Complete mitochondrial genomes of three neobatrachian anurans: a case study of divergence time estimation using different data and calibration settings.

We sequenced the whole mitochondrial (mt) genomes of three neobatrachian species: Japanese tree frog Hyla japonica, Japanese common toad Bufo japonicus, and narrow-mouthed toad Microhyla okinavensis. The gene arrangements of these genomes diverged from that of basal anurans (suborder Archaeobatrachia), but are the same as that of the members of derived frogs (i.e., superfamily Hyloidae and Ranoidae in suborder Neobatrachia), suggesting the one-time occurrence of a gene rearrangement event in an ancestral lineage of derived anurans. Furthermore, several distinct repeat motifs including putative termination-associated sequences (TASs) and conserved sequence blocks (CSBs) were observed in the control regions (CRs) of B. japonicus and H. japonica, while no repeat motifs were found in that of M. okinavensis. Phylogenetic analyses using both nucleotide and amino acid data of mt genes support monophyly of neobatrachians. The estimated divergence time based on amino acid data with multiple reference points suggests that the three living amphibian orders may have originated in the Carboniferous period, and that the divergences of anurans had occurred between the Permian and Tertiary periods. We also checked the influence of the data types and the settings of reference times on divergence time estimation. The resultant divergence times estimated from several datasets and reference time settings suggest that the substitution saturation of nucleotide data may lead to overestimated (i.e., older) branching times, especially for early divergent taxa. We also found a highly accelerated substitution rate in neobatrachian mt genes, and fast substitution possibly resulted in overestimation. To correct this erroneous estimation, it is efficient to apply several reference points among neobatrachians.

Complete nucleotide sequence of the mitochondrial genome of a Malagasy poison frog Mantella madagascariensis: evolutionary implications on mitochondrial genomes of higher anuran groups.

We determined the complete nucleotide sequence of the mitochondrial (mt) genome of a Malagasy poison frog, Mantella madagascariensis (family Mantellidae), and partial sequences of two Mantella (M. baroni and M. bernhardi) and two additional mantellid species (Boophis madagascariensis and Mantidactylus cf. ulcerosus). The M. madagascariensis genome was shown to be the largest (23kbp) of all vertebrate mtDNAs investigated so far. Furthermore, the following unique features were revealed: (1) the positions of some genes and gene regions were rearranged compared to mitochondrial genomes typical for vertebrates and other anuran groups, (2) two distinct genes and a pseudogene corresponding to transfer RNA gene for methionine (tRNA-Met) were encoded, and (3) two control regions with very high sequence homology were present. These features were shared by the two other Mantella species but not the other mantellid species, indicating dynamic genome reorganization in a common ancestor linage before divergence of the Mantella genus. The reorganization pathway could be explained by a model of gene duplication and deletion. Duplication and deletion events also seem to have been responsible for concerted sequence evolution of the control regions in Mantella mt genomes. It is also suggested that the pseudo tRNA-Met gene sustained for a long time in Mantella mt genomes possibly functions as a punctuation marker for NADH dehydrogenase subunit (ND) 2 mRNA processing. Phylogenetic analyses employing a large sequence data set of mt genes supported the monophyly of Mantellidae and Rhacophoridae and other recent phylogenetic views for ranoid frogs. The resultant phylogenetic relationship also suggested parallel occurrence of two tRNA-Met genes, duplicated control regions, and ND5 gene translocation in independent ranoid lineages.