in the South China Sea (Chen et al. 2019; Ke et al. 2022), which also has a similar coarse, regular, lattice-like sculpture. In P fenestrata, however, the spiral and axial ribs are of similar strength and spiral ribs do not form raised keels; nodes at the intersection between the two nodes are also lacking in P fenestrata (Chen et al. 2019). In individuals of P fenestrata with 2 spiral ribs, it is always the mid-whorl spiral rib that is missing, whereas the missing spiral keel is always the posterior-most shoulder keel in P exquisita sp. nov. Furthermore, the periostracum of P fenestrata is yellowish green compared to golden brown in P exquisita sp. nov. The radulae of the two species are similar, although in P fenestrata the central and lateral teeth have sharper cusps and the marginals are less serrated (12-14 vs 22-24 denticles). A number of other Provanna species also exhibit reticulate shell sculpture, including P. clathrata Sasaki, Ogura, Watanabe & Fujikura, 2016 from Okinawa Trough vents, Provanna pacifica (Dall, 1908) from seeps in Gulf of Panama and Oregon Margin, Provanna muricata Warén & Bouchet, 1986 from Galapagos Rift vents, P admetoides Waren & Ponder, 1991 from Florida Escarpment seeps, P segonzaci Warén & Ponder, 1991 from Lau Basin vents, Provanna buccinoides Warén & Bouchet, 1993 from Lau and North Fiji vents, and Provanna reticulata Warén & Bouchet, 2009 from seeps off West Africa. However, compared to P exquisita sp. nov. all of these species exhibit much weaker spiral sculpture (Warén and Bouchet 1986, 2001, 2009; Warén and Ponder 1991; Sasaki et al. 2016). The spiral ribs of P muricata are much weaker than the axial ones, which is opposite to the pattern seen in P exquisita sp. nov. (Warén and Bouchet 1986). The radulae of P pacifica and P admetoides exhibit slender, reduced central teeth and are very different from the solid central tooth in P exquisita sp. nov. (Warén and Bouchet 1986; Warén and Ponder 1991). Molecular support The phylogenetic tree of the superfamily Abyssochrysoidea reconstructed with Bayesian inference using the mitochondrial COI gene is shown in Fig. 4. The phylogeny recovered all seven currently recognised genera in Abyssochrysoidea (Provanna, Desbruyeresia, Abyssochrysos, Cordesia, Alviniconcha, Ifremeria, Rubyspira) as monophyletic clades with weak to full support (Bayesian posterior probability (BPP) between 0.46-1). Provanna which was fully supported (BPP = 1) as the earliest-branching genus within the superfamily. Within Provanna, P exquisita sp. nov. was recovered sister to a weakly supported clade (BPP = 0.48) containing three species known from Okinawa Trough and South China Sea, including P fenestrata, P clathrata, and P subglabra Sasaki, Ogura, Watanabe & Fujikura, 2016 (Xu et al. 2016; Ogura et al. 2018; Chen et al. 2019; Ke et al. 2022). The clade containing these four species, including P exquisita sp. nov., was also only weakly supported (BPP = 0.58). The K2P genetic distances of P exquisita sp. nov. from P clathrata, P subglabra, and P fenestrata over a 357 bp alignment of the COI gene were 6.65%, 6.60%, and 7.40%, respectively. 132 Chong Chen & Hiromi Kayama Watanabe / ZooKeys 1112: 123-137 (2022) 0.61 Provanna fenestrata Chen, Watanabe & Sasaki, 2019 (MK560877) Provanna subglabra Sasaki et al., 2016 (AB810080) Provanna Clathrata Sasaki et al., 2016 (AB810212) Provanna exquisita sp. nov. (ON324570) _ Paratype #2 Provanna glabra Okutani, |suchida & Fujikura, 1992 (AB810156) aie Provanna laevis Warén & Ponder, 1991 (GQ290594) cae Provanna lomana (Dall, 1918) (KF467665) - Provanna sculpta Warén & Ponder, 1991 (GQ290595) sya_Provanna kuroshimensis Sasaki et al., 2016 (AB810187) Provanna aff. ios Warén & Bouchet, 1986 (GQ290586) Provanna ios Warén & Bouchet, 1986 (GQ290585) Provanna n. sp. 2 sensu Johnson et al., 2010 (GQ290578) Provanna shinkaiae Okutani & Fujikura, 2002 (LC095875) 0.61 Provanna aff. pacifica (Dall, 1918) (GQ290588) o.99-—= Provanna beebei Linse, Nye, Copley & Chen, 2019 (MK790057) 0.96 Provanna cooki Linse, Nye, Copley & Chen, 2019 (MK790059) 1 Provanna cingulata Chen, Watanabe & Ohara, 2018 (LC094443) Provanna lucida Sasaki et al., 2016 (AB810182) Provanna macleani Warén & Bouchet, 1989 (GQ290579) Provanna n. sp. 1 sensu Johnson et al., 2010 (GQ290577) 0.95 Abyssochrysos melvilli (Schepman, 1909) (GQ290611) 0.87 Abyssochrysos sp. seusu Johnson et al., 2010 (GQ290609) Cordesia atlantica Souza et al., 2020 (MN850533) Rubyspira goffrediae Johnson et al., 2010 (GQ290574) Rubyspira osteovora Johnson et al., 2010 (GQ290568) Desbruyeresia melanoides Warén & Bouchet, 1993 (GQ290596) 1 0.95 Desbruyeresia chamorrensis Chen, Ogura & Okutani, 2016 (LC090201) Oe Desbruyeresia cancellata Warén & Bouchet, 1993 (LC090203) Desbruyeresia armata Chen, Watanabe & Sasaki, 2019 (MK560875) 0.92 0.95-- Desbruyeresia costata Chen, Watanabe & Sasaki, 2019 (MK560876) 1 Alviniconcha adamantis Johnson et al., 2014 (AB856040) Alviniconcha boucheti Johnson ef al., 2014 (AB235212) Alviniconcha marisindica Okutani in Johnson et al., 2014 (AB235213) 0.91= Alviniconcha hessleri Okutani & Ohta, 1988 (AB235216) 077 ™ Alviniconcha kojimai Johnson et al., 2014 (AB23521 1) Alviniconcha strummeri Johnson et al., 2014 (AB235215) Ifremeria nautilei Bouchet & Warén, 1991 (GQ290603) Cerithium zonatum (Wood, 1828) (MN249983) Bittiolum varium (Pfeiffer, 1840) (MH235831) Echinolittorina vidua (Gould, 1859)(Gould, 1859) (AM157047) Littorina littorea (Linnaeus, 1758) (EU876332) = 0.4 | Provanna Dall, 1908 ABYSSOCHRYSOIDEA 0.91 0.98 Outgroup Figure 4. Bayesian phylogenetic reconstruction of Abyssochrysoidea using a 530-bp alignment of partial COI sequences. Node values are Bayesian posterior probabilities, those less than 0.6 not shown. Scale bar indicates substitutions per site. Discussion The phylogenetic reconstruction herein recovered Provannidae as a paraphyletic clade, agreeing with previous studies (Johnson et al. 2010; Linse et al. 2019; Souza et al. 2020). Abyssochrysidae, as defined in Souza et al. (2020) to contain Abyssochrysos and Cordesia, was recovered as monophyletic. The position of genera in our COI tree generally agreed with the 2-gene tree of Souza et al. (2020), with the exception that the symbiotic genera Alviniconcha and Ifremeria came out as sisters in our tree. Both these trees suggested Provanna as the earliest-branching genus in Abyssochrysoidea, but this differs from the 10-gene (3 mitochondrial and 7 nuclear) tree by Breusing et al. (2020), which suggests that the earliest split in the superfamily is between the symbiotic Alviniconcha—Ifremeria clade and all the rest. This sister-relationship between the two symbiotic genera found by Breusing et al. (2020) was also recovered in our tree. Though the nodes of the Breusing et al. (2020) tree were much better supported, Cordesia was not included in the analyses. ‘The true phylogenetic relationships among abyssochrysoid genera, therefore, require further studies using at least multiple genes New Provanna from NW Eifuku Lad from all seven genera, as well as more outgroup taxa, to include potentially closely re- lated lineages from Cerithoidea and Littorinimorpha. Detailed anatomy at the species and genus level may shed further lights on their evolutionary relationships. Provanna exquisita sp. nov. is a large species for its genus. If the spires of specimens examined were intact it would be similar in size or even exceed that of the largest known Provanna species, P cingulata Chen, Watanabe & Ohara, 2018 from a ser- pentinite-hosted system in Mariana Trench, which is known to reach 16.5 mm with a slightly corroded spire (Chen et al. 2018). Although the Golden Lip site where the ex- amined specimens were recovered from is 40 m away from the centre of venting activ- ity and CO, flux at the Champagne vent (Rossi 2016), it still shares with Champagne a similar acidic environment with pH of 5.78 (Rossi 2016; Rossi and Tunnicliffe 2017). This explains the very corroded spire of P exquisita