Distinct Beak Morph-variation Between The Ground Tit And The Great Tit:the Molecular Basis

Vertebrates, particularly birds, show extremely variable species-specific morphology in craniofacial bone structures. Beak morphological variation is considered as a sub-product under the "ecological force" in birds, and the species-specific beak morphology is vital for their life style and survival mode. Cranial neural crest cells give rise to all the cartilage and bone of the face, and transplantation experiment between duck and quail has shown that these cells contain species-specific patterning information. The expression levels of BMP4, CALM1, TGFBR2, CTNNB1and DKK3were considered to regulate the development of FNP, which is the immediate cause for beak variation, a part of beak structure. The relationship between morphology and underlying developmental causes is high flexibility, where different developmental programs can generate identical shapes, and similar developmental programs can also shape different patterns. Due to the current limitations of less groups and only focused on gene expression levels in previous researches, this article selected the Parus humilis and P. major, and explored their beak variation direction with the geometrical morphology method, and used the RNA-Seq to excavate the molecular basis of beak variation from both expression level and structure variation.1) The standardization with the cube root of weight is the purpose for eliminating the effects of weight on the beak size. The violin plot, t-test and PCA analysis of the linear measurement data showed that P. humilis has longer and relatively more pointed beak than P. major. Then, the geometric morphometrics revealed that the beak shape of P. humilis is more curved. Comprehensive analysis on beak size and shape told that the beak varied from long, pointed, curved ones to short, blunt and straight ones. In view of the differences of habitats and behaviors between P. humilis and P. major, we speculated that the beak had been lengthened and curved for adapting to the foraging and the digging behavior in the alpine meadow habitat of the high Tibetan Plateau. 2) To reduce interference of the false positives, the intersection of Cuffdiff2and DESeq was in favour of analyzing differentially expression.292differentially expressed genes were screened out with the cut-off value of P-value>0.01and|log2(fold change)|>1. Through the function enrichment and clustering analysis, the final selected41differentially expressed genes possibly associated with beak morphological variation, of which15are up-regulate genes and26are down-regulate genes. These genes belong to8signaling pathways which are Wnt, TGF-β,MAPK, Calcium, OCD, Hedgehog, Notch and RAC. And some regulatory genes of these pathways and some TFs that regulate gene expression are inclusive.3) The SNP calling detected359,748SNPs. The coding region possesses51,461SNPs, accounts for only14.305%of the whole.15,371non-synonymous substitution sites and35,168synonymous substitution sites were statisticed. A total of3,963genes with non-synonymous substitution sites were filted out. And Ka/Ks ratio of205genes is more than1calculated with Codeml and MYN methods. Functional enrichment presented26genes suffered positive selection, distribute in Wnt, TGF-β, MAPK, Calcium, Hedgehog, Notch and RAC, and contain some genes regulated the development of locomotive organs.Inter-proteins interactions between26differentially expressed genes and12positive selected genes uncovered that the structure variation of PDGFRA, PDGFC, PLCG1and GLI2regulate the TFs and some signaling pathways including MAPK, OCD and Wnt. This model resulted in the specific beak morphology in P. humilis and P. major. FGF17, FGF13, OTX2, DLX2, SOX1/2, ASCL1, FOXG1and IRX6constitute the up-regulated genes that promote the beak longer and more curved. However, the down-regulated genes consist of WNT5A, WIF1, FRZB, PRICKLE1, CALM3, ADCY2, NFATC1, ITGB3, BLNK, ZEB2and MEOX2, which restrain the beak to become shorter and straighter.