Fig. 1. Reproductive isolation between P. pacificus and P. exspectatus.

a, Phylogeny of the pacificus complex with the outgroup species P. maxplancki. The tree is a subtree of the reported phylogeny based on phylotranscriptomics³⁸. The reproductive mode of each species is shown as symbol. ⚥♂, androdioecy; ♀♂, dioecy. b, Crossing experiments between P. pacificus male and females of related species. The species of the pacificus complex and P. maxplancki were crossed with P. pacificus. The four numbers at the arrows indicate the results of four-replicate crosses of three females and six males. The presence/absence of F1 progeny was first tested. If F1 were produced, F1 siblings were allowed to mate with each other, and their ability to propagate in one month (~six generations) was tested. c–k, Results of quantitative reproduction tests of hybrid crosses between P. pacificus and P. exspectatus (c–e), backcrosses with F1 animals produced by a cross between a P. exspectatus female and a P. pacificus male (f–h), backcrosses with F1 animals produced by a cross between a P. pacificus hermaphrodite and a P. exspectatus male (i–k). The proportion of crosses with progeny (c,f,i), the number of the progeny (d,g,j) and the proportion of immature progeny (e,h,k) are compared between different crosses. The first column of the backcrosses is the result for F1 females without males to test fertility in selfing. P and E indicate P. pacificus and P. exspectatus, respectively. For the number of progeny and proportion of immature progeny, all replicates are shown as jitter plots while the box plot (upper whisker, the largest data point less than the third quartile + 1.5 × interquartile range; upper bound, the third quartile; centre line, median; lower bound, the first quartile; lower whisker, the smallest data point more than the first quartile − 1.5 × interquartile range) are shown when at least five data points exist. Old dams (four days after J4 stage) were used for the hybrid cross whereas young dams (J4 stage) were used for the backcross. The sample number is shown in Supplementary Table 1.