sp. nov., similar to shell dissolution seen in the co-occurring mussel Bathymodiolus septemdierum (Tunnicliffe et al. 2009) and shows that Provanna is evidently capable of living in such acidic conditions. Nev- ertheless, increased energetic demands associated with high pCO, may have impacted its capacity in shell repair and maintenance. The discovery of Provanna exquisita sp. nov. from the Mariana Arc adds to the diversity of known abyssochrysoid from hydrothermal vents on the IBM Arc. In contrast, on the Izu-Ogasawara (Bonin) Arcs, only two provannid genera, Desbruyeresia and Alviniconcha, have been reported despite considerable sampling efforts (Fujiwara et al. 2013; Chen et al. 2019; Watanabe et al. 2019; Giguére and Tunnicliffe 2021). The three segments of the IBM Arc are separated by the Sofugan Tectonic Line (29°30'N) between Izu and Ogasawara arcs and the conjunction with the West Mariana Ridge (23°N) between Ogasawara and Mariana arcs (Stern et al. 2003). Previous research has shown that the Sofugan Tectonic Line acts as a boundary for faunal subdivision (Watanabe et al. 2019), and it is possible that the conjunction with the West Mariana Ridge also acts similarly, preventing the dispersal of some taxa like Provanna with lecithotrophic development (Warén and Bouchet 1993; Chen et al. 2018). Nevertheless, even on the Mariana Arc, Provanna is so far only recorded from NW Eifuku and nowhere else (Giguére and Tunnicliffe 2021), and it is possible that further exploration of the IBM Arc vents will reveal other populations and species. Despite being one of the better explored regions of the world in terms of hydrothermal vent biodiversity, new discoveries like P exquisita sp. nov. continue to remind us that some of the species diversity remains undocumented at western Pacific vents—despite many endemic species there being threatened from anthropogenic impacts such as deep-sea mining (Thomas et al. 2022). Acknowledgements We thank the captains and crew of R/V “Roger Revelle” and R/V “Thomas G. Thomp- son” for their excellent support of scientific activity during the SROF 2014 expedition (cruise RR1413) and SROF 2004 expedition (cruise TN167), respectively. We extend this thanks to the pilots and technical teams of ROVs JASON I and ROPOS, as well as all on-board scientists. The SROF 2014 expedition was funded by the United States 134 Chong Chen & Hiromi Kayama Watanabe / ZooKeys 1112: 123-137 (2022) National Oceanic and Atmospheric Administration (NOAA) Earth-Oceans Interac- tion Program and the SROF 2004 expedition was funded through the NOAA Ocean Exploration Program, the NOAA Vents Program. Verena Tunnicliffe (University of Victoria) is gratefully acknowledged for collecting the specimens examined and for providing them to us for study. We appreciated helpful comments from Winston Pon- der (The Australian Museum) and Anders Warén (Swedish Museum of Natural His- tory) which improved an earlier version of this paper. CC conceived and designed the study. CC conducted morphological examination and microscopy. HKW carried out DNA sequencing, the molecular data obtained was analysed by CC and HKW. CC interpreted the data and drafted the original manuscript, to which HKW contributed. Both authors approved the submission and agreed to its publication in the present form. Newly obtained COI sequence was deposited in GenBank (accession number ON324570). All specimens used in the present study were deposited in the Museum national dHistoire naturelle (MNHWN), Paris, France (MNHN-IM-2000-37945, MNHN-IM-2000-37946) or the National Museum of Nature and Science, Tsukuba, Japan (NSMT-Mo 79360, NSMT-Mo 79361). References Beaulieu SE, Szafranski K (2020) InterRidge Global Database of Active Submarine Hydrother- mal Vent Fields, Version 3.4. 