Glass scallop genome reveals key adaptations to deep-sea environments and ectosymbiosis

Domain: physics
Published: 2026-04-01
Editorial outcome: minor_revision

Abstract
Chemosynthetic symbioses enable many deep-sea animals to flourish, yet the genomic basis of ectosymbiosis in deep-sea bivalves is poorly understood. We present a chromosome-level genome for the glass scallop Catillopecten margaritatus, the only scallop known to host sulphur-oxidising bacteria on its gills. The genome comprises a conserved set of 19 chromosomes shared with true scallops, and evolutionary analyses place the lineage split in the Early Devonian, predating the establishment of ectosymbiosis. Integrating genome, gene-expression, and shell chemistry data, we identify adaptations to deep-sea life and symbiosis: loss of vision, enhanced mantle sensing, reduced shell calcification, immune mechanisms that recognise and accommodate symbionts, robust sulphide detoxification, and host provisioning of metabolites to the bacteria. The species also retains predatory feeding, indicating mixotrophy. These results clarify how this species colonised chemosynthetic habitats, broaden the spectrum of symbiotic strategies in bivalves, and provide a genomic framework for testing transitions from asymbiosis to symbiosis.

Introduction
Hydrothermal vents and hydrocarbon seeps are extreme deep-sea environments characterised by perpetual darkness, high hydrostatic pressure, elevated concentrations of toxic compounds, and a limited input of photosynthetically derived organic matter from surface waters1,2. Despite these harsh conditions, they harbour diverse communities of specialised organisms, such as siboglinid tubeworms, vesicomyid clams, bathymodiolin mussels, and alviniconch snails2–4. These invertebrates form symbioses with chemoautotrophic bacteria that oxidise reducing chemicals (e.g., hydrogen sulphide, methane) in vent and seep fluids to fuel primary production that sustains their hosts5–7. Studies of endosymbiotic associations in tubeworms, snails, mussels, and clams have greatly advanced understanding of holobiont (host-symbiont) evolution and adaptation7–11. However, ectosymbiotic systems have been overlooked at the genomic level, limiting insights into the biology of many marine invertebrate taxa and obscuring our understanding of the evolutionary transition from asymbiosis to symbiosis12–15.

The superfamily Pectinoidea, which comprises four families, Pectinidae (common scallops), Propeanmussidae (glass scallops), Spondylidae (thorny oysters), and Cyclochlamydidae (cyclochlamydids)16,17, epitomises this research void. Genomic studies have predominantly focused on shallow-water and commercially relevant species from the family Pectinidae18,19, leaving a gap in our understanding of the evolutionary trajectories and adaptive mechanisms of pectinoids. The recent discovery of the deep-sea glass scallop Catillopecten margaritatus (Propeamussiidae) at a hydrocarbon seep (Supplementary Fig. 1) offers a critical opportunity to address this disparity. This species represents the only documented pectinoid to establish chemosynthesis, hosting sulphur-oxidising bacteria on its gill surfaces20. Analogous to ectosymbiotic wood- and bone-associated bathymodiolin mussels (e.g., Idas washingtonia and Adipicola iwaotakii), which are proposed evolutionary intermediates between shallow-water ancestors and specialised deep-sea vent/seep-endemic lineages21-23, C. margaritatus and its ectosymbiont provide a model to investigate the mechanisms of deep-sea colonisation by shallow-water species and a comparative framework to explore the various spectrum of symbiotic strategies from ectosymbiosis to endosymbiosis in Bivalvia24-26. While genomic analyses of its bacterial partner have revealed substantial nutritional contributions to the host20, the corresponding adaptations of C. margaritatus, including genomic innovations enabling deep-sea survival and symbiont maintenance, remain unresolved.

Here, we report a high-quality, chromosomal-level genome assembly of C. margaritatus, employ comparative genomic analyses with Pectinidae species in a phylogenetic context, and utilise multi-tissue transcriptomic data to unravel metabolic interdependencies between the host and symbiont, while shell elemental profiling is conducted to contrast biomineralisation strategies with shallow-water scallops. By integrating these approaches, we elucidate the adaptive mechanisms underpinning C. margaritatus’ survival in deep-sea chemosynthetic environments and its role in ecto-chemosymbiosis, focusing on sensory system evolution (e.g., photoreception loss, enhanced environmental sensing), biomineralisation trade-offs, immune mechanisms facilitating symbiosis establishment, sulphide detoxification pathways, and nutritional reciprocity. Our study advances understanding of how symbiotic plasticity enables bivalves to thrive in diverse ecological niches and provides resources for studies of their genome evolution.

Results
High-quality genome assembly
The assembly of PacBio HiFi sequences produced a draft genome of C. margaritatus, consisting of 1,173 contigs with a total length of 1.1 Gb and an N50 of 1.6 Mb (Supplementary Note 1 & Supplementary Data 1-2). Incorporation of the Hi-C data resulted in a final assembly with 19 chromosomes (Fig. 1a, Supplementary Fig. 2, & Supplementary Data 2). Compared to shallow-water scallops, this deep-sea species has a similar genome size and identical chromosome number (Supplementary Data 3)19. The assembly has 25,153 protein-coding genes; among them, 98.88% were annotated with at least one hit against public databases (Supplementary Fig. 2 & Supplementary Data 2-4). Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment27 revealed 97.3% and 98.3% completion for the genome assembly and annotation, respectively (Supplementary Data 2-3), indicating the assembly is of high quality. The repeat content is high, accounting for 50.64% of the assembly. The transposable elements (TEs) load in C. margaritatus is similar to that of Placopecten magellanicus but is larger than that of other shallow-water Pectinidae. Specifically, the DNA transposons (DNAs) in the deep-sea scallop account for 91.82 Mbp, while those in other species range from 22.36 to 52.06 Mbp (Supplementary Fig. 3 & Supplementary Data 5). A comparison of the estimated insertion times for four TE classes among the scallops reveals that the C. margaritatus genome has had a similar TE insertion history to that of Chlamys farreri and P. magellanicus. Notably, DNA transposons have maintained consistently high levels of insertion activity since 150 million years ago, peaking around 60 to 40 million years ago (Supplementary Note 1 & Supplementary Fig. 3).

Conserved karyotype and extensive intrachromosomal variation
The Hox and ParaHox gene clusters are developmental regulators that determine the anterior-posterior body axis in bilaterians28. The C. margaritatus genome includes complete Hox and ParaHox clusters, featuring the same gene order as that of the inferred lophotrochozoan ancestor and other Bivalvia, including deep-sea species (Supplementary Fig. 4)8,29,30. Chromosome-scale macrosynteny based on ancestral gene families shows that C. margaritatus, M. yessoensis, and Pecten maximus preserve nearly all 20 molluscan linkage groups (MLGs)31,32, with a fusion of MLG16 and MLG12, and the conservation indices (CI 0.96–0.97) significantly higher than those of other bivalves (Fig. 1b & Supplementary Fig. 7). Consistent with the widely conserved 19-chromosome scallop karyotype31,32, the chromosome of C. margaritatus corresponds one-to-one with those of M. yessoensis, indicating minimal inter-chromosomal rearrangement and a karyotype largely inherited from the scallop ancestor (Fig. 1c). In contrast, intra-chromosomal synteny analysis reveals extensive structural variation across most chromosomes between C. margaritatus and Pectinidae scallops and within Pectinidae (Fig. 1d), underscoring dynamic genome reorganisation. Such variation may reflect the high transposable-element content in C. margaritatus, particularly DNA transposons (Supplementary Fig. 3 & Supplementary Data 5)30, or the distant phylogenetic relationships between the Propeamussiidae and Pectinidae families20,33. Additionally, chromosomal rearrangements can drive adaptation by suppressing allelic recombination in locally adapted gene complexes, disrupting reading frames to create gene functions, and modifying gene expression34-36, potentially contributing to traits linked to deep-sea ectosymbiosis. Distinguishing phylogenetic divergence from adaptive change will require comparative genomes across multiple propeamussiids occupying different habitats.

Phylogenomic relationship and divergent time
A phylogenetic tree constructed from 391 single-copy orthogroups of 29 molluscs, along with an annelid and a brachiopod as outgroups, reveals a topology that aligns with previous phylogenomic studies of Mollusca, with C. margaritatus as the sister to Pectinidae and Ctenoides ales as the sister to Pectinida (Fig. 2, Supplementary Fig. 8, & Supplementary Data 6)30,37. Calibration of the tree using fossils and geological events (Supplementary Data 7) indicates that the common ancestor of Propeamussiidae and Pectinidae diverged around 410.6 million years ago (MYA) during the Early Devonian (Fig. 2a). The oldest fossil records of the Propeamussiidae were found in the Middle Triassic in 242.0–237.0 MYA, while an occurrence date of Carboniferous was calibrated using the age of Pectinidae based on the fossil of Praechlamys16,38,39. Our calibration using the Givetian fossil records of Pectinidae indicated that the ancestor of Propeamussiidae might have emerged earlier than previously thought16. Additionally, most extant Propeamussiidae species are found in deep water40, and fossils of several Parvamussium species from the Dolomites in northern Italy and the Montagne des Voirons in southeastern France suggested that propeamussiids were already adapted to life at greater water depths during the Early Cretaceous41,42. During the Late Jurassic, the sea level generally rose, reaching a peak of approximately 150 meters above the current level in the late Kimmeridgian and early Tithonian intervals43. This increase in sea level may have prompted propeamussiids to colonise deeper waters before the Early Cretaceous, but more evidence is required. Previous research has classified the propeamussiid Catillopecten vulcani from deep-sea vents as asymbiotic based on the absence of specialised morphological features in its flat and homorhabdic gill20,44. Therefore, the ancestor of Catillopecten was pre-adapted to the deep-sea environment before C. margaritatus established extracellular symbiosis with thiotrophic bacteria in seep habitats. However, the time of establishing the symbiosis cannot be determined based on current data. Nevertheless, the same hypothesis has been proposed for the deep-sea Vesicomyidae and shallow-water Veneridae, whose ancestors were inferred to have invaded the deep sea around 128 MYA, predating the divergence between non-symbiotic vesicomyids and symbiotic pliocardiines, which occurred later than 48 MYA based on fossil records30,45.

