Fig 1.
Hierarchical clustering using Spearman’s correlation on RNAseq gonad samples.
Samples are divided into two clusters based on their contig expression pattern, discriminating male (blue) and female gonads (red). (M/F)E: male/female at early stage; (M/F)I: male/female at intermediate stage; (M/F)M: male/female at mature stage; (M/F)R: male/female at regression stage; purple or Inv: gonads in sexual inversion; grey or Und: gonad sex is undetermined.
Table 1.
Histological description of the gonadic categories of the female sexual pathway of P. margaritifera.
Table 2.
Histological description of the gonadic categories of the male sexual pathway of P. margaritifera.
Fig 2.
Histological features of gonads in sexual inversion in P. margaritifera.
Two types of sexual inversion are distinguished: sexual inversion from male to female (InvF, Table 1) (A), and sexual inversion from female to male (InvM, Table 2) (B). Og: ovogonia; O: oocytes; Dg: degenerating oocytes; Spg: spermatogonia; T: connective tissue; Spc: spermatocytes; Spz: spermatozoids.
Table 3.
List of the candidate marker genes of the sexual pathway of P. margaritifera.
Fig 3.
RNAseq expression of the nine selected genes of the sexual pathway of P. margaritifera.
Heat map of the RNAseq expression in the new gonad categories of the nine selected genes. Color represents the normalized expression after variance-stabilizing transformation (DESeq). Expression levels are depicted with a color scale, in which shades of red represent higher expression and shades of green represent lower expression. FP: the overall female pathway, eFP: early female pathway, F: female in active gametogenesis, MP: the overall male pathway. M or F: male or female in gametogenesis; (M/F)R: male/female at regression stage; Und(M/F): Undifferentiated gonad at the cellular level beginning a male or a female determination/differentiation at the molecular level; Inv(M/F): oyster in sexual inversion female to male or male to female; MRF: male at regression stage at the cellular level beginning a female differentiation at the molecular level.
Fig 4.
Relative expression of the nine selected marker genes of the female pathway of P. margaritifera.
Relative expression profiles (real-time PCR) of pmarg-foxl2 (A), pmarg-c43476 (B), pmarg-c45042 (C), pmarg-c19309 (D), pmarg-c54338 (E), pmarg-vit-6 (F), pmarg-zglp1 (G), pmarg-dmrt (H), and pmarg-fem1-like (I) in gonad categories (new set of samples). Different letters indicate statistically significant differences determined by ANOVA. M or F: male or female in gametogenesis; (M/F)R: male/female at stage of regression; Und: Undetermined sex.
Fig 5.
Hierarchical clustering using Spearman’s correlation on gonad samples.
Samples are divided into two main clusters based on their gene expression ratio pattern (36 variables), discriminating male and female pathways. Each cluster is divided into two sub-clusters discriminating oysters in early male or early female pathway (eMP or eFP) and oysters in the course of male or female pathway (MP or FP). M or F: male or female in gametogenesis; (M/F)R: male/female at regression stage; Und: gonad sex undetermined.
Fig 6.
Multivariate regression tree of oyster sexual pathway categories.
Candidate predictor/explanatory variables are the relative expression ratio of the nine potential marker genes (36 variables), and the response variables are the four sub-clusters of gonad samples (n = 44) determined by hierarchical clustering analysis (eMP or eFP: oyster on the early male or female pathway; MP or FP: oyster in the course of male or female pathway). This tree explained 100% of the total variance. The bar graphs at the end of each leaf represent the number of samples of the corresponding group. Statistics of the MRT are presenting at the left of the figure. Error: Residual error; CV Error: Cross-validation error; and SE: Standard error.
Table 4.
Results of the predictive MRT model on RNAseq sample (data from [29]).
Fig 7.
The histo-molecular model of the reproduction of P. margaritifera.
M or F: male or female in gametogenesis; (M/F)E: male/female at early stage; (M/F)I: male/female at intermediate stage; (M/F)M: male/female at mature stage; (M/F)R: male/female at regression stage; Und(M/F): gonad undifferentiated at the cellular level, beginning a male or a female differentiation at the molecular level; Inv(M/F): oyster in sexual inversion female to male or male to female; MRF: male in regression stage at the cellular level beginning a female differentiation at the molecular level; and FRM: female in regression stage at cellular level beginning a male differentiation at the molecular level.