Zoosyst. Evol. 100 (1) 2024, 111-118 | DOI 10.3897/zse.100.115692 Gp MuseuM TOR BERLIN Molecular characterization and phylogenetic position of the giant deep-sea oyster Neopycnodonte zibrowii Gofas, Salas & Taviani, 2009 Matteo Garzia', Daniele Salvi! 1 Department of Health, Life & Environmental Sciences - University of L’Aquila, Via Vetoio snc, 67100 L’Aquila-Coppito, Italy https://zoobank. org/7DB92668-7DCA-4F 0D-B6AB-8C74C3EC6B89 Corresponding author: Daniele Salvi (danielesalvi.bio@gmail.com) Academic editor: M. Glaubrecht # Received 13 November 2023 Accepted 3 January 2024 ¢ Published 26 January 2024 Abstract The giant deep-sea oyster Neopycnodonte zibrowii Gofas, C. Salas & Taviani, 2009 is a keystone deep-sea habitat builder species. Discovered about fifteen years ago in the Azores, it has been described and assigned to the genus Neopycnodonte Fischer von Waldheim, 1835 based on morphological features. In this study, we generated DNA sequence data for both mitochondrial (COI and 16S) and nuclear (ITS2 and 28S) markers based on the holotype specimen of N. zibrowii to establish a molecular phylogenetic framework for the systematic assessment of this species and to provide a reliable (1.e., holotype-based) reference sequence set for multilocus DNA barcoding approaches. Molecular data provide compelling evidence that the giant deep-sea oyster is a distinct spe- cies, rather than a deep-water ecophenotype of Neopycnodonte cochlear (Poli, 1795), with extremely high genetic divergence from any other gryphaeid. Multilocus phylogenetic analyses place the giant deep-sea oyster within the clade “Neopycnodonte/Pycnodon- te” with closer affinity to NV. cochlear rather than to P. taniguchii Hayami & Kase, 1992, thus supporting its assignment to the genus Neopycnodonte. Relationships within this clade are not well supported because mitochondrial variation is inflated by saturation that eroded phylogenetic signal, implying an old split between taxa within this clade. Finally, the set of reference barcode sequences of N. zibrowii generated in this study will be useful for a wide plethora of barcoding applications in deep-sea biodiversity surveys. Molecular validation of recent records of deep-sea oysters from the Atlantic Ocean and the Mediterranean Sea will be crucial to clarify the distribution of N. zibrowii and assess the phenotypic variation and ecology of this enigmatic species. Key Words Azores, DNA sequences, Gryphaeidae, holotype, molecular systematics, Mollusca, multilocus phylogeny, Natural History Museum Introduction ola et al. 2016; Everett and Park 2018). However, the low number of reference sequences taxonomically validated Deep-sea is the Earth’s largest biome but it is still one of the most underexplored regions (Ramirez-Llodra et al. 2010). Deep-sea biodiversity is mostly unknown due to the extreme environmental conditions that limits sampling capabilities (Rogers et al. 2015; Sinniger et al. 2016; Woodall et al. 2018). Along with advances in ex- ploration technologies (Feng et al. 2022), new molecular technologies such as high-throughput sequencing and the molecular identification of multiple species in environ- mental DNA (eDNA metabarcoding; Taberlet et al. 2012) have boosted deep-sea biodiversity assessments (Guardi- in online repository databases (e.g. GenBank) limits the identifications of MOTUs (Molecular Operational Tax- onomic Unit), thus reducing the taxonomic resolution of eDNA studies (Ruppert et al. 2019). Studies using an integrative taxonomic approach — combining molecular, morphological and environmental data — on new deep- sea taxa have been carried out in several groups of or- ganisms, such as Anthozoa (Lopez-Gonzalez et al. 2022), Mollusca (Xu et al. 2019), and some others (Silva et al. 2016; Blazewicz et al. 2019). However, while molecular data are still not available for a great portion of known Copyright Garzia, M. & Salvi, D. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Ll deep-sea biodiversity, deep-sea exploration has contin- ued, contributing to the discovery of new benthic eco- systems and associated communities. Therefore, there is a great need to constantly improve with reliable reference sequences the taxonomic coverage of deep-sea taxa in re- pository databases. In this study, we focused on a keystone deep-sea hab- itat builder species discovered about fifteen years ago in the Azores Archipelago: the giant deep-sea oyster Neopycnodonte zibrowii Gofas, C. Salas & Taviani, 2009 (Gryphaeidae Vialov, 1936) (Wisshak et al. 2009b). This reef-forming oyster was first observed during a submers- ible dive along the Faial Channel (480-500 meter depth) (Wisshak et al. 2009b). Deep-sea reefs of N. zibrowii are built by both stacked living and dead specimens on ver- tical rocky substrate of seamounts, escarpments and in canyons (Beuck et al. 2016), and host peculiar deep-sea communities. Benthic associations between N. zibrowii and the cyrtocrinid Cyathidium foresti Cherbonnier & Guille, 1972 have been documented in the Atlantic Ocean (Wisshak et al. 2009a), and between N. zibrowii and cold-water corals in both the Atlantic Ocean (Van Rooy et al. 2010) and the Mediterranean Sea (Taviani et al. 2017, 2019). Recently, new records and observations on N. zibrowii in the Atlantic Ocean allowed updating its ecology and distribution (Beuck et al. 2016). The gi- ant deep-sea oyster has been meticulously described in terms of external morphology, microstructures of shell and anatomy (Wisshak et al. 2009b). The systematic placement of this species in the genus Neopycnodonte was based on morphological characteristics such as the circular muscle scar, the enlarged vermiculate chomata (see ‘neopycnodontine chomata’ in Harry 1985) and the vesicular structures in the inner shell layer. Neopycno- donte zibrowii is morphologically different from the only extant congeneric species Neopycnodonte cochlear (Poli, 1795) in several characters such as the shell architecture and outline, the absence of the resilifer bulge in the lat- ter species and the shape and thickness of the vesicular microstructures. On the other hand, 15 years on from its discovery, molecular data are still not available for this species, thus limiting the assessment of its phylogenetic position and systematic placement. The taxonomic assessment of oysters based on mor- phology can be challenging due to a high shell variability and a low number of diagnostic characters (Lam and Mor- ton 2006; Raith et al. 2015; Salvi et al. 2021). Molecular data have a key role in species delimitation and taxonom- ic identification of oyster species (Lam and Morton 2003; Bieler et al. 2004; Kirkendale et al. 2004; Al-Kandari et al. 2021; Salvi et al. 2022) and would provide compelling evidence that the giant deep-sea oyster N. zibrowii is a distinct species rather than a deep-water ecophenotype of N. cochlear (Wisshak et al. 2009b). In this study, we generated DNA sequence data of the giant deep-sea oyster N. zibrowii for both mitochondrial and nuclear markers based on the holotype and performed zse.pensoft.net Garzia, M. & Salvi, D.: Systematics of Neopycnodonte zibrowii a multilocus phylogenetic analyses to establish its rela- tionships with other gryphaeids. The main aims of this study are