A single-cell atlas of the oyster mantle and spatial-temporal distribution of shell-forming cells.

(A) UMAP visualization of mantle cells, with clusters colored according to their cell identities. A schematic illustration of the internal anatomy of the oyster and H&E staining of the mantle are shown at the top. Abbreviations: of, outer fold; mf, middle fold; if, inner fold. (B) Dot plot of marker gene expression for each cell cluster shown in (A). Marker genes were retrieved from published studies (Supplementary file 1). Clusters lacking characterized functional marker genes were labeled as “Unknown” or classified as mixed cell populations. (C) Spatial distribution of the five SEC types across mantle regions, as indicated by their marker genes. Top: schematic cross-sections of the mantle showing the expression locations of five representative marker genes corresponding to the SEC types. SEC1 and SEC3-5 are highlighted in red, and SEC2 in yellow. Bottom: expression plots of the same SEC marker genes. (D) UMAP visualization of six major cell types used for pseudotime analysis. Red lines indicate the inferred trajectories. SEC3_27 and SEC3_29 represent clusters 27 and 29 within the SEC3 type, respectively. (E) Pseudotime trajectories of SEC and proliferative cell populations. Proliferative cells were used as the origin for the pseudotime analysis. Red arrows indicate predicted differentiation paths, and black lines show the inferred trajectories. (F) Density distribution of the five SEC types on the pseudotime trajectory. (G) Schematic summary of inferred differentiation trajectories from proliferative cells to SEC populations. Two major paths were identified: Path 1, proliferative cells → SEC3_27 → SEC1 → SEC2 → SEC5; and Path 2, proliferative cells → SEC2 → SEC3_29.

Distinct shell-forming cell types between larval and adult stages in C. gigas.

(A) UMAP visualization of integrated single-cell transcriptomes from gastrula and trochophore stages. Different colors indicate the distribution of shell gland cell populations in the schematic of the trochophore dorsal side (top left). (B) Separate UMAP plots of gastrula (left) and trochophore (right) cells. (C) Proportions of major cell types from gastrula (left) to trochophore (right). (D) Pairwise comparison of marker-gene overlap of larval (gastrula and trochophore) and adult (mantle) cell types. For each pair, the box is shaded by statistical significance (-log10 scale of P values by hypergeometric test). Shell-forming cell types are highlighted by grey backgrounds, and dashed boxes indicate the comparisons between larval and adult shell-forming cell types. (E) Scatter plot showing significantly enriched GO terms (q value < 0.01, FDR-adjusted) for marker genes of SEC (blue) and shell gland (purple) cell types. GO terms shared between the two cell types are highlighted in green.

Transcriptome age indices (TAI) for larval (gastrula and trochophore) (A) and adult (mantle) (B) cell types.

Lower TAI values correspond to “older” gene ages. Shell-forming cell types are highlighted in red.

Cross-species cell-type alignment between adult C. gigas and other spiralians.

(A-D) Pairwise SAMap alignments between C. gigas and (A) the annelid P. leidyi, (B) the flatworm D. japonica, (C) the flatworm S. mediterranea, and (D) the chaetognath P. gotoi. The scale bar represents the SAMap alignment score, defined as the average number of mutual nearest cross-species neighbors for each cell relative to the maximum possible number. Dashed boxes highlight the alignment with SECs of C. gigas. (E) Dot plots showing the expression of highly expressed TF genes in SECs, as well as their orthologous expression patterns in aligned cell types of other spiralians.

Conserved cell types and gene co-option underpin spiralian morphological innovations.

(A) Hypergeometric enrichment of genes that originated at successive phylostrata in broad cell types of the oyster mantle. Asterisks indicate significant enrichment (P < 0.01). SECs and significantly enriched phylostrata are highlighted in red. (B) Summary of SAMap comparisons between the oyster mantle and representative spiralian species. Dot plots show the number of conserved cell-type genes between C. gigas and each species. (C) Evolutionary model illustrating the evolutionary relationships among spiralian cell types. Conserved ancestral cell types such as muscle, neural, and proliferative cells are widely shared across Spiralia, whereas lineage-specific innovations such as molluscan carbonate shells, annelid chaetae, planarian secretory cells, and chaetognath chaetae and epidermis evolved independently through co-option of ancestral genes and expression programs. Question marks indicate two unresolved evolutionary problems in spiralian characters: one concerns the brachiopods, where the homology between their calcium phosphate shells and molluscan carbonate shells, as well as the relationship between brachiopod and other spiralian chaetae, remain unclear; the other relates to the Cambrian fossil Pelagiella exigua, a potential stem-group mollusc that possibly possessed both chaetae and a carbonate shell, leaving open whether it represents the ancestral molluscan morphology or an independent lineage that evolved both traits convergently.

Expression patterns of larval and adult shell matrix protein (SMP) genes in C. gigas.

(A, D) Heatmaps showing the expression dynamics of larval (A) and adult (D) SMP genes across developmental stages and adult tissues. (B, E) Dot plots showing the expression of larval (B) and adult (E) SMP genes in each cell type from single-cell transcriptomic data of the adult mantle. Shell-forming cell types are highlighted with gray backgrounds. (C, F) Dot plots showing the expression of larval (C) and adult (F) SMP genes in each cell type from single-cell transcriptomic data of the gastrula and trochophore stages. Shell-forming cell types are highlighted with gray backgrounds. Abbreviations: TC, two-cell; FC, four-cell; EM, early morula; M, morula; B, blastula; RM, rotary movement; FS, free swimming; EG, early gastrula; G, gastrula; T1-T5, trochophore stages; ED1-ED2, early D-shape larva stages; D1-D7, D-shape larva stages; EU1-EU2, early umbo stages; U1-U6, umbo stages; LU1-LU2, late umbo stages; P1-P2, pediveliger stages; S, spat; J, juvenile; ME, mantle edge; MC, mantle center; SH, shell; He, hemolymph; AM, adductor muscle; Gi, gill; LP, labial palp; DG, digestive gland; MG, male gonad; FG, female gonad; Re, remaining tissues; SEC: Shell-secreting epithelial cells.

Pairwise comparisons of cell types between larval (gastrula and trochophore) and adult (mantle) single-cell transcriptomes based on Pearson correlation (upper left), Spearman correlation (upper right), Jensen-Shannon distance (JSD) (lower left), and Jaccard index (lower right).

Shell-forming cell types are marked in red. Red dashed boxes indicate the comparisons between larval and adult shell-forming cell types. Abbreviations: SEC, shell-secreting epithelial cells.

Gene age analyses for different developmental stages and adult tissues of C. gigas.

(A, B) Hypergeometric enrichment of genes that originated at successive phylostrata in larval (A) and adult (B) cell types of oysters. Shell-forming cell types are marked in red. Asterisks indicate significant enrichment (P < 0.01). (C, D) Transcriptome Age Index (TAI) across oyster ontogeny (C) and adult tissues (D), computed using published bulk transcriptomes. Shaded areas indicate ± standard error. Abbreviations are as in Figure 2-figure supplement 2.

Re-analyses of cell atlases of two spiralian species.

(A, C) Two-dimensional uniform manifold approximation and projection (UMAP) visualizations of cell clusters in the annelid worm P. leidyi (A) and the chaetognath P. gotoi (C). (B, D) Dot plots showing the expression of selected cell-type marker genes for each cluster in P. leidyi (B) and P. gotoi (D).