The khdrbs genes are evolutionarily conserved. We found that khdrbs date far back to the basal metazoans such as placozoa and cnidarian. Except for fruit fly, all the invertebrates analyzed possess a single khdrbs gene in each species. In fruit fly, there are five khdrbs genes, including qkr54B, qkr58E-1, qkr58E-2, qkr58E-3, and nsr (Ding et al., 2010; Volk, 2010), which are all localized on a region of the chromosome 2R, and clustered into one branch (Supplementary Figure 3), suggesting that they were generated by local tandem gene duplication of the ancestral khdrbs gene. Based on their expression patterns and tandem duplicated gene traits (Fan et al., 2008; Zhou et al., 2008; Volk, 2010), we speculated that these five genes perform similar biological functions. In contrast, three khdrbs genes, khdrbs1, khdrbs2, and khdrbs3, are identified in all the vertebrates examined, including lamprey (Xu et al., 2016). The birth of these three khdrbs gene clades coincides with the origin of vertebrates. As there existed two rounds (2R) of WGD happened in the vertebrate ancestors, it is thus expected that four khdrbs genes would be generated by these WGD. Interestingly, there are only three khdrbs genes identified so far in vertebrates. It is thus highly likely that only one copy out of the two khdrbs genes generated by the first round WGD underwent further duplication, thereby leading to the appearance of three khdrbs genes in vertebrates. This seems supported by the facts that KHDRBS2 and KHDRBS3 were grouped together, forming a sub-clade in the phylogenetic trees (Figure 1), and shared higher identity (Figure 2). However, the possibility cannot be ruled out that this may be due to gene loss as observed in Amazon molly. Additionally, we found duplication of khdrbs1 in teleost lineage. This duplication happened between the split of spotted gar and bony fish, agreeing with the third rounds (3R) of WGD in bony fish (Braasch et al., 2016).