2022: InterRidge Global Database of Active Submarine Hy- drothermal Vent Fields, Version 3.4. http://vents-data.interridge.org/ [accessed 2022.4.22] Breusing C, Johnson SB, Tunnicliffe V, Clague DA, Vrijenhoek RC, Beinart RA (2020) Allopat- ric and sympatric drivers of speciation in Alviniconcha hydrothermal vent snails. Molecular Biology and Evolution 37(12): 3469-3484. https://doi.org/10.1093/molbev/msaal77 Chen C, Watanabe HK, Ohara Y (2018) A very deep Provanna (Gastropoda: Abyssochrysoidea) discovered from the Shinkai Seep Field, Southern Mariana Forearc. Journal of the Marine Biological Association of the United Kingdom 98(3): 439-447. https://doi.org/10.1017/ S0025315416001648 Chen C, Watanabe HK, Sasaki T (2019) Four new deep-sea provannid snails (Gastropoda: Abyssochrysoidea) discovered from hydrocarbon seep and hydrothermal vents in Japan. Royal Society Open Science 6(7): e190393. https://doi.org/10.1098/rsos. 190393 Corliss JB, Dymond J, Gordon LI, Edmond JM, von Herzen RP, Ballard RD, Green K, Wil- liams D, Bainbridge A, Crane K, van Andel TH (1979) Submarine thermal sprirngs on the Galapagos Rift. Science 203(4385): 1073-1083. https://doi.org/10.1126/sci- ence.203.4385.1073 Embley RW, Baker ET, Butterfield DA, Chadwick Jr WW, Lupton JE, Resing JA, de Ronde CEJ, Nakamura K-i, Tunnicliffe V, Dower JE Merle SG (2007) Exploring the Submarine Ring of Fire: Mariana Arc - Western Pacific. Oceanography 20(4): 68-79. https://doi. org/10.5670/oceanog.2007.07 Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3(5): 294-299. New Provanna from NW Eifuku dS Fujiwara Y, Okutani T, Kimura H (2013) First occurrence of Alviniconcha from Japanese waters (Gastropoda: Provannidae). Venus 71: 217-219. Giguére TN, Tunnicliffe V (2021) Beta diversity differs among hydrothermal vent systems: Implications for conservation. PLoS ONE 16(8): e0256637. https://doi.org/10.1371/ journal.pone.0256637 Johnson S, Warén A, Lee R, Kano Y, Kaim A, Davis A, Strong E, Vrijenhoek R (2010) Rubyspira, new genus and two new species of bone-eating deep-sea snails with an- cient habits. The Biological Bulletin 219(2): 166-177. https://doi.org/10.1086/ BBLv219n2p166 Ke Z, Li R, Chen Y, Chen D, Chen Z, Lian X, Tan Y (2022) A preliminary study of mac- rofaunal communities and their carbon and nitrogen stable isotopes in the Haima cold seeps, South China Sea. Deep-sea Research. Part I, Oceanographic Research Papers 184: e103774. https://doi.org/10.1016/j.dsr.2022.103774 Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16(2): 111-120. https://doi.org/10.1007/BF01731581 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547-1549. https://doi.org/10.1093/molbev/msy096 Lanfear R, Senfeld T, Frandsen PB, Wright AM, Calcott B (2016) PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphologi- cal phylogenetic analyses. Molecular Biology and Evolution 34: 772-773. https://doi. org/10.1093/molbev/msw260 Limén H, Juniper S (2006) Habitat controls on vent food webs at Eifuku Volcano, Mariana Arc. Cahiers de Biologie Marine 47: 449-455. Linse K, Nye V, Copley JT, Chen C (2019) On the systematics and ecology of two new species of Provanna (Gastropoda: Provannidae) from deep-sea hydrothermal vents in the Carib- bean Sea and Southern Ocean. The Journal of Molluscan Studies 85(4): 425-438. https:// doi.org/10.1093/mollus/eyz024 Lupton J, Butterfield D, Lilley M, Evans L, Nakamura K-i, Chadwick Jr W, Resing J, Embley R, Olson E, Proskurowski G, Baker E, de Ronde C, Roe K, Greene R, Le- bon G, Young C (2006) Submarine venting of liquid carbon dioxide on a Mariana Arc volcano. Geochemistry, Geophysics, Geosystems 7(8): Q08007. https://doi. org/10.1029/2005GC001152 Ogura T, Watanabe HK, Chen C, Sasaki T, Kojima S, Ishibashi J-i, Fujikura K (2018) Popula- tion history of deep-sea vent and seep Provanna snails (Mollusca: Abyssochrysoidea) in the northwestern Pacific. Peer] 6: e5673. https://doi.org/10.7717/peerj.5673 Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic Biology 67(5): 901-904. https://doi. org/10.1093/sysbio/syy032 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic infer- ence and model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sysbio/sys029 136 Chong Chen & Hiromi Kayama