to provide: (1) a molecular phylogenetic frame- work for the systematic assessment of the giant deep-sea oyster, and (11) a reliable (1.e., holotype-based) reference sequence set for multilocus DNA barcoding approaches. Materials and methods Specimens and sequence data gathering We gathered tissue samples for molecular analyses from museum collections and by field collection. The holotype of N. zibrowii (MNHN-IM-2000-20888) and the speci- men of Hyotissa numisma (Lamarck, 1819) (MNHN- IM-2013-13700) are deposited at the National Museum of Natural History (MNHN) of Paris, while the specimen of Pycnodonte taniguchii Hayami & Kase, 1992 (UF 280382) is preserved in the collection of Florida Muse- um of Natural History (FLMNH). Neopycnodonte co- chlear (OS239) was collected during scuba diving off the coast of Civitavecchia (nearby Rome, Italy) and stored in pure ethanol. Total genomic DNA was extracted from adductor muscles following standard high-salt protocols (Sambrook et al. 1989). We amplified two mitochondri- al — cytochrome oxidase subunit I (COI) and 16S rRNA (16S) — and two nuclear — 28S rRNA (28S) and ITS2 rRNA (ITS2) — gene fragments by polymerase chain re- action (PCR). Primers and conditions used for the am- plification are reported in Table 2. Sequencing of PCR products was carried out by the company Genewiz® (https://www.genewiz.com), using the same primers em- ployed for amplification. Sequences generated from these specimens were complemented with sequences obtained from GenBank for additional gryphaeid species. Locali- ties and GenBank accession numbers of sequences used for molecular analyses are shown in Table 1. GenBank sequences were selected in order to minimise the use of chimeric sequences 1n concatenated alignments (i.e., se- quences of different gene fragments obtained from differ- ent voucher specimens), therefore whenever possible for each species we selected mitochondrial (COI and 16S) and nuclear (28S and ITS2) sequences from the same voucher. Three specimens (Hyotissa hyotis #2, Hyotissa imbricata and N. cochlear #1) have GenBank sequences from different vouchers (chimeric concatenated sequenc- es). We validated the taxonomic identification of each of these vouchers based on single-gene NJ trees. First, we built four single-gene datasets (COI, 16S, 28S and ITS2) including all the sequences of Gryphaeidae species in GenBank and our sequences. Then for each marker we selected GenBank sequences that clustered within the same clade of conspecific vouchers we sequenced (H. hyotis #1 and N. cochlear #1) or that have a congru- ent phylogenetic placement among the four single-gene datasets (H. imbricata) (results not shown). Zoosyst. Evol. 100 (1) 2024, 111-118 tiple} Table 1. Details on the species and DNA sequence data used in this study. Asterisks indicate specimens sequenced in this study. Gen- Bank data are as follows: ': Matsumoto 2003; 7: Matsumoto and Hashimoto unpublished; 3: Kirkendale et al. 2004; +: Plazzi and Pas- samonti 2010; °: Kim et al. 2009; °: Plazzi et al. 2011; 7: Li et al. unpublished; *: Ren et al. 2016; °: Salvi et al. 2014; !°: Ip et al. 2022. Specimen Locality Genbank accession number col 16S 28S ITS2 Hyotissa hyotis #1 Madagascar GQ166583° GQ166564° - - Hyotissa hyotis #2 Singapore (COl); Maldives (16S and ITS2) OM946450!