Phylogenetic taxonomy of the Zambian Anopheles coustani group using a mitogenomics approach

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Gebhardt, Limonty Simubali, Kochelani Saili, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5976492/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Malaria Journal → Version 1 posted 7 You are reading this latest preprint version Abstract Background Mosquito species belonging to the Anopheles coustani group have been implicated in driving residual malaria transmission in sub-Saharan Africa and are regarded as an established primary vector in Madagascar. The morphological identification of mosquitoes in this group is challenging due to cryptic features and their molecular confirmation is difficult due to a paucity of reference sequence data representing all members of the group. Conventional molecular barcoding with the cytochrome oxidase I (COI) gene and the internal transcribed spacer 2 (ITS2) region targets is limited in their discrimination and conclusive identification of members of species complexes. In contrast, complete mitochondrial genomes (mitogenomes) have demonstrated much improved power over barcodes to be useful in rectifying taxonomic discrepancies in Culicidae. Methods We utilized a genome skimming approach via shallow shotgun sequencing on individual mosquito specimens to generate sequence reads for mitogenome assembly. Bayesian inferred phylogenies and molecular dating estimations were perfomed on the concatenated protein coding genes using the Bayesian Evolutionary Analysis by Sampling Trees 2 (BEAST 2) platform. Divergence estimates were calibrated on published calucations for Anopheles - Aedes . Results This study generated 17 new complete mitogenomes which were comprable to reference An. coustani mitogenomes in the GenBank repository by having 13 protein coding, 22 transfer RNA and 2 ribosomal RNA genes, with an average length of 15,400 bp and AT content of 78.3%. Bayesian inference using the concatenated protein coding genes from the generated and publicly available mitogenomes yielded six clades: one for each of the four taxa targeted in this study, the GenBank references, and a currently unknown species. Divergence times estimated that the An. coustani group separated from the An. gambiae complex approximately 110 million years ago (MYA), and members within the complex diverged at times points ranging from~34 MYA to as recent as ~7 MYA. Conclusions These findings demonstrate the value of mitochondrial genomes in differentiating cryptic taxa and help to confirm morphological identities of An. coustani s.s. , An. paludis , An. zeimanni and An. tenebrosus . Divergence estimates with the An. coustani group are similar to those for well-studied anopheline vector groups. These analyses also highlight the likely prescence of other cryptic An. coustani group members circulating in Zambia. Anopheles coustani mitochondrial genome phylogeny Zambia malaria Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Vector control methods like indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) have been instrumental in progress toward malaria elimination [ 1 , 2 ]. Primary, well-studied vectors like Anopheles gambiae and An. funestus , which typically engage in endophagic and endophilic behaviors by seeking human hosts indoors, are the focus of these key intervention measures [ 1 , 2 ]. However, selection pressure driven by the broad deployment of IRS and LLINs have either reduced these populations, driven insecticide resistance, yielded shifts in vector species composition and/or resulted in changes in biting and resting behaviors [ 2 – 7 ]. Shifts to outdoor biting or having a high plasticity in this behavior, and the existence of other exophagic malaria vectors have been identified as a significant barriers to malaria control and elimination [ 3 , 8 , 9 ]. Though frequently collected, exophagic anopheline mosquitoes such as members of the An. coustani group [ 10 – 12 ], An. squamosus , and An. rufipes [ 14 ] are understudied despite contributing to malaria transmission in sub-Sahran Africa. The Anopheles coustani group is widely distributed throughout sub-Saharan Africa and the Middle East, with members typically exhibiting zoophilic and outdoor foraging behaviors [ 11 ]. Within the group, morphologically similar species including An. coustani, An. zeimanni , An. paludis , and An. tenebrosus , have demonstrated opportunistic