Genomic restructuring for aphotic and sensory adaptation
Eye loss in pteriomorphians is associated with lineages that transitioned to burrowing lifestyles or deep-sea habitats46. Five distinct types of mantle photoreceptor organs have evolved independently in pteriomorphians: pigmented cups or compound eyes in Arcida, cap eyespots in Ostreida, invaginated eyes in Limida, and concave mirror eyes in Pectinida16,46,47. Among Pectinida, concave mirror eyes originated once in the common ancestor of Pectinoidea and are retained in all Pectinidae and Spondylidae species, as well as select Propeamussiidae such as Cyclopecten hoskynsi, Similipecten nanus, and Parvamussium pourtalesianum. In contrast, most propeamussiids (e.g., Propeamussium spp.) lack eyes entirely46. The retention of eyes in the three Propeamussiidae species may reflect their broad habitat ranging from intertidal zones to abyssal depths and reliance on photoreception in shallow waters48,49. Conversely, Propeamussium and Catillopecten species are restricted to depths of around 300-6000 m, where light is very dim to absent50,51. Our analysis of C. margaritatus revealed no eyes but identified elongated tentacles (Fig. 3a-b), suggesting a shift from visual to sensing as an adaptation to perpetual deep-sea darkness. In the shallow-water pectinids, eye morphogenesis and function are mainly regulated by the paired box 6 (pax6)-independent pathway, and their genomes encode 18 opsin and 5-6 cryptochrome genes for photoreception29,47. We found that C. margaritatus genome encodes both pax6-independent and pax6-dependent pathways52, as evidenced by the retention of key transcription factors and regulatory genes involved in eye morphogenesis, including paired box (pax6 and pax2/5/8), homeobox protein (six1/2, six3/6, and six4/5), POU domain transcription factor 3 (brn3), LIM homeobox transcription factor 1-beta-like (lmx1b), eyes absent (eya), dachshund (dach), rentinal homeobox (rx), orthodenticle-related homeobox (otx1 and otx2), and microphthalmia-associated transcription factor (mitf) (Fig. 3c & Supplementary Data 8). However, most opsin and cryptochrome genes were absent, with only a melanopsin, a rhodopsin, and two cryptochrome-II (CRY-II) genes retained (Fig. 3c & Supplementary Data 8). Expression pattern analysis revealed that most of the retained genes did not show specifically high expression levels in the mantle tissues containing the mantle edge compared to other tissues, except for brn3, dach, and lmx1b, which also were reported to perform pleiotropic roles beyond visual function53-55 (Fig. 3b-c). Overall, these results suggest that C. margaritatus lost its ability for photoreception as an adaptation to constant darkness in the deep sea46,56.

Genome-wide gene family expansion screening (Fig. 2, Supplementary Fig. 9, & Supplementary Data 9) of C. margaritatus identified two lectin gene families, including ten ficolin and 20 galactose-binding lectin (gal-lectin) genes. Subsequent transcriptomic analysis demonstrated that these expanded gene families show tissue-specific expression, with markedly elevated transcription levels in the mantle (Fig. 4). Ficolin and gal-lectin function as pattern-recognition molecules that identify carbohydrate structures on pathogens, playing a crucial role in bacterial adhesion and phagocytosis, which are important for immune response57,58. Our Gene Ontology (GO) functional enrichment analysis of positively selected genes identified that the biological processes related to stimulus responses were positively selected in C. margaritatus compared to shallow-water scallops (Supplementary Data 10-12). Additionally, transcriptomic analysis of the mantle tissue indicated that C. margaritatus allocates more highly expressed genes than M. yessoensis to signalling and responding to biotic and abiotic stimuli, environmental stress, and substances (Fig. 3d & Supplementary Fig. 10-11). These results suggest a refined sensory capacity in C. margaritatus59. In conclusion, the high expression of environmental response genes and the expanded ficolin and gal-lectin genes suggest that the mantle tissue serves as a primarily sensory and defensive organ for C. margaritatus. This unique adaptation may have helped the deep-sea scallop to thrive in its extreme habitat.

Reduced calcification and shell environmental responses
Biomineralisation by the mantle underpins bivalve shell formation60. Our gene expression profiling showed that, relative to other tissues, the mantle is enriched for GO terms linked to calcium metabolism and biomineralisation, including regulation of calcium transport, regulation of biomineralisation, inorganic ion transportation, and biomineral tissue development (Supplementary Fig. 12). To explore functional implications, we analysed shell chemistry by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and identified 11 isotopes with distinct compositional patterns (Fig. 3e & Supplementary Fig. 13). Calcium dominated all examined shells, consistent with other scallops61, yet C. margaritatus had significantly lower calcium concentrations than M. yessoensis (Fig. 3e & Supplementary Fig. 13). Moreover, the relative abundances of Ca44 and Ca43 were markedly reduced in the right valve of C. margaritatus (Supplementary Fig. 13), indicating a lower overall calcium incorporation in this valve compared to the left, consistent with its thinner structure compared to the left valve62. In the deep sea, calcium carbonate saturation declines with depth because low temperature and high pressure reduce carbonate ion availability, increasing the energetic cost of calcification and risking partial dissolution of existing shell63,64. The thin, fragile, and transparent shells of C. margaritatus contrast with the thick, robust, and opaque shells of M. yessoensis, and we hypothesize that C. margaritatus may reduce shell calcification as a response to deep-sea calcium carbonate saturation state and an energy-conserving strategy65,66. Additionally, zinc (Zn) and boron (B) concentrations are comparable between the two species, with higher Zn but lower B in the right valves (Fig. 3e & Supplementary Fig. 13). Zinc was reported to play a role in the formation of shells by supporting the function of carbonic anhydrase, which promotes the precipitation of calcium carbonate, while the absorption of B is related to the acidity67,68. The discrepancy of Zn and B abundances among the left and right valves of scallops might be driven by their different environmental exposure69. Furthermore, C. margaritatus shells display lower Sr/Ca ratios than M. yessoensis (Fig. 3e & Supplementary Fig. 13). Despite elevated strontium levels in the Haima seep due to pore water effusion, Sr/Ca ratios are primarily governed by stable environmental factors like temperature, widely serving as a reliable temperature proxy in organisms such as corals and sclerosponges70,71. Additionally, shell Sr/Ca ratios were found to co-vary significantly with calcification temperature in shallow-water scallop Pecten maximus72. Therefore, the lower Sr/Ca ratios in C. margaritatus shells likely reflect the cold temperature in the Haima seep. Notably, shells of C. margaritatus exhibit significantly higher levels of iron (Fe), manganese (Mn), magnesium (Mg), barium (Ba), chromium (Cr), and copper (Cu) compared to M. yessoensis, with the right valve, directly contacting the substrate, showing elevated Fe, Mn, Ba, Cr, and Cu than the left (Fig. 3e & Supplementary Fig. 13). Deep-sea vents and seeps are rich in these heavy metals73,74, and the enrichment of these elements in C. margaritatus shells likely mirrors the metal-rich conditions of the hydrocarbon seep, serving as potential biomarkers for studying seep fluid elemental dynamics75. Collectively, reduced calcification underscores the evolutionary adaptations to the deep sea of C. margaritatus, while distinct elemental compositions highlight the intimate correlations between environmental conditions and shell formation.

Innate immune system-mediated symbiotic establishment
The ectosymbionts of C. margaritatus are horizontally transmitted, so each host generation acquires bacteria from the environment containing diverse microbes, making precise symbiont recognition and calibrated immune responses essential for establishing ectosymbiosis while avoiding pathogens20,76,77. Weighted correlation network analysis (WGCNA) identified three gill-positive correlated modules in C. margaritatus (Fig. 5a & Supplementary Fig. 14-15) enriched for immune and defence functions. By contrast, gill-positive modules in the asymbiotic M. yessoensis are dominated by transport and intracellular organelle functions (Fig. 5b & Supplementary Data 13-14), indicating that C. margaritatus gills uniquely engage with their microbiome. Pattern recognition receptors (PRRs) mediate immune recognition and response and often show elevated expression in chemosymbiotic invertebrate tissues78,79. Among PRRs, peptidoglycan recognition proteins (PGRPs) and Toll-like receptors (TLRs) can influence symbiont establishment, including binding to bacterial peptidoglycan8. Additionally, C-type lectins (C-lectins) support immune recognition and phagocytosis in deep-sea invertebrates80. Moreover, C-type lectins help ectosymbiotic mussels and shrimps recognise and attach to ectosymbionts, acting as scaffolds for PRRs81,82. In C. margaritatus, we identified five PGRP genes, 12 TLR genes (TLR1, TLR2, and TLR13), and one C-lectin gene that are positively correlated with, and highly expressed in, gills relative to other tissues (Fig. 5b), consistent with roles in symbiont management. Furthermore, C. margaritatus shows expansion of immune response gene families linked to symbiosis (Fig. 4 & Supplementary Data 8-9). Notably, the expanded gene endothelin-converting enzyme (ECE), which belongs to the neprilysin family, is involved in various biological functions83. This gene family is known to modulate the response of immune effector cells in mussels and immunity against bacterial, fungal, and protozoan infections in insects84,85. We found that the four copies of ECE genes are specifically highly expressed in the gill tissue of C. margaritatus (Fig. 4d). Additionally, mucin-3A-like protein (MUC3A) genes are also expanded (Fig. 4a), though their expression levels are specifically associated with all examined tissues. MUC3A is part of a group of membrane-bound glycoproteins that form epithelial protective barriers and contribute to antibacterial defence, suggesting its potential role in shaping host-symbiont interactions86,87. Together, the presence and gill-specific expression of pathogen-defence genes, coupled with expansions of innate immune families, point to adaptations that support the glass scallop’s ectosymbiotic lifestyle. Nevertheless, studies in other symbiotic organisms have highlighted complex interactions between host immunity and symbiont establishment88,89. Therefore, future work should test how specific gene family expansions, like ECE and MUC3A, modulate colonisation and long‑term symbiont stability in the glass scallop.

