Molecular Evolutionary Relationships of Side-Necked Turtle Hemoglobin:cDNA-Derived Primary Structures and Computer-Assisted Tertiary Structuresof α<sup>A</sup>, α<sup>D</sup>, and β Globin Chains

Abstract

Extant side-necked turtles consist of three families: Chelidae, Pelomedusidae, and Podocnemididae. The lasttwo families (the subject of this study) include five genera, three of which are endemic to Africa (Pelomedusa andPelusios) and Madagascar (Erymnochelys), while the other two (Podocnemis and Peltocephalus) are restrictedto South America. This study was undertaken to determine the molecular relationships of side-necked turtlesbased on the primary and tertiary structures of α^A, α^D, and β globin chains as deduced by cDNA sequencing and amachine-learning algorithm for protein structure prediction, AlphaFold2. Analyses of the globin primary structures of four species from different genera provide three major insights. (1) The genera Pelomedusa and Pelusiosmight be endemic taxa that diversified in Africa after the Gondwana separation. (2) Although the extant species ofErymnochelys and Podocnemis are restricted to Africa and South America, respectively, they are closely related toeach other, and thus, these genera apparently diversified during the breakup of Gondwana. Thus, the colonizationof the ancestral population of Podocnemis unifilis in South America might have coincided with continental drift.(3) The divergence times of the four species based on globin genealogy were estimated to have begun 120–11million years ago (mya), which generally coincides with the break-up of Gondwana 135–65 mya. In addition,tertiary structures predicted by AlphaFold2 were compared using a PyMOL overlaying technique. An in-depth examination of the four α^A globin chains revealed considerable consistency among species, and a similar pattern wasobserved for α^D and β globin chains. Additionally, species-specific differences were recognized among the primarystructures, and their higher-order structures (i.e., tertiary structures) were consistent, offering new information onthe molecular phylogeny of side-necked turtles.