Cluster analyses using the amino acid content predicted from the coding regions (13 genes) of complete vertebrate mitochondrial genomes as traits grouped selected vertebrates into two clusters, terrestrial and aquatic vertebrates. Exceptions were the hagfish (Eptatretus burgeri), thought to be an early ancestor of vertebrates, and the black spotted frog (Rana nigromaculata), which is terrestrial as an adult and aquatic as a larva. These two species fall into the terrestrial and aquatic clusters, respectively. Using the nucleotide (G, C, T and A) content in the coding and non-coding regions, and in the complete genome as traits, similar results were obtained but with some additional exceptions. In addition, phylogenetic analyses of 16S rRNA sequences produced a consistent result. The results of this study indicated that vertebrate evolution is controlled by natural selection under both an internal bias as a result of nucleotide replacement genomic rules, and an external bias caused by environmental biospheric conditions.
The concept of natural selection was established by Charles Darwin and Alfred Wallace 150 years ago. This theory was derived from specific differences or similarities in the phenotypes of organisms that lived on geologically isolated islands. The theory of biological evolution has been further developed by palaeontology [
Sorimachi [
It has been reported that in complete single-stranded DNA genomes, the nucleotide content alternations were strictly controlled, and were expressed by linear regression lines of nucleotide content relationships in both coding and non-coding regions [
Mitochondrial genome data were obtained from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/sites). The organisms examined has been described previously [
In the analysis of amino acid compositions in the human (Homo sapiens) mitochondrial genome, the Leu content was extremely high compared with the other amino acids (
Met contents were significantly inverse compared with their content in human mitochondria. The relationships between the amino acid contents of human and bony fish (O. latipes) were used to represent the characteristics of terrestrial and aquatic vertebrates (
The black spotted pond frog (Rana nigromaculata), which is terrestrial as an adult and aquatic as a larva (tadpole) had an amino acid content pattern that was midway between the terrestrial and aquatic patterns (
matched that of the terrestrial group (
The coelacanth (Latimeria chalumnae) is thought of as a living fossil but the amino acid composition of the mitochondrial genome was similar to that of human mitochondria, although the Asp, Glu and Ser content pattern was intermediate between that of human and bony fish (
Hagfish (Eptatretus burgeri) are thought to be the origin of vertebrates [
The relationships among the amino acid contents of Asp, Glu and Ser, between Thr and Ala, and between Val and Met resembled the terrestrial vertebrate pattern. An exception was the high Pro content, which was a characteristic of the hagfish mitochondrial genome.
In previous studies [12,18], 12 eubacteria were classified into two groups, G type representing Staphylococcus aureus and E type representing Escherichia coli, based on their amino acid compositions [
Using the amino acid composition as the trait, the vertebrates examined in this study were separated into two major clusters (
Using the G, C, A and T content of the coding regions of the mitochondrial genomes as the trait for cluster
analyses, the vertebrates again separated into the two major clusters (
In the non-coding regions of the mitochondrial genomes, some of the terrestrial vertebrates clustered with the major aquatic group (
Using the G, C, T and A content of the entire genome as the trait, two major clusters were formed (
A consistent result was obtained by the Neighbourjoining method [
Using the nucleotide contents as traits, the vertebrates in the sample were classified into two major clusters, terrestrial and aquatic; however, some exceptions were observed (Figures 2-7). These results indicated that alternations in nucleotide content were controlled by internal biases that, in the cluster analyses, resulted in the formation of several clusters. Because nucleotide alternations are strictly governed by linear formulae in both the coding and non-coding regions [
separation between the terrestrial and aquatic vertebrates might not have been observed in the cluster analyses. Using amino acid content, the vertebrates in the sample were similarly classified into two major clusters, terrestrial and aquatic. The exceptions were the hagfish (E. burgeri) and the frog (R. nigromaculata) that clustered with the terrestrial and aquatic vertebrates, respectively (Figures 2-7). In the cluster analyses using the amino acid compositions at traits, the decrease in the number of exceptions might be because of the inclusion of phenotype expressions that were reflected in the amino acid composition [
Although the frog (R. nigromaculata) was consistently grouped with the aquatic vertebrates, this may reflect the conservation of tadpole characteristics after metamorphosis. The clustering of the hagfish (E. burgeri) with the terrestrial vertebrates may reflect the controversy over the classification of this fish [
line better than the invertebrate regression line, and the vertebrate mitochondria diverged from the same origin of invertebrate mitochondria. If the hagfish truly belongs to the terrestrial group it suggests that hagfish still possess some primitive vertebrate mitochondrial characteristics that were present before its evolution. Terrestrial and aquatic vertebrates have evolved independently, and vertebrate evolution under natural selection has occurred based on both nucleotide alternation rules and on biosphere biases. In this study, the vertebrates were classified completely into the terrestrial and aquatic groups indicating that the barriers between these two biospheres have been definitive for vertebrate evolution. Because the mitochondrial function is not linked directly with either the genomic rules or with the biospheres, the present findings reflect vertebrate evolution itself.
Vertebrates were classified into two clusters, terrestrial and aquatic vertebrates, in the phylogenetic trees based on the amino acid content predicted from the complete mitochondrial genomes, nucleotide content of the complete mitochondrial genomes and 16s rRNA sequence homologies, with some exceptions. The hagfish (Eptatretus burgeri), which is thought to be an early vertebrate,
falls into the terrestrial group. Thus, vertebrate evolution is controlled by natural selection under both an internal bias as a result of nucleotide replacement rules, and an external bias caused by environmental biospheric conditions.