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Complete Mitochondrial DNA Sequences of Six Snakes: Phylogenetic Relationships and Molecular Evolution of Genomic Features

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Abstract

Complete mitochondrial DNA (mtDNA) sequences were determined for representative species from six snake families: the acrochordid little file snake, the bold boa constrictor, the cylindrophiid red pipe snake, the viperid himehabu, the pythonid ball python, and the xenopeltid sunbeam snake. Thirteen protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 control regions were identified in these mtDNAs. Duplication of the control region and translocation of the tRNALeu gene were two notable features of the snake mtDNAs. The duplicate control regions had nearly identical nucleotide sequences within species but they were divergent among species, suggesting concerted sequence evolution of the two control regions. In addition, the duplicate control regions appear to have facilitated an interchange of some flanking tRNA genes in the viperid lineage. Phylogenetic analyses were conducted using a large number of sites (9570 sites in total) derived from the complete mtDNA sequences. Our data strongly suggested a new phylogenetic relationship among the major families of snakes: ((((Viperidae, Colubridae), Acrochordidae), (((Pythonidae, Xenopeltidae), Cylindrophiidae), Boidae)), Leptotyphlopidae). This conclusion was distinct from a widely accepted view based on morphological characters in denying the sister-group relationship of boids and pythonids, as well as the basal divergence of nonmacrostomatan cylindrophiids. These results imply the significance to reconstruct the snake phylogeny with ample molecular data, such as those from complete mtDNA sequences.

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Acknowledgments

We thank Mr. K. Yagi, Drs. M. Nishida and D. Wake, Remix Peponi Co., and the Museum of Vertebrate Zoology, University of California at Berkeley, for providing animal samples. We also thank Dr. T. Nishikawa and Nagoya University Museum for the curation of our specimens and Ms. C. Aoki for her excellent experimental assistance. Gratitude is extended to Dr. H. Shimodaira, two anonymous reviewers, and the Associate Editor for valuable comments on the phylogenetic tests. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Nos. 12640680 and 14540641).

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Correspondence to Yoshinori Kumazawa.

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Appendix

Appendix

Complete mtDNA Sequencing for Six Snakes

We employed an efficient and accurate method for sequencing reptile mtDNAs using the long-and-accurate polymerase chain reaction (LA-PCR) amplification of mtDNAs followed by sequencing and assembling nested PCR products with a number of reptile-oriented primers (Kumazawa and Endo 2004). Using the LA-PCR technology, we amplified mtDNA segments for each taxon with the combinations of primers shown in the note to Table A1. Because snake mtDNAs typically contain two CRs, inclusion of two CRs in the same PCR target may cause the jumping PCR (Pääbo et al. 1990) to give rise to artifactually shorter products. This problem was carefully avoided in this study by separating mtDNA sequences into three to five portions for the amplification targets.

More than 85% of the whole mtDNA sequences for the six snakes were successfully amplified and sequenced with the reptile-oriented primers (Kumazawa and Endo 2004). In general, primers designed for some extremely variable regions (e.g., CRs and 3′ end portion of ND5 gene to ND6 gene) were less effective in the PCR amplification. These gaps could be readily filled by the primer walking strategy. The boa constrictor and the sunbeam snake had long tandem repeats within CRs and sequencing from surrounding primers did not extend over the repeated region. We estimated the length of the repeated region from the size of an amplified product using these primers and then carefully assembled sequences obtained from these primers in light of the length of a repeat unit.

Table A1 Taxon-specific and common primers designed for initial amplification of mtDNA segments

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Dong, S., Kumazawa, Y. Complete Mitochondrial DNA Sequences of Six Snakes: Phylogenetic Relationships and Molecular Evolution of Genomic Features. J Mol Evol 61, 12–22 (2005). https://doi.org/10.1007/s00239-004-0190-9

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