foraging towards anthropophilic and endophilic feeding [ 10 , 15 ]. Little is known about the basic biology, ecology and behaviors of most of these species. This knowledge gap is particularly noteworty given members of the group have been implicated as established vectors with a key role in sustaining residual malaria transmission in Kenya, Madagascar, Ethiopia, Cameroon, Mozambique and Zambia [ 10 , 15 – 20 ]. Members of this group present an imminent threat to malaria elimination efforts due to inherent plasticity in their foraging behaviors, which enable them to evade many of the existing vector control strategies that target endophagic and endophilic mosquitoes [ 3 , 21 – 23 ]. Morphological and molecular techniques have proved to be challenging for identification of species in this group due to cryptic features, damaged specimens which obscures key morphological attributes [ 23 – 25 ], and the paucity of reference molecular data for comparison in genomic repositories [ 26 ]. Additionally, the well-established cytochrome oxidase I gene (COI) and the internal transcribed spacer 2 (ITS2) molecular barcodes commonly used for species confirmation have limited power in delineating phylogenetic disparities in cryptic species groups [ 23 , 27 , 28 ]. Though limited in number, published genetic and molecular studies have highlighted cryptic members within the An. coustani group [ 29 – 31 ]. Early studies using chromosomal inversion analyses identified An. coustani and An. crypticus as separate species [ 29 , 30 ]. Genetic diversity analyses in Zambia and the Democratic Republic of the Congo also reported two distinct phylogenetic groups of An. coustani populations [ 31 ] in 2020, and definitive species identification remained unverified based on conventional barcoding methods in Mozambique in 2024 for An. tenebrosus and An. zeimanni [ 18 ]. Mitochondrial genomes (mitogenomes) are circular, double stranded DNA molecules with high copy numbers, low incidence of recombination, absence of introns, and maternal inheritance [ 32 – 34 ]. These characteristics facilitate utility for inferring phylogenies, addressing species identification, and evolutionary studies in a range of organisms including metazoans [ 35 – 37 ]. The mitogenome encodes for 13 protein coding genes (PCGs), 22 transfer RNA (tRNA), 2 ribosomal RNA (rRNA) and a non-coding control region [ 38 ]. Developments in computational and sequencing technologies enable more datasets to include chromosomal and mitochondrial reference genomes for mosquito species, where both data are available [ 32 , 36 , 39 , 40 ]. However, sequencing efforts to date have been biased toward well-studied and defined species groups such as An. gambiae [ 41 – 43 ] and An. funestus [ 44 , 45 ]. At the present time, there are five mitochondrial and two chromosomal genomes collectively available in the GenBank databse for An. coustani sensu stricto and An. ziemanni [ 46 – 49 ]. Generating additional reference mitogenomes for members of the An. coustani group would prove beneficial for phylogenetic analyses and these data can inform taxonomic classification, mosquito diversity, and evolutionary history in relation to malaria transmission of this understudied group [ 50 , 51 ]. Although full genomes would be ideal for these tasks, mitochondrial genomes can be sequenced and assembled quickly and inexpensively compared to full nuclear genome sequencing and annotation. Given that accurate species identification is crucial for vector incrimination and the development and evaluation of vector control strategies, the taxonomic resolution of species in the An. coustani group is essential for malaria control efforts [ 23 ]. Additionally, it is not plausible to generate significant inferences regarding population and evolutionary histories or actual taxonomic species boundaries based on currently available evidence. This study aims to contribute complete reference mitochondrial genomes for members of the An. coustani group in Zambia and delineate the phylogenetic taxonomy for this epidemiologically important mosquito complex. Methods Mosquito collection and morphological identification. Outdoor mosquito collections were carried out in Zambia as part of the Southern and Central Africa International Centers of Excellence for Malaria Research (ICEMR). Specimen collections were performed in 2023–2024 using standard Centers for Disease Control and Prevention (CDC) miniature