A New Paralepetopsis Limpet from a South China Sea Seep Hints at a Paraphyletic Neolepetopsidae

© 2023 Academia Sinica, Taiwan Open Access A New Paralepetopsis Limpet from a South China Sea Seep Hints at a Paraphyletic Neolepetopsidae Chong Chen1,* , Zhaoyan Zhong2, Jian-Wen Qiu3, and Jin Sun2 1X-STAR, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2–15 Natsushima, Yokosuka 237–0061, Japan. *Correspondence: E-mail: [email protected] (Chen) 2Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China. E-mail: [email protected] (Zhong); [email protected] (Qiu) 3Department of Biology and Hong Kong Branch of the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong Baptist University, 224 Waterloo Road, Hong Kong. E-mail: [email protected] (Sun) Received 20 June 2022 / Accepted 21 February 2023 / Published 26 May 2023 Communicated by Benny K.K. Chan Neolepetopsidae is a little-studied true limpet family only known from deep-sea chemosynthetic ecosystems, containing just over a dozen species in three genera: Neolepetopsis, Paralepetopsis, and Eulepetopsis. Although considered monophyletic by a recent phylogenetic analysis, a lack of Paralepetopsis sequence linked to morphology casts some uncertainty. Here, we discovered a new species of Paralepetopsis from the Haima methane seep in the South China Sea, described as Paralepetopsis polita sp. nov. The new species is distinct from all other described Paralepetopsis by its smooth and semi-transparent shell, combined with a radula exhibiting pluricuspid teeth with two cusps. We tested its relationship with other neolepetopsids using a molecular phylogeny reconstructed from the mitochondrial COI gene, revealing a surprising position nested within Lepetidae, a family with a very different radula morphology. The clade containing lepetids and our new species was recovered sister to other neolepetopsids with sequence data available. This hints at a paraphyletic Neolepetopsidae, and suggests the neolepetopsid-type radula might not be exclusive to one monophyletic group of limpets. Key words: Cold seep, Deep sea, Gastropoda, Mollusca, Phylogenetics. BACKGROUND The true limpet family Neolepetopsidae is a small, little-studied group containing just 16 species (Chen et al. 2021), and is the only patellogastropod family restricted to chemosynthesis-based ecosystems (McLean 1990 2008; Warén and Bouchet 2001). The family has contained three genera since the original description, including Neolepetopsis, Eulepetopsis, and Paralepetopsis. Neolepetopsis is mostly known from inactive sulfides at the periphery of active vent fields, though there is one species known from whale bones and a record is present from a seep off Peru (Van Dover 2019; Warén and Bouchet 2001; Chen et al. 2021). Paralepetopsis inhabits a wide-range of environments from hydrothermal vents to hydrocarbon seeps to whale bones (Beck 1996; McLean 2008), while Eulepetopsis is only known from active chimneys near hightemperature venting (Warén and Bouchet 2001; Chen et al. 2021). Eulepetopsis is characterised in having a lowprofile shell that is fully transparent due to the lathic calcite composition (McLean 1990); Neolepetopsis and Paralepetopsis are more similar but the shell of Neolepetopsis carries strongly cancellate sculptures (McLean 1990). The most distinctive feature of Neolepetopsidae among Patellogastropoda is the unusual radula with articulate shafts, containing a robust central tooth, two Citation: Chen C, Zhong Z, Qiu JW, Sun J. 2023. A new Paralepetopsis limpet from a South China Sea seep hints at a paraphyletic Neolepetopsidae. Zool Stud 62:26. doi:10.6620/ZS.2023.62-26. Zoological Studies 62:26 (2023) doi:10.6620/ZS.2023.62-26 1 © 2023 Academia Sinica, Taiwan dissimilar inner laterals, a strong and broad pluricuspid formed by the fusing of multiple outer laterals, and two marginal teeth (McLean 1990 2008). The radula is variable in the extent of mineralisation, ranging from barely mineralised (typical for Paralepetopsis) to relatively well-mineralised (more common in Neolepetopsis). The condition of the neolepetopsidtype radula in having a well-developed rachidian tooth is only mirrored in Patellidae among patellogastropod families, but patellids have three pairs of marginals and the teeth are usually strongly mineralised (McLean 1990). This radula type is shared among all three genera and is considered to be an evidence of monophyly for the family (McLean 1990). Nevertheless, some differences are observed among genera, such as the outermost marginal in Eulepetopsis being