Sulphide detoxification mitigates thiotrophic negative effects
The ectosymbionts of C. margaritatus depend on thiotrophy for energy, utilizing thiosulfate and hydrogen sulphide through rhodanese-like proteins, a highly expressed sulphur-oxidizing (SOX) multienzyme complex, and a reverse dissimilatory sulphite reduction (rDSR) pathway20. However, hydrogen sulphide is highly toxic because it binds tightly to cytochrome c oxidase, disrupting electron transport and aerobic ATP production90. To mitigate sulphide toxicity, animals employ various detoxification strategies. For instance, thiotrophic clams from Vesicomyidae, Lucinidae, and Solemyidae produce sulphur-binding proteins in their blood or haemolymph to transport sulphide safely from the environment to the symbionts91,92. Some seep vestimentiferan tubeworms and the gutless clam Solemya reidi have developed specialised organelles known as sulphide-oxidising bodies in their integument or gill cells92,93. Additionally, vestimentiferans and bathymodiolines eliminate sulphide through sulphide-oxidising pathways or by the symbionts’ accumulation of elemental sulphur87,89,94. In the genome of C. margaritatus, we identified genes involved in sulphur oxidisation, including eukaryotic sulphide quinone oxidoreductase (SQOR), sulphur dioxygenase (ETHE1), and sulphite oxidase (SUOX). These proteins catalyse the conversion of hydrogen sulphide to sulphite and sulphate, along with thiosulfate/3-mercaptopyruvate sulfurtransferase (TST) for thiosulfate oxidisation and hydrogen sulphide detoxification95,96. Notably, our positive selection analysis indicated that the gene of SQOR oxidising sulphide was positively selected in C. margaritatus compared to shallow-water scallops, with a substitution of N270L in the FadH2 domain, which is also seen in thiotrophic clams Archivesica marissinica and Conchocele bisecta (Fig. 2b). This substitution occurs in the FAD binding site of the Rossmann fold, potentially positively affecting the enzyme’s kinetics, although further evidence is needed95,97,98. Analysis of gene expression revealed that SQOR is highly expressed in the gill tissues of C. margaritatus, ranking in the top 5.2% of all genes in the gills, compared to the top 12.8% in M. yessoensis gills (Supplementary Data 16). Both ETHE1 and TST showed higher expression levels in C. margaritatus gills than in M. yessoensis gills (Supplementary Data 16), suggesting that detoxification through sulphur-oxidisation is active. We found that glutathione S-transferase (GST), gamma-glutamyltranspeptidase (GGT), and glutathione peroxidase (GPx) genes were among the gill-positive correlated genes in C. margaritatus (Fig. 4a & 5c). Beyond their defensive role as antioxidants, these genes are believed to facilitate sulphide detoxification as their product, glutathione, can bind to persulfate produced during sulphide oxidisation99,100. Previous studies have shown high accumulations of thiotaurine and hypotaurine in tubeworms, clams, snails, and mussels from vent and seep environments, linked to sulphide contraction101,102. A proposed mechanism for thiotaurine synthesis involves the use of hypotaurine and sulfhydryl groups from glutathione-SSH, aiding in sulphide transport within symbiotic tissues101. We identified expansions in the C. margaritatus genome for the neurotransmitter transporter of taurine (SLC6A6) and chondroitin 4-sulfotransferase (CHST11) genes, with eight and five copies, respectively (Fig. 4a). Taurine transporters are known to transport thiotaurine, hypotaurine, and taurine into cells in chemosymbiotic mussels, while CHST11 catalyses the conversion of sulphate to organic sulphur for the detoxification in vent shrimp and squat lobsters103,104. Additionally, reduced sulphur released from thiotaurine may serve as an energy source for thioautotrophic symbionts105. Overall, the processes of sulphur oxidisation, glutathione metabolism, thiotaurine synthesis, and related transporters likely constitute a comprehensive detoxification system that alleviates the negative effects of thiotrophy.

Host’s nourishing and harvesting of symbionts
A previous study suggested that the ectosymbionts of C. margaritatus cannot synthesise the TCA-cycle intermediates oxaloacetate, fumarate, and succinate, which are likely supplied by the host20. Our positive selection analysis supports and extends this hypothesis20 by identifying five host genes involved in producing these intermediates exhibit strong signals of positively selection, including ATP citrate (pro-S)-lyase (ACLY), pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PCK), succinyl-CoA synthetase (LSC), and succinate dehydrogenase (SDH), with substitutions in their functional domains (Fig. 2c-d). Selection on these key enzymes may reflect evolutionary tuning of the TCA cycle to enhance metabolic flux toward oxaloacetate, succinate, and fumarate for export to ectosymbionts. However, these genes do not show strong tissue-bias expression, and therefore, the direct biochemical measurements of enzyme activities are needed to test this hypothesis. Additionally, chemosynthetic symbionts often lack complete biosynthetic repertoires, and most cannot produce all required nutrients89,106. Symbiont locations can influence the nutrient exchange mechanisms: in endosymbiosis, where symbionts reside within host cells, metabolites are commonly transferred across a symbiont-containing vacuolar membrane via symbiont-encoded transporters13, whereas extracellular symbionts may require transfer across both the host apical membrane and the symbiont cell membrane, often through more complex and context-dependent pathways77. Prior work indicated that ectosymbionts associated with C. margaritatus cannot synthesise cysteine and threonine, and gill transcriptomes suggested host delivery via exosomes20. Consistent with this, we found an expansion of the solute carrier family 7 L-type amino acid transporter member 8 (SLC7A8) to seven copies (Fig. 4a). This Na+‐independent transporter mediates the efflux of neutral amino acids across membranes107,108. Our genomic analysis further indicates that the host can synthesise the neutral amino acids cysteine and threonine (Fig. 6a & Supplementary Data 17), supporting a model in which gill epithelial cells export these amino acids to ectosymbionts.

Hosts can also acquire nutrients from their chemosymbionts through a variety of mechanisms that vary across different symbiotic systems. For intracellular associations, reported pathways include host cell apoptosis109 and phagolysosomal digestion110,111 of siboglinid tubeworms, and release of metabolites via outer membrane vesicles in the flatworm Paracatenula112. For extracellular symbioses, nutrient transfer may occur via active secretion7, diffusion of small molecules113, host endocytosis and digestion114, or “milking” strategies as seen in stilbonematine nematodes115. In C. margaritatus, pathways related to endosome, lysosome, and vesicle show signals of positive selection (Supplementary Data 11), including genes for endosome/lysosome assembly and recycling and proteases such as cathepsins (CTSB, CTSL), and legumain (Supplementary Data 12). Our WGCNA analysis revealed gill-specific co-expression of genes involved in vesicles, endocytosis, lysosomes, and endosomes, while these patterns are not observed in asymbiotic M. yessoensis (Fig. 5, Supplementary Fig. 15, & Supplementary Data 13-14). We also found 12 lysosome cathepsin genes (CTSB, CTSC, CTSD, CTSF, CTSL, CTSO, and CTSZ) with gill-specific high expression in C. margaritatus, whereas these enzymes are not highly expressed in M. yessoensis gills relative to its other tissues (Fig. 6d & Supplementary Fig. 15-17). Cathepsins are known to mediate intracellular digestion of symbionts in multiple systems13,30,77. Given that the ectosymbiont lacks of most amino acid transporters and host gill endocytosis has been observed20, we propose that C. margaritatus harvests ectosymbionts via endocytosis followed by intracellular digestion to obtain energy and nutrients, particularly the seven amino acids and five vitamins it cannot synthesise (Fig. 6a). This host-regulated harvesting strategy, mirrors convergent solutions in other chemosynthetic animals, such as bathymodioline mussels and vestimentiferan tubeworms, which digest endosymbionts to supplement nutrition77,110,111, underscoring the effectiveness of this mechanism for thriving in nutrient-poor deep-sea environments.

Mixotrophy supports the holobiont
An examination of the holobiont’s nutrient biosynthesis pathways revealed that neither C. margaritatus nor its ectosymbiont can produce ubiquinone (Fig. 6a & Supplementary Data 17), and the symbiont’s nitrogen assimilation pathway is incomplete, indicating a reliance on additional nutrient sources. These results are consistent with the observation that members of deep-sea Propeamussiidae adopt predation targeting meiobenthos, epibenthic planktonic larvae, and crustaceans, with the prey captured by their lips transported into a buccal pouch and stuffed into the mouth by the foot116,117. A metagenomic study of the digestive gland showed that C. margaritatus might use the small debris of marine molluscs and fishes as the primary food sources, including some deep-sea organisms like bathymodiolines (Fig. 6b-c). These results indicated it may retain predatory traits to supplement the nutrients apart from chemosymbionts, but it is important to note that the results were generated from one sample only. Furthermore, several host’s duplicated genes were highly expressed in the digestive glands compared to other tissues, including 13 copies of DBH-like monooxygenase (EDBH), seven of transmembrane protein 79 (TMEM79), eight of cytochrome P450 family 2 subfamily D (CYP2D), five of cytochrome P450 family 4 subfamily B1 (CYP4B1), and five of glutathione S-transferase (GST) (Fig. 4). These genes play crucial roles in responding to oxidative stress and are involved in mucosal repair within the digestive system118–121. Additionally, some immune-related genes showed specifically high expression in the digestive glands (Fig. 5c). Together, these results indicate that the digestive system is active in the deep-sea glass scallop and that mechanisms for combating oxidative stress play a crucial role. The digestive glands use different cathepsins than the gill, with six copies of the CTSL gene tandemly duplicated and specifically highly expressed in the digestive gland tissues (Fig. 6d & Supplementary Fig. 17). While most cathepsins are lysosomal proteases, CTSL can be secreted from cells and digest food extracellularly122,123. Secreted CTSL also mediates microenvironment and inflammatory responses124,125. The fact that the gills and the digestive gland tissues express different cathepsins suggests distinct functions and substrates, with the specific expression of CTSL in the digestive gland indicating active extracellular digestion and heterotrophy. Mixotrophy is common in symbiotic organisms. Unlike obligate chemosymbiotic tubeworms and clams, which have reduced digestive systems9,106,126, chemosynthetic bathymodiolines, especially those hosting ectosymbionts, retain their functional digestive systems and heterotrophic capabilities25,127. Therefore, mixotrophy may enable C. margaritatus to avoid over-reliance on sulphur compounds and seek an optimal environment, enhancing its adaptability and survival in the deep sea. Further studies investigating stable carbon and nitrogen isotopes would be needed to shed light on the relative contribution of symbiont- and food-derived resources to C. margaritatus nutrition and the interdependence of this unique symbiosis.

In summary, we present the chromosome-level genome of the deep-sea glass scallop Catillopecten margaritatus, with the elucidation of its evolutionary trajectory and adaptations to the deep sea and chemosymbiosis, uncovering intricate host-ectosymbiont interactions. The genomic resource generated in this study advances our understanding of the diversity of symbioses in Bivalvia and their transitions from shallow waters to the deep sea, and establishes a foundation for comparative studies on genome evolution.

Methods
Sample collection
All samples used in this study were collected with appropriate permits and all procedures used complied with institutional and national guidelines. Four C. margaritatus individuals were collected from the Haima seep (16°54.04’N, 110°28.47’E) in the South China Sea during a an ROV (Pioneer) dive from the R/V Xiangyanghong 01 at 1,433–1,441 m depth in September 202220 (Supplementary Fig. 1). Specimens were dissected onboard. Shells were preserved at -20 °C for laser-induced breakdown spectroscopy (LIBS; results presented in this Supplementary Information),and the laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Tissues were stored at −80 °C for DNA and RNA extractions.

