Codon usage bias is a common feature in most organism genome including prokaryotes and eukaryotes, and may contribute to genome evolution in a profound manner (Behura and Severson 2013; Zhou et al. 2016). The two widely referred theories that explained the foundation of codon usage bias in organisms are neutral theory and selection-mutation drift model (Duret 2002; Deb et al. 2020). According to neutral theory, silent mutations in the codons` third position rise the phenomenon of codon preference (Subramanian 2008). Whereas, selection-mutation drift model suggest that, CUB occurs as an equilibrium event to balance natural selection, mutation pressure and genetic drift (Bulmer 1991; Duret 2002). Individual genes show variation in codon usage pattern; however, even within a gene the pattern of codon usage may vary depending on the position of the codon (Gerdol et al. 2015). The coding sequences of a gene and/or genome provide information on gene function and hence evolution of the organism (Behura and Severson 2013; Zhou et al. 2016). Thus, studies on the patterns of codon usage including their causes, consequences, as well as selective forces shaping their evolution are significant in understanding genome biology and population diversification.
In this study, compositional aspects and codon usage properties for MT-COI gene in Cerithidea, Cerithideopsilla, Cerithideopsis and Terebralia genera of Potamididae were analyzed. Findings from the present study have demonstrated that both natural selection and mutation pressure were contributing factors on CUB and evolutionary variations among Potamidids. From PR2 plot (Fig. 4) it was found that, gene spots deviated from base composition rule (Chargaff`s second parity rule) that is, A and T were not proportionate with C and G (Fig. 4). This suggested that a combinatorial action of mutation pressure and natural selection influenced CUB in the species studied. Although parity plot reflected the main factors influencing codon usage bias in different genera of Potamidids, there was no precise estimation of whether mutation pressure or natural selection was the most significant factor. From neutrality plots for all genera (Fig. 5), negative correlation between GC3% and GC12% was found, corroborating that natural selection was significant factor to evolutionary changes and CUB for MT-COI gene. However, the exception was in genus Cerithidea, where positive correlation between GC3% and GC12% was observed suggesting that other evolutionary forces like mutation pressure, genetic drift and non-random mating were contributing factors to evolutionary changes and CUB for MT-COI gene. Narrow GC content, which was less than 50%, further suggests that natural selection was the most significant factor for codon usage pattern of MT-COI gene in Potamidids. In support of these findings, other studies in reptiles (Chakraborty et al. 2022) and amphibians (P. A. Barbhuiya et al. 2021) concluded that natural selection and mutation pressure were the major and minor evolutionary forces respectively, responsible for shaping CUB in mitochondrial genes.
Furthermore, other compositional features parameters that explain possible codon usage pattern of MT-COI gene among different species of the family Potamididae were found to be interesting. For example, third position of codons was mostly occupied by nucleobase T suggesting that codons ending with T were more preferred as compared to other nucleotides. It is known that third codon position is subjected to silent mutations pressure (Chamary and Hurst 2009; Alberts 2015) therefore, when studied in details such investigations play a vital role in interpretation of codon trend of the various genes. In the present study, silent mutations at third codon positions involving T nucleobase was observed to be common as a result codon ending with T nucleobase were highly favored. Additionally, slight difference was observed in positional GC contents (GC1, GC2 and GC3) among the four genera of Potadimidae (Fig 02). These findings agree with the postulate that compositional properties might have effects on the codon usage pattern of a gene (Deka and Chakraborty 2014; Choudhury et al. 2018; Barbhuiya et al. 2020). Moreover, codon usage bias of MT-COI gene was found to be weak because the ENC values in the present study was greater 35, but was less 50. These findings are supported by earlier investigations which revealed low CUB in mitochondrial ATP, ND and CO genes of amphibians, arthropods and reptiles respectively (R. I. Barbhuiya et al. 2019; P. A. Barbhuiya et al. 2021; Chakraborty et al. 2022).
The coding sequences of MT-COI gene were evaluated for their relative synonymous codon usage (RSCU). There was variability in RSCU values among and between four genera of Potamididae in the present study. Interestingly, some codons were overrepresented across different genera while others were underrepresented (Table 3). Specifically, five codons (CUU, GUU, UCU, ACU, and AGG) were found to be commonly overrepresented across all four genera of family Potamididae, and six codons (GGA, CGA, GAG, UAC, CUG, CUA) were commonly underrepresented across the four different genera in the family Potamididae (Table 3). It has been reported that similar codon usage patterns including over and underrepresented codons in genomes ensures translational efficiency (Tuller et al. 2010), thus, the observed similar pattern in codon preference might be due to translational selection. Further, a multivariate statistical approach of correspondence analysis (CoA), utilizing RSCU values, was performed to clearly define the varying trends in codon usage among potamidids. A chunk of codons were situated close to the axes and some concentrated in the center of the plot proposing that nucleotide compositional constraints under mutation pressure might associated with the CUB of MT-COI gene. Earlier investigations on codon usage bias in Harpago chiragra and Lambis lambis (Gastropoda: Stromboidea) also revealed that the mitochondria genes were subjected to different mutational pressures which signified compositional constraints (Jiang et al. 2019). Similar findings were observed in reptiles (Chakraborty et al. 2022), cotton species (Wang et al. 2018), and bivalves (Gerdol et al. 2015).
This study was performed to deduce factors that might favor compositional properties of MT-COI gene in family potamidids. Furthermore, the evolutionary pattern of MT-COI gene in the family Potamididae was evaluated. Low level of CUB was observed in MT-COI gene of potamidids. The codon usage pattern was different across the different genera. The two evolutionary forces, mutation pressure and natural selection were found to be significant in determining the codon usage bias of the gene under study. However, natural selection had a dominant role while mutation pressure had a submissive role in influencing codon usage bias of MT-COI gene in different genera within the family Potamididae.