extremely reduced compared to the other genera and the lateral cusps being typically narrow and sharply pointed in Neolepetopsis but overhanging with broad, blunt cusps in Paralepetopsis (McLean 2008). The phylogenetic position of Neolepetopsidae, its monophyly, and the relationships among the three genera remain unsettled. Earlier work recovered the family as sister to Acmaeidae using a fragment of the 18S rRNA gene (Harasewych and McArthur 2000), whereas later work with better taxon sampling using the mitochondrial cytochrome oxidase c subunit I (COI) gene and the nuclear histone H3 gene recovered it as sister to Lepetidae (Nakano and Sasaki 2011). A recent phylogenetic reconstruction (Chen et al. 2021) was the first to include supposed members from all three neolepetopsid genera, recovering a monophyletic Neolepetopsidae and again a sister relationship to Lepetidae, though not with strong support. However, the two sequences of Paralepetopsis included in these phylogenetic studies lack morphological support (Aktipis and Giribet 2010; Goffredi et al. 2017) and are only provisionally assigned to this genus; whether they truly represent members of Paralepetopsis remains questionable. As such, molecular data from a specimen morphologically confirmed as Paralepetopsis is much needed to confirm the phylogenetic position of that genus. Paralepetopsis currently contains seven described species, the type species being P. floridensis McLean, 1990 from a hydrocarbon seep in Florida Escarpment (McLean 1990). Two further seep species are known, including Paralepetopsis lepichoni Warén & Bouchet, 2001 from Nankai Trough, Japan and Paralepetopsis sasakii Warén & Bouchet, 2009 from a West African seep. Three vent species have been described, including Paralepetopsis rosemariae Beck, 1996 from the Edison Seamount in the western Pacific, Paralepetopsis ferrugivora Warén & Bouchet, 2001 from Mid-Atlantic Ridge, and Paralepetopsis tunnicliffae McLean, 2008 from Juan de Fuca Ridge. The last species, Paralepetopsis clementensis McLean, 2008, was found on a whale fall off California. Haima seep is a cold seep located on the northwestern slope of the South China Sea, with a lush chemosynthetic ecosystem fuelled by rich deposits of methane hydrates (Chen et al. 2018; Feng et al. 2018). During a research cruise to the Haima seep, a hitherto undescribed species of Paralepetopsis was found living on the shells of the large vesicomyid clam Archivesica marissinica Chen et al., 2018. Here, we describe and characterise this new species, and test the monophyly of Neolepetopsidae using a molecular phylogeny with the first reliable genetic data from Paralepetopsis. MATERIALS AND METHODS Sample Collection Specimens of limpets were collected with several individuals of the vesicomyid clam Archivesica marissinica Chen et al., 2018 in September 2020 from the Haima cold seep (16°41.6561'N, 110°23.8165'E, 1361 m depth; Fig. 1) by the remotely operated vehicle (ROV) Haima II using a scoop-net, on-board R/V Haiyang #6. The clams were immediately placed in a -80°C deep-freezer after the ROV returned on deck. Later in the shore-based laboratory, limpets were removed from the frozen clams and placed into 99% ethanol before further investigation. Morphology Limpet specimens were observed and dissected under a stereo dissecting microscope (Olympus SZX9), with shell lengths (SL), shell widths (SW), and shell heights (SH) measured using digital callipers to the nearest one decimal point. Soft parts were carefully removed from its shell using tungsten needles and rehydrated in a graded ethanol-MilliQ water series (99%, 90%, 70%, 50%, 30%, 10%, 0% ethanol) for one hour each and left to sit in MilliQ water overnight prior to observation. Detailed anatomical investigation was not possible due to the poor preservation of tissue. Specimens investigated herein were deposited into either the Tropical Marine Biodiversity Collections of the South China Sea, Chinese Academy of Sciences, Guangzhou, China (TMBC) or the National Museum of Nature and Science, Tsukuba, Japan (NSMT). For scanning electron microscopy (SEM) of the radula, fine forceps and tungsten needles were used to dissect the radula ribbon from a limpet, which was then page 2 of 11Zoological Studies 62:26 (2023) © 2023 Academia Sinica, Taiwan Fig. 1. The Haima methane seep in the South China Sea. A: Map showing the location of Haima seep southeast off Hainan Island. B: Photograph of the type locality showing Paralepetopsis polita sp. nov. (arrows) on the shells of the vesicomyid clam