Complete mtgenome sequences of Anopheles peditaeniatus and An. nitidus and phylogenetic relationships of the genus Anopheles based on mtgenome sequences (Diptera: Culicidae: Anophelinae)

Background: Despite the medical importance of the genus Anopheles in the transmission of malaria and other human diseases, its phylogenetic relationships are not unsettled and the characteristics of mitochondrial genome (mtgenome) are not well understood. Methods: The present study sequenced and analyzed the complete mtgenomes of An. peditaeniatus and An. nitidus, and investigated the characteristics and phylogenetic relationships of 76 complete mtgenome sequences in the genus Anopheles using Illumina sequencing and bioinformatics techniques. Results: The complete mtgenomes of An. peditaeniatus and An. nitidus are 15416 and 15418 bp long, respectively, and include 13 PCGs, 22 tRNAs, two tRNAs and one control region (CR). These 76 mtgenomes are similar as earlier reports in insects in general characteristics, and however the trnR and trnA have a reversal arrangement to form “trnR-trnA” as reported in other mosquito genera. Their variations mainly occur in CR with a length of 493 - 886 bp, and six repeat unit types are identied for the rst time and demonstrate some evolutionary signals. The subgenera Lophopodomyia, Stethomyia, Kerteszia, Nyssorhynchus, Anopheles and Cellia, are proposed to be monophyletic with the phylogenetic relationships of (Lophopodomyia + ((Stethomyia + Kerteszia) + (Nyssorhynchus + (Anopheles + Cellia)))). Four series Neomyzomyia, Pyretophorous, Neocellia and Myzomyia in Cellia, and two series Arribalzagia and Myzorhynchus in Anopheles are proposed to be monophyletic, and three sections Myzorhynchella, Argyritarsis and Albimanus and their subdivisions in Nyssorhynchus all appear polyphyletic or paraphyletic. Conclusions: The study comprehensively uncovered the characteristics of mtgenome and the phylogenetics based on mtgenomes in the genus Anopheles, and provided an information frame for further study on the mtgenomes, phylogenetics and taxonomic revision of the

The classi cation of genus Anopheles started more than 100 years ago [6], in which it was treated as one of 18 genera in the Anophelinae, and Cellia, Nyssorhynchus, Stethomyia and Kerteszia were also treated as independent genera based on the morphological characteristics. Subsequently, the ve genera were successively degraded as subgenera of the genus Anopheles based on the number and location of the specialized setae on male genital gonocoxites and other characteristics [7][8][9], and three additional subgenera, Lophopodomyia, Baimaia and Christya were established for the genus Anopheles [10][11][12]. Due to the diversity of species contained in subgenus Anopheles, Cellia and Nyssorhynchus, taxonomists divided some species into informal categories such as Sections, Series and Groups. Earliest phylogenetic studies for the genus Anopheles were mainly based on morphological characters and individual genes, different data sets and phylogenetic inference methods often lead inconsistent results, and therefore the phylogenetic relationship of Anopheles have not been well settled.
There have been a number of representative phylogenetic studies on the genus Anopheles. An analysis including 63 species in Anophelinae based on 163 morphological characters suggested the monophyly of the subgenera Cellia, Nyssorhynchus, StethomyiaKerteszia and Lophopodomyia. In Nysorhynchus, the three sections Albimanus, Argyritarsis and Myzorhynchella were suggested to be paraphyly; in Cellia, only series Cellia was considered to be monophyly; and in Anopheles, series Arribalzagia and Lophoscelomyia were considered to be monophyly, while series Cycloleppteron+Arribalzagia was nested in series series Myzorhynchus [13]. Some further morphology-based studies also suggested the monophyly of subgenera Nyssorhynchus, Cellia and Kerteszia, and displayed the sister relationships between subgenera Kerteszia and Nyssorhynchus [12,14,15]. An analysis based on COX1 + ITS2 dataset suggested the monophyly of the subgenus Anopheles (16 species included) and Cellia (18 species), and the analysis using ITS2 dataset alone resulted in the same conclusion but not for COX1 dataset alone [16]. Two studies based on the nucleotides of 13 protein-coding genes of mtgenomes, including 50 and 33 species, both also supported the monophyly of subgenera Anopheles, Nyssorhynchus, Cellia and Keterszia [17,18]. Generally, the monophyly of the subgenera Anopheles, Nyssorhynchus, Cellia, Stethomyia, Keterszia and Lophopodomyia have been suggested by most nowadays studies; however, the sections and series in the subgenera Anopheles, Nyssorhynchus and Cellia have not been well determined. There is a need to elucidate the phylogeny of the genus Anopheles using more species, more data and updated phylogenetic analysis approaches.
Mitochondria is a very important organelle in eukaryotic cells, which has a genome independent of the nuclear genome, namely "mitochondrial genome" (mtgenome) [19]. Mtgenome has the characteristics of small genome size, low level of recombination and maternal inheritance, and therefore it has been widely used as a molecular marker for identi cation of species, evolution, phylogenetic inference and population structure research [20,21]. Since the publication of the rst insect mtgenome (Drosophila yakuba) in 1985 [22], the number of insect mtgenomes have increased rapidly. Phylogenetic studies based on insect mtgenomes have shown good results in Diptera [23], Orthoptera [24], Coleoptera [25] and Hymenoptera [26]. So far, NCBI has housed the complete mtgenomes of 125 species in Culicidae, of which 74 species belong to the genus Anopheles. The Diptera mtgenome is mostly 14-20 kb long, including 37 genes: 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes and a control region (CR), and these genes are arranged in a compact circular genome [27]. The gene number and structure in all reported mosquito mtgenomes are similar to the typical mtgenomes of Diptera, and however, trnA and trnR of mosquitoes are rearranged to form "trnR-trnA" arrangement [17,18,22].
In the present study, we sequenced and annotated the complete mitogenomes of An. peditaeniatus and An. nitidus in the genus Anopheles, and comparatively analyzed the characteristics of 76 species of mtgenome sequences in the genus Anopheles. More importantly, we constructed and discussed the phylogenetic relationships of these 76 known mtgenome sequences. The study provided new insight of the mtgenomes characteristics and phylogenetic relationships in the genus Anopheles.

Methods
Sample collection and DNA extraction Specimens of An. peditaeniatus and An. nitidus were collected from Yadong County (29°11'46"N, 95°12'11"E), Tibet, China in July 2014, and Tiebei County, Jilin Province, China (42°27′21″N, 128°06′18″E) in July 2013, respectively. All collected samples were preserved in individual vials in silica. After morphological identi cation in laboratory [28], these samples were stored in 100% alcohol, and housed at -20℃ until the DNA extraction. Total DNA was extracted from the individual adult mosquito using the Qiagen Genomic DNA Kit [29], and used for 350 bp library construction and Illumina high throughout sequencing of mitochondrial genome in Shenzhen Huitong Biotechnology Co. Ltd..

Mtgenomes assembly, annotation and characteristics analysis
The mtgenomes of An. peditaeniatus and An. nitidus assemblied and annotated using Mitos (http://mitos.bioinf.unileipzig.de/index.py) [30]. The annotation of 13 PCGs and two rRNA genes wase con rmed in reference of known mosquito mtgenomes, and corrected using Geneious v4.8.5 [31]. The secondary structures of tRNAs were predicted using tRNAscan-SE 2.0 [32], and the structure map of the mtgenomes were visualized using OGDRAW1.3.1 [33]. The base composition, codon usage, relative synonymous codon usage (RSCU), and amino acid content were computed with MEGA v.7.0.26 software [34]. The nucleotide composition bias was calculated using the formulas AT skew = [A − T] / [A + T] and GC skew = [G − C] / [G + C] [35], and the Three-dimensional scatterplot of the AT-Skew, GC-Skew and AT% was drawn using Origin Pro v.9.0 [36]. The selection pressure of 13 PCGs during the evolution process was analyzed by calculating Ka and Ks values using DnaSP v6.12.03. Sequence motifs in the CR were identi ed using the Tandem Repeats Finder program [37].

Phylogenetic analysis
Multiple sequence alignment of 13 PCGs was performed on the Translator-X Server (http://translatorx.co.uk/), in which MAFFT was used to align the amino acid sequences of 13 PCGs, and Gblocks was used to remove poorly aligned sites. Finally, the individual alignments were connected together using SequenceMatrix [38] to obtain the amino acid tandem sequence of 13 PCGs. The best-t substitution model for nucleotide datasets was selected by PartitionFinder 2 [39].
Phylogenetic analyses of the 76 Anopheles species of mtgenomes (two sequenced in the study and 74 known) were performed using the Maximum likelihood (ML) analysis in IQ-TREE 1.6.10 [40], and the Bayesian Inference (BI) analysis in MrBayes v.3.2.7a [41] using Culex pipiens pallens as the outgroup ( Table 1). The bootstrap values were calculated with 1000 replicates for ML, and for BI, performed two independent runs, each with four chains, and these chains ran simultaneously for 1,000,000 generations, and the tree being sampling every 1000 steps with 25% burn-in rate. The phylogenetic tree was drawn using FigTreev.1.4.4 (http://tree.bio.ed.ac.uk/software/ gtree/).

Nucleotide composition and genome organization
The complete mtgenomes of An. peditaeniatus (MT822295) and An. nitidus (MW401801) are both circular, closed and double-stranded structures, with full lengths of 15,416 and 15,418 bp, respectively ( Fig. 1). Both are composed of 37 genes (including 13 PCGs, 22 tRNA genes and two rRNA genes) and one control region (CR). There are 22 genes (nine PCGs and 13 tRNAs) located on the majority coding strand (J-strand), while the other 15 genes (four PCGs, nine tRNAs and two rRNAs) are on the minority strand (N-strand). Compared with the typical Diptera mtgenome (Drosophila yakuba), both An. peditaeniatus and An. nitidus have "trnR-trnA" rearrangements. The AT content of the mtgenomes of the two species are as high as 78.32% and 78.26%, respectively, which are signi cantly higher than their GC content (21.68%, 21.74%), showing obvious AT bias (Additional le 1: Table S1). The AT-skew of An. peditaeniatus (0.0322) is higher than the average AT-skew of all investigated mosquito mtgenomes (0.0283), whereas the AT-skew of An. nitidus mtgenome (0.0266) is lower than the average AT-skew value. The GC-skew in An. peditaeniatus(-0.1587) and An. nitidus (-0.1536) are higher than the average GC-skew value in mosquitoes investigated (-0.16048).
The three-dimensional scatter plot of the AT content, AT-skew and GC-skew of 76 mtgenomes in the genus Anopheles is shown in Fig. 2. The AT-skew with the range of variation from 0.005 for An. gilesi to 0.043 for An. christyi. However, all mtgenomes display negative GC-skews ranging from -0.207 for An. parvus to -0.136 for An. punctulatus. Most of the species of the subgenera Nyssorhynchus and Cellia have similar AT content and AT/GC-skew, which are closely distributed in the Three-dimensional scatter plot, whereas the species of the subgenera Lophopodomyia, Stethomyia, Kerteszia and Anopheles are widely distributed in the plot for AT content, AT-skew and GC-skew.

Protein-coding genes
The total nucleotide lengths of the 13 PCGs of An. peditaeniatus and An. nitidus is 11,223 and 11,168 bp, respectively. In the An. peditaeniatus, ATN is used as the start codon, except for COX1 and ND5 which use TCG and GTG as the start codon, respectively, and in the An. nitidus, 13 PCGs initiate with ATN as the start codon, but COX1 uses TCG as a start codon ( Table 2).
The RSCU values of 76 species of mtgenomes in the genus Anopheles are presented in Additional le 2: Table S2. The mtgenomes of the Anopheles have relatively different usage frequencies of synonymous codons. In the 76 species, UUA is the most frequently used codon, followed by CGA, GGA, GCU. The amino acid Leu has the highest usage percentage for all 76 mtgenomes investigated with an average of 16.37%, followed by Phe (9.69%), Ile (9.31%) and Ser (8.48%), whereas Cys has the lowest percentage (0.99%). The usage percentages of amino acids seem no obvious difference among different subgenera (Fig. 3).
The non-synonymous (Ka) and synonymous (Ks) substitution ratio (Ka/Ks) of 13 PCGs are shown in the  Figure S1). In the two newly sequenced mtgenomes, rrnL is located between trnL2 and trnV, and rrnS between trnV and CR. The length of the rRNAs is 2125 bp, with an AT content of 81.36% in An. punctulatus; 2122 bp, with an AT content of 81.39%% in An. nitidus.
The control regions (CRs) of the mtgenomes are both located between rrnS and trnI with their lengths of 575 and 580 bp, and their AT content of 94.43% and 93.62% (the highest among all mtgenome regions), respectively in An. peditaeniatus and An. nitidus. Six repeat unit types are identi ed in the CRs of the 74 species of mtgenomes in Anopheles (Additional e 4: Fig S2). All species have the repeat unit type of 15-27 bp poly-T Stretch, which is located in front of other repeat unit types and just after 140-212 bp of conserved sequence. The poly-T Stretch is adjacently connected with the conserved motif 5′-CCCCTA-3′ in the conserved sequence in 68 species, whereas the motif was substituted by 5′-ATTGTA-3′ in An. cracens and An. dirus, and 5′-TTCCCC-3′ in An. kompi, An. nimbus, An. gilesi and An. pseudotibiamaculatus. The second type is a 12-55 bp sequence with 2-6 repeats, which is just after the poly-T Stretch and exists in 54 species. The third type ([TA(A)]n Stretch) contains 22-91 repeats, which exists in 36 species. The fourth type is a 12-38 bp sequence with 2-5 repeats which are near trnI and exist in 40 species. The remaining two repeat unit types are found in only a few species, one of them is a 15-36 bp sequence which after the second type and exists in 5 species; and the last one is a 108-171 bp sequence, which is longest one among all six types and only exists in four species.

Phylogenetic relationships
Bayesian inference (BI) and Maximum-likelihood (ML) analyses produced two same topology of phylogenetic trees in the subgenus-level (Fig. 5-6). The six subgenera investigated, Lophopodomyia, Stethomyia, Kerteszia, Nyssorhynchus, Anopheles and Cellia all seem to be monophyly in both analyses, with posterior probability (pp) = 1 for every subgenus in BI (Fig 5) and bootstrap values (bv) ranging from 99% to 100% in ML analysis (Fig. 6). The subgenus Lophopodomyia is located at the base of these six subgenera, and the branch comprising the remaining ve subgenera has the support of pp = 1 and bv = 71%. The two subgenera Stethomyia and Kerteszia form a monophyly with pp = 1 and bv = 89%, which was earliest derived but the Lophopodomyia. The branch containing the Nyssorhynchus, Anopheles and Cellia possess the support of pp = 1 and bv = 68%. The subgenus the Nyssorhynchus seems to be sister group with the monophyly Anopheles + Cellia that has pp = 1 and bv = 99%.
In the subgenus Cellis, four series investigated, Myzomyia, Neocellia, Pyretophorus and Neomyzomyia each seem monophyletic with pp = 1 and bv = 100% for all of these monophylies. The series Neomyzomyia would be earliest derived and sister with remaining three seriers, and the series Pyretophorus would be sister with series Myzomyia and Neocellia. In the subgenus Anopheles, two sections Angusticorn and Laticorn both seem polyphyletic, and in section Laticorn both series Arribalzagia (pp = 1 and bv = 96%) and Myzorhynchus (pp = 1 and bv = 100%) seem monophyletic. In the subgenus Nyssorhynchus, three sections investigated Myzorhynchella, Argyritarsis and Albimanus all seem polyphyletic, and in the section Argyritarsis, two series Argyritarsis and Albitarsis both seem polyphyletic as well.
On the other hand, internal relationships of the Kerteszia are different of BI tree and Ml tree: An. homunculus branched out earlier than An. bellator in BI-tree (Fig. 5), however, in ML-tree, An. bellator branched out earlier than An. homunculus (Fig. 6).

Characteristics of the mtgenome sequences of the genus Anopheles
The length of 76 mtgenomes in the genus Anopheles ranges from 15,573 bp to 15,803 bp, and the length variation mainly occurred in the CRs, which is similar as earlier reported mtgenomes in insects [42,43]. Each mtgenome sequence includes 37 genes, and the trnR and trnA have a reversal arrangement to form "trnR-trnA" in comparison of Drosophila yakuba, as those reported in other genera in Culicidae [22,45]. All tRNA genes can form a complete clover secondary structure, except for trnS2 that lacks the DHU arm, which seem to be a common feature of metazoans [43]. The nucleotide composition for all species exhibits high AT bias with AT-skew values all positive and GC-skew all negative, similar as earlier reports in insects. The 13 PCGs mainly use ATN as the start codon and TAA as the stop codon, which is similar as other mtgenome sequences in insects [43]. The usage frequencies of synonymous codons and amino acids vary with the codon UAA having the highest usage frequency, followed by CGA and GGA, and the amino acid Leu to be most used, followed Phe and Ile. This is the detailed analysis for the usage frequency for the rst time, and may potentially contribute the biochemical and functional characteristics of mitochondrial genes.
The lengths of CRs are quite variable with range from 493 bp to 886 bp. The present study identi ed six repeat unit types for the rst time. All mtgenome sequences investigated have the poly-T stretch, which may involve in the identi cation of the replication origin of mtDNA [44]. The remaining ve repeat unit types vary in length and position among species, some of them seem different among subgenera to some extent. For example, the third type ([TA(A)]n Stretch) were not found in subgenera Anopheles, Lophopodomyia and Stethomyia, and the longest type only found in subgenera Lophopodomyia and Kerteszia. The CRs have been reported to be taxon-speci c and of evolutionary information, and was used as an important evidence in the inference of phylogenetics in genus Culex and Lutzia and taxon [46]. However, the evolutionary information carried in the genus Anopheles does not seem stable and reliable.
A phylogeny study based on 163 morphological characters for 64 species in the subfamily Anophelinae with Approximations Weighting (AW) method in 2000 showed that the subgenera Lophopodomyia, Stethomyia, Kerteszia, Nyssorhynchus and Cellia were monophyletic, whereas the subgenus Anopheles polyphyletic. These two subgenera Lophopodomyia and Stethomyia were separately linked inside the subgenus Anopheles [13]. A further morphology-based phylogenetics analysis published in 2005 used 167 characters for 66 species in the Anophelinae with both Equal Weighting (EW) and Implied Weighting (IW) methods, which got the same results as described above [15]. All analyses from these three methods showed that the two subgenera Nyssorhynchus and Kerteszia were sister-group, and the AW and EW methods suggested a relationship (Nyssorhynchus + Kerteszia) + (Cellia + (Lophopodomyia + Stethomyia + Anopheles)), whereas the IW method suggested (Anopheles + Lophopodomyia + Stethomyia) + (Cellia + (Kerteszia + Nyssorhynchus)). For molecular-based phylogenetic analysis, a study using COI, COII and 5.8S rRNA for 47 species in the genus Anopheles with ML method in 2015 suggested the monophyly of the subgenus Stethomyia, Kerteszia, Nyssorhynchus, Anopheles and Cellia with the phylogenetic relationships Anopheles +(Cellia + (Nyssorhynchus + (Stethomyia + Kerteszia))) [48]. A study using a.a. sequences of 1,085 single-copy orthologous genes for 18 species of the subgenera Nyssorhynchus, Anopheles and Cellia with ML method in 2015 proposed that all of these three subgenera are monophyletic with the relationships (Nyssorhynchus + (Anopheles + Cellia)) relationship [49]. Our earlier study using all PCG nucleotide sequences of 50 mtgenomes in Culicidae with ML and BI method in 2017 showed that the subgenera Nyssorhynchus, Anopheles and Cellia are monophyletic with the relationships (Nyssorhynchus + (Anopheles + Cellia)) [17].
All these six subgenera included in these comprehensive phylogenetic analyses above were suggested to be monophyly except for the subgenus Anopheles, which was recognized as a polyphyly in two morphology-based inferences while as a monophyly in three molecular-based inferences. Importantly, the study based on 18 whole nuclear genomes showed that the subgenus Anopheles is monophyletic [49]. This present study supported the monophyly of these six subgenera, resulting from these molecularbased inferences. The studies based on 18 whole nuclear genomes [50] and 50 whole mtgenomes [17] suggested that the subgenus Nyssorhynchus be sister group with (Anopheles + Cellia), and the study supports the result. The study based on COI, COII and 5.8S rRNA suggested the sister relationship of the subgenera Stethomyia and Kerteszia [48], and the study supports the result. The subgenus Lophopodomyia were grouped with the subgenera Anopheles and Stethomyia in two morphology-based inferences [13,15], whereas it was not included in the molecular-based inferences [17,48,49]. This study suggests that the subgenus Lophopodomyia be the sister with other ve subgenera together. In general, the phylogenetic relationships constructed between morphology-based and molecular-based inference are quite different, and there is need of further studies with inclusion of more species and data to elucidate the among-subgenera relationships.
For the subgenus Cellia, four series Neomyzomyia, Pyretophorous, Neocellia and Myzomyia investigated all appear to be monophyletic (pp = 1 and bv = 100% for their clades), with the phylogenetic relationships of Neomyzomyia + (Pyretophorous + (Neocellia + Myzomyia)). The results are completely consistent with those of our earlier study that was also based on whole mtgenomes [17], and almost consistent with the phylogenetic study based on 18S, 28S, COl and COII data in monophyly and relationship [47]. However, the early morphology-based study in 2000 treated the four series as paraphyly [13]. These suggest that results stemmed from molecular and morphology are often con icting as discussed above.
For the subgenus Anopheles, the two sections Angusticorn (only series Anopheles included) and Laticorn (two series Myzorhynchus and Arribalzagia included) both seem to be polyphyletic. The two series Myzorhynchus and Arribalzagia would be monopheletic (pp = 1 and bv ³ 96% for their clades), and if An. lindesayi were excluded, the series Anopheles would also be monopheletic (pp = 0.92 and bv = 85%), with the relations of (Anopheles + (Myzorhynchus + Arribalzagia)). The phylogenetic study based on COI, COII and 5.8S rRNA suggested the sections Laticorn and Angusticorn be polyphyletic, and inside the two series Anopheles and Myzorhynchus involved also be polyphyletic. In two morphology-based studies, one based on 163 morphological characters proposed the sections Laticorn and Angusticorn to be polyphyletic, the series Arribalzagia to be monophyletic, and the two series Myzorhynchus and Anopheles to be paraphyletic [13]. The another based on 167 morphological characters proposed the section Laticorn to be monophyletic, the section Angusticorn to be polyphyletic, the two series Arribalzagia and Myzorhynchus to be monphyletic, and the series Anopheles to be polyphyletic [15]. All of these four studies suggested that the section Angusticorn be polyphyletic, in which the series Anopheles be polyphyletic, and most of these studies proposed that the section Laticorn be polyphyletic, in which the series Arribalzagia be monophyletic and the series Myzorhynchus may be monophyletic.
For the subgenus Nyssorhynchus, three sections Myzorhynchella, Argyritarsis and Albimanus investigated, and their subdivisions in the three sections all appear polyphyletic or paraphyletic. The morphology-based study based on 163 morphological characters data suggested the three sections Albimanus, Argyritarsis and Myzorhynchella were paraphyletic [13]. In two molecular-based study, one based on white and ND6 for 21 species in the Nyssorhynchus with BI method in 2010 [50] suggested the three sections be not monophyletic, and a another one based on white, CAD and COI for 32 species in Nyssorhynchus with BI method in 2013 showed the three sections to be polyphyletic, and the three series also to polyphyletic [51]. All of these four studies demonstrate that the taxonomy and phylogenetics of the subgenus are quite con icted, and there is more necessarity to reconstruct the taxonomic system of the subgenus along the phylogenetic study.

Conclusions
This study sequenced and analyzed the complete mtgenomes of An. peditaeniatus and An. nitidus, and investigated the characteristics and phylogenetic relationships of 76 complete mtgenome sequences in the genus Anopheles. These mtgenomes are of general characteristics similar as earlier reports in insects, and however the trnR and trnA have a reversal arrangement to form "trnR-trnA" in comparison of Drosophilayakuba mtgenomes as those reported in other genera in Culicidae. Their variations mainly occur in CR regions with length from 493 bp -886 bp, and six repeat unit types are identi ed for the rst time, which demonstrate the evolutionary importance among subgenera to some extent. The subgenera Lophopodomyia, Stethomyia, Kerteszia, Nyssorhynchus, Anopheles and Cellia are all proposed to be monophyletic with the phylogenetic relationships of Lophopodomyia + ((Stethomyia + Kerteszia) + (Nyssorhynchus + (Anopheles + Cellia)))). Four series Neomyzomyia, Pyretophorous, Neocellia and Myzomyia in the subgenus Cellia, are proposed to be monophyletic, two series Arribalzagia and Myzorhynchus in the subgenus Anopheles are proposed to be monophyletic while the series Anopheles seems polyphyletic, and three sections Myzorhynchella, Argyritarsis and Albimanus and their subdivisions in the subgenus Nyssorhynchus all appear polyphyletic or paraphyletic. In general, there is need of further studies with inclusion of more species and data to elucidate the phylogenetic relationships in the genus.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.
Availability of data and materials All data are available as tables and gures in the main document and its additional les. The GenBank accession numbers for the two mtgenomes produced in the present study are MW401801 and MT822295.

Competing interests
The authors declare that they have no competing interests. Tables Table 1 Detailed sequence information of mtgenomes used in the present phylogenetic analysis.  Figure 1 Mtgenome structure of An. peditaeniatus and An. nitidus.

Figure 2
Three-dimensional scatter plot of the AT-Skew, GC-Skew and AT% of 76 mtgenome sequences in the genus Anopheles.    Table 1.