Genomic sequencing
DNA extraction. Genomic DNA for Illumina (non-Hi-C) and PacBio HiFi sequencing was extracted from mantle tissue and digestive gland (stomach and gut; Illumina sequencing only) using the CTAB method128. DNA quality was assessed by 1.0% agarose gel electrophoresis and quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA).
Genome survey and metagenomics. To estimate genome size and assemble the mitochondrial genome (mitogenome), a 350 bp insert library was prepared from mantle DNA using the TruSeq Nano DNA HT Sample Prep Kit (Illumina, USA) and sequenced on an Illumina NovaSeq 6000 sequencer (Novogene, China), yielding 64.88 Gb of 150 bp paired-end raw data (432.52 million reads; Supplementary Data 1). To investigate the potential diet, a metagenomic library (350 bp insert) from digestive gland DNA was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, USA) and sequenced on a NovaSeq 6000 sequencer (Novogene, China), yielding 10.63 Gb of raw data (70.89 million reads; Supplementary Data 1).
PacBio HiFi sequencing. A PacBio HiFi library from mantle DNA was constructed with the SMRTbell Express Template Prep Kit 2.0 and sequenced on a PacBio Revio system (Novogene, China) for one SMRT Cell, targeting ~30× depth based on the Illumina-derived genome size estimate (Supplementary Fig. 2). Sequencing produced 31.73 Gb of raw data (2.09 million single reads; read N50 = 16,623 bp; Supplementary Data 1).
High-throughput chromosome conformation capture (Hi-C) sequencing. Hi-C data were generated to scaffold the draft assembly to the chromosomal level. After removing gills and shells, the remaining tissues of a C. margaritus individual were used to construct a 350 bp insert library with the Truseq Nano DNA HT Sample Preparation Kit (Illumina, USA). The library was sequenced on an Illumina NovaSeq 6000 sequencer (Illumina, USA) to produce 150-bp paired-end reads, yielding 235.43 Gb of raw data with 784.78 million reads (Supplementary Data 1).

RNA sequencing
Total RNA was extracted from six tissues (adductor muscle, digestive gland, foot, gill, mantle, and gonad) of three individuals using Trizol (TAKARA, Japan). RNA quality and quantity were examined as for DNA. Ribosomal RNA was removed using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), RNA was fragmented to 250-300 bp and reverse-transcribed to cDNA, and libraries were sequenced on an Illumina NovaSeq 6000 sequencer (Illumina, USA) to produce 150 bp paired reads. On average, about 11 Gb of raw data with 79 million reads per sample were generated (Supplementary Data 1).

Draft genome, mitogenome, and metagenomic assembly
Genome size estimation and draft genomic assembly. Illumina raw reads (Supplementary Data 1) were trimmed using Trimmomatic v0.39129 (quality score < 30, length < 40 bp). Clean data were used to generate a 21-mer histogram using Jellyfish v2.2.028130, and GenomeScope v2.0131 was used to estimate the genome size. Genomic ploidy was predicted using Smudgeplot v0.2.5131 based on a k-mer of 21. PacBio HiFi reads (Supplementary Data 1) were assembled de novo with HiFiasm v0.18.9132 (coverage = 32). Putative alternative heterozygous contigs were removed using Purge_Haplotigs v1.1.3133 (l = 5, m = 70, h = 180) based on a read-depth histogram. Assembly statistics were evaluated with QUAST v5.2.0134, and completeness (scaffold-level assembly) was assessed with Compleasm v0.2.6135 against Metazoa_odb10 (Supplementary Data 3).
Mitogenome assembly. Two million clean Illumina reads from mantle tissue were used to assemble the mitogenome using NOVOPlasty v3.2136, using C. margaritatus cox1 and mitogenome (GenBank OQ434059.1) as the seed and reference, respectively. Annotation was performed with MITOS2 v2.1.9137, and manually curated using GenBank references and the invertebrate mitogenomic codon.
Metagenomic assembly, binning, and annotation. Adaptors and low-quality reads from the metagenomic sequencing of the digestive gland were removed using Trimmomatic v0.39129 (LEADING:15, TRAILING:15, SLIDINGWINDOW:4:20, MINLEN:40). Clean reads were assembled with SPAdes v3.14.1138 in metagenomic mode (k = 21, 33, 55, 77, 99, 127). Scallop contigs were removed via BLASTx v2.11.0139 (99% identity) against a custom database of Pectinidae genomes and our C. margaritatus assembly (Supplementary Data 4). Remaining contigs were taxonomically annotated using BLASTx v2.11.0139 against the NT database ( E-value 1e-10; best hit retained). Assembled contigs from the digestive gland were also used to recover the bacterial genomes using MetaWRAP v1.2.1140 with “Binning”, “Bin refinement”, and “Reassemble bins” modules, generating a gammaproteobacterial bin and an alphaproteobacterial bin. Clean reads were mapped to reassembled bins to calculate the relative abundance of each bin using SAMtools v1.11141. Bin completeness and contamination were assessed with CheckM2 v1.0.1142. Bacterial genomes were annotated using Prokka v1.14.6143 and functionally annotated using EggNOG-MAPPER v5.0144. Average nucleotide identity (ANI) between the Gammaproteobacteria bin and the gill ectosymbiont was measured using the JSpeciesWS Online Server v4.1.1145 under the default settings.

Hi-C analysis and chromosome assembly
Hi-C reads (Supplementary Data 1) were trimmed using Trimmomatic v0.39129 (quality score < 30, length < 40 bp), processed using HiC-Pro v2.10146 to identify high-quality reads, and duplicates were removed using Juicer v1.5147. Filtering generated 155.4 million valid reads (112.1 Gb). Scaffolding was performed using the 3D de novo assembly pipeline148 under a haploid genome model. Nineteen pseudo-chromosomal linkage groups were inspected and corrected in Juicebox v1.11.08149 to ensure Hi-C contact consistency. Assembly completeness (chromosomal-level) was assessed with Compleasm v0.2.6135 against Metazoa_odb10. QUAST v5.2.0150 was used to obtain assembly statistics.

Gene model prediction and functional annotation
The final genome was soft-masked with RepeatMasker v4.1.2 (http://www.repeatmasker.org/) using RepBase v20181026151 and species-specific repeats built using RepeatModeler v2.0.3152. MAKER v3.0153 was applied to predict gene models, informed by transcriptomic and protein evidence. The RNA sequencing reads were trimmed (quality score < 30, length < 40 bp) with Trimmomatic v0.39129. Clean reads from all tissues were pooled for de novo assembly with Trinity v2.8.5154. Genome-guided assembly was performed with Trinity, with HISAT v2.1.0155 to align RNA data to the genome. The two transcriptomes were merged using the PASA pipeline v2.2.0156. Protein evidence comprised sequences from 28 molluscs (Supplementary Data 4). Augustus v3.1157 was used for de novo gene prediction on the repeat-masked genome. EVidenceModeler v1.1.1158 was used to integrate predictions (Augustus and Maker). PASA v2.2.0156 was used to refine models and add UTR annotations. Gene models were filtered using gFACs v1.1.2159 (min-exon-size 20, min-intron-size 20, min-CDS-size 150, rem-5prime-3prime-incompletes, rem-all-incompletes, unique-genes-only, in-frame stop codons not allowed). Completeness was assessed with Compleasm v0.2.6135 ( MetazoaS_odb10; protein model). Functionally annotation using EggNOG-MAPPER v5.0144 (default settings) and BLASTP of Diamond v0.9.24160 against custom (Supplementary Data 4) and NR databases (E-value of 1e-10).

Phylogenomic analysis and time estimation
Orthologous groups (OGs) across 31 genomes, including one annelid, one brachiopod, and 29 molluscs (Supplementary Data 4), were inferred using OrthoFinder v2.5.5161 (“more-sensitive”, E-value threshold 1e-10). Single-copy OGs with ≥80% taxonomic representation (25 species) in OGs, including 391 single-copy OGs, were used. Protein sequences were aligned using MAFFT v7.520162 under the “auto” option and in “normal alignment” mode. Gblocks v0.91b163 was applied to remove ambiguously aligned regions, with missing protein or alignment gaps filled with “-”. Alignments with 104,936 distinct alignment patterns, including missing sequences, were concatenated. Maximum-likelihood (ML) analyses were conducted using IQ-TREE v2.1.3164 with the MFP option for model selection and 1,000,000 ultrafast bootstraps. Divergence times, based in ML tree, were estimated with MCMCtree implemented in PAML v4.9h165, using fossil records or geological events to calibrate30 (Supplementary Data 7). The LG model was employed per partition. Burn-in was 1,000,000, sampling every 1,000 for 10,000 samples, over 10,000,000 MCMC generations.

Insertion times of transposable elements
Insertion time estimation followed a previous study79. Briefly, lineage-specific nucleotide substitution rates were estimated with the free-ratio model in codeml (PAML v4.8165) using codon alignments from the 391 OGs. Protein sequences from the phylogenomic analysis were converted to codon alignments using pal2nal.pl166 and concatenated and used as input for codeml under seqtype = 1 to estimate lineage-specific substitution rates. For transposable elements (TEs) dating, divergences from consensus sequences were extracted from RepeatMasker outputs and corrected for multiple substitutions using the Jukes-Cantor formula: K = − 300/4 × Ln (1 − D × 4/300), where D is the distance between the fragments and consensus30. Insertion times (T) were calculated as T = K/2r167, where r is the lineage-specific nucleotide substitution rate.

Macro- and microsynteny analysis
Macrosynteny comparisons among the 20 presumed molluscan linkage groups (MLGs)32, M. yessoensis, C. margaritatus, and other selected bivalves were made with PanSyn under default settings31. Microsynteny analysis between C. margaritatus, shallow-water scallops, and Ctenoides ales was conducted using MCScanX168 and visualised using NGenomeSyn v1.41169 under default settings.

Gene family expansion, contraction, and functional convergence
CAFÉ v4.2.1170 was used to identify gene family expansions and contractions based on gene families (OGs) determined by OrthoFinder v2.5.5161 during the phylogenomic analysis. Gene families for each species were filtered to remove those with > 100 genes in any species. The filtered gene count Data and the ultrametric tree of 31 species served as input for CAFÉ, with parameters “load -p 0.05 -t 10 -r 1000 -filter” and -t parameter in the lambda command set to 1. GO and KEGG enrichment of expanded and contracted gene families was performed using TBtools v2.112171 against the embedded go-basic.obo and KEGG Backend databases, with a false discovery rate (FADR) < 0.05 considered significantly enriched.
R package constellatoR172 was used to detect functional groups amongst genes exclusively (compared with asymbiotic organisms) expanded or contracted in four deep-sea symbiotic bivalve lineages: Lucinida (Conchocele bisecta), Venerida (Archivesica marissinica), Pectinida (C. margaritatus), and Mytilida (Gigantidas platifrons, Bathymodiolus brooksi, and B. septemdierum). Exclusively expanded and contracted OGs in each lineage were extracted, and a pairwise similarity matrix of each OG was calculated based on the GO annotations using GOSemSim v.3.16173 under the Wang semantic similarity metric174. The Affinity Propagation cluster algorithm was applied to group OGs based on semantic similarity175. Multidimensional scaling was used to reduce dimensionality and produce constellation plots172.

