Result and Discussion
Genome organization
The complete mitochondrial genome of Chitala chitala was found to be 16,381 in length which is within the range of other teleost mitogenomes. The genome size was found to be similar with other osteoglossiformes as shown in Table no 2. The genome has 37 protein coding genes just like other animals mtDNAs. There is no substantial variation from the general organization of mitochondrial genome. The genome consists of 13 protein coding genes, 22tRNAs (transfer RNAs), 2rRNAs (ribosomal RNAs) and a D-loop (Boore 1999; Kilpert et al, 2006; Gissi et al, 2008). (Figure 1) (GenBank accession no. _____).
Twelve out of thirteen protein coding genes are encoded on the heavy strand that is typically observed for vertebrate mitochondrial genomes, whereas only ND6 is located on the light strand (Table 1). Two overlapping regions were found namely between ATPase8 – ATPase6 and COI-tRNA Ser. ATpase8 and ATPase6 had the largest overlap of 9 nucleotides.
Overall Base composition
Generally AT skew, GC skew and A+T content are used in the investigation of the nucleotide- compositional behavior of the mitochondrial genomes (Hassanin et al., 2005; Wei et al., 2010). The nucleotide composition was calculated by Mega 6 software. The overall base composition of C. chitala mitogenome is 32.1% for A, 27.7% for C, 15.3% for G and 24.9% for T with A+T bias of 57% as shown in Table 3.
The H strand composition was 30.7% for A, 29.5% for C, 13.8% for G and 25.7% for T with AT biasness of 56.4%, whereas the L strand showed 43.8% for A, 29.9% for C, 12.6% for G and 13.7% for T with AT biasness of 57.5%. D-loop showed the highest AT content of 70.2%.
The rRNA genes, tRNA genes and protein coding genes showed positive AT skew values unlike D-loop which showed negative value. The overall AT skew value was 0.13 and GC skew value was -0.29 showing biasness towards cytosine residue.
Protein coding genes
The total length of 13 protein coding genes is 11,098 bp accounting for 67.7% of the complete genome. The base composition was 31.4% for A, 13.8% for G, 29.5% for C and 25.3% for T. In all the positions of codons (1st, 2nd and 3rd), G was found to be the least frequent nucleotide. The A+T content of protein coding genes were found to be 56.7%. These genes ranged in size from 168bp (ATPase8) to 1839bp (ND5). No obvious deviation was seen from the general organization of mitochondrial genome.
The total number of codons found in protein coding genes of chitala chitala was 3696 including the stop codons. AAU (N) Asparagine (4.56%) is the most frequently found codon among others followed by CCU (P) Proline (4.2%), ACA (T) Threonine (3.35%), AUU (I) Isoleucine (3.27%) and AGC (S) Serine (3.24%). On the other hand GUG (V) Valine (0.08%) and GCG (A) Alanine (0.14%) showed the lowest frequency of codons. All the amino acids were coded by two, three or four different codons except for Leucine and Arginine which were coded by six different codons. (Table 4)
Evolutionary rates of protein coding genes
In protein coding genes synonymous substitutions (Ks) occur more frequently than the non-synonymous substitutions (Ka) (Anton et al., 2002). In this study, few genes namely ND4L, ND5, ND6 and Cyt b did not support the above statement as they showed lower Ks values than Ka. The average Ka/Ks values of thirteen protein coding genes for the species Chitala chitala were <1 except for three genes (ND5, ND6 and Cytb). The genes which show values <1 indicate the existence of purifying selection. The values of Ka/Ks varied from 0.0 (ND4L) to 2.84 (ND5).Most showed values lower than 0.5 except for CO3, ND4, ND5, ND6 and Cytb.
ND5 and ND6 showed the highest values for Ka/Ks depicting that the selection pressures were not dependent on which strand the gene is located. The forces that are not able to adapt like random drift and mutation pressure frame the necessary base for genome evolution. However, the stronger the functional constraint is, the slower its rate of substitution will be so in a way functional constraint forces a burden on mutation. (Michael et al, 2006)
The conservation of mitochondrial genes was studied based on the p-genetic distance among fifteen Osteoglossiformes species which are Chitala chitala, Chitala ornate, Chitala blanci, Chitala lopis, Notopterus notopterus (Thai), Notopterus notopterus (India), Papyrocranus congoensis, Xenomystus nigri, Brienomyrus niger, Gnathonemus petersii, Gymnarchus niloticus, Scleropages formosus, Osteoglossum bicirrhosum, Heterotis niloticus and Arapaima gigas.