light traps in Choma and Nchelenge Districts (Fig. 1 ). Larvae were collected in the Chilubi and Mbala Districts and were reared to adults at the Tropical Diseases Research Centre (TDRC), Ndola, Zambia. Mosquitoes were sorted and identified using a morphological key [ 52 ] by members of the ICEMR team. Specimens morphologically identified as An. coustani , An. ziemanni , An. tenebrosus , and An. paludis were stored in tubes containing silica gel and shipped to the Johns Hopkins Bloomberg School of Public Health (Maryland, USA) for molecular analysis. The specimens with intact morphological characteristics that allowed clear identification as An. coustani , An. tenebrosus, An. paludis and An. ziemanni , were molecularly confirmed and selected for sequencing and downstream analysis. Specimens that could not be further keyed to species type due to damage or cryptic features were labelled as An. coustani sensu lato (s.l.). DNA extraction, sequencing, mitogenome assembly and annotation. Single mosquito specimens were homogenized in a mixture containing 98 µL of PK buffer (Applied Biosystems, Waltham, MA) and 2 µL of proteinase K (Applied Biosystems, Waltham, MA) followed by an incubation at 56 o C for 2.5 hours [ 53 ]. After incubation, DNA was extracted using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions. Using the Qubit dsDNA assay kit (Thermo Fisher Scientific, Waltham, MA) the extracted DNA was quantified and stored at -20 o C. Extracted DNA was shipped to SeqCenter (Pittsburgh, USA) for library construction and Illumina sequencing. Libraries were 150 bp paired end sequenced to a depth of 13.3 million reads. Using NOVOPlasty [ 54 ] (RRID:SCR_017335) version 4.3.5, the mitochondrial genomes were assembled with k-mer set at 39 and reference mitogenomes (MT_806097, NC_064609, NC_064611) as seed sequences. The generated contigs were automatically annotated using the MITOchondrial genome annotation (MITOS) [ 55 ] galaxy tool under the invertebrate genetic code with default settings. Using reference An. coustani mitochondrial genomes as guides, start and stop codon positions were manually modified in Geneious Prime (RRID:SCR_010519) version 2023.2.1 (Biomatters, Auckland, Australia). Resulting sequences and their corresponding annotations were uploaded to the GenBank database. Phylogenetic analysis and divergence time estimation The protein coding genes of the mitogenomes constructed in this study and those from An. coustani (MT_806097, NC_064611, OX_030899), An. ziemanni (NC_064609, OX_030922), An. gambiae (NC_083487), An. arabiensis (NC_028212), An. pharoensis (PP_068257), An. rufipes (PP_068269) and Ae. aegypti (NC_035159) reference sequences were imported from the GenBank repository, aligned, and exported in nexus format using the MAFFT amino acid alignment mode in Geneious Prime (RRID:SCR_010519) version 2023.2.1 (Biomatters, Auckland, Australia). Using jModelTest (v2.1.10) software [ 56 ] with default settings. The best fit base pair substitution model for the aligned sequence matrix was identified based on the Bayesian information criterion (BIC) and the Akaike information criterion (AIC). Bayesian inference analysis and node age calculations were performed in Bayesian Evolutionary Analysis by Sampling Trees (BEAST) version 2.7.6 [ 57 ] using the GTR + G + I substitution model with three independent runs as described [ 58 ]. An application of 20% burn-in rate was implemented for tree building purposes and FigTree v.1.4.4 ( http://tree.bio.ed.ac.uk/software/figtree/ ) was used to visualize trees. Molecular dating time estimations were inferred alongside the previously mentioned parameters using Aedes-Anopheles divergence time as the calibration point. The Aedes-Anopheles divergence was set as a prior with normal distribution around 154.7 million years ago (MYA) [ 59 ]. Pairwise genetic distances between representative groups were computed in the MEGA X 10.0.5 software [ 60 ] using the exported MAFTT amino acid alignment from Geneious Prime. Results Mitochondrial genome characteristics Review of collections from 2023–2024 provided 81 putative An. coustani group specimens. From these, 17 specimens passed morphological and molecular confirmation, and were sequenced and annotated. The 17 novel mitogenomes produced in this study were arranged similarly to the reference An. coustani and An. ziemanni mitochondrial genomes available in the GenBank database, with lengths ranging from 