First Description of the Mitogenome and Phylogeny of Culicinae Species from the Amazon Region

Abstract

The Culicidae family is distributed worldwide and comprises about 3587 species subdivided into the subfamilies Anophelinae and Culicinae. This is the first description of complete mitochondrial DNA sequences from Aedes fluviatilis, Aedeomyia squamipennis, Coquillettidia nigricans, Psorophora albipes, and Psorophora ferox. The mitogenomes showed an average length of 15,046 pb and 78.02% AT content, comprising 37 functional subunits (13 protein coding genes, 22 tRNAs, and two rRNAs). The most common start codons were ATT/ATG, and TAA was the stop codon for all PCGs. The tRNAs had the typical leaf clover structure, except tRNA^Ser1. Phylogeny was inferred by analyzing the 13 PCGs concatenated nucleotide sequences of 48 mitogenomes. Maximum likelihood and Bayesian inference analysis placed Ps. albipes and Ps. ferox in the Janthinosoma group, like the accepted classification of Psorophora genus. Ae. fluviatilis was placed in the Aedini tribe, but was revealed to be more related to the Haemagogus genus, a result that may have been hampered by the poor sampling of Aedes sequences. Cq. nigricans clustered with Cq. chrysonotum, both related to Mansonia. Ae. squamipennis was placed as the most external lineage of the Culicinae subfamily. The yielded topology supports the concept of monophyly of all groups and ratifies the current taxonomic classification.

Keywords: Culicidae; mitochondrial genome; molecular taxonomy; sequencing.

Figure 1

Structural representations of the Ps. albipes, Ps. ferox, Ae. fluviatilis, Cq. nigricans and Ad. squamipennis mitogenomes. The internal values indicate the overall content of the nucleotide bases. The blue, pink, and green blocks indicate PCGs, tRNAs, and rRNAs, respectively. Each tRNA is identified by a unique letter abbreviation. The genes arranged in the outer circle are located in the J strand (Forward), and the genes arranged in the inner circle are located in the N strand (Reverse).

Figure 2

Information on AT/GC contents, and AT/GC-skews of the investigated mitogenomes. (A) AT contents (%). (B) AT-skews. (C) GC-skews.

Figure 3

Relative synonymous codon usage (RSCU) of the obtained mitogenomes. RSCU values are represented on the y-axis, and families of synonymous codons and their respective amino acids are indicated on the x-axis. The synonymous codon families observed in Psorophora albipes (which had its RSCU graph enlarged to make the other graphs easier to read) are the same observed for the other mosquito species.

Figure 4

Proportions between rates of non-synonymous (dN) and synonymous (dS) nucleotide substitutions (dN/dS). (A) Bar chart for pairwise proportions of dN/dS for each of the mitochondrial subunits of the investigated species. (B) Box chart illustrating the averages for pairwise proportions of dN/dS for each of the mitochondrial subunits of the investigated species. In the graphs, the dN/dS ratios are plotted on the y-axis, and the PCGs are plotted on the x-axis.

Figure 5

Nucleotide diversity (π) among the obtained mitogenomes in this study. Blue lines indicate nucleotide diversity values based on evaluation of sequences from the tribe Mansoniini, including Cq. nigricans. Red lines indicate nucleotide diversity values based on evaluation of sequences from the Aedini tribe, including Ae. fluviatilis, Ps. albipes, and Ps. ferox. The purple lines indicate the nucleotide diversity values based on the evaluation of the Aedeomyia squamipennis (Aedeomyiini) sequence compared to the Mansoniini and Aedini sequence groups. The π values were calculated from a 200 bp sliding window analysis in 25 bp steps and are plotted on the y-axis. The length values of the aligned sequences of the three evaluated groups are plotted on the x-axis. The limits of each gene are indicated in the representation above the graph: vertical black bars indicate tRNAs, and white rectangles indicate PCGs and rRNAs.

Figure 6

Phylogenetic reconstruction by maximum likelihood based on the 13 PCGs concatenated of the species sequenced in this study (marked with the red asterisk) and 42 other taxa with data available on the GenBank and EMBL databases. The values of support for bootstrapping (BPP) are shown on the left, in each node. Dash lines were drawn for viewing purpose.

Figure 7

Phylogenetic reconstruction by Bayesian Inference based on the 13 PCGs concatenated of the species sequenced in this study (marked with the red asterisk) and 42 other taxa with data available on the GenBank and EMBL databases. The values of support Bayesian probabilities (BP) are shown on the left, in each node. Dash lines were drawn for viewing purpose.