Recombination and evolution of duplicate control regions in the mitochondrial genome of the Asian big-headed turtle, Platysternon megacephalum

Abstract

Complete mitochondrial (mt) genome sequences with duplicate control regions (CRs) have been detected in various animal species. In Testudines, duplicate mtCRs have been reported in the mtDNA of the Asian big-headed turtle, Platysternon megacephalum, which has three living subspecies. However, the evolutionary pattern of these CRs remains unclear. In this study, we report the completed sequences of duplicate CRs from 20 individuals belonging to three subspecies of this turtle and discuss the micro-evolutionary analysis of the evolution of duplicate CRs. Genetic distances calculated with MEGA 4.1 using the complete duplicate CR sequences revealed that within turtle subspecies, genetic distances between orthologous copies from different individuals were 0.63% for CR1 and 1.2% for CR2app:addword:respectively, and the average distance between paralogous copies of CR1 and CR2 was 4.8%. Phylogenetic relationships were reconstructed from the CR sequences, excluding the variable number of tandem repeats (VNTRs) at the 3' end using three methods: neighbor-joining, maximum likelihood algorithm, and Bayesian inference. These data show that any two CRs within individuals were more genetically distant from orthologous genes in different individuals within the same subspecies. This suggests independent evolution of the two mtCRs within each P. megacephalum subspecies. Reconstruction of separate phylogenetic trees using different CR components (TAS, CD, CSB, and VNTRs) suggested the role of recombination in the evolution of duplicate CRs. Consequently, recombination events were detected using RDP software with break points at ≈290 bp and ≈1,080 bp. Based on these results, we hypothesize that duplicate CRs in P. megacephalum originated from heterological ancestral recombination of mtDNA. Subsequent recombination could have resulted in homogenization during independent evolutionary events, thus maintaining the functions of duplicate CRs in the mtDNA of P. megacephalum.

Figure 1. Locations of duplicate CRs and primers used for amplification.

G17/G20 are outer primers for CR1; G21/G22 are outer primers for CR2. These primer pairs were used to determine the presence of CRs in the mtDNA of individual turtles. Inner primers, CR1S/CR1X and CR2S/CR2X, are designed to amplify specific duplicate CRs. The names of tRNA genes are shown using abbreviations of amino acids (T: tRNA^Thr, P: tRNA^Pro, F: tRNA^Phe, V: tRNA^Val, L: tRNA^Leu, I: tRNA^Ile, H: tRNA^His, S: tRNA^Ser, Q: tRNA^Gln, M: tRNA^Met). Gray boxes represent non-coding spaces between two genes. CR1 and CR2 are indicated with slashes.

Figure 2. Structures of duplicate CRs in individuals.

Three functional regions, TAS, CD, and CSB, were detected in both CRs in all individuals. The core sequences of these regions were found to be identical in CR1 and CR2. Variable numbers of tandem repeats (VNTRs) were detected only in CR1.

Figure 3. Heterology of VNTRs in different subspecies.

The line at the bottom of the image indicates the lengths of VNTRs. The numbers above the dots represent the copy numbers of motifs.

Figure 4. Phylogenetic trees reconstructed using (a) neighbor-joining, (b) maximum likelihood and Bayesian methods.

Three clades were grouped. These contained the three subspecies of Platysternon megacephalum: (A) P.m. megacephalum, (B) P.m. shiui, and (C) P.m. peguense.

Figure 5. Phylogenetic trees reconstructed using (a) TAS, CD, and CSB domains and (b) VNTRs.

All three phylogenetic trees constructed separately based on TAS, CD, and CSB shared similar topology and therefore only one tree is presented here.

Figure 6. Assessment of recombination in duplicate CRs detected by RDP software in an individual, AH2.

Green shade indicates pairwise identity between orthologous CR2 genes in individuals, TP and AH2. Purple shade depicts pairwise identity between paralogous CRs from AH2. Similar analysis was performed for all individuals.