Saxicola is a genus of insectivorous birds widely distributed across Eurasia and Africa. Their mitochondrial genomes (mitogenomes), classification, phylogeny, and biogeography are underresearched. Here, we sequenced and/or assembled the mitogenomes of S. stejnegeri, S. torquatus, S. maurus, S. rubicola hibernans, S. r. rubicola and S. dacotiae for detailed comparative analysis, integrating them with published mitogenomes of members of Muscicapidae for phylogenetic reconstruction. Additionally, we estimated the divergence times and ancestral ranges for Saxicola using ND2 and Cytb. These mitogenomes, spanning 16,764 bp to 16,804 bp, exhibit typical gene arrangement, AT bias, and A/C skew tendencies. Among the 13 protein-coding genes, ND3 evolved the fastest, whereas COX1 evolved the slowest. The 22 tRNAs form the classic cloverleaf structure except trnS1 (AGY). The control region has three domains with 10 conserved elements. Mitogenomic phylogenetic analysis revealed the topology: (((((S. r. hibernans + S. r. rubicola) + S. dacotiae) + S. maurus) + S. torquatus) + S. stejnegeri), supporting the independent species status of S. stejnegeri rather than a subspecies of S. maurus. Ancestral range reconstruction indicated that Saxicola originated in the Indian subcontinent and mainland Southeast Asia during the late Miocene. These findings enhance our understanding of the classification, evolution, and geographical origin of Saxicola.

The control region (CR) regulates the replication and transcription of the mitochondrial genome (mitogenome). Some avian mitogenomes possess two CRs, and the second control region (CR2) may enhance replication and transcription; however, the CR2 in lark mitogenome appears to be undergoing loss and is accompanied by tandem repeats. Here, we characterized six lark mitogenomes from Alaudala cheleensis, Eremophila alpestris, Alauda razae, and Calandrella cinerea and reconstructed the phylogeny of Passerida. Through further comparative analysis among larks, we traced the evolutionary process of CR2. The mitochondrial gene orders were conserved in all published lark mitogenomes, with Cytb-trnT-CR1-trnP-ND6-trnE-remnant CR2 with tandem repeat-trnF-rrnS. Phylogenetic analysis revealed Alaudidae and Panuridae are sister groups at the base of Sylvioidea, and sporadic losses of CR2 may occur in their common ancestor. CR sequence and phylogeny analysis indicated CR2 tandem repeats were generated within CR2, originating in the ancestor of all larks, rather than inherited from CR1. The secondary structure comparison of tandem repeat units within and between species suggested slipped-strand mispairing and DNA turnover as suitable models for explaining the origin and evolution of these repeats. This study reveals the evolutionary process of the CR2 containing tandem repeat in Alaudidae, providing reference for understanding the evolutionary characteristics and dynamics of tandem repeats.

Wolf (Canis lupus) is a species included in appendices of CITES and is often encountered in cases of alleged poaching and trafficking of their products. When such crimes are suspected, those involved may attempt to evade legal action by claiming that the animals involved are domestic dogs (C. l. familiaris). To respond effectively to such claims, law enforcement agencies require reliable and robust methods to distinguish wolves from dogs. Reported molecular genetic methods are either unreliable (mitogenome sequence based), or operationally cumbersome and require much DNA (un-multiplexed microsatellites), or financially expensive (genome wide SNP genotyping). We report on the validation of a panel of 12 ancestral informative single nucleotide polymorphism (SNP) markers for discriminating wolves from dogs. A SNaPshot multiplex genotyping system was developed for the panel, and 97 Mongolian wolves (C. l. chanco) and 108 domestic dogs were used for validation. Results showed this panel had high genotyping success (0.991), reproducibility (1.00) and origin assignment accuracy (0.97 ± 0.05 for dogs and 1.00 ± 0.03 for wolves). Species-specificity testing suggested strong tolerance to DNA contamination across species, except for Canidae. The minimum DNA required for reliable genotyping was 6.25 pg/μl. The method and established gene frequency database are available to support identification of wolves and dogs by law enforcement agencies.