Structure and compositional characteristics of mitochondrial genome
The full-length mitochondrial genome sequence of albino
E. eleusis determined in this study was 16,347 bp, containing 37 coding genes and a control region (OH, OL) (Fig 2A), which was similar to the genome length of other normal
Eothenomys species that have been published. Moreover, the structure, composition and distribution pattern of the genome also showed a high degree of consistency, further confirmed its high conservation in the evolutionary process
(Yang et al., 2012; Chen et al., 2015; Mu et al., 2019; Zhu et al., 2023). Specifically, among the 37 genes, the “-” chain contains 9 genes, with 1 gene responsible for encoding proteins and the remaining 8 genes involved in the synthesis of tRNA, while the “+” chain contains 28 genes, with 12 PCGs, 14 tRNAs and 2 rRNAs. It’s worth noting that there were intervals or overlaps phenomenon between different genes and control regions in the genome, namely 16 gene intergenic regions (1-416 bp), the longest between
trnP and OH and 8 gene overlap regions (1-43 bp), the longest between
trnK and
atp8 (Table 1).
In addition, among the base compositions and skewness of all genes, the A + T content of the whole genome was 59.19%, much higher than the G + C content of 40.8% and the AT_skew was 0.115, indicating a significant. preference for AT. Moreover, all genes except for OH had a positive AT_skewand all genes except for OL had a negative GC_skew (Fig 2A, Table 2).
This preference was similarly present in other species of
Eothenomys, with only the specific AT content and skewness ratios varying slightly due to differences among species
(Mu et al., 2019; Zhu et al., 2023). However, compared to the impact of codon preference on translation efficiency, the differences in base composition of the mitochondrial genome were more closely related to the specificity of the coding chains
(Niu et al., 2024).
PCGs, RSCU and RNAs
The 13 PCGs of albino
E. eleusis typically followed standard genetic code rules when encoding proteins, using ATN (including ATT, ATG, ATA) as the start codon and TAN (including TAA, TAG) as the stop codon. However, there were also some exceptions, such as
nad1 gene employed an atypical GTG as the start codon, while
nad1,
cox3 and
nad4 genes end their coding sequences with T (
i.e. AA) as the incomplete stop codon (Table 1). Further analysis of the codon preference, or RSCU, of these PCGs discovered that 25 codons had RSCU values > 1, of which 14 ended in A/U, indicating that the third position of the codon tended to preferentially use A/U bases. Among these codons, the most commonly used codons were CUA, AUC and AUA, with RSCU values of 2.95, 1.16 and 1.61, respectively (Fig 3A).
Among all encoded amino acids, Leu1 (12.68%), Ile (9.96%) and Thr (8.08%) had relatively high contents, while Arg (1.72%), Ser1 (1.29%) and Cys (0.82%) had relatively low contents (Fig 3B). These commonly used codons and amino acids further reinforce the clear AT preference of this genome. Specifically, the stop codons UAA and UAG didn’t correspond to the translation of any amino acid and the codons AGA and AGG weren’t used in the translation of Arg in albino
E. eleusis (Fig 3A). In addition, the gene lengths of
rrnS and
rrnL were 948 bp and 1,565 bp, respectively. The gene length of 22 tRNAs ranged from 59 bp (
trnS1) to 75 bp (
trnL2) (Table 1) and except for the
trnS1 gene, which lacked DHU arm, all the other genes exhibited a standard clover structure. Meanwhile, many U-G mismatches were also observed in the secondary structure of tRNAs (Fig 4), but these mismatches didn’t impact subsequent transcriptional functions.
On the contrary, they may play a crucial role in maintaining the secondary structural stability of tRNA (Varani and McClain, 2000;
Chen et al., 2024).
Phylogenetic and evolutionary rates analyses
Based on the constructed maximum likelihood tree, the results indicated that albino
E. eleusis was a sister branch of normal
E. eleusisand was grouped with
E. miletus,
E. cachinus and
E. melanogaster. This phenomenon has been validated through high bootstrap values (Fig 2B), which was consistent with the latest research results of
Abramson et al., (2021), Wang et al., (2022) and
Zhu et al., (2023) on
Eothenomys. In addition, mitochondrial genomes often exhibit adaptive evolution under multiple selection pressures, providing necessary energy for organisms to adapt to changing environments
(Liu et al., 2023). So, for the analysis of the evolutionary rates of the 13 PCGs in
Eothenomys,
atp8 and
nd4l genes had higher dN/dS mean values, suggesting that they may had undergone rapid evolution or some changes in function. On the contrary,
cox1 and
cox2 genes had lower dN/dS mean values, indicating that their evolution was relatively slow and their functions were relatively conserved. This was basically consistent with previous studies by
Sun et al., (2023), Chen et al., (2024) and
Ghosh et al., (2024). Subsequently, further statistical analysis showed that there were also significant differences in the evolutionary rate between different genes (P<0.05), among which the differences between
atp8 and
nd4l genes were not significant, but significant differences between
atp8 and other genes (Fig 5).
Therefore,
cox1 and
cox2 genes can be used as molecular markers for phylogenetic reconstruction and species identification of
Eothenomys.