° LM993886° - LM993876° Hyotissa imbricata Japan: Okinawa (COI and ITS2); ABO76917! KC847136’ KC847157’ AB1027582 China: Beibu Bay (16S and 28S) Hyotissa numisma #1 Guam - AY376598* AF137035° - Hyotissa numisma #2 * Papua New Guinea: Rempi Area - PPO70396 PPO70400 - Neopycnodonte cochlear #1 Italy: Mediterranean Sea (COI, 16S and |ITS2) JF496772° JF496758° - LM9938 782 Neopycnodonte cochlear #2 * Italy: Civitavecchia PP069758 PPO70397 PPO70401 PPO74322 Neopycnodonte zibrowii * Azores: Faial Channel PP069759 PPO/70398 PPO/70402 PPO7/4323 Pycnodonte taniguchii #1 Japan: Okinawa AB0O76916! - AB102759 - Pycnodonte taniguchii #2 * Indonesia: Sulawesi Island PP069760 PPO70399 PPO/70403 PP082050 Magallana gigas (outgroup) Japan (COI, 16S and 28S); South Korea (ITS2) KJ8552418 KJ855241® AB1027572 EU072458> Table 2. Primers used in this study: forward primers are listed above and reverse primers below. For the COI and ITS2 gene frag- ments we designed new primers specific to Ostreoidea Rafinesque, 1815, and we used the following PCR cycling conditions: dena- turation step: 94 °C /3 min; 35 cycles of: 94 °C / 60 s, T° annealing (COI: 49 °C; ITS2: 50 °C) / 60 s, 72 °C / 60 s; final extension: 10 min at 72 °C. Gene Primer Sequence Reference Notes COl Moll-F 5’ — ATAATYGGNGGNTTTGGNAAYTG — 3’ This study Dr Zuccon D. (MNHN), pers. comm. osHCO998-R 5’ — ACRGTIGCIGCICTRAARTAAGCICG — 3’ Salvi et al., in prep 16S 16Sar-L 5’ — CGCCTGTTTATCAAAAACAT - 3’ Salvi et al. (2010) 16Sbr-H 5’ - CCGGTCTGAACTCAGATCAC — 3’ 28S D1F-OS 5’ — GAGACTACGCCCTGAACTTAAGCAT — 3’ This study D6R-OS 5’ — GCTATCCTGAGGGAAACYTCAGAGG - 3’ Salvi et al. (2022) ITS2 its3d-OS 5’ - GGGTCGATGAAGARCGCAGC - 3’ This study Modified from Oliverio and Mariottini (2001) its4r-OS 5’ —- CCTAGTTIAGTTTCTTTTCCTGC — 3’ Phylogenetic analyses Newly generated sequences for each marker were used as query in BLAST searches (blastn algorithm) using default settings to evaluate contaminants and to con- firm the identification of the specimens from family to species level. Multiple sequence alignments of each marker were performed with MAFFT v.7 (Katoh et al. 2019) using the G-INS-I iterative refinement algorithm for the COI and the E-INS-i iterative refinement al- gorithm for the rRNA markers. GBlocks (Castresana 2000) was used to remove poorly aligned and ambigu- ous position of the hypervariable regions of the rRNA alignments using a relaxed selection of blocks (Tala- vera and Castresana 2007). Single-gene alignments were concatenated using the software SequenceMatrix (Vaidya et al. 2011). Phylogenetic relationships were inferred using Max- imum Likelihood (ML) and Bayesian Inference (BI) methods. We used the oyster Magallana gigas (Thun- berg, 1793) as outgroup based on previous phyloge- netic studies (Témkin 2010; Plazzi et al. 2011). ML analyses were performed in the W-IQ-TREE web serv- er v.1.6.12 [http://iqtree.cibiv.univie.ac.at/; (Trifino- poulos et al. 2016)] based on a partitioned substitution model. For each gene partition, the best substitution model was calculated by the ModelFinder module (Kalyaanamoorthy et al. 2017) using an edge-linked model and the BIC criterion (COI: TPM2ut+F+G4; 16S: HKY+F+G4; 28S: TN+F+G4; ITS2: K2P+G4). ML analysis was performed with 1,000 pseudo-rep- licates of ultrafast bootstrapping [uBS; (Minh et al. 2013)]. Bayesian analyses (BA) were carried out with MrBayes v.3.2.7 (Ronquist et al. 2012), using the sub- stitution models selected by ModelFinder for each gene partition. We ran two Markov chains of two million generations each, with a sample frequency of 200 gen- erations. Convergence of the runs (ESS values > 200) were checked with Tracer 1.7 (Rambaut et al. 2018) after a burn-in of 25%. Nodal support was estimated as Bayesian posterior probability (BPP). FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualize both ML and BI trees. Genetic divergence between species at each marker (COI, 16S, 28S and ITS2) were calculated