Watanabe / ZooKeys 1112: 123-137 (2022) Rossi GS (2016) The reproductive and physiological condition of a deep-sea mussel (Bathymo- diolus septemdierum Hashimoto & Okutani, 1994) living in extremely acidic conditions. MSc Thesis, University of Victoria, Victoria, Canada. Rossi GS, Tunnicliffe V (2017) Trade-offs in a high CO, habitat on a subsea volcano: Condi- tion and reproductive features of a bathymodioline mussel. Marine Ecology Progress Series 574: 49-64. https://doi.org/10.3354/meps12196 Sasaki T, Warén A, Kano Y, Okutani T, Fujikura K, Kiel S (2010) Gastropods from recent hot vents and cold seeps: systematics, diversity and life strategies. In: Kiel S (Ed.) Topics in Geobiology 33: the Vent and Seep Biota. Springer Netherlands, Dordrecht, 169-254. https://doi.org/10.1007/978-90-48 1-9572-5_7 Sasaki T, Ogura T, Watanabe HK, Fujikura K (2016) Four new species of Provanna (Gas- tropoda: Provannidae) from vents and a seep off Nansei-shoto area, southwestern Japan. Venus (Tokyo) 74: 1-17. Souza BHM, Passos FD, Shimabukuro M, Sumida PYG (2020) An integrative approach dis- tinguishes three new species of Abyssochrysoidea (Mollusca: Caenogastropoda) associated with organic falls of the deep south-west Atlantic. Zoological Journal of the Linnean Soci- ety 191(3): 748-771. https://doi.org/10.1093/zoolinnean/zlaa059 Stern RJ, Fouch MJ, Klemperer S (2003) An overview of the Izu-Bonin-Mariana subduction factory. Geophysical Monograph 138: 175—222. https://doi.org/10.1029/138GM10 Thomas EA, Bohm M, Pollock C, Chen C, Seddon M, Sigwart JD (2022) Assessing the extinc- tion risk of insular, understudied marine species. Conservation Biology 36(2): e13854. https://doi.org/10.1111/cobi.13854 Tunnicliffe V, Davies KTA, Butterfield DA, Embley RW, Rose JM, Chadwick Jr WW (2009) Survival of mussels in extremely acidic waters on a submarine volcano. Nature Geoscience 2(5): 344-348. https://doi.org/10.1038/nge0500 Warén A, Bouchet P (1986) Four new species of Provanna Dall (Prosobranchia, Cerithiacea?) from East Pacific hydrothermal sites. Zoologica Scripta 15(2): 157-164. https://doi. org/10.1111/j.1463-6409.1986.tb00218.x Warén A, Bouchet P (1993) New records, species, genera, and a new family of gastropods from hydrothermal vents and hydrocarbon seeps. Zoologica Scripta 22(1): 1-90. https://doi. org/10.1111/}.1463-6409.1993.tb00342.x Warén A, Bouchet P (2001) Gastropoda and Monoplacophora from hydrothermal vents and seeps; new taxa and records. The Veliger 44: 116-231. Warén A, Bouchet P (2009) New gastropods from deep-sea hydrocarbon seeps off West Africa. Deep-sea Research. Part II, Topical Studies in Oceanography 56(23): 2326-2349. https:// doi.org/10.1016/j.dsr2.2009.04.013 Warén A, Ponder WF (1991) New species, anatomy, and systematic position of the hydrother- mal vent and hydrocarbon seep gastropod family Provannidae fam. n. (Caenogastropoda). Zoologica Scripta 20(1): 27-56. https://doi.org/10.1111/j.1463-6409.1991.tb00273.x Watanabe HK, Shigeno S, Fujikura K, Matsui T, Kato S, Yamamoto H (2019) Faunal composi- tion of deep-sea hydrothermal vent fields on the Izu—Bonin—Mariana Arc, northwestern Pacific. Deep-sea Research. Part I, Oceanographic Research Papers 149: 103050. https:// doi.org/10.1016/j.dsr.2019.05.010 New Provanna from NW Eifuku 137 Wolff T (2005) Composition and endemism of the deep-sea hydrothermal vent fauna. Cahiers de Biologie Marine 46: 97-104. Xu TJ, Sun J, Chen C, Qian P-Y, Qiu J-W (2016) The mitochondrial genome of the deep-sea snail Provanna sp. (Gastropoda: Provannidae). Mitochondrial DNA. Part A, DNA Map- ping, Sequencing, and Analysis 27(6): 4026-4027. https://doi.org/10.3109/19401736.2 014.1003827 Supplementary material | KML file of the type locality of Provanna exquisita sp. nov. Authors: Chong Chen, Hiromi Kayama Watanabe Data type: Occurence Explanation note: KML file of Golden Lip site, NW Eifuku Volcano, the type locality of Provanna exquisita sp. nov. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1112.85950.suppl1