Mitogenome annotation and sequence analysis

The newly assembled mitogenomes of D. arrosor and D. aspersus were annotated using the software of Sequin (version 15.10, http://www.ncbi.nlm.nih.gov/Sequin/). The boundaries of protein-coding and ribosomal RNA genes were performed using NCBI-BLAST (http://blast.ncbi.nlm.nih.gov). Transfer RNA genes were manually plotted, according to the secondary structure predicted by the MITOS Web Server³⁵ and tRNAscan-SE 1.21³⁶. The control region was determined by the locations of adjacent genes. Finally, circular mitogenome maps of D. arrosor and D. aspersus were drawn with the BLAST Ring Image Generator (BRIG) v0.95³⁷.

The base composition and relative synonymous codon usage (RSCU) were obtained using MEGA X³⁸. The strand asymmetry was calculated using the following formulas: AT-skew = (A − T)/(A + T); GC-skew = (G − C)/(G + C)³⁹. Furthermore, we chose the complete mitogenome of Pagurus filholi as the reference genome for comparative genomic analysis. Genome sequence similarity among the 15 Paguroidea species was carried out using the BRIG tool. Synteny analysis between the genomes was performed using Mauve v2.4.0⁴⁰. To estimate the evolutionary-selection constraints on 13 PCGs in the superfamily Paguroidea, the nonsynonymous (dN) and synonymous (dS) substitution rates were calculated using Mega X. The genetic distances of 13 PCGs were also estimated using Mega X based on the Kimura 2-parameter (K2P) substitution model.