Summary: | The mustache toads <i>Leptobrachium boringii</i> and <i>Leptobrachium liui</i> are two attractive species in Megophryidae, in which adult males have mustache-like keratinized nuptial spines on their upper lip. However, both are under threat due to multiple factors, of which scientific studies are still very limited. In this study, two new complete mitochondrial genomes of <i>L. boringii</i> and <i>L. liui</i> were sequenced, assembled, and annotated based on next-generation sequencing. The mitogenome lengths of <i>L. boringii</i> and <i>L. liui</i> were found to be 17,100 and 17,501 bp, respectively, with both containing 13 protein coding genes, 23 tRNAs, 2 rRNAs, and 1 non-coding control region. Nucleotide diversity analyses indicate that <i>atp8</i>, <i>atp6</i>, and <i>nad2</i> showed higher nucleotide diversity than <i>cox1</i>, <i>cox3,</i> and <i>cytb</i>. The intraspecific genetic distances among three different populations of <i>L. boringii</i> exceed 4%, and those between two populations of <i>L. liui</i> reach 7%. Phylogenetic relationships support their division into two subfamilies of Megophryidae (Leptobrachiinae and Megophryinae) as well as two species groups within <i>Leptobrachium,</i> corresponding to the number of keratinized nuptial spines (10–48 in the <i>L. boringii</i> species group vs. 2–6 in the <i>L. liui</i> species group). The two new mitogenomes reported in this study provide valuable data for future molecular evolutionary and conservation studies of the genus <i>Leptobrachium</i> and other Megophryidae toads.
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