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To explore the characteristics of mitogenomes and discuss the phylogenetic relationships within the genus Prosopocoilus , the mitogenomes of two species ( P. castaneus and P. laterotarsus ) were newly sequenced and comparatively analyzed. The arrangement of the mitogenome in these two lucanid beetles was the same as that in the inferred ancestral insect, and the nucleotide composition was highly biased towards A + T as in other lucanids. The evolutionary rates of 13 protein-coding genes (PCGs) suggested that their evolution was based on purifying selection. Notably, we found evidence of the presence of a large IGS between trnI and trnQ genes, whose length varied from 375 bp (in P. castaneus ) to 158 bp (in P. laterotarsus ). Within the large IGS region, a short sequence (TAAAA) was found to be unique among these two species, providing insights into phylogenomic reconstruction. Phylogenetic analyses were performed using the maximum likelihood (IQ-TREE) and Bayesian (PhyloBayes) methods based on 13 protein-coding genes (PCGs) in nucleotides and amino acids (AA) from published mitogenomes ( n = 29). The genus Prosopocoilus was found to constitute a distinct clade with high nodal support. Overall, our findings suggested that analysis of the characteristics of the large IGS (presence or absence, size, and location) in mitogenomes of the genus Prosopocoilus may be informative for the phylogenetic and taxonomic analyses and for evaluation of the genus Prosopocoilus , despite the dense sampling materials needed.

Phylogenetic Implication of Large Intergenic Spacers: Insights from a Mitogenomic Comparison of Prosopocoilus Stag Beetles (Coleoptera: Lucanidae)