Multi-omic insights into the formation and evolution of a novel shell microstructure in oysters

Background Molluscan shell, composed of a diverse range of architectures and microstructures, is a classic model system to study the relationships between molecular evolution and biomineralized structure formation. The shells of oysters differ from those of other molluscs by possessing a novel microstructure, chalky calcite, which facilitates adaptation to the sessile lifestyle. However, the genetic basis and evolutionary origin of this adaptive innovation remain largely unexplored. Results We report the first whole-genome assembly and shell proteomes of the Iwagaki oyster Crassostrea nippona. Multi-omic integrative analyses revealed that independently expanded and co-opted tyrosinase, peroxidase, TIMP genes may contribute to the chalky layer formation in oysters. Comparisons with other molluscan shell proteomes imply that von Willebrand factor type A and chitin-binding domains are basic members of molluscan biomineralization toolkit. Genome-wide identification and analyses of these two domains in 19 metazoans enabled us to propose that the well-known Pif may share a common origin in the last common ancestor of Bilateria. Furthermore, Pif and LamG3 genes acquire new genetic function for shell mineralization in bivalves and the chalky calcite formation in oysters likely through a combination of gene duplication and domain reorganization. Conclusions The spatial expression of SMP genes in the mantle and molecular evolution of Pif are potentially involved in regulation of the chalky calcite deposition, thereby shaping the high plasticity of the oyster shell to adapt to a sessile lifestyle. This study further highlights neo-functionalization as a crucial mechanism for the diversification of shell mineralization and microstructures in molluscs, which may be applied more widely for studies on the evolution of metazoan biomineralization. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-023-01706-y.

). Pif and LamG3 genes of oysters are marked with red color.Yellow, orange, and blue solid circles represent Pif, ancestral Pif, and LamG3 genes, respectively.Red solid squares after gene IDs indicate the genes highly expressed in the mantle of selected molluscan species, while the red hollow squares indicate genes that are not highly expressed in the mantle.Red branches on the clades represent three groups of genes which are all highly expressed in the mantle tissue.Numbers on the nodes are bootstrap values (>50%).

Figure S1 .
Figure S1.Genome assembly of C. nippona.(a) The Hi-C heatmap of genome assembly.The righ axis represents the chromosome number.(b) Estimated genome size of C. nippona based on K-mer analysis.

Figure S2 .
Figure S2.Distribution of TEs in the C. nippona genome.(a) History of TE accumulation in the C. nippona genome.Historical TE divergence was calculated by the Kimura distance-based copy divergence analysis.(b) Proportions of Helitrons, DNA transposons, LTR, LINE and SINE retrotransposons in the C. nippona genome.

Figure S4 .
Figure S4.Expansion of tyrosinase gene family in Protostomia.The black, blue, and green triangles respectively represent the SMPs identified in the prismatic, foliated, and chalky layer.(a) Phylogeny of protostomian tyrosinases demonstrates oyster-specific expansions.(b) Expression pattern of oyster-specific expanded tyrosinase genes in C. nippona.The number after the abbreviation of tissue represents biologically independent individuals (N = 3).Abbreviations: AM, adductor muscle; D, digestive gland; G, gill; H, hemolymph; MC, mantle center; ME, mantle edge.

Figure S5 .
Figure S5.Expansion of peroxidase gene family in Protostomia.The black, blue, and green triangles respectively represent the SMPs identified in the prismatic, foliated, and chalky layer.(a) Phylogeny of protostomian peroxidases demonstrates oyster-specific expansions.(b) Expression pattern of oyster-specific expanded peroxidase genes in C. nippona.The number after the abbreviation of tissue represents biologically independent individuals (N = 3).Abbreviations: AM, adductor muscle; D, digestive gland; G, gill; H, hemolymph; MC, mantle center; ME, mantle edge.

Figure S6 .
Figure S6.Expansion of tissue inhibitor of metalloproteinase (TIMP) gene family in Protostomia.The black and green triangles respectively represent the SMPs identified in the prismatic and chalky layer.(a) Phylogeny of protostomian TIMPs demonstrates oyster-specific expansions.(b) Expression pattern of oyster-specific expanded TIMP genes in C. nippona.The number after the abbreviation of tissue represents biologically independent individuals (N = 3).Abbreviations: AM, adductor muscle; D, digestive gland; G, gill; H, hemolymph; MC, mantle center; ME, mantle edge.

