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Archaea are ubiquitous with key role in the biogeochemical cycles of the earth. Archaea are assigned a third domain of life in addition to Bacteria and Eukarya, based on universal small sub unit ribosomal RNA (SSU rRNA) and protein trees. The Archaea look like organisms that use eukaryotic-like proteins in a bacteria like context. The identification of archaeal genomic signatures gives a measure of the distinctiveness of Archaea as a coherent group, although these signatures can differ according to the degree of stringency. A compaartive phylogenetic study using highyly conserved 16s rRNA and the RadA genes may reveal the distinctiveness of the Archae.
Keywords: Archaea, Ubiquitous, Genomic signatures, NJ method, Bootstrap.
Microbiologists and Ecologists have always perceived Archaea as strange, highly typical bacteria. The Archaea are presently known to be metabolically diverse organisms coexisting with Bacteria and Eukarya in the majority of Earth Environments, both terrestrial and aquatic, are including extreme ones, such as high or low pH, low temperature, High salinity or pressure. This fundamental distinction of the archaea from the eubacteria is justified by the unique nature of their lipids, by their lack of murein cell walls, by features of the translation machinery [1-4], by their RNA polymerases [5-6] and even by some metabolic features[7-8]. In accordance to comparative cataloging of 16S rRNA sequences, the kingdom of the archaea consists of a group of related phyla comprising the strictly anaerobic methanogens and the aerobic extreme halophiles on the one hand, and two rather isolated phyla, represented by the extremely thermo acidophilic aerobic genera Thermoplasma and Sulfolobus, on the other hand.
The assignment of Archaea to a third domain of life in addition to Bacteria and Eukarya, based on universal small sub unit ribosomal RNA (SSU rRNA)  and protein trees. Archaeal molecular systems generally show a level of complexity in terms of number of components-half way through that of bacterial and eukaryal ones. A number of ribosomal proteins are uniquely shared between Archaea and Eukarya, while none is uniquely shared with Bacteria or between Bacteria and Eukaryota . The same tendency is found in other molecular systems linked to informational processes such as transcription, protein co-translational targeting and RNA metabolism. However, Archaea are also remarkably similar to Bacteria in many respects, such as the size and organization of their chromosomes, the presence of poly cistronic transcription units and the utilization of Shine-Dalgarno sequences for the initiation of translation . To sum up, the Archaea look like organisms that use eukaryotic-like proteins in a bacterial-like context [12, 13, 14]. Nevertheless, a growing number of studies of archaeal molecular biology are unveiling a rather sophisticated level of complexity, such as the combination of multiple origins for chromosome replication , so far not known in any Bacteria, and the possible implication of chromatin proteins in transcription regulation . Finally, the identification of archaeal genomic signatures gives a measure of the distinctiveness of Archaea as a coherent group, although these signatures can differ according to the degree of stringency [17, 18].
rRNAs are at present the most useful and most used of the molecular chronometers. They show a high degree of functionally constancy, which assures relatively good clocklike behavior . Their sizes are large and they consist of many domains. There are about 50 helical stalks in the 16S rRNA secondary structure and roughly twice that number in the 23S rRNA [20, 21], which makes them accurate chronometers on two counts. According to analysis Carl Woese the Archaea display own characteristic version of the 16S rRNA . The Rad A or the Rec A protein plays the key role in the DNA repair and recombination process. The functional domains of the protein are highly conserved from the lower orders of Prokaryotes to the higher orders of Eukaryotes.
Materials and methods
Data mining of Sequences
Up to date 71 Archaea Genomes have been sequenced and are annotated. These are made available at the NCBI Genome Database. Using the web the DNA sequences coding for the 16SrRNA and the RadA genes are retrieved in the Fasta format. The above mentioned sequences are retrieved for the Escherichia coli (Eubacteria), Chlamydomonas reinhardtii (Protozoa), Saccharomyces cerevisiae (Eukaryote) are used to study the evolution of these forms from the Archaea group.
The multiple sequence alignment of the 74 retrieved sequences is performed with the ClustalW software. Using the multiple sequence alignment of the ClustalW the Phylogenic trees are constructed by the Neighbor joining method in the Molecular Evolutionary Genetics Analysis (MEGA)  software. The further analysis of the genes is carried out using the Distance and Pattern analysis tool in the MEGA software.
Results and discussion
Fig 1: Phylogenic tree constructed using the 16S rRNA gene sequences
Fig 2: Phylogenic tree constructed using the RadA gene sequences
The evolutionary history was inferred using the Neighbor-Joining method . The bootstrap consensus tree inferred from 5000 replicates  is taken to represent the evolutionary history of the taxa analyzed . Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (5000 replicates) is shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method  and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 676 positions in the final dataset for 16SrRNA and 441 positions for RADA gene. Phylogenetic analyses were conducted in MEGA4.