Archivesica marissinica. page 3 of 11Zoological Studies 62:26 (2023) © 2023 Academia Sinica, Taiwan placed in diluted commercial bleach (1:10 dilution) for cleaning. As neolepetopsid radulae are prone to disintegration (McLean 1990), the radula was monitored using the dissecting microscope and immediately moved into MilliQ water upon sufficient cleaning. The radula was washed in fresh MilliQ water and again in 99% ethanol before being mounted onto a SEM stub using carbon tape. To observe the surface sculpture of the shell, the shell of a limpet was placed into the same diluted commercial bleach after removing the soft parts, cleaned briefly using soft brushes, then washed twice in MilliQ water and twice in 99% ethanol before being mounted onto SEM stubs using carbon tape. The SEM stubs were observed using a Hitachi TM-3000 table-top SEM uncoated at 15 kV. Genetics Two limpet specimens were used for DNA sequencing. Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA) following manufacturer’s protocols. The barcoding fragment of the mitochondrial COI gene was amplified and sequenced using the universal primer pair HCO2198–LCO1490 (Folmer et al. 1994), using methods as reported in previous studies (Zhou et al. 2018; Chen et al. 2021). Geneious R11 (https://www.geneious.com) was used to assemble the consensus sequence for each limpet individual, prior to downstream analyses. New sequences generated herein have been deposited into NCBI GenBank under the accession numbers ON810774–ON810775. Bayesian inference was used for phylogenetic reconstruction as implemented in the software MrBayes v3.2.6 (Ronquist et al. 2012), using a 472 bp alignment of the COI gene following a previous study (Chen et al. 2021). A selection of patellogastropod COI sequences was downloaded from GenBank, including representatives of all eight currently recognised families: Acmaeidae, Eoacmaeidae, Lepetidae, Lottiidae, Nacellidae, Neolepetopsidae, Patellidae, and Pectinodontidae (Nakano and Ozawa 2007; Nakano and Sasaki 2011; Chen et al. 2021). Two vetigastropod sequences from Fissurellidae were selected as the outgroup, following Nakano and Ozawa (2007) and Chen et al. (2021). These sequences were aligned in Geneious R11. As the third codon position of COI was suggested to be problematic for the group from a previous comprehensive phylogenetic analysis of Patellogastropoda (Nakano and Ozawa 2007), this position was removed from downstream analyses. The most suitable substitution model was found to be GTR+I+G for both the first and second codon positions using the PartitionFinder v.2.1.1 software (Lanfear et al. 2017). In MrBayes, Metropolis-coupled Monte Carlo Markov chains were run for one million generations with topologies sampled every 100 generations. Tracer v1.6 (Rambaut et al. 2013) was used to monitor and select a suitable burn-in, determined as 3000 in our case. RESULTS SYSTEMATICS Subclass Patellogastropoda Lindberg, 1986 Family Neolepetopsidae McLean, 1990 Genus Paralepetopsis McLean, 1990 Type species: Paralepetopsis floridensis McLean, 1990 (by original designation). Paralepetopsis polita sp. nov. (Figs. 2–4) urn:lsid:zoobank.org:act:B202762C-E2B1-42E1-86447B7DFCC556BF Type locality: On the shell of Archivesica marissinica, Haima methane seep (16°41.6561'N, 110°23.8165'E, 1361 m depth) in the South China Sea (Fig. 1B). Type material: Holotype (TMBC031004; Fig. 2A); SL 7.9 mm, SW 5.6 mm, SH 2.4 mm; a piece of mantle removed for DNA sequencing. Paratype 1 (NSMT Mo79367; Fig. 2B); SL 7.0 mm, SW 4.7 mm, SH 2.8 mm. Paratype 2 (NSMT Mo-79368; Fig. 2C); SL 6.4 mm, SW 4.3 mm, SH 2.3 mm. Paratype 3 (NSMT Mo79369; Fig. 2D); SL 5.6 mm, SW 4.0 mm, SH 2.0 mm. Paratype 4 (TMBC031005; Fig. 3); SL 5.7 mm, SW 4.0 mm, SH 1.6 mm; a piece of foot used for DNA sequencing, shell and radula used for SEM. All collected from the type locality, taken using a scoop-net by ROV Haima II in September 2020 on-board R/V Haiyang #6 during cruise HYDZ6-202005. They were originally frozen in -80°C while still attached to Archivesica clams and later transferred into 99% ethanol. Etymology: ‘Politus’ (Latin), polished or smooth, named for its smooth teleoconch surface devoid of major concentric or radial sculpture. Diagnosis: A medium-sized Paralepetopsis (SL up to 7.9 mm) with semi-transparent shell lacking any significant sculpture except very fine concentric growth lines, apex located on the mid-line at the anterior third of the shell. Central tooth with finely pointed cusp, other teeth with quadrangular cusps. All teeth exhibiting sturdy, well-developed shaft; each teeth significantly descend in position relative to the central tooth. page 4 of 11Zoological Studies 62:26 (2023) © 2023 Academia Sinica, Taiwan Description: Shell (Figs. 2, 3A–B) thin, semitransparent, with a very thin layer of transparent periostracum where not corroded. Maximum shell length among specimens available 7.9 mm (holotype). Elliptic in outline, narrower at anterior end than posterior end, especially obvious in large specimens (Fig. 2A). Apex on midline, located at approximately one-third from anterior edge. Apex strongly corroded, inner surface of protoconch sealed. Protoconch unknown. Shell profile moderately high, margin aligned along one plane in specimens examined. Anterior margin straight to concave (Fig. 2A–B), posterior margin convex. Shell surface nearly completely smooth except very fine concentric growth lines. Muscle scars indistinct. Radula (Fig. 3C–D) with two laterals, pluricuspid tooth, two marginals on either side of rachidian, no clear evidence of significant mineralisation. Each tooth from rachidian outwards descent significantly in position, each outer tooth diverging posteriorly, resulting in teeth on each row aligned like inverted ‘V’. Rachidian narrow but sturdy, well-developed, with finely pointed cusp. Shaft of rachidian slender with narrow lateral ridge on each side, narrower than cusp where they connect. First inners lateral twice as broad as rachidian, with broad, overhanging, truncated quadrangular cutting edge. Shaft of first inner lateral sigmoidal in outline on outer side. Second inner lateral similar but with strong lateral ridge near base to accommodate base of first inner lateral. Pluricuspid robust, twice as broad as inner laterals, with one very broad, overhanging rounded inner cusp plus one small, narrow, poorly formed outer cusp. Shaft with lateral mid-shaft projection from where shaft tapers towards base. Inner marginals with narrow shaft, broadening apically to form rounded, spoonlike overhanging cusps. Shaft of inner marginals most Fig. 2. Paralepetopsis polita sp. nov.. A: Holotype (TMBC031004). B: Paratype 1 (NSMT Mo-79367). C: Paratype 2 (NSMT Mo-79368). D: Paratype 3 (NSMT Mo-79369). Scale bars = 2 mm. page 5 of 11 Zoological Studies 62:26 (2023) © 2023 Academia Sinica, Taiwan narrow at centre, with lateral projection near base. Outer marginals two-thirds as long as inner marginals, with broader shafts. Cusps spoon-like, less developed than inner marginals. Soft parts (Fig. 4). Cephalic tentacles simple, conical, tapered, lacking appendages. Eyes appear lacking from external examination. Oral disc with muscular outer lip, labial lobe poorly developed. Clear groove separates oral disc from foot. Foot sole oval, large, with unciliated rim, epipodium lacking. Shell muscle U-shaped, comprising numerous muscle bundles along posterior third of body, length of bundles increases posteriorly. Mantle edge with numerous papillae, presumably sensory. Mantle cavity extending to just over one-third of body length. Gonad located along mid-body ventrally posterior or pericardium, partly visible through mantle roof. Ctenidium lacking. Intestine and stomach embedded within digestive glad which extends extensively as seen through mantle roof. Intestine loops twice prior to emerging as rectum. Anus located on right side of mantle roof, just left of urogenital papillae. Right kidney sizeable, at posterior end near shell muscles. Distribution: Only known from the Haima methane seep area in the South China Sea. Remarks: The shell lacking cancellate sculpture, as well as a radula with broad, overhanging lateral cusps lacking noticeable mineralisation places P. polita sp. nov. in Paralepetopsis rather than Neolepetopsis. Two features together separate P. polita sp. nov. from all other described Paralepetopsis species, including the semi-transparent shell lacking any significant sculpture and the pluricuspid tooth with two cusps. Although species such as P. tunnicliffae and P. rosemariae also exhibit little shell sculpture, they still show weak radial striae (Beck 1996; McLean 2008) which are lacking in P. polita sp. nov. Paralepetopsis clementensis is the only described congener lacking radial sculpture, Fig. 3. Paralepetopsis polita sp. nov., scanning electron micrographs of paratype 4 (TMBC031005). A: Apex with arrows indicating the region lost by corrosion. B: Shell surface. C–D: Radula. Scale bars: A = 200 μm, B: 1 mm, C–D = 50 μm. page 6 of 11Zoological Studies 62:26 (2023) © 2023 Academia Sinica, Taiwan but that species has a clearly convex anterior margin (McLean 2008) as opposed to straight to concave in P. polita sp. nov. The only other neolepetopsid exhibiting a pluricuspid with two clearly separated cusps is Neolepetopsis gordensis McLean, 1990 which also has a much larger, major inner cusp and a smaller outer cusp (McLean 1990 2008). Although the cutting edge of the pluricuspid tooth in P. ferrugivora also exhibits two to three blunt tubercles, they are not separated out to form individual cusps (Warén and Bouchet 2001). A key to the described Paralepetopsis species, with information on the known distribution of each, is shown in table 1. Molecular Phylogeny We used the first and second codon positions of the COI gene to reconstruct the phylogeny of Patellogastropoda and to assess the systematic position of Paralepetopsis polita sp. nov., with the consensus tree shown in figure 5. In our tree, the two sequences of P. polita sp. nov. included (from the holotype and paratype 4) were recovered together with full support (Bayesian posterior probability, BPP = 1). They were, however, not clustered with other sequences assigned to Neolepetopsidae which formed a weakly supported clade (BPP = 0.6) – including two described species of Neolepetopsis and two described species of Eulepetopsis, as well as two sequences of undescribed preliminarily assigned to Paralepetopsis but without published data on their morphology (Aktipis and Giribet 2010; Goffredi et al. 2017). Within this clade, the two Neolepetopsis species formed a strongly supported clade (BPP = 0.98), as did the two Eulepetopsis species (BPP = 0.97); these two genera were recovered as Fig. 4. Paralepetopsis polita sp. nov. (paratype 1, NSMT Mo-79367), external anatomy. Left: dorsal view after removing the shell, right: ventral view. Abbreviations: a, anus; dg, digestive gland; f, foot; gr, groove separating the foot’s sole from peripheral flange; i, intestine; ll, labial lappet; m, mouth; me, mantle edge; ol, outer lip of the mouth; pc, pericardium; r, rectum; rk, right kidney; s, stomach; sm, shell muscles; sp, sensory papillae; t, cephalic tentacle; te, testis; up, urinogenital papilla. page 7 of 11Zoological Studies 62:26 (2023) © 2023 Academia Sinica, Taiwan Fig. 5. Reconstructed consensus tree of Patellogastropoda using Bayesian inference, based on 472 bp of the mitochondrial COI gene. Node values indicate Bayesian posterior probabilities, only values > 0.6 are shown. GenBank accession numbers of the sequences are indicated in parentheses the species names. Table 1. Key to the described Paralepetopsis species, including information on their distribution 1 a Shell with radial sculpture Go to 2 bShell lacking any significant sculpture Go to 7 2 a Radial sculpture consisting of fine striation Go to 3 b Radial sculpture consisting of strong ribs Go to 4 3 a Rachidian and laterals with triangular cusps P. floridensis (Atlantic, Florida Escarpment, 3270 m, seep) b Rachidian and laterals with square cusps P. rosemariae (Pacific, Edison Seamount, 1483 m, vent) 4 a Shell surface with raised nodules where ribs cross Go to 5 b Shell surface lacking raised nodules Go to 6 5 a Rachidian and laterals with triangular cusps P. lepichoni (Pacific, Nankai Trough, 2140 m, seep) b Rachidian and laterals with round cusps P. sasakii (Atlantic, West Africa off Congo River, 3150 m, seep) 6 a Shafts of marginals well-developed P. ferrugivora (Atlantic, Mid-Atlantic Ridge, 1665–1728 m, vent) bShafts of marginals not differentiated from base P. tunnicliffae (Pacific, Juan de Fuca Ridge, 2145 m, vent) 7 a Anterior slope of shell convex P. clementensis (Pacific, Off California, 1800 m, whale fall) b Anterior slope of shell straight to concave P. polita sp. nov. (Pacific, South China Sea, 1361 m, seep) page 8 of 11Zoological Studies 62:26 (2023) © 2023 Academia Sinica, Taiwan sisters with weak support (BPP = 0.43). This clade was in turn sister to a moderately supported clade (BPP = 0.66) containing the two undescribed species assigned to Paralepetopsis, but only with moderate support (BPP = 0.66). Surprisingly, we found P. polita sp. nov. nested within two sequences of species in Lepetidae, where it was sister to Lepeta caeca (Müller, 1776) with moderate support (BPP = 0.63). These two species were in turn found to be closely related to Lepeta kuragiensis (Yokoyama, 1920) with strong support (BPP = 0.93). The clade containing these three species was found to be sister of the clade containing the abovementioned six neolepetopsid species, a relationship that was moderately supported (BPP = 0.65). The other six currently accepted patellogastropod families were all recovered as monophyletic with various levels of support (BPP = 0.69–1), the relationships among which were the same as a previously published phylogeny by Chen et al. (2021). DISCUSSION The phylogenetic position of Paralepetopsis polita sp. nov. recovered in our phylogenetic analysis (Fig. 5) is surprising, and suggests a paraphyletic Neolepetopsidae where Paralepetopsis is paraphyletic, with P. polita sp. nov. nested within species currently assigned to Lepetidae with strong support. Though P. polita sp. nov. appeared to nest within Lepeta, that genus has been suggested to be paraphyletic (Nakano and Sasaki 2011). The phylogenetic position of P. polita sp. nov. is in strong contrast to the morphology where P. polita sp. nov. is clearly assignable to Neolepetopsidae on the basis of the characteristic radula type (McLean 1990 2008). Lepetidae is a strictly subtidal family, representing another little-studied deep-water group of true limpets. Lepetids, including Lepeta, are characterised by a very different radula type lacking the rachidian and with fused inner laterals as well as the loss of outer laterals, resulting in a narrow radula with only two laterals and two marginals being present (McLean 1990; Nakano and Ozawa 2007). This is strikingly different from the radula of P. polita sp. nov. Considering that Neolepetopsidae and Lepetidae have been repeatedly recovered as sister families in molecular phylogenies (Nakano and Sasaki 2011; Chen et al. 2021), a condition which we also recovered in our tree, it appears likely that the two are indeed the most closely related groups among living patellogastropods. Since the articulating teeth and the presence of radula are considered to be plesiomorphic characters of Patellogastropoda (Fretter 1990), it seems likely that the shared ancestor of Neolepetopsidae and Lepetidae possessed a neolepetopsid-type radula. If this is the case, perhaps Paralepetopsis represents a lineage of Lepetidae retaining the plesiomorphic radula type, and in this scenario Neolepetopsidae, as currently considered, would indeed be paraphyletic. Another possibility is that the Paralepetopsislike radula may be present in two evolutionarily distinct lineages, one in Neolepetopsidae and one in Lepetidae. This is suggested by the two sequences of ‘Paralepetopsis’ in Aktipis and Giribet (2010) and Goffredi et al. (2017), which clustered with other neolepetopsids, unlike P. pollita sp. nov. – but as these sequences are not linked to described species or morphological data it is unclear whether they truly are Paralepetopsis. Testing these hypotheses would require sequences from other described neolepetopsid species and especially P. floridensis, the type species of Paralepetopsis. If P. floridensis is clustered with other neolepetopsids, then P. polita sp. nov. may actually represent a new genus of Lepetidae with an unusual radula. Either way, our present results strongly suggest the clade containing Neolepetopsidae and Lepetidae requires revision. Lacking genetic data to test the relationship with P. floridensis, we retain P. polita sp. nov. in Paralepetopsis for the time being. An improved phylogenetic reconstruction of Neolepetopsidae, using multiple genetic markers and including data from the type species of each genus, is warranted in the future. In addition to both being deep-water clades, Neolepetopsidae and Lepetidae also share a number of characters such as the reduction of mantle tentacles, the lack of eyes, and interestingly the absence of basal plate in the radula (McLean 1990; Nakano and Ozawa 2007). Unfortunately, the specimens of P. polita sp. nov. available herein were first frozen and then transferred to high-grade ethanol, rendering the tissue unsuitable for detailed examination of fine internal anatomy. Future collections of fresh individuals fixed in a manner suitable for anatomical investigations, such as formalin or glutaraldehyde fixation, would be very useful in clarifying the systematic position of P. polita sp. nov. from an anatomical point of view. CONCLUSIONS A new limpet discovered from the Haima methane seep in the South China Sea morphologically matched the true limpet genus Paralepetopsis in the family Neolepetopsidae and is described as P. polita sp. nov. Phylogenetic reconstruction of Patellogastropoda including the new species revealed a surprising position where it was nested within Lepetidae instead page 9 of 11Zoological Studies 62:26 (2023)