The Molecular Mechanism Of The Evolution Of Whale Brain

Cetaceans are a mysterious group of secondary-adapted marine mammals, the common ancestor of which was diverged from terrestrial artiodactyls approximately 53 million years ago (Mya). Fossil and anatomical evidence showed that cetacean brain size expansion throughout its evolutionary history. Toothed whales especially for family delphinoidea had increased relative brain size (measured by "encephalization quotient", abbreviation for EQ). The brain mass of mysticetes increased whereas their body mass increased at a much rapid rate, leading to a decrease in EQ. Previous ecological studies had shown that odontocetes have high degrees of encephalization primarily as an adaptation for living in complicated social groups, as asserted by "social brain hypothesis", yet genetic basis of brain size evolution in cetaceans remains poorly explored. Primary microcephaly (MCPH) genes play key roles during brain development due to the fact that MCPH gene mutations can cause a severely developmental defect of cerebral cortex. In the present study, evolution of seven MCPH genes (MCPH1-7:MCPH1, WDR62, CDK5RAP2, CEP 152, ASPM, STIL and CENPJ) was investigated in representative species of major cetacean lineages. First, we explored selective pattern of seven MCPH genes acted on cetacean lineages and tested whether different lineages evolved under different selection pressure. Second, we explored the putative association between the evolutionary rate of MCPH genes and some morphological variables of cetacean brains. Thirdly, the correlation between MCPHs evolution and group size was examined to test whether there was evidence to support the "social brain hypothesis" at the molecular level. Finally, we tested whether adaptive functional changes of such MCPHs under positive selection occured in the cetaceans.A total of 17 representative cetacean species (3 mysticetes and 14 odontocetes) were used in our study. All the exons of seven MCPHs were obtained by PCR amplified and downloaded from NCB1. Protein sequences of MCPH1 were aligned by MEGA6.0. Inactivating mutations (frameshift insertion/deletion, premature stop codon, disrupted intron splice sites) were found at MCPH] in 10 cetacean species. More importantly, some pseudogenized mutations in cetacean lineages located in the functional domains of the MCPH1 gene. All the evidence of inactivating mutation implied that MCPH1 might be pseudogene in major cetacean lineages. This suggestion was further supported that relaxed selective pressure was examined at the pseudogenized cetacean lineages and the functional constraints on cetacean MCPHl were not completely removed. Sequences of six genes (MCPH2-MCPH7) from the cetacean species were intact, without premature stop codons or frameshift mutations, suggesting the presence of a functional protein in each gene in cetaceans. For cetaceans, site-specific model that incorporate selection fitted the data significantly better than did the neutral model at five MCPH genes, i.e. WDR62, CDK5RAP2, CEP152, ASPM, and STIL (P< 0.001), suggesting that the five MCPH genes were subjected to positive selection. In addition, positive selected sites were identified by three maximum likelihood (ML) methods, i.e.M8, FEL and REL. Sixty positively selected sites were picked out by two ML methods, whereas only 11 codons were picked out by three methods. The result of TreeSAAP revealed that 88.33% sites were examined to be radical changes at the protein-level. We predicted the three-dimensional (3D) structures of MCPH by the I-TASSER, and mapped all the positive selected sites identified by at least two ML methods that occured radical changes onto the 3D structures. Notably, up to 50% of these positively selected sites were localized in the functional region of the five MCPH genes. Lineage-specific models test revealed extensive positive selection at CDK5RAP2 along lineages with both increased and decreased EQ. In contrast, only odontocetes with expansed EQ were identified to be under positive selection at ASPM, STIL and WDR62. More importantly, a significantly positive association between evolution rate (root-to-tip ω) and relative brain size (expressed by EQ) was identified at both CDK5RAP2 and ASPM (R2 CDK5RAP2= 0.397, P CDK5RAP2= 0.022; R2 ASPM= 0.547, P ASPM= 0.006), suggesting the both genes might contribute to the brain size expansion in cetaceans. We performed GST-pulldown assay and Octet protein-protein interaction between IQ motif including one positively selected site 1684 of the ASPM and CaM, the results showed that the odontocetes with highest EQs had stronger effect on calmodulin binding than for the mysticetes, which suggested that adaptive functional changes might occur in lineage with expansion EQ. Interesting, significantly or nearly significantly positive associations between evolution rate and mean group size (as one measure of social complexity) were examine at the ASPM, CDK5RAP2, and CEP 152 (R2CDK5RAP2= 0.213, PCDK5RAP2= 0.055; R2CEP152= 0.167, PCEP152= 0.082; R2ASPM= 0.315, P aspm= 0.022). The result was consistent with the social brain hypothesis that cetaceans evolved large brains to manage their unusually complex social systems.