Simple sequence repeats (SSRs) are known as microsatellites, and consist of tandem 1–6-base motifs. They have become one of the most popular molecular markers, and are widely used in molecular ecology, conservation biology, molecular breeding, and many other fields. Previously reported methods identify monomorphic and polymorphic SSRs and determine the polymorphic SSRs via experimental validation, which is potentially time-consuming and costly. Herein, we present a new strategy named insertion/deletion (INDEL) SSR (IDSSR) to identify polymorphic SSRs by integrating SSRs with nucleotide insertions/deletions (INDEL) solely based on a single genome sequence and the sequenced pair-end reads. These INDEL indexes and polymorphic SSRs were identified, as well as the number of repeats, repeat motifs, chromosome location, annealing temperature, and primer sequences, enabling future experimental approaches to determine the correctness and polymorphism. Experimental validation with the giant panda demonstrated that our method has high reliability and stability. The efficient SSR pipeline would help researchers obtain high-quality genetic markers for plants and animals of interest, save labor, and reduce costly marker-screening experiments. IDSSR is freely available at https://github.com/Allsummerking/IDSSR.

Ants (Formicidae) are the most diverse eusocial insects in Hymenoptera, distributed across 17 extant subfamilies grouped into 3 major clades, the Formicoid, Leptanilloid, and Poneroid. While the mitogenomes of Formicoid ants have been well studied, there is a lack of published data on the mitogenomes of Poneroid ants, which requires further characterization. In this study, we first present three complete mitogenomes of Poneroid ants: Paraponera clavata, the only extant species from the subfamily Paraponerinae, and two species (Harpegnathos venator and Buniapone amblyops) from the Ponerinae subfamily. Notable novel gene rearrangements were observed in the new mitogenomes, located in the gene blocks CR-trnM-trnI-trnQ-ND2, COX1-trnK-trnD-ATP8, and ND3-trnA-trnR-trnN-trnS1-trnE-trnF-ND5. We reported the duplication of tRNA genes for the first time in Formicidae. An extra trnQ gene was identified in H. venator. These gene rearrangements could be explained by the tandem duplication/random loss (TDRL) model and the slipped-strand mispairing model. Additionally, one large duplicated region containing tandem repeats was identified in the control region of P. clavata. Phylogenetic analyses based on protein-coding genes and rRNA genes via maximum likelihood and Bayes methods supported the monophyly of the Poneroid clade and the sister group relationship between the subfamilies Paraponerinae and Amblyoponinae. However, caution is advised in interpreting the positions of Paraponerinae due to the potential artifact of long-branch attraction.