Figure S7 .
Figure S7.Real-time PCR results showing gene expression patterns among tissues of C. nippona (N = 3 biologically independent individuals).Statistical significance (P < 0.05) shown by different letters was determined using the LSD test in the R package agricolae (v 1.3-5).

Figure S9 .
Figure S9.Base peak chromatogram of three types of protein sample of the C. nippona shell.Black color indicates protein from the prismatic layer; Red color: foliated layer; Green color: chalky layer.

Figure S10 .
Figure S10.Expression patterns of genes encoding shell matrix proteins (SMPs) in six types of tissues of C. nippona.Abbreviations: AM, adductor muscle; D, digestive gland; G, gill; H, hemolymph; ME, mantle edge; MC, central mantle.(a) Distribution of expression levels of genes encoding SMPs in six types of tissues.Red dots indicate SMPs involved in the chalky layer.Blue dots indicate SMPs which are not identified in the chalky layer.(b) Tissue-specific expression of SMPs in C. nippona.Heatmap shows the normalized expression profiles of SMPs in different tissues.Mantle-specific genes encoding SMPs are marked with green color on the right, while other genes are marked in dark pink.The number after the abbreviation of tissue represents biologically independent individuals (N = 3).

Figure S12 .
Figure S12.Cartoon representation indicating domain structures of four SMPs in Figure 2e.The red box represents signal peptide (SP).The unfilled box indicates the functional domain and the domain ID.

Figure S15 .
Figure S15.Flower plot comparing orthologous groups among seven species.Two conserved orthogroups are showed in the center of flower.Numbers show the species-specific SMPs of each species, while percentages in brackets indicate the proportion of species-specific SMPs in each species.

Figure S17 .
Figure S17.Maximum likelihood (ML) tree of Pif and LamG3 in seven molluscs with transcriptome data (Additional file 2: TableS15).Pif and LamG3 genes of oysters are marked with red color.Yellow, orange, and blue solid circles represent Pif, ancestral Pif, and LamG3 genes, respectively.Red solid squares after gene IDs indicate the genes highly expressed in the mantle of selected molluscan species, while the red hollow squares indicate genes that are not highly expressed in the mantle.Red branches on the clades represent three groups of genes which are all highly expressed in the mantle tissue.Numbers on the nodes are bootstrap values (>50%).

Figure S18 .
Figure S18.Genomic arrangement of Pif, ancestral Pif, and LamG3 genes in mollusks.Arrows indicate the direction of the transcripts.Dashed lines represent the long gaps (> 1 Mb) in the genome.

Figure S20 .
Figure S20.Spatial expression patterns of Pif and LamG3 genes in C. nippona mantle.White arrows symbolize the end of the gene expression region.Full view and partial enlargement show positive cells stained in blue by in situ hybridization of Pif (a), LamG3 (c), and Ostreidae-specific LamG3 (e) genes, respectively.Shell layers: prismatic layer (PL), foliated layer (FL), chalky layer (CL).Mantle folds: outer fold (OF), middle fold (MF), inner fold (IF) (scale bars: 200 µm).On the right side, the representative models of mantle show the spatial expression patterns of Pif (b), LamG3 (d), and Ostreidae-specific LamG3 (f) genes, respectively.Yellow color in the representative model of mantle indicates the expression regions of Pif (CNI_013832-RA), while blue color indicates the expression regions of LamG3 (CNI_014385-RA and CNI_014386-RA).Asterisks indicate the periostracal groove.
Tissue expression patterns and protein domain structures of Pif_LamG3_cluster members in bivalves.(a) Expression heatmaps of Pif_LamG3_cluster members in different tissues of bivalves.Mantle tissues are marked by red font.The number after tissue name represents the biological duplicate.Abbreviations: AM, adductor muscle; D, digestive gland; E, eyes; F, foot; FG,