Complete Mitochondrial Genome of a Tiny Chinese Bitterling Rewrites the Fish Family Tree

Deep in the tributaries of China’s Yangtze and Yellow River systems lives a small, unassuming freshwater fish that has just delivered a surprisingly big evolutionary message. Acheilognathus imberbis, a benthopelagic cyprinid known as a bitterling, has had its complete mitochondrial genome sequenced and characterized for the first time using a rigorous primer-walking strategy, and the resulting data are forcing scientists to rethink how the bitterling family tree is drawn. The study, published in Acta Ichthyologica et Piscatoria, sequenced the entire circular mitochondrial genome of A. imberbis and used it, together with 31 other bitterling species, to resolve long-standing phylogenetic debates within the subfamily Acheilognathinae.

The mitochondrial genome of A. imberbis spans 16,576 base pairs, a length squarely within the typical 16 to 17 kilobase range for vertebrates. It contains the standard vertebrate mitogenomic toolkit: 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding control region. Nucleotide composition analysis revealed a strongly AT-rich genome, with adenine and thymine together accounting for 58.93 percent of all bases. Guanine was distinctly deficient at just 15.88 percent, a bias consistent with what has been observed in typical bony fish mitochondrial genomes and a hallmark of vertebrate mitogenomic architecture.

Gene arrangement followed the conserved pattern seen across most Cyprinidae. Of the 37 encoded genes, 28 sit on the heavy strand while nine, including the ND6 gene and eight tRNA genes, occupy the light strand. Four gene overlaps were identified, three between protein-coding genes, namely ATP8/ATP6 at 6 base pairs, ND4L/ND4 at 7 base pairs, and ND5/ND6 at 4 base pairs, plus a 2-base-pair overlap between tRNA-Ile and tRNA-Gln. Twelve intergenic spacers ranging from 1 to 31 base pairs were found, with the longest gap between tRNA-Asn and tRNA-Cys. The control region, or D-loop, measured 915 base pairs and showed a pronounced A+T preference of 65 percent, reinforcing the anti-guanine tendency typical of fish mitochondrial DNA.

Among the 13 protein-coding genes, COI stood out by using GTG, which encodes valine, as its initiation codon, a feature prevalent among non-tetrapod vertebrates. The remaining 12 genes all began with ATG, encoding methionine. Stop codons varied considerably: complete TAA codons appeared in seven genes, TAG in two, while ND4 carried the incomplete TA and COII, COIII, and Cyt b ended with a single T, presumably completed by post-transcriptional polyadenylation. Codon usage analysis revealed a preference for leucine and alanine, and the relative synonymous codon usage showed that codons ending in A at the third position were used more frequently than expected by chance.

The 22 tRNA genes, totaling 1,561 base pairs, ranged from 67 to 76 base pairs in length and folded into the classic cloverleaf secondary structure with amino acid-accepting, dihydrouridine, anticodon, and TΨC arms plus a variable loop. Two copies each of tRNA-Ser and tRNA-Leu were present, while all other tRNAs appeared once. Notably, tRNA-Ser (AGY) lacked the DHU arm entirely, accompanied by UG and UC mismatches, a nucleotide-economy feature also seen in other cyprinids. The two rRNA genes, 12S at 963 base pairs and 16S at 1,683 base pairs, sat between tRNA-Phe and tRNA-Leu, separated by tRNA-Val.

Within the control region, the team identified several conserved motifs critical for mitochondrial replication and transcription, including the extended termination-associated sequence, the central conserved domain containing CSB-F, CSB-E, and CSB-D, and the conserved sequence blocks CSB-1, CSB-2, and CSB-3. A short TA tandem repeat was detected, and a brief AT duplication occurred between positions 798 and 812. Such insertions and deletions can generate different haplotypes, hinting at the structural plasticity that makes the control region a hotspot for evolutionary studies.

To place A. imberbis in its evolutionary context, the researchers constructed a maximum likelihood phylogenetic tree using concatenated sequences of all 13 protein-coding genes from 37 species, including five outgroup taxa. The best-fitting substitution model, TIM2+F+R4, was selected under the Bayesian Information Criterion, and branch confidence was assessed with 5,000 ultrafast bootstrap replicates. The tree robustly confirmed the monophyly of Acheilognathinae, which split into two primary clades: one containing Acheilognathus and the other comprising the Tanakia–Rhodeus complex. This two-clade structure provides a clearer framework than earlier analyses based on short mitochondrial fragments, which had yielded inconsistent results for the genera Tanakia and Rhodeus.

However, the phylogeny also exposed cracks in current taxonomy. Rhodeus ocellatus and Rhodeus lighti clustered together as a strongly supported sister group that diverged before the split between the core Tanakia and Rhodeus lineages, while Pseudorhodeus tanago nested within the Rhodeus/Pseudorhodeus clade. Most strikingly, Rhodeus albomarginatus was placed within the Acheilognathus clade rather than grouping with other Rhodeus species, an anomalous position the authors urge readers to interpret cautiously given that only a single complete mitochondrial genome of this species is currently available. The monophyletic status of Rhodeus received limited nodal support overall, suggesting that generic boundaries within the subfamily may not fully reflect evolutionary history and that taxonomic revisions could be on the horizon.

Genetic distance analysis using the Tamura–Nei model revealed substantial heterogeneity across the subfamily, with pairwise distances ranging from 0.0071 to 0.2600. The closest relative to A. imberbis was Acheilognathus meridianus at a distance of 0.1153, followed by A. yamatsutae at 0.1461. More provocatively, Acheilognathus gracilis and Acheilognathus rhombeus clustered together on the tree yet showed a genetic distance of only 0.0071, far below the 0.0406 threshold generally associated with significant species or subspecies differentiation in mitochondrial cytochrome b studies. Similarly low divergences appeared between Rhodeus suigensis and R. notatus (0.0162) and between R. suigensis and R. fangi (0.0153). These findings suggest that some nominal bitterling species may warrant re-evaluation using integrative taxonomic approaches that combine molecular, morphological, and ecological evidence.

Positive selection analysis of the ND2 gene, conducted across 29 species using six codon-based site models in PAML, detected no statistically significant codon positions under adaptive evolution. Likelihood ratio tests between nested models showed that while the discrete and beta-plus-omega models fit better than simpler alternatives, the Bayes Empirical Bayes analysis yielded no sites with posterior probabilities of omega exceeding 1 above the 0.95 significance threshold. This absence of positively selected sites indicates that ND2 has evolved predominantly under purifying selection within Acheilognathinae, consistent with the functional constraints on oxidative phosphorylation genes and the relatively stable thermal and ecological conditions these fish inhabit. The result underscores the conservative nature of mitochondrial energy metabolism genes and suggests limited lineage-specific adaptive divergence in this group. Taken together, the complete mitogenome of A. imberbis enriches the molecular resources available for bitterling systematics, sharpens the subfamily’s phylogenetic framework, and highlights both the power and the limits of mitochondrial inference, pointing toward future studies that incorporate nuclear genomic data and broader taxon sampling to achieve a more stable understanding of bitterling evolution.

Subject of Research: Mitogenomics and phylogeny of the bitterling fish Acheilognathus imberbis

Article Title: Mitogenomic architecture of Acheilognathus imberbis ( Actinopterygii , Cypriniformes , Cyprinidae ): Implications for Acheilognathinae phylogeny

Article References: Liu, G., Peng, D., Zhang, H., Song, D., Liu, B., & Cheng, Q. (2026). Mitogenomic architecture of Acheilognathus imberbis (Actinopterygii, Cypriniformes, Cyprinidae): Implications for Acheilognathinae phylogeny. Acta Ichthyologica et Piscatoria, 56, 181-194. https://doi.org/10.3897/aiep.56.181178

Image Credits: AI Generated

DOI: 10.3897/aiep.56.181178

Keywords: mitochondrial genome, Acheilognathus imberbis, bitterling, phylogenetics, Acheilognathinae, Cyprinidae, genetic distance, positive selection, ND2 gene, taxonomic revision, Mitogenomic, architecture