De Novo Transcriptome Assembly and Sex-Biased Gene Expression in the Cyclical Parthenogenetic Daphnia galeata

Abstract

Daphnia species have become models for ecological genomics and exhibit interesting features, such as high phenotypic plasticity and a densely packed genome with many lineage-specific genes. They are also cyclic parthenogenetic, with alternating asexual and sexual cycles and environmental sex determination. Here, we present a de novo transcriptome assembly of over 32,000 D. galeata genes and use it to investigate gene expression in females and spontaneously produced males of two clonal lines derived from lakes in Germany and the Czech Republic. We find that only a low percentage (18%) of genes shows sex-biased expression and that there are many more female-biased gene (FBG) than male-biased gene (MBG). Furthermore, FBGs tend to be more conserved between species than MBGs in both sequence and expression. These patterns may be a consequence of cyclic parthenogenesis leading to a relaxation of purifying selection on MBGs. The two clonal lines show considerable differences in both number and identity of sex-biased genes, suggesting that they may have reproductive strategies differing in their investment in sexual reproduction. Orthologs of key genes in the sex determination and juvenile hormone pathways, which are thought to be important for the transition from asexual to sexual reproduction, are present in D. galeata and highly conserved among Daphnia species.

Keywords: crustaceans; environmental sex determination; genetic variation; juvenile hormone pathway; males; sexual dimorphism.

Fig. 1.—

Number of orthologous groups per species that are shared between Daphnia and insects, are shared between multiple species within either the insects or Daphnia or contain only proteins from one species (species-specific).

Fig. 2.—

Heatmap showing all sex-biased genes with more than a 2-fold expression difference between males and females in the two-factor (combined) analysis. Clustering is based on Euclidean distances of RPKM values.

Fig. 3.—

Results of the one-factor analysis showing sex-biased genes in each clone separately. (A) Dot plot showing the ratio of male/female expression for the J2 clone and the M10 clone, highlighting those genes that show differences in sex bias in the one-factor analysis compared with the two-factor (combined) analysis. Genes identified as sex-biased in the two-factor analysis or in the one-factor analysis for both clones are depicted in gray. Genes detected as sex-biased only in the J2 clone are shown in orange, whereas those detected as sex-biased only in the M10 clone are shown in yellow. Green dots indicate genes that are significantly female-biased in one clone, but significantly male-biased in the other. (B) Dot plot showing the ratio of M10/J2 expression for females and males. Genes identified as clone-biased in the two-factor analysis or in the one-factor analysis in both sexes are depicted in gray. Genes detected as clone-biased only in females are shown in orange, whereas those detected as clone-biased only in males are shown in yellow. Green dots indicate genes that are significantly M10-biased in one sex, but significantly J2-biased in the other. (C) Proportional Venn diagram for female-biased genes in the two-factor analysis and in each of the clones in the one-factor analysis. (D) Proportional Venn diagram for male-biased genes in the two-factor analysis and in each of the clones in the one-factor analysis.

Fig. 4.—

Divergence between clones J2 and M10. (A) Expression correlation between females of clone J2 and clone M10 including Spearman’s ρ. (B) Expression correlation between males of clone J2 and clone M10 including Spearman’s ρ. (C) Expression correlation between clones for male-biased, female-biased, and unbiased genes as a measure of gene expression divergence. Error bars indicate 95% confidence intervals for 1000 bootstrap replicates. Significance was determined by a test of the equivalence of two correlation coefficients after Fisher Z-transformation. *P< 0.05, **P < 0.01, ***P< 0.001.

Fig. 5.—

Divergence between Daphnia galeata and D. pulex measured as Ka/Ks ratios between 1:1 orthologs. (A) Sequence divergence for male-biased, female-biased, and unbiased genes. (B) Sequence divergence for the different categories of sex-biased genes according to the degree of sex bias. Outlier genes with values > 0.8 are not shown on the plot, but were included in the statistical analysis. Significance was determined by a Wilcoxon test. *P< 0.05, **P < 0.01, ***P< 0.001.