Positive selection analysis
We used Orthofinder v2.5.2161 (parameters: -S blast -M msa -l 1.5) to infer gene orthogroups for six species, including Catillopecten margaritatus, Patinopecten yessoensis, Placopecten magellanicus, Amusium japonicum, Amusium pleuronectes, and Pecten maximus, retaining the longest isoform per gene. Alignments were generated using MAFFT v.7.490162 (--localpair --maxiterate 1000) and trimmed using Gblocks v0.91b163. Approximately maximum-likelihood gene trees were constructed using FastTree v2.1.10176 under the default Jukes-Cantor + CAT model. Single-copy orthologs were obtained using PhyloPyPruner (https://pypi.org/project/phylopypruner; parameters: --min-len 100 –min-support 80 –prune MI), then aligned using MAFFT v.7.490162 and trees constructed using FastTree v2.1.10176. Rapidly evolving genes and positively selected genes were detected with branch and branch-site models implemented in codeml (PAML v4.8165). Likelihood ratio tests (LRT) were used to determine model performance. Log-likelihood times two (2ΔlnL) were compared χ2 distributions with 2 df165. GO enrichment for positively selected genes (both models) was performed using TBtools v2.112171 against the embedded databases (FDR < 0.05). Selected protein structures were predicted using AlphaFold (https://alphafoldserver.com/) and visualised using UCSF ChimeraX v1.8177.

Gene expression level quantification and transcriptomic analysis
RNA sequencing reads from C. margaritatus generated in this study and M. yessoensis (SRR8469068, SRR8469071, SRR8469070, SRR18361759, SRR18361758, SRR18361757, SRR18361912, SRR18361911, SRR18361910, SRR18361892, SRR18361891, SRR18361890, SRR18361879, SRR18361878, and SRR18361877, including three replicates of adductor muscle, foot, gill, gonad, and mantle tissues) were mapped to the transcripts of each gene model of the two scallops, and their expression levels were quantified as transcripts per million (TPM) and read count using the RSEM in Trinity v2.8.5154. Read count data was used to determine differentially expressed genes (DEGs) between the tissues using DESeq2 v1.30.0178. Gill-positively related gene modules were identified by weighted correlation network analysis (WGCNA) using WGCNA-shinyApp v0.0.6.230121 (https://github.com/ShawnWx2019/WGCNA-shinyApp) within TBtools v2.112171 (sample percentage 0.8, expression cutoff 10, power selection 7, min module size 30, module cuttree height 0.25). GO enrichment for selected modules was performed using TBtools v2.112171 against the embedded databases (FDR < 0.05). Because mantle-associated WGCNA modules for M. yessoensis were not significant, mantle-highly expressed genes (log fold-change > 2 vs. other tissues; q < 0.05) identified by DESeq2 in both species were subjected to GO enrichment as above. GOplot v1.0.2179 was used to visualise enrichment for gill-positive modules in C. margaritatus..

