Comparative mitogenomic analysis provides evolutionary insights into Formica (Hymenoptera: Formicidae)

Abstract

Formica is a large genus in the family Formicidae with high diversity in its distribution, morphology, and physiology. To better understand evolutionary characteristics of Formica, the complete mitochondrial genomes (mitogenomes) of two Formica species were determined and a comparative mitogenomic analysis for this genus was performed. The two newly sequenced Formica mitogenomes each included 37 typical mitochondrial genes and a large non-coding region (putative control region), as observed in other Formica mitogenomes. Base composition, gene order, codon usage, and tRNA secondary structure were well conserved among Formica species, whereas diversity in sequence size and structural characteristics was observed in control regions. We also observed several conserved motifs in the intergenic spacer regions. These conserved genomic features may be related to mitochondrial function and their highly conserved physiological constraints, while the diversity of the control regions may be associated with adaptive evolution among heterogenous habitats. A negative AT-skew value on the majority chain was presented in each of Formica mitogenomes, indicating a reversal of strand asymmetry in base composition. Strong codon usage bias was observed in Formica mitogenomes, which was predominantly determined by nucleotide composition. All 13 mitochondrial protein-coding genes of Formica species exhibited molecular signatures of purifying selection, as indicated by the ratio of non-synonymous substitutions to synonymous substitutions being less than 1 for each protein-coding gene. Phylogenetic analyses based on mitogenomic data obtained fairly consistent phylogenetic relationships, except for two Formica species that had unstable phylogenetic positions, indicating mitogenomic data are useful for constructing phylogenies of ants. Beyond characterizing two additional Formica mitogenomes, this study also provided some key evolutionary insights into Formica.

Fig 1. The size of protein-coding genes (PCGs), tRNA, rrnL, rrnS, and control region (CR) sequences among Formica mitochondrial genomes.

Ant species names are abbreviated as follows: Formica candida, Fc; Formica fusca, Ff; Formica glauca, Fg; Formica moki, Fm; Formica neogagates, Fn; Formica selysi, Fs; Formica sinae, Fi.

Fig 2. Putative secondary structures of the 22 tRNA genes found in the Formica candida mitogenome.

All tRNA genes are shown in the order of occurrence in the mitochondrial genome starting from trnL2. Completely conserved sites within the twelve species are shown as white nucleotide abbreviations within red spheres. Bars indicate Watson–Crick base pairings or G and U pairs. Unpaired bases are represented as dots.

Fig 3. AT% versus AT-skew and GC% versus GC-skew in the 12 Formica mitochondrial genomes.

Measured in bp percentage (y-axis) and level of nucleotide skew (x-axis). Values are calculated for J-strands in full-length mitochondrial genomes. A, A + T% vs AT-skew; B, G + C% vs GC-skew.

Fig 4. Evaluation of codon bias in the mitochondrial genomes of 12 Formica species.

G + C%, G + C content of all codon positions; (G + C)3%, G + C content of the third codon positions; ENC, effective number of codons; CBI, codon bias index.

Fig 5. The correlation between effective number of codons (ENC) and G + C content of the third codon positions (GC3) for 12 Formica species.

The colored dots correspond to those in Fig 3. (A) The solid line represents the relationship between ENC and GC3 content. (B) The solid line represents the relationship between GC12 and GC3 content, whereas the dotted line indicates y = x. GC12, G + C content of the first and second positions.

Fig 6. Sequence alignment of the intergenic spacer between trnS2 and nad1 for Formica mitochondrial genomes.

Fig 7. A + T contents of the mitochondrial protein-coding genes in Formica mitochondrial genomes.

Fig 8. Evolutionary rates of 13 protein-coding genes in the mitochondrial genomes of 12 species of Formica.

The left y-axis shows the substitution rate of mitochondrial genes, while the right y-axis shows the G + C content. Synonymous nucleotide substitutions per synonymous site (K_s) and nonsynonymous nucleotide substitutions per nonsynonymous site (K_a) were calculated using the Kumar method. The standard error estimates were obtained by a bootstrap procedure (1,000 replicates).

Fig 9. Two phylogenies of 16 Formicinae species from four genera based on two datasets (P123 dataset and P123AA dataset) and three analytical methods (Bayesian inference [BI], neighbor-joining [NJ], and maximum likelihood [ML]).