The invertebrate KHDRBS proteins formed a single clade, positioned at the base of vertebrate KHDRBS proteins. The overall phylogenetic relationship of KHDRBS well reflects the classic phylogeny of metazoan taxa. In contrast to only one KHDRBS group in invertebrates, there were three distinct clades of KHDRBS proteins in vertebrates, KHDRBS1, KHDRBS2, and KHDRBS3 (Figure 1), consistent with recent reports that humans, mice, and birds have three KHDRBS genes (Artzt and Wu, 2010). Apparently, the emergence of these three clades occurred around the split of subphylum Vertebrata, implicating that the three KHDRBS clades originated from sequential WGD events. Notably, absence of KHDRBS3 was also observed in Amazon molly (Poecilia formosa), suggesting that gene loss event may have happened during evolution process. In addition, KHDRBS protein sequences alignment of major vertebrates (Figure 2) showed that KHDRBS2 and KHDRBS3 shared higher identity (range from 44.5 to 69.8%) than that between KHDRBS1 and KHDRBS2 (range from 43.6 to 65.9%) or KHDRBS1 and KHDRBS3 (range from 39.6 to 61.3%), which meant KHDRBS2 and KHDRBS3 formed a more close evolutionary relationship.