Shell elemental relative quantification
LA-ICP-MS was performed using an Applied Spectra J200 213 nm Laser Ablation System (Applied Spectra, USA), coupled to Agilent 8900 ICP-QQQ (Agilent Technologies, USA). Three shell samples for each species were treated with 90% acetic acid for 50 s and rinsed with distilled water. In each sample, 6-8 spots per valve were randomly selected and analysed with three technical replicates. Instrument parameters are in Supplementary Data 19. The Wilcoxon rank-sum test180 was applied to assess elemental concentration differences among four sample groups. Data were transformed using the inverse hyperbolic sine function. Pairwise comparisons were conducted, and the Bonferroni correction was applied to control for false discovery (p < 0.001). Analyses were performed in R, with visualizations produced using ggplot2181 and ggpubr182. To compare left and right valves of C. margaritatus, laser-induced breakdown spectroscopy (LIBS) of a pair of shells was conducted using a mLIBScope system. Shells were embedded in epoxy resin AB and sectioned along the centre line of the shell. Cross-sections were scanned using microscopic LIBS in two-dimensional mode with a detection laser energy of 15 mJ, a delay of 1.28 µs, and an integration time of 1.05 ms183.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The work by Lin et al., describe a chromosomal level assembly of the glass scallop Catillopecten margaritatus. From a\nmethodological perspective, the bioinformatic methods are robust and well written. Sometimes it is taken for granted how\nchallenging could be generating and annotating an animal genome, and the quality of the described genomic resource, its\nannotation and comparative omics analyses employed to dissect the evolution of the glass scallop is good. While most of\nthe results and the authors’ interpretation are well supported throughout the manuscript, there are some parts of the work that\nneed a more thoughtful consideration and discussion (please check my specific comments below). Particularly, I found\ndifficult to connect sometimes the references with the statements from the authors, this deserves a better care. Additionally, I\nnoticed some striking similarities between some sections of the methodology described in this paper to an already published\nwork. This is worrying and must be amended by the authors. Overall, the authors did a thorough and compelling work\nexploring the genome of the glass scallop and providing many substantiated hypotheses about its adaptation to the deep-\nsea environment and symbiotic lifestyle. This work surely open new venues for the exploration of this system in future\nresearch.\nComments:\nLines 55 – 59: The authors claimed that ectosymbioses is overlooked in contrast to endosymbiosis in the hydrothermal vent\ncontext, however, this is not entirely correct (see Duperron, Laurent, Gaill, & Gros, 2008; Fujiwara et al., 2010; O. Gros &\nGaill, 2007; Olivier Gros, Guibert, & Gaill, 2007). Are the authors referring to whole genome information of hydrothermal\nanimals engaged in ectosymbioses in this context?\nLines 66 – 68: How is the reference Distel et al. (2000) connected to the presence of chemosymbiotic bacteria in C.\nmargaritatus?\nLines 71 – 73: It could be my interpretation, but here it reads to me that endosymbiosis might be an evolutionary end-goal for\nhost-microbe associations. This could be misleading to some readers, since ecto and endosymbioses are just different\nevolutionary strategies, and the transition to ecto to endo is one of many possible pathways. This same line of thought is\npresented in the abstract. Is there any evidence in the literature describing the shift from ecto- to endosymbionts in scallops\n(or even in mussels’ species) to assume that this can be a viable evolutionary trend in this context?\nIn the lines 125 to 126 the authors conclude that “Genomic studies on other propeamussiids are needed to investigate\nintrachromosomal structure variations linked to deep-sea adaptation and symbiosis”, but, for me it is still uncertain how\nintrachromosomal structural variations have functional importance to deep-sea adaptation and symbiosis in the current\ninvestigated species C. margaritatus. Could the authors please, as initial step, first explain/hypothesise the impacts of these\nrearrangements in the light of the C. margaritatus evolution before suggesting the impacts of these phenomenon in other\nclosely-related species?\nLines 144 – 147: I failed to identify where in the references provided by the authors it is described that M. vulcani individuals\ndo not harbour any symbiotic bacteria based on amplicon sequencing. Lin et al., (2024) describes the presence of\nThiodubiliella in all analysed samples of M. margaritatus, and Beninger et al. (2003) hypothesised that this is the case based\non the gill morphology of M. vulcani.\nLines 172 – 175: Please make it explicit, as it is in the Sup. Table 8, that these genes are involved in the eye development\npathway. This is not clear based on the current text.\nLines 173 – 175: The row Z-scores showed in the figure tell us how the expression of the selected genes in the different\ntissues deviates (in terms of SD) from their own mean. This is not a plot showing, for example, absolute expression levels,\nand thus, it is insufficient to actually show if the interrogated genes have low gene expression compared to others. If the\nauthors want to corroborate their statement, I suggest an alternative normalisation method (normalising the expression of\nthese genes against a background of constitutively expressed house-keeping genes), or even rank-based statistics to know\nexactly where those genes fall in relation to all other expressed genes in the tissues. The same argument applies for other\nsimilar plots in the manuscript.\nLines 177 – 179: Tissue specificity is difficult to be investigated based purely on genomic data. I guess the authors meant\nthat these gene families are expanded (i.e., CAFE analysis) and the gene expression based on comparative transcriptomics\nof these expanded elements showed tissue specificity. This sentence needs to be rewritten for better clarity.\nLine 258: I am struggling to connect some of the references with the statements described by the authors in the manuscript.\nThe reference 78 describes, using FISH and TEM, how gill tissues in Bathymodiolus mussels and one vesicoymid clam\nchange when challenged with the presence of new symbionts. At any point of this specific MS genomic analyses were\nconducted and it is even stated in the conclusion of the work that “Comprehensive morphological and molecular analyses of\nsymbiont colonization of bivalve gills as well as studies of the cellular and immune-related processes that occur during gill\ncell development are needed to better understand how host cell differentiation, symbiont source and other factors contribute\nto symbiont acquisition in bivalve gills.“ So, how this work by Wentrup et al. (2014) is giving any support to the claim that\n„The expansion of the ECE and MUC3A genes in deep-sea scallops may reflect enhanced immune defences against\nsymbiont infection“. My same argument applies for the reference 79. Could the authors briefly explain how these studies\nsupport their statement?\nLines 270 – 272: It is well-characterised in recent genome publications that sulphide detoxification by a simultaneous non-\ncompetitive and reversible binding of oxygen and sulphide in also present in respiratory pigments of tubeworms like Riftia,\nLamellibrachia and Paraescarpia (Li et al., 2019; Sun et al., 2021; De Oliveira et al., 2022), in addition to bivalves. Did the\nauthors check for the globin genes in the symbiont and asymbiont scallop genomes? If so, are there any differences in gene\nexpression patterns in these animals and also across M. margaritatus tissues?\nLines 307 - 312: This same hypothesis has been presented in Lin et al., (2024) and this needs to be more correctly cited in\nthis particular paragraph. What would be the functional relevance of the positively selected genes in this regard? Are the\npositively selected genes boosting the intracellular pool of TCA intermediates compared to other scallop systems? What are\nthe expectations?\nLines 315 – 317: this sentence is overly broad and needs some rewriting. The mode of how compounds are exchanged\nbetween host and symbionts are context-dependent and not easily generalisable by the location of the symbionts in the host\nbody plan.\nLines 327 - 331: I will expand on my previous comment. Again, these statements are again too broad and could be\nmisleading for researchers not involved in the symbiosis field. The mechanisms underpinning host nutrition in symbiotic\nanimals can vary a lot. While it is true that apoptosis of symbiont-housing cells, e.g., in Riftia, Lamellibrachia trophosome,\ncan be involved in energy acquisition, there are genomic, transcriptomic and proteomic evidence (Li et al., 2019; Hinzke et\nal., 2019; De Oliveira et al., 2022) pointing to phagolysosomal-type of digestion. This is also the case for shallow water\ngutless oligochaetes and their extracellular endosymbionts (i.e. Olavius) (Wippler et al., 2016). The exchange of nutrients via\n“milking” mediated by outer membrane vesicles (OMVs), is also described in intracellular endosymbionts in the flatworm\nParacatenulla (Jäckle et al., 2019). Alternatively, ectosymbionts can also be digested and farming could be as prevalent as\n“milking” in that regard (check the literature on chemosynthetic nematodes Stilbonematinae). I suggest the authors to add\nthese nuanced characteristics into their text, since this journal is quite broad and interdisciplinary.\nLines 341 to 349: this mechanism, as previously mentioned, has been already described in other chemosynthetic\norganisms, and this is a chance for the authors to expand their discussion beyond their researched organism and highlight\nthe convergent evolutionary strategies used by different animals with intimate symbioses (briefly).\nComments regarding the methodology:\nThe bioinformatic methods are sound and robust. The tools together with their versions are included in the text, and when\nnon-default parameters were employed, the authors properly described them. On a more negative note, I was surprised by\nsome quite remarkable similarities (i.e., the structure and content of the sentences) in some sections of the work with\nprevious published works. E.g.:\nThis work: The nucleotide substitution rates of bivalves were estimated using a free-ratio model implemented in the codeml\nprogram of PAML v4.840. Divergences of TEs from the consensus sequences extracted from RepeatMasker results were\nadjusted for multiple substitutions using the Jukes-Cantor formula K = − 300/4 × Ln (1 − D × 4/300), where D represents the\ndistance between the fragmented repeat and the consensus sequence41. Insertion times of TEs were estimated using the\nequation T = K/2r42, where T is the insertion time and r is the nucleotide substitution rate for each bivalve species.\nFrom Sun et al. (2020): The nucleotide substitution rates of the three species were estimated using a free-ratio model\nimplemented in the codeml script in PAML v4.8 (Yang 2007). The divergences of TEs from the consensus sequences\nextracted from RepeatMasker results were adjusted for multiple substitutions using the Jukes–Cantor formula K = −300/4 ×\nLn (1 − D × 4/300), where D represents the distance between the fragmented repeat and the consensus sequence. The\ninsertion times of TEs were estimated using the equation T = K/2r (Kimura 1980), where T is the insertion time and r is the\nnucleotide substitution rate for each species.\nI am completely aware that the methods and pipelines used in metazoan genome papers are similar, however, there is a\nminimum rewording here when compared to an already published paper (which was not properly acknowledged). I haven’t\ndone a thorough check of whole methodological part, and to be fair I only recognised this instance, because I have read the\nprevious Sun et al., (2020) paper recently, so I cannot assess the extent of this behaviour throughout the manuscript. As a\ngeneral advice for the authors, I would recommend when reusing published standard methods, to properly cite the original\narticles and/or slightly changing the text. The given example below is almost literally a copy-and-paste version of an already\npublished paper with the “bivalve” word added.\n- I have a question about using codeml to calculate the free-ratio model of nucleotide sequences. Normally, codeml is used\nfor coding sequencing only based on codon (seqtype = 1) or aminoacid (seqtype = 2) alignments. The authors describe the\nnucleotide substitution rates, and based on the way it is written, no methodological approach was used to generate a codon-\nalignment or protein alignment. Could the authors please clarify their approach considering what was previously argued?\n- I am curious why the authors used PhyloPyPrunner route to identify single-copy orthogroups from a relatively few species\nof closely-related scallops with well-behaved genomes (i.e., generally conserved synteny and not elevated number of\nparalogous genes). Could the authors, please, share the number of single copy orthogroups identified by orthofinder before\napplying the PhyloPyPrunner pipeline? From a methodological perspective, both approaches are valid, however, the\nnumber of false positive is likely increased with the PhyloPyprunner approach. What was the rationale behind this decision?", "reviewer_2": "This manuscript describes the first chromosomal-level genome assembly from Propeamussiidae and the first Pectinida\ndemonstrated to harbor thioautotrophic symbionts, providing convincing evidence of changes in photoreceptor,\nbiomineralization, sulfide detoxification, and immune and nutritional interactions with symbionts in comparison to a shallow\nwater, non-symbiotic relative. This manuscript will be of high interest to those investigating invertebrate-bacteria symbioses\nand deep-sea invertebrate biology. The work largely supports the conclusions, with some minor caveats described below,\nwhere claims exceed the evidence. The methods and data analyses appear sound and meet current standards in the field.\nAll relevant data is provided in public repositories.\nComments:\n56: Ectosymbiotic systems are marginally less well studied, but certainly not overlooked!\n201: Please provide a brief explanation of the theory underlying the interpretation of the significance of Ca44 and Ca43\nisotope levels.\n205 and 391: The authors claim that there is limited calcium availability in the deep sea. It is my understanding that calcium\nconcentrations do not differ substantially with depth in the ocean. Are the authors referring to reduced calcium carbonate\nsaturation in the deep sea due to pressure and temperature? Please clarify or substantiate this claim.\n325-326: The authors state: \"Such specific and active nutritional contributions of the host to the symbiont would be\nremarkable\". Unless the authors explain why they make this value judgement, this statement remains an opinion that adds\nnothing to the manuscript.\n351: An article appears to be missing before \"lips\".