Of the 13 protein coding genes CO1 (0.046) showed the lowest value and ND6 (0.335) the highest, based on 1st and 2nd codon. According to the 3rd codon Cytb (0.04) has the lowest value and ATPase6 (0.472) has the highest value, whereas the full sequence analysis revealed CO3 (0.168) to have the lowest value and ATPase8 (0.264) to have the highest. The overall p-genetic distance was comparatively higher for 3rd codon as compared to 1st and 2nd codon or the full sequence which is in line with the fact that in fishes, most of the differences when it comes to protein coding genes occur at the third codon position. (Zhuang et al, 2013).
Heat Map for codon usage bias
The codon usage bias is an important feature that reflects the evolutionary pattern of genome that has been reported in various organisms (Sharp et al, 1988). The codon usage bias was compared between 30 species in which 15 species belong to the order osteoglossiformes forming the ingroup and rest are species from orders Hiodontiformes, Cypriniformes, Clupeiformes, Gonorynchiformes, Salmoniformes, Anguilliformes and Polypteriformes, forming the outgroup. The genetic code plays a crucial role in all living cells. The codon usage biases are affected by nucleotide composition (Osawa et al, 1988), tRNA abundance (Ikemura et al, 1981), protein structure (OreÅ¡iÄ et al, 1998), length (Moriyama et al, 1998), gene function (Chiapello et al, 1998), translation processes (Sharp et al, 1986), environment temperature (Sau et al, 2009), hydrophobicity (Romero et al, 2000) and other factors.
Codons that have RSCU values less than 0.1 were classified as rare codons. Codon usage patterns of protein coding genes for the mitochondrial genomes were investigated by calculating the RSCU values (Supplementary table). The RSCU is the observed frequency of a codon divided by the expected one. If the RSCU values are close to 1 then the synonymous codons are used without any apparent biases. Whereas, if the RSCU values are greater or less than 1, then the codons in question are used more or less frequently than expected, respectively. Bigger RSCU values are represented by darker shades of red and green represents the lower RSCU values as shown in the heat map (Figure 2).
Non-coding region
The control region or D-loop was located between the tRNA Pro and tRNA-Phe. The control region was determined to be 716bp in length and had an overall base composition that was rich in A and T (A+T= 70.2%). The D-loop composition was 34.2% for A, 16.6% for C, 13.1% for G and 36.0% for T.
rRNA genes and tRNA genes
The 12s and 16s rRNA gene of C. chitala is 956bp and 1704bp respectively. These rRNA genes are located between tRNA-Phe and tRNA-Leu and are separated by the tRNA-Val gene. The A+T content of rRNA genes were found to be 54.8%. The rRNA composition was 34.8% for A, 24.8% for C, 20.3% for G and 20.0% for T.
The twenty-two tRNA genes of mitochondrial genome were found to be 1553bp in size. They are found to be scattered between the rRNA and protein coding genes and range in size from 66-75bp. The A+T content of tRNA genes were found to be 56.5%. The tRNA composition was 31.7% for A, 25.0% for C, 18.5% for G and 24.8% for T.
Phylogenetic analysis
Phylogenetic relatedness was studied using the mitogenomes of 29 related fish species taken from NCBI. The phylogenetic tree includes 15 osteoglossiformes species representing 5 families and 11 genera. Other orders which were used as outgroups were Hiodontiformes (1), Gonorynchiformes (1), Cypriniformes (2), Salmoniformes (3), Clupeiformes (2), Anguilliformes (3) and Polypteriformes (3). Total number of species which were taken for phylogenetic analysis was 30 including outgroups.
The results obtained by both the methods i.e. NJ and ML were phylogenetically similar (Figure 3 and 4). Most of the nodes were statistically supported by high posterior probability and bootstrap values. The tree was rooted by a fish species belonging to the order Polypteriformes. Two species Chitala chitala and Chitala lopis are shown to be in the most evolved clade. C. chitala forms a monophyletic group with other species namely Chitala lopis, Chitala ornate, Chitala blanci, Notopterus notopterus Thai and Notopterus notopterus India. This comparative analysis sheds light on the evolutionary history of C. chitala