15,404 bp ( An. tenebrosus ) to 15,425 bp ( An. paludis ) and an average AT content of 78.3% (Table S1 ). The An. coustani group mitogenomes comprised of 13 PCGs, 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs) as shown in Fig. 2 . Phylogenetic and Divergence time analysis The aligned and concatenated protein coding sequences from the 25 mitogenomes (24 Anopheles and 1 Aedes mosquito species as an outgroup) resulted in a matrix of 11,023 bp, which was included in the Bayesian analyses for the phylogenetic tree construction and molecular dating. Bayesian inferences resulted in well supported phylogenies with posterior probabilities close to or at one for the mitogenomes generated in this study. Six main clades were identified. Five clades represent four taxa ( An. tenebrosus , An. coustani , An. ziemanni , An. paludis ) from the An. coustani group and an ‘unspeciated’ group comprised of specimens morphologically identified as An. coustani s.l. (Fig. 3 ). The sixth clade is comprised of the GenBank reference sequences labeled as An. coustani and An. ziemanni as identified in GenBank. The most recent common ancestor (MRCA) of all Anopheles was dated at 109.77 MYA (Fig. 4 ) with a 95% confidence interval spanning from 68.4 to 157.02 MYA (Table 1 ), using the Anopheles - Aedes divergence period set at 154.7 MYA [ 59 ]. The MRCA for An. coustani s.l. and An. ziemanni within the An. coustani group dates to 10.4 MYA, with a credibility interval that spans from 0.7 to 14.3 MYA. This MRCA is more recent than those determined for the unspeciated group and An. paludis from other members of the An. coustani group, estimated at 15.9 and 34.4 MYA respectively (Fig. 4 and Table 1 ). The pairwise genetic distance matrix calculations (Table S2) between representatives of each group/clade ranged from 0.0008–0.0217, except for An. paludis which resulted in a much wider genetic distance. Table 1 Divergence estimations output from BEAST v 2.7.6 for Anopheles species including the mitochondrial genomes generated in this study. Selected nodes Mean ages 95% credibility interval Aedes/Anopheles 155.02 150.00 -158.02 Anopheles 109.77 68.40–157.02 An. paludis/ An. coustani group 34.40 17.40–57.00 Unspeciated group/ An. coustani group 15.90 7.20 − 24.10 An. coustani s.s./ An. ziemanni 10.40 0.70 − 14.30 An. coustani s.s./ An. tenebrosus 7.06 3.00 -12.50 Discussion This study generated 17 new full-length mitochondrial genomes for members of the An. coustani group from Zambia that improve the resolution of within-group species taxonomy and provide insight into the species group’s complexity. Bayesian analyses using the concatenated PCGs from the mitogenomes generated in this study supported phylogenies and separated the specimens into distinct taxonomic groups including An. coustani s.s., An. tenebrosus , An. paludis and An. ziemanni . These new phylogenies have better taxonomic resolution and stronger branch support when compared to earlier studies in Zambia using the COI and ITS2 molecular barcodes [ 31 , 61 ]. Those studies separated An. coustani s.l. specimens into two general groups, An. coustani clade 1 or 2 [ 31 , 61 ], or undefined Anopheles species groups [ 61 ]. Furthermore, a subset of the An. coustani s.s. specimens in this study formed a separate clade from the GenBank reference genome sequences identified as An. coustani and An. ziemanni , an indication of additional complexity within the An. coustani species group or perhaps, morphological misidentification prior to sequencing. This study highlights the significance of anopheline morphological data and molecular verification for identifying both known and unknown anopheline species, especially those implicated as malaria vectors. Though previous studies have shed light on mosquitoes in the An. coustani group and their association with malaria transmission [ 20 , 21 , 23 , 31 ], there remains a paucity of sequence data corresponding to well-curated specimens which can be used to accurately speciate members of this group. As a result, the majority of available COI and ITS2 sequences are categorized as ‘ An. coustani s.l.’, rather than to specific species within the group [ 18 , 24 , 61 ]. Despite the increased taxonomic power the data in this study provided, there were some limitations to identification of all specimens. In the absence of voucher specimens available for sequencing or genomic data for other members of the group such as An. caliