Mitochondrial genome sequencing and phylogeny of Haemagogus albomaculatus, Haemagogus leucocelaenus, Haemagogus spegazzinii, and Haemagogus tropicalis (Diptera: Culicidae)

Abstract

The genus Haemagogus (Diptera: Culicidae) comprises species of great epidemiological relevance, involved in transmission cycles of the Yellow fever virus and other arboviruses in South America. So far, only Haemagogus janthinomys has complete mitochondrial sequences available. Given the unavailability of information related to aspects of the evolutionary biology and molecular taxonomy of this genus, we report here, the first sequencing of the mitogenomes of Haemagogus albomaculatus, Haemagogus leucocelaenus, Haemagogus spegazzinii, and Haemagogus tropicalis. The mitogenomes showed an average length of 15,038 bp, average AT content of 79.3%, positive AT-skews, negative GC-skews, and comprised 37 functional subunits (13 PCGs, 22 tRNA, and 02 rRNA). The PCGs showed ATN as start codon, TAA as stop codon, and signs of purifying selection. The tRNAs had the typical leaf clover structure, except tRNA^Ser1. Phylogenetic analyzes of Bayesian inference and Maximum Likelihood, based on concatenated sequences from all 13 PCGs, produced identical topologies and strongly supported the monophyletic relationship between the Haemagogus and Conopostegus subgenera, and corroborated with the known taxonomic classification of the evaluated taxa, based on external morphological aspects. The information produced on the mitogenomes of the Haemagogus species evaluated here may be useful in carrying out future taxonomic and evolutionary studies of the genus.

Figure 1

Structural representations of the Hg. albomaculatus, Hg. leucocelaenus, Hg. spegazzinii, and Hg. tropicalis mitogenomes. The internal values indicate the content of the nucleotide bases. The blue, red, and yellow 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 (Foward), and the genes arranged in the inner circle are located in the N strand (Reverse). The structural representations of the genomes in this figure were generated using GenomeVx (https://wolfe.ucd.ie/GenomeVx/) and edited using Inkscape 0.92.4 (https://inkscape.org/).

Figure 2

Information on lengths, AT contents, and AT/GC-skews of the investigated mitogenomes. AT contents (%) (a). AT-skews (b). GC-skews (c). All graphs in this figure were generated using GraphPad Prism 8.0.1 (https://www.graphpad.com/scientific-software/prism/) and edited using Inkscape 0.92.4 (https://inkscape.org/).

Figure 3

Relative synonymous codon usage (RSCU) of the sequenced 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. All graphs in this figure were generated using GraphPad Prism 8.0.1 (https://www.graphpad.com/scientific-software/prism/) and edited using Inkscape 0.92.4 (https://inkscape.org/).

Figure 4

Proportions between rates non-synonymous (d_N) and synonymous (d_S) nucleotide substitutions (d_N/d_S) in 05 Haemagogus mitogenomes. The ratios were calculated in paired analyzes between the 13 PCGs of the sequenced mitogenomes, together with the data already available for Hg. janthinomys. The d_N/d_S ratios are plotted on the y-axis, and the PCGs are plotted on the x-axis. Abbreviations: HA (Hg. albomaculatus), HJ (Hg. janthinomys), HL (Hg. leucocelaenus), HS (Hg. spegazzinii), and HT (Hg. tropicalis). The graph in this figure was generated using GraphPad Prism 8.0.1 (https://www.graphpad.com/scientific-software/prism/) and edited using Inkscape 0.92.4 (https://inkscape.org/).

Figure 5

Nucleotide diversity among the mitogenomes of the four species evaluated, with Hg. janthinomys (GenBank ID: NC28025). The π values were calculated from a sliding window analysis of 200 bp in 25 bp steps, and are represented on the y-axis. The length values of the aligned sequence are represented on the x-axis. The limits of each gene are indicated in the representation above: vertical black bars indicate tRNAs and white rectangles indicate PCGs and rRNAs. The graph of this figure was generated using DnaSP v.6.12.03 (https://www.ub.edu/dnasp/) and edited using Inkscape 0.92.4 (https://inkscape.org/).

Figure 6

Phylogenetic reconstruction by the Bayesian Inference and Maximum Likelihood methods, based on the concatenation of the 13 PCGs of the Haemagogus species sequenced in this study and 23 other taxa with data available on GenBank database. The subsequent Bayesian probabilities (BP), and the values of support for bootstrapping (BPP) for 1,000 replicates, respectively, are shown on the left, in each node. The complementary lines dotted in red indicate the newly-sequenced species. The GenBank database accession numbers and references of mitogenomes sequences of the taxa used are listed in Supplementary Table 2. The topologies were visualized using FigTree v.1.4.4 (https://tree.bio.ed.ac.uk/software/figtree/) and edited using Inkscape 0.92.4 (https://inkscape.org/).