Abstract
Megalobrama is China's most economically valuable fish genera. Four species make up this genus: M. amblycephala (MA), M. terminalis (MT), M. pellegrini (MP), and M. hoffmanni (MH). Many researchers have investigated the genetic relationship of Megalobrama based on mitochondrial DNA (mtDNA) and discovered that the branches of the phylogenetic tree for MT and MP are intertwined. We hypothesized that this occurs because mitogenomic heteroplasmy is overlooked when working with mtDNA, which causes MP and MT positions to intersect in phylogenetic trees. To eliminate the influence of nuclear mitochondrial DNA fragments (NUMTs) before analyzing mitogenomic heteroplasmy, we used PLastZ to identify NUMTs, which were then removed from the samples for the subsequent heteroplasmy analysis. Using the heteroplasmy caller icHET, we discovered 126, 339, 135, and 203 heteroplasmic variants in six MA, MT, MP, and MH samples. We reconstructed the Megalobrama fish genus's phylogenetic tree using the RY coding method and rejecting the third position on codons, which improved the performance of the phylogenetic tree by increasing the ratio of treeness to relative component variability from 100.02 ± 1.76 to 688.59 ± 190.56. Despite this, the RY coding method cannot alter the intersection of MP and MT positions in phylogenetic trees. We hypothesize that gene flow between MT and MP leads to intertwining mtDNA-based phylogenetic trees. In conclusion, our findings on the mitogenomic heteroplasmy of Megalobrama provide new insights into mtDNA-based phylogenetic studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-023-03523-0.