Evolution of male fitness diminishes the opportunity for reproductive assurance. (A) Experimental evolution of male fitness in 305 mM NaCl of dioecious populations cultured for 50 generations under the sudden regime. There was a significant increase in male fitness during evolution (LMM: |z| =2.8, P-value =0.006, residual d.f. =30). (B) Experimental evolution of fertility rates in the sudden dioecious populations. After 50 generations there was an increased proportion of females that were fertile after development from the L1 larval stage (24 ± 2 hours) to the time of reproduction (96 ± 2 hours) in 305 mM NaCl (GLMM: |z| =2.3, P-value =0.03, residual d.f. =5). Differences in fertility rates between the evolved G50 dioecious population and the ancestral lab-adapted androdioecious population are not significant (grey line; GLM: |z| =1.6, P-value =0.112, residual d.f. =5). For (A) and (B), bars and errors show ordinary least-square estimates. *, ** indicates, respectively, P-values of <0.05, <0.01. d.f., degrees of freedom; GLMM, generalised linear mixed effects models; LMM, linear mixed effects model.