\nThe authors present their conclusions in well-justified and measured terms in the main text but make unsupported claims in\nthe conclusion section. The first paragraph of the conclusion is redundant with the preceding text. The second paragraph\nrestates hypotheses proposed in the preceding text as if they have been tested empirically rather than inferred\nbioinformatically. For example:\n396-397: \"Catillopecten margaritatus appears to selectively recognise ectosymbionts and facilitate their colonisation on\ngills\".\nand\n399-402: The host employs a “farming” strategy: it supports essential amino acids and metabolites from gill epithelial cells to\nsupport symbiont growth, subsequently harvesting energy and nutrients (including seven essential amino acids and five\nvitamins) via endocytosis and intracellular digestion.\nAlthough loosely suggested by the genome analyses, neither of these claims was tested. These claims should be restated\nas untested hypotheses inferred from sequence analyses.\nIn conclusion, this is a well-written manuscript describing important work and should be published after addressing these\nminor issues described."}, "author_response": "Overall responses to the reviewers:\nWe are deeply grateful to the reviewers for their rigorous review and invaluable\nfeedback on our manuscript. Your insightful comments have helped substantially\nstrengthen the scientific accuracy, clarity, and overall impact of our work. In response,\nwe have undertaken a comprehensive revision of the manuscript, addressed all\nmethodological concerns, and revised the format in line with formatting requirements.\nKey improvements include refining interpretations and citations, enhancing\nmethodological transparency and rigour, and providing more precise and measured\nlanguage. We believe the revised manuscript is now substantially improved and fully\naddresses the points raised by the reviewers. As detailed below, we have carefully\nresponded to each comment and revised the text accordingly.\nPoint-to-Point responses to reviewers:\nReviewer #1 (Remarks to the Author)\nThe work by Lin et al., describe a chromosomal level assembly of the glass scallop\nCatillopecten margaritatus. From a methodological perspective, the bioinformatic\nmethods are robust and well written. Sometimes it is taken for granted how\nchallenging could be generating and annotating an animal genome, and the quality of\nthe described genomic resource, its annotation and comparative omics analyses\nemployed to dissect the evolution of the glass scallop is good. While most of the\nresults and the authors’ interpretation are well supported throughout the manuscript,\nthere are some parts of the work that need a more thoughtful consideration and\ndiscussion (please check my specific comments below). Particularly, I found difficult\nto connect sometimes the references with the statements from the authors, this\ndeserves a better care. Additionally, I noticed some striking similarities between some\nsections of the methodology described in this paper to an already published work.\nThis is worrying and must be amended by the authors. Overall, the authors did a\nthorough and compelling work exploring the genome of the glass scallop and\nproviding many substantiated hypotheses about its adaptation to the deep-sea\nenvironment and symbiotic lifestyle. This work surely open new venues for the\nexploration of this system in future research.\nComments:\nLines 55 – 59: The authors claimed that ectosymbioses is overlooked in contrast to\nendosymbiosis in the hydrothermal vent context, however, this is not entirely correct\n(see Duperron, Laurent, Gaill, & Gros, 2008; Fujiwara et al., 2010; O. Gros & Gaill,\n2007; Olivier Gros, Guibert, & Gaill, 2007). Are the authors referring to whole\ngenome information of hydrothermal animals engaged in ectosymbioses in this\ncontext?\nResponses: We have revised the statement following the reviewer’s suggestion to\nclarify that genome-level studies of ectosymbiosis are limited to avoid exaggeration\n(lines 70-71).\nLines 66 – 68: How is the reference Distel et al. (2000) connected to the presence of\nchemosymbiotic bacteria in C. margaritatus?\nResponses:We apologise for this incorrect citation. This reference pertains to the\nsubsequent sentence. We have corrected the citation placement (lines 83-86).\nLines 71 – 73: It could be my interpretation, but here it reads to me that\nendosymbiosis might be an evolutionary end-goal for host-microbe associations. This\ncould be misleading to some readers, since ecto and endosymbioses are just different\nevolutionary strategies, and the transition to ecto to endo is one of many possible\npathways. This same line of thought is presented in the abstract. Is there any evidence\nin the literature describing the shift from ecto- to endosymbionts in scallops\n(or even in mussels’ species) to assume that this can be a viable evolutionary trend in this\ncontext?\nResponses: We thank the reviewer for highlighting this issue. Beyond the species\nstudied here, there is currently no research on chemoautotrophic symbiosis in other\nscallops. Within bathymodiolines, Fujiwara et al. (2010) suggested that the symbiosis\nin A. pacifica may represent an earlier stage in evolution than other endosymbiotic\nmussels.Additionally, Smith (1979) proposed that host-symbiont associations can be\narranged along a continuum of increasing morphological integration of the symbiont\ninto the host cell from extracellular and intracellular symbionts. Although these\nstudies imply a possible evolutionary transition from ectosymbiosis to endosymbiosis\nin some lineages, they do not provide conclusive evidence. We have therefore revised\nthe Abstract and Introduction to avoid any implying evolutionary end-point and to\npresent potential misunderstandings (lines 23-57, 87-89).\nFujiwara, Y. et al. Extracellular and mixotrophic symbiosis in the whale-fall mussel\nAdipicola pacifica: a trend in evolution from extra- to intracellular symbiosis.\nPLoS One5, e11808 (2010).\nSmith, D. C. From extracellular to intracellular: The establishment of a symbiosis.\nProc. R. Soc. Lond. B. Biol. Sci. 204, 115–130 (1979).\nIn the lines 125 to 126 the authors conclude that “Genomic studies on other\npropeamussiids are needed to investigate intrachromosomal structure variations linked\nto deep-sea adaptation and symbiosis”, but, for me it is still uncertain how\nintrachromosomal structural variations have functional importance to deep-sea\nadaptation and symbiosis in the current investigated species C. margaritatus.\nCould the authors please, as initial step, first explain/hypothesise the impacts of these\nrearrangements in the light of the C. margaritatus evolution before suggesting the\nimpacts of these phenomenon in other closely-related species?\nResponses: We agree and have revised the manuscript to first outline specific\nhypotheses for how intrachromosomal structural variations might contribute to deep-\nsea adaptation and symbiosis inC. margaritatus. We then suggest comparative studies\nacross propeamussiids as a logical step based on these hypotheses, rather than\npreceding them (lines 151-161).\nLines 144 – 147: I failed to identify where in the references provided by the authors it\nis described that M. vulcani individuals do not harbour any symbiotic bacteria based\non amplicon sequencing. Lin et al., (2024) describes the presence of Thiodubiliella in\nall analysed samples of M. margaritatus, and Beninger et al. (2003) hypothesised that\nthis is the case based on the gill morphology of M. vulcani.\nResponses:We have removed the incorrect statement (lines 181-182).\nLines 172 – 175: Please make it explicit, as it is in the Sup. Table 8, that these genes\nare involved in the eye development pathway. This is not clear based on the current\ntext.\nResponses: We have now specified the genes involved in the eye development\npathway, as suggested (lines 208-213).\nLines 173 – 175: The row Z-scores showed in the figure tell us how the expression of\nthe selected genes in the different tissues deviates (in terms of SD) from their own\nmean. This is not a plot showing, for example, absolute expression levels, and thus, it\nis insufficient to actually show if the interrogated genes have low gene expression\ncompared to others. If the authors want to corroborate their statement, I suggest an\nalternative normalisation method (normalising the expression of these genes against a\nbackground of constitutively expressed house-keeping genes), or even rank-based\nstatistics to know exactly where those genes fall in relation to all other expressed\ngenes in the tissues. The same argument applies for other similar plots in the\nmanuscript.\nResponses: We thank the reviewer for this insightful comment on gene expression\ndata. The reviewer is correct that row Z-scores show relative expression patterns\nacross tissues and do not directly reflect absolute expression levels compared to all\nother genes. Our primary objective was not to demonstrate absolute gene expression,\nbut to test whether these eye development genes exhibit tissue-specific high\nexpression in the mantle, where eyes form in shallow-water relatives. Row Z-scores\nare suited for this purpose, as they highlight tissue-specific upregulation or\ndownregulation relative to each gene’s own expression in other tissues. Many of these\ngenes are pleiotropic beyond visual function;for example, brn3 contributes to\nauditory, balance, and somatosensory functions (Leyva‐Díaz et al., 2020), so absolute\nexpression in a single tissue may not fully capture their functional context. The cross-\ntissue comparison therefore better illustrates that most vision-associated genes are not\nspecifically highly expressed in the mantle ofC. margaritatus, supporting the\nhypothesis that this tissue does not serve a visual role in this deep-water species.\nAdditionally, as noted by the reviewer, Fig. 3c displays normalised TPM values for\neach gene, which readers can consult for absolute expression levels. Taken together,\nthese visualisations provide a more comprehensive view of the data.\nWe acknowledge that our original description of these genes as being expressed at\n“low levels” was misleading. Accordingly, we have revised the text to state that they\n“did not show tissue-specific high expression in the mantle” (lines215-219). For\nother transcriptomic heatmaps (Figs. 4d, 5c, and 6d), the goal is likewise to compare\nexpression patterns across tissues; thus, the use of row Z-scores is considered\nappropriate. To prevent misunderstanding, we have added clarifying descriptions at\nthe corresponding locations that these plots display relative expression patterns (lines\n225-227, 309, 317, 319, 323, 395, 441-444, 472, 477, 481, 485).\nLeyva‐Díaz, E., Masoudi, N., Serrano‐Saiz, E., Glenwinkel, L., & Hobert, O.\nBrn3/POU‐IV‐type POU homeobox genes-Paradigmatic regulators of neuronal\nidentity across phylogeny. Wiley Interdiscip. Rev. Dev. Biol. 9, e374 (2020).\nLines 177 – 179: Tissue specificity is difficult to be investigated based purely on\ngenomic data. I guess the authors meant that these gene families are expanded (i.e.,\nCAFE analysis) and the gene expression based on comparative transcriptomics of\nthese expanded elements showed tissue specificity. This sentence needs to be\nrewritten for better clarity.\nResponses: We have rewritten the sentences for accuracy and clarity, as suggested\n(lines 223-224).\nLine 258: I am struggling to connect some of the references with the statements\ndescribed by the authors in the manuscript. The reference 78 describes, using FISH\nand TEM, how gill tissues in Bathymodiolus mussels and one vesicoymid clam\nchange when challenged with the presence of new symbionts. At any point of this\nspecific MS genomic analyses were conducted and it is even stated in the conclusion\nof the work that “Comprehensive morphological and molecular analyses of symbiont\ncolonization of bivalve gills as well as studies of the cellular and immune-related\nprocesses that occur during gill cell development are needed to better understand how\nhost cell differentiation, symbiont source and other factors contribute to symbiont\nacquisition in bivalve gills.“ So, how this work by Wentrup et al. (2014) is giving any\nsupport to the claim that „The expansion of the ECE and MUC3A genes in deep-sea\nscallops may reflect enhanced immune defences against symbiont infection“. My\nsame argument applies for the reference 79. Could the authors briefly explain how\nthese studies support their statement?\nResponses: We thank the reviewer for this critical comment and apologise for the\ninappropriate citations. We intended to use these two references to provide context for\nthe complex immune-symbiont interactions during gill colonisation and to highlight\nknowledge gaps in this field, thereby positioning ECE and MUC3A as promising\ntargets for future research. We have now revised the text prevent misinterpretation,\nand removed any implication that these specific studies directly support our claim\n(lines 319-336).\nLines 270 – 272: It is well-characterised in recent genome publications that sulphide\ndetoxification by a simultaneous non-competitive and reversible binding of oxygen\nand sulphide in also present in respiratory pigments of tubeworms like Riftia,\nLamellibrachia and Paraescarpia (Li et al., 2019; Sun et al., 2021; De Oliveira et al.,\n2022), in addition to bivalves. Did the authors check for the globin genes in the\nsymbiont and asymbiont scallop genomes? If so, are there any differences in gene\nexpression patterns in these animals and also across M. margaritatus tissues?\nResponses: As suggested, we examined globin genes in C. margaritatus and M.\nyessoensis based on functional annotations. We identified only one copy of\nhaemoglobin in each genome, although we found multiple copies of neuroglobin in\nboth species (to our knowledge, neuroglobin is not implicated in sulphide\ndetoxification). The haemoglobin of C. margaritatus (Cma03G001244) showed low\nexpression in mantle replicate 2 (TPM = 10.8), but was not expressed in other tissues,\nincluding gill (TPM = 0). In M. yessoensis, haemoglobin (Miz0086170) also exhibited\nvery low expression across tissues (TPM 0–1.022). These observations suggest that\nhaemoglobin-mediated sulphide detoxification is unlikely to occur in the deep-sea\nscallop symbiotic system.\nLines 307 - 312: This same hypothesis has been presented in Lin et al., (2024) and\nthis needs to be more correctly cited in this particular paragraph. What would be the\nfunctional relevance of the positively selected genes in this regard? Are the\npositively selected genes boosting the intracellular pool of TCA intermediates compared\nto other scallop systems? What are the expectations?\nResponses: We have corrected the citation to Lin et al. (2024) and clarified the\nproposed mechanism in the text. While our genomic data provide evidence for\npositive selection, indicating that these genetic changes may increase the flux of TCA\nintermediates will require direct biochemical measurements.We therefore identify\nfunctional validation of this hypothesis as a key priority for future work (lines 382-\n397).\nLines 315 – 317: this sentence is overly broad and needs some rewriteing. The mode\nof how compounds are exchanged between host and symbionts are context-dependent\nand not easily generalisable by the location of the symbionts in the host body plan.\nResponses:We have revised the text to tone down the generalisation and to state\nmore accurately that symbiont location can influence, rather than determine, the mode\nof metabolite exchange, acknowledging the context-dependent nature of these\nprocesses (lines 399-407).\nLines 327 - 331: I will expand on my previous comment. Again, these statements are\nagain too broad and could be misleading for researchers not involved in the symbiosis\nfield. The mechanisms underpinning host nutrition in symbiotic animals can vary a\nlot. While it is true that apoptosis of symbiont-housing cells, e.g., in Riftia,\nLamellibrachia trophosome, can be involved in energy acquisition, there are genomic,\ntranscriptomic and proteomic evidence (Li et al., 2019; Hinzke et al., 2019; De\nOliveira et al., 2022) pointing to phagolysosomal-type of digestion. This is also the\ncase for shallow water gutless oligochaetes and their extracellular endosymbionts (i.e.\nOlavius) (Wippler et al., 2016). The exchange of nutrients via “milking” mediated by\nouter membrane vesicles (OMVs), is also described in intracellular endosymbionts in\nthe flatworm Paracatenulla (Jäckle et al., 2019). Alternatively, ectosymbionts can also\nbe digested and farming could be as prevalent as “milking” in that regard (check the\nliterature on chemosynthetic nematodes Stilbonematinae). I suggest the authors to add\nthese nuanced characteristics into their text, since this journal is quite broad and\ninterdisciplinary.\nResponses: We have thoroughly revised the paragraph to reflect the diversity of\nnutritional mechanisms in chemosymbiotic animals. The text now acknowledges a\nbroader spectrum of strategies, including phagolysosomal digestion, nutrient\nexchange via OMVs (“milking”), and the “farming” of ectosymbionts, with the key\nreferences provided by the reviewer cited to support these points (lines 420-431).\nLines 341 to 349: this mechanism, as previously mentioned, has been already\ndescribed in other chemosynthetic organisms, and this is a chance for the authors to\nexpand their discussion beyond their researched organism and highlight the\nconvergent evolutionary strategies used by different animals with intimate symbioses\n(briefly).\nResponses:We have expanded the paragraph to highlight the convergent evolution of\nthis nutritional strategy across chemosymbiotic systems, as suggested (lines 451-459).\nComments regarding the methodology:\nThe bioinformatic methods are sound and robust. The tools together with their\nversions are included in the text, and when non-default parameters were employed,\nthe authors properly described them. On a more negative note, I was surprised by\nsome quite remarkable similarities (i.e., the structure and content of the sentences)\nin some sections of the work with previous published works. E.g.:\nThis work: The nucleotide substitution rates of bivalves were estimated using a free-\nratio model implemented in the codeml program of PAML v4.840. Divergences of\nTEs from the consensus sequences extracted from RepeatMasker results were\nadjusted for multiple substitutions using the Jukes-Cantor formula K = − 300/4 × Ln\n(1 − D × 4/300), where D represents the distance between the fragmented repeat and\nthe consensus sequence41. Insertion times of TEs were estimated using the equation T\n= K/2r42, where T is the insertion time and r is the nucleotide substitution rate for\neach bivalve species.\nFrom Sun et al. (2020): The nucleotide substitution rates of the three species were\nestimated using a free-ratio model implemented in the codeml script in PAML v4.8\n(Yang 2007). The divergences of TEs from the consensus sequences extracted from\nRepeatMasker results were adjusted for multiple substitutions using the Jukes–Cantor\nformula K = −300/4 × Ln (1 − D × 4/300), where D represents the distance between\nthe fragmented repeat and the consensus sequence. The insertion times of TEs were\nestimated using the equation T = K/2r (Kimura 1980), where T is the insertion time\nand r is the nucleotide substitution rate for each species.\nI am completely aware that the methods and pipelines used in metazoan genome\npapers are similar, however, there is a minimum rewording here when compared to an\nalready published paper (which was not properly acknowledged). I haven’t done a\nthorough check of whole methodological part, and to be fair I only recognised this\ninstance, because I have read the previous Sun et al., (2020) paper recently, so I\ncannot assess the extent of this behaviour throughout the manuscript. As a general\nadvice for the authors, I would recommend when reusing published standard methods,\nto properly cite the original articles and/or slightly changing the text. The given\nexample below is almost literally a copy-and-paste version of an already published\npaper with the “bivalve” word added.\nResponses: Does the reviewer mean the study by Sun et al. (2021), titled “Genomic\nsignatures supporting the symbiosis and formation of chitinous tube in the deep-sea\ntubeworm Paraescarpia echinospica” which we referred to? The methodology\ndescribed in that study aligns with the information provided by the reviewer. We\nregret this oversight and have thoroughly revised this methodological description,\nwith appropriate citation to Sun et al. (2021) (Supplementary Information, track-\nchanged, lines 179). We have also carefully reviewed and edited the entire Materials\nand Methods to ensure academic rigour and integrity, and to avoid textual similarity.\nThe updated version is provided in the Supplementary Information.\n- I have a question about using codeml to calculate the free-ratio model of nucleotide\nsequences. Normally, codeml is used for coding sequencing only based on codon\n(seqtype = 1) or aminoacid (seqtype = 2) alignments. The authors describe the\nnucleotide substitution rates, and based on the way it is written, no methodological\napproach was used to generate a codon-alignment or protein alignment. Could the\nauthors please clarify their approach considering what was previously argued?\nResponses: We have added a detailed description of the data sources and processing\nprocedures in the relevant section (Supplementary Information, track-changed,\nlines 179-187), clarifying how coding sequence alignments were generated for\ncodemal analyses.\n- I am curious why the authors used PhyloPyPrunner route to identify single-copy\northogroups from a relatively few species of closely-related scallops with well-\nbehaved genomes (i.e., generally conserved synteny and not elevated number of\nparalogous genes). Could the authors, please, share the number of single copy\northogroups identified by orthofinder before applying the PhyloPyPrunner pipeline?\nFrom a methodological perspective, both approaches are valid, however, the\nnumber of false positive is likely increased with the PhyloPyprunner approach. What\nwas the rationale behind this decision?\nResponses: We thank the reviewer for raising this methodological point. Our decision\nto employ PhyloPyPruner for orthology inference was based on the following\nrationale:\n1. PhyloPyPruner is a widely adopted method for orthology inference, used in\ndiversee evolutionary studies, including phylogenomic tree reconstruction and\npositive selection analyses (Martínez‐Redondo et al., 2023; Christodoulides et al.,\n2024; Zhou et al., 2025).\n2. While OrthoFinder is highly effective at identifying 1:1 orthologs, scallop genomes\nexhibit notable gene duplication and loss. A strict 1:1 requirement would inevitably\nexclude many legitimate orthologues that have undergone lineage-specific change.\n3. In our analysis, OrthoFinder identified 6,486 single-copy orthogroups, whereas\nPhyloPyPruner recovered 11,063, nearly twice as many. To comprehensively detect\npositively selected genes, we prioritised the recoverying a larger orthologue set for\ndownstream analyses. To mitigate false positives, we applied multiple conservative\nfilters in PhyloPyPruner: --min-support 80 (retain only well-supported relationships),\n--min-len 100 (exclude short and potentially unreliable sequences), and --prune MI\n(remove inconsistent sequences using the most inconsistent pruning method).\n4. Among orthogroups under significant positive selection (p < 0.05; 1,634 and 426 in\nbranch-site-model and branch-model, respectively), 98 (6.0%) and 37 (8.7%),\nrespectively, were uniquely recovered by PhyloPyPruner approach. To further validate\nthe reliability of the PhyloPyPruner-derived ortholog set, we performed synteny\nanalysis between C. margaritatus and M. yessoensis using MCScanX. The\nbackground genomic synteny rate was 37.4% (9,401 of 25,153 genes). Among\nstringent 1:1 single-copy orthologues under positive selection, synteny rates were\n64.3% (1,051 of 1,634) and 73.9% (315 of 426), respectively, as expected for\nevolutionarily stable genes. For the positively selected genes uniquely identified by\nPhyloPyPruner, synteny rates were51.0% (50 of 98) and 51.3% (19 of 37) for the two\nmodels, both substantially higher than the genomic background (37%). This supports\nthe conslusion that the expanded ortholog set captured genuine orthologous\nrelationships and contributed meaningfully to the inference of positively selection.\nMartínez‐Redondo, G. I. et al. Parallel duplication and loss of aquaporin‐coding genes\nduring the “out of the sea” transition as potential key drivers of animal\nterrestrialization. Molecular Ecology.32, 2022-2040 (2023).\nChristodoulides, N., Urgiles, V. L., Guayasamin, J. M., & Savage, A. E. Selection and\ngene duplication associated with high-elevation diversification in Pristimantis,\nthe largest terrestrial vertebrate genus. Genome Biology and Evolution. 16,\nevae167 (2024).\nZhou, Y. et al. Two routes to land: Genomic underpinnings of parallel aerial egg\ndeposition in aquatic Old-World Pila and New-World Pomacea (Ampullariidae).\nbioRxiv. 2025:2025-11.\nReviewer #2 (Remarks to the Author):\nThis manuscript describes the first chromosomal-level genome assembly from\nPropeamussiidae and the first Pectinida demonstrated to harbor thioautotrophic\nsymbionts, providing convincing evidence of changes in photoreceptor,\nbiomineralization, sulfide detoxification, and immune and nutritional interactions with\nsymbionts in comparison to a shallow water, non-symbiotic relative. This manuscript\nwill be of high interest to those investigating invertebrate-bacteria symbioses and\ndeep-sea invertebrate biology. The work largely supports the conclusions, with some\nminor caveats described below, where claims exceed the evidence. The methods and\ndata analyses appear sound and meet current standards in the field. All relevant data is\nprovided in public repositories.\nComments:\n56: Ectosymbiotic systems are marginally less well studied, but certainly not\noverlooked!\nResponses: We have revised the statement following the reviewer’s suggestion to\nnote that whole-genome studies of ectosymbioses are limited, avoiding confusion\n(lines 70-71).\n201: Please provide a brief explanation of the theory underlying the interpretation of\nthe significance of Ca44 and Ca43 isotope levels.\nResponses: We have added a brief explanation to clarify the interpretation of Ca44\nand Ca43 isotope data (lines 251-255).\n205 and 391: The authors claim that there is limited calcium availability in the deep\nsea. It is my understanding that calcium concentrations do not differ substantially with\ndepth in the ocean. Are the authors referring to reduced calcium carbonate saturation\nin the deep sea due to pressure and temperature? Please clarify or substantiate this\nclaim.\nResponses: We thank the reviewer for this precise correction.We intended to refer to\nthe challenging chemical conditions for calcification in the deep sea. As highlighted\nby the reviewer, this is primarily due to the low calcium carbonate saturation state (Ω)\nresulting from high pressure and low temperature (Liu et al., 2022), which makes\ncalcium carbonate precipitation more energetically demanding and can lead to shell\ndissolution (Waldbusser et al., 2015). To avoid confusion, we have replaced “limited\ncalcium availability” with “low calcium carbonate saturation state” and clarify the\npoint in the Results and Discussion (lines 255-260).\nLiu, X., Luo, Y., & Boudreau, B. P. Effects of deep circulation on CaCO3 dissolution\nand accumulation in the Southwestern Atlantic Ocean. Geophys. Res. Lett.49,\ne2021GL095020 (2022).\nWaldbusser, G. G. et al. Saturation-state sensitivity of marine bivalve larvae to ocean\nacidification. Nat. Clim. Change5, 273–280 (2015).\n325-326: The authors state: \"Such specific and active nutritional contributions of the\nhost to the symbiont would be remarkable\". Unless the authors explain why they\nmake this value judgement, this statement remains an opinion that adds nothing to the\nmanuscript.\nResponses: We have removed this statement, as suggested (lines 418-419).\n351: An article appears to be missing before \"lips\".\nResponses: We have added “their” before “lips” (line 466).\nThe authors present their conclusions in well-justified and measured terms in the main\ntext but make unsupported claims in the conclusion section. The first paragraph of the\nconclusion is redundant with the preceding text. The second paragraph restates\nhypotheses proposed in the preceding text as if they have been tested empirically\nrather than inferred bioinformatically. For example:\n396-397: \"Catillopecten margaritatus appears to selectively recognise ectosymbionts\nand facilitate their colonisation on gills\".\nand 399-402: The host employs a “farming” strategy: it supports essential amino acids\nand metabolites from gill epithelial cells to support symbiont growth, subsequently\nharvesting energy and nutrients (including seven essential amino acids and five\nvitamins) via endocytosis and intracellular digestion.\nAlthough loosely suggested by the genome analyses, neither of these claims was\ntested. These claims should be restated as untested hypotheses inferred from sequence\nanalyses. In conclusion, this is a well-written manuscript describing important work\nand should be published after addressing these minor issues described.\nResponses: We have condensed the entire Conclusion section into a concise summary\nparagraph to enhance clarity and align with Nature Communications’ editorial policy,\nwhich does not permit a separate “Conclusion” section. The two claims noted by the\nreviewer have been removed in the revised version, and the summary text now\nreflects the status of these ideas as hypotheses inferred from genomic analyses rather\nthan empirically tested conclusions (lines 495-535)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I appreciate the effort made by the authors to address each specific point I raised during the first round of revisions. The\nauthors clarified all my question/doubts, and revised the text taking into consideration my concerns. I am satisfied with the\nchanges made and I have no more scientific concerns with the work. I recommend this work for publication."}, "author_response": ""}]

Decision letter