Phylogenetic tree constructed with 16S rRNA genes shows a division of the Euryarchaeota and Crenarcheota and also the other groups. There is no such clear division within the tree constructed by using the RadA genes. A similarity of 54.9% is observed on comparing both the trees using Compare 2 trees .
Fig 3: Comparison of the two phylogenetic trees using the Compare 2 Trees. Dark lines
show the conserved branches and the light lines shows variable ones.
In both the phylogenetic trees the Clades of the taxonomic groups Methanocellales, Methanobacteriales, Halobacteriales, Thermoplasmata, Methanococcales and Thaumarchaeota have been unaltered showing a variable rates in the evolution of the gene sequences. The Clade of the Desulfurococcales in the RadA phylogenetic tree has been introduded by Metallosphaera sedula DSM 5348 showing a close relationship of the organism with the Desulfurococcales. Similarly the clade of the Methanosarcinales branches away at Methanococcoides burtonii DSM 6242 to give rise to the Methanocellales which might have a common ancestor in their evolutionary history.
According to the 16S rRNA the Thermococcales of Euryarchaeota might have evolved primitively from a common ancestor and forms primitive ancestors. The RadA gene might have originated in the Thermoproteales of Crenarchaeota and remains highly conserved all over the groups. This gene of Saccharomyces cerevisiae (Eukaryota) is more closely related to that of Archae than that of Eubacteria and Protozoa. Thus this gene might have been highly conserved in the Eukaryotes.
From the phylogenetic trees it can be inferred that the Cenarchaeum symbiosum A belonging to Thaumarchaeota might have evolved into higher Eukaryota and remains closely related to higher organisms of Protozoa and Eubacteria.
The calculation of distances in the Table 1 shows that the evolutionary distances of the Thaumarchaeota group members showing that the high rate of base substitutions might have occurred in this group thus making the group members closely relating to higher organisms.
Table 1: Statistics of the divergence of selected groups of Archaea.
Mean within groups
Mean diversity within Subpopulations
Mean diversity for entire population
Mean Inter population Diversity
Coefficient of Differentiation
The nucleotide frequencies of 16S rRNA gene sequences are 0.231 (A), 0.172 (T/U), 0.254 (C), and 0.344 (G). The transition/transversion rate ratios are k1 = 1.857 (purines) and k2 = 4.082 (pyrimidines). The overall transition/transversion bias is R = 1.581.
Table 2: Base substitution patterns in the 16S rRNA gene sequences
Maximum Composite Likelihood Estimate of the Pattern of Nucleotide Substitution of 16S rRNA gene sequences. 
The nucleotide frequencies for the RadA are 0.299 (A), 0.215 (T/U), 0.227 (C), and 0.259 (G). The transition/transversion rate ratios are k1 = 1.726 (purines) and k2 = 2.458 (pyrimidines). The overall transition/transversion bias is R = 0.928.
Table 3: Base substitution patterns in the RadA gene sequences
Maximum Composite Likelihood Estimate of the Pattern of Nucleotide Substitution of the RadA gene Sequences.
The transition/transversion bias of the RadA genes is less than the same of the 16S rRNA sequences implying that the RadA sequences have shown a less rate of substitutions in the selected nucleotide bases. High rate C-T transitions are observed in both the gene sets.
From the Tables 4 and Table 5 the Tajima test statistic (D) value for the RadA gene shows value greater than one making the phylogenetic prediction using the RadA genes less reliable than that of 16S rRNA.
Table 4: Results from Tajima's Neutrality Test  for 16S rRNA sequences.
Table 5: Results from Tajima's Neutrality Test  for RadA sequences.
m = number of sites, S = Number of segregating sites, ps = S/m, Θ = ps/a1, and π = nucleotide diversity. D is the Tajima test statistic
The two unrooted trees drawn by NJ method using the MEGA tool predicts that the Archaea might have been evolved from a common unknown ancestor. The Thermococcales or the Thermoproteales might have evolved primarily later followed by other groups. The misposition of the clades of Desulfurococcales by Sulfolobales and Methanocellales by Methanosarcinales shows that they might have evolved from a closely related ancestor. The RadA gene remains highly conserved from Archaea to Eukaryota but shows variance in the Eubacteria and the Protozoa. The taxon Thaumarchaeota shows a high level of variance in both the 16S rRNA and the RadA gene sequences. The species Cenarchaeum symbiosum A shows a high level of similarity with the sequences of higher organisms which shows that it has branched away to higher organisms from a closely related ancestor.