using both uncorrected genetic distance (p-distance) and genetic distance corrected under the Kimura 2-paramer model (K2P-distance) using the software Megall and the op- tion “Compute Between Groups Mean Distance” (Tamura et al. 2021). zse.pensoft.net 114 Results BLAST searches using mitochondrial sequences (COI and 16S) of the newly sequenced specimens of H. nu- misma, N. cochlear and P. taniguchii confirmed the taxo- nomic identifications of these species (sequence identity of 99-100%). BLAST searches using the mitochondrial sequences generated from the holotype of N. zibrowii re- covered as best hits sequences belonging to Gryphaeidae species (COI: sequence identity of 73.2%/72.5%/73.1% with GenBank sequences of Hyotissa sp./Neopycnodonte sp./Pycnodonte sp. respectively; 16S: sequence identity of 87.3%/87.5% with GenBank sequences of Hyotissa sp./Neopycnodonte sp. respectively). This confirms the lack of contamination during the amplification and the affiliation of this species to Gryphaeidae. The concatenated dataset included 2409 positions (COI: 455, 16S: 449, 28S: 1078, ITS2: 427 positions) and among the 828 variable positions 436 were phylogeneti- cally informative (1.e., parsimony informative). Maximum likelihood and Bayesian trees show two main clades: one including Hyotissa species (UBS = 95; BPP = 1), and the other one including Pycnodonte and Neopycnodonte species (UBS = 83; BPP = 0.94) (Fig. 1). Neopycnodon- te zibrowii is nested within the second clade with a sister relationship with N. cochlear (uBS = 56; BPP = 0.72), whereas P. taniguchii is sister to Neopycnodonte species. Hyotissa imbricata Hyotissa hyotis #1 Hyotissa hyotis #2 Neopycnodonte cochlear #2 Neopycnodonte zibrowii [HOLOTYPE MNHN-IM-2000-20888] Pycnodonte taniguchii #1 Garzia, M. & Salvi, D.: Systematics of Neopycnodonte zibrowii The COI genetic distances (K2P/p-distance) between N. zibrowii and N. cochlear and between N. zibrowii and P. taniguchii are respectively 35.8%/28.2% and 35%/27.6% (Table 3). The 16S genetic distances (K2P/p- distance) between N. zibrowii and either N. cochlear or P. taniguchii are 13.5%/12.1% (Table 3). The mean in- terspecific genetic distances (K2P/p-distance) among the six gryphaeid species are 33.7% + 4.6% / 26.8% + 3% at the COI and 15.5% + 4.6%/13.7% + 3.7% at the 16S. The 28S genetic distances (K2P/p-distance) between N. zibrowii and N. cochlear and between N. zibrowii and P. taniguchii are respectively 2.5%/2.4% and 9%/8.4% (Table 4). The ITS2 genetic distances (K2P/p-distance) between N. zibrowii and N. cochlear and between N. zi- browii and P. taniguchii are respectively 15.8%/14.9% and 38.2%/29.6% (Table 4). The mean interspecific ge- netic distances (K2P/p-distance) among the six gryphaeid species are 5.5% + 2.5% / 5.1% + 2.2% at the 28S and 27.4% + 16.8% / 22.0% + 11.9% at the ITS2. Discussion Benthic organisms such as oysters, with extensive pheno- typic variation and few diagnostic characters, are prone to misidentification in morphological assessments. The util- ity of molecular characters for taxonomic identification Hyotissa numisma #1 Hyotissa numisma #2 Neopycnodonte cochlear #1 Pycnodonte taniguchii #2 So « _«$._ TTT Magallanaa gigas [Ostreidae] 0.02 Figure 1. Bayesian phylogenetic tree of six Gryphaeidae species based on COI, 16S, 28S and ITS2 markers. Nodal supports indicate the values of uBS (upper) and the BPP (lower). The tree is rooted with Magallana gigas which belongs to the sister family Ostreidae Rafinesque, 1815. Specimens sequenced in this study are highlighted in bold. zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 111-118 lis ke Table 3. Mean genetic distance based on COI (lower triangular matrix) and 16S (upper triangular matrix) DNA sequences, calculat- ed using the K2P model (first value) and uncorrected (p-distance: value inside brackets). The COI and 16S dataset are composed by 2 sequences for each species, except for NV. zibrowii (one sequence for each marker), H. imbricata (one COI and one 16S sequence) and P. taniguchii (one 16S sequence), see Table 1; n. a.: not available. Neopycnodonte Neopycnodonte Pycnodonte Hyotissa hyotis Hyotissa Hyotissa zibrowii cochlear taniguchii numisma imbricata Neopycnodonte zibrowii - 13.5% 13.5% 15.1% 23.5% 14.9% (12.1%) (12.1%) (13.4%) (19.9%) (13.3%) Neopycnodonte cochlear 35.8% - 11.2% 15.5% 22.9% 14.9% (28.2%) (10.3%) (13.8%) (19.5%) (13.4%) Pycnodonte taniguchii 35.0% 35.4% — 14.9% 22.3% 14.2% (27.6%) (28.1%) (13.3%) (19.0%) (12.7%) Hyotissa hyotis 33.3% 39.6% 32.7% - 15.6% 5.3% (26.7%) (30.5%) (26.5%) (13.9%) (5.1%) Hyotissa numisma n. a. n. a. n. a. n. a. - 15.1% (13.6%) Hyotissa imbricata 34.0% 37.0% 31.8% 22.4% n. a. - (27.2%) (28.8%) (25.8%) (19.1%) Table 4. Mean genetic distance based on 28S (lower triangular matrix) and ITS2 (upper triangular matrix) DNA sequences, calcu- lated using the K2P model (first value) and uncorrected (p-distance: value inside brackets). The 28S and ITS2 dataset are composed by 2 sequences for each species, except for N. zibrowii (one sequence for each marker), H. hyotis (one ITS2 sequence and no 28S sequence), N. cochlear (one 28S sequence) and P. taniguchii (one ITS2 sequence), see Table 1. The ITS2 sequence of H. imbricata was not used for genetic distance calculation because it was too short; n. a.: not available. Neopycnodonte Neopycnodonte Pycnodonte Hyotissa hyotis Hyotissa Hyotissa zibrowii cochlear taniguchii numisma imbricata Neopycnodonte zibrowii - 15.8% 38.2% 23.8% n. a. n. a. (14.9%) (29.6%) (20.4%) Neopycnodonte cochlear 2.5% - 43.5% 20.9% n. a. n. a. (2.4%) (33.0%) (19.4%) Pycnodonte taniguchii 9.0% 4.4% - 48.4% n. a. n. a. (8.4%) (4.2%) (35.4%) Hyotissa hyotis n. a. n. a. n. a. - n. a. n. a. Hyotissa numisma 6.8% 4.4% 7.3% n. a. - n. a. (6.5%) (4.2%) (6.9%) Hyotissa imbricata 6.8% 4.6% 7.7% n. a. 1.2% - (6.5%) (4.4%) (6.3%) (1.2%) and systematic assessment of these organisms cannot be overstated and has been proven over and over by studies on true oysters (Lam and Morton 2006; Raith et al. 2015; Salvi et al. 2021), pearl oysters (Cunha et al. 2011), tree oysters (Garzia et al. 2022) as well as gryphaeid oysters (Li et al. 2023). Conchological convergence, phenotypic plasticity, and the occurrence of cryptic species make mo- lecular taxonomic validation of new oyster species nec- essary to accurately estimate the diversity of these taxa. Our molecular phylogenetic results clearly demon- strate that N. zibrowii is a distinct species with extreme- ly high genetic divergence from any other gryphaeid at all the markers analysed (Tables 3, 4). Neopycnodonte zibrowii is nested within the clade “Neopycnodonte/ Pycnodonte” with closer affinity to N. cochlear rather than P. taniguchii (Fig. 1), and thus supporting its as- signment to the genus Neopycnodonte Fischer von Wald- heim, 1835 based on morphological features (Wisshak et al. 2009b). Phylogenetic relationships within this clade are not well-supported, like in a previous phylogenetic study including N. cochlear and P. taniguchii and based on COI and 28S markers (Li et al. 2021). However, the extended dataset of our study improved nodal support and allowed us to clarify the source of phylogenetic uncertainty. Indeed, at both mitochondrial markers, val- ues of pairwise genetic distance between N. cochlear / N. zibrowii / P. taniguchii are similar and remarkably high (COI: 35.0-35.8%; 16S: 11.2-13.5%); whereas at nucle- ar markers the genetic distance between P. taniguchii and N. zibrowii is two-three times higher than between the latter and N. cochlear (Table 4). Such a pattern suggests that mitochondrial variation is inflated by saturation that eroded phylogenetic signal, implying an old split between taxa within this clade. Wisshak et al. (2009b) highlighted a low number of morphological and ecological differenc- es between the genus Neopycnodonte and Pycnodonte Fischer von Waldheim, 1835 and pointed out the need for a systematic revision of the genera. Our results highlight that nuclear data will have a key role in further systematic assessment of these genera. zse.pensoft.net 116 The availability of taxonomically validated reference sequence is a premise for DNA barcoding and metabar- coding approaches for large-scale, fast, and cost-effective molecular taxonomic identification (Hebert et al. 2003; Moritz and Cicero 2004; Schindel and Miller 2005; Salvi et al. 2020). The mitochondrial COI and 16S are the most common markers in DNA barcoding studies on Ostreidae and Gryphaeidae (Lam and Morton 2003, 2004, 2006; Kirkendale et al. 2004; Liu et al. 2011; Hsiao et al. 2016; Salvi et al. 2021). However, also nuclear rRNA mark- ers such as 28S (Mazon-Suastegui et al. 2016) and ITS2 (Salvi et al. 2014; Salvi and Mariottini 2017) have proven useful for molecular taxonomic identification of oysters. Moreover, rRNA markers are frequently selected as tar- get genes in eDNA metabarcoding projects (Ruppert et al. 2019). In this respect, the set of four reference (holo- type-based) barcode sequences of N. zibrowii provided in this study will be useful for a wide plethora of barcoding applications in deep-sea biodiversity surveys. During the last decade, deep-sea oysters from a mounting number of regions across the Atlantic Ocean and the Mediterranean Sea have been morphologically identified as N. zibrowii: from Bay of Biscay (Van Roojj et al. 2010), Gulf of Ca- diz (Gofas et al. 2010), Celtic Sea (Johnson et al. 2013), Angola and Mauritania (Beuck et al. 2016), southern Sar- dinia (Taviani et al. 2017), Sicilian Channel (Rueda et al. 2019) and Gulf of Naples (Taviani et al. 2019). Molec- ular validation of these records will be crucial to clarify the distribution of N. zibrowii and assess the phenotypic variation and ecology of this enigmatic species. Finally, given the high prevalence of cryptic species in oysters, it is not unlikely that future molecular assessments of deep- sea oysters will disclose new species. Acknowledgements The authors wish to thank Philippe Bouchet (Muséum National d’Histoire Naturelle, Paris) and Serge Gofas (Universidad de Malaga) for the access to holotype mate- rial, Paolo Mariottini for the specimen of Neopycnodonte cochlear and to Gustav Paulay (FLMNH) for the speci- mens of Pycnodonte taniguchii. MG 1s supported by the SYNTHESYS Project “Synthesys FR-TAF Call3_040” at The Muséum National d’ Histoire Naturelle (MNHN, Par- is) and the research grant of the Symposium in honour of Philippe Bouchet (World Congress of Malacology 2022 - LMU Munich). This work is part of the PhD thesis of MG under the supervision of DS (“Health and Environmental Sciences” PhD Program, University of L’ Aquila). References Al-Kandari M, Oliver PG, Salvi D (2021) Molecular and morphological systematics of a new, reef forming, cupped oyster from the north- ern Arabian Gulf: Zalonostrea salpinx new species. 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