Replication slippage footprint in the mitochondrial non-coding region: extreme structural variation and GC skew gradient analysis in conspecific Dactylogyrus from two geographic origins

Abstract Background Tandem repeats in the mitochondrial non-coding region (NCR) are a major driver of genome size variation. Although the genus Dactylogyrus has been used as a model for studying host-parasite coevolution, intraspecific structural variation of its mitochondrial NCR remains largely unexplored. Methods We sequenced and compared the complete mitochondrial genomes of one representative individual each of Dactylogyrus baueri (both parasitizing Carassius auratus ) from two independent river systems, the Lancang River (Nanjian) and the Irrawaddy River (Ruili). Stepwise PCR amplification combined with Sanger sequencing was employed. Sliding window analysis was used to quantify GC content and GC skew. Tandem repeats were identified using TRF, and secondary structures were predicted with UNAFold. Results The coding regions of the two specimens were highly conserved, but the NCR length showed an extreme 3.7-fold difference. The Ruili NCR contains a large-scale 681 bp tandem repeat unit arranged in head-to-tail fashion as approximately 5.1 copies, whereas the Nanjian NCR harbors only a highly similar single-copy ancestral sequence. GC skew sliding window analysis revealed five periodic troughs in the Ruili NCR that precisely match the five complete repeat units (681 bp), a pattern consistent with replication slippage-driven expansion; the Nanjian NCR showed only a single trough. A 33 bp stem-loop is structurally conserved in both specimens and maintains high GC skew values, suggesting it is a candidate structurally conserved motif that could potentially be involved in replication-origin regulation, although experimental validation is needed. Conclusions This study reports the first case of NCR length variation consistent with replication slippage at the intraspecific level in monogeneans, identifying a large-scale 681 bp tandem repeat unit that is rarely documented in flatworms and substantially larger than previously reported analogous structures. The high similarity of coding regions contrasts with extreme NCR variation, suggesting that NCR structural variation may precede coding region divergence between individuals from different geographic origins. Our findings reveal structural plasticity of the Dactylogyrus mitochondrial NCR and highlight the necessity of employing segmented Sanger sequencing or long-read technologies for resolving complex repetitive regions. However, it should be noted that this study is based on one complete mitogenome per locality; therefore, the findings represent a comparison between two individuals rather than a population-level characterization. Additional sampling is required to assess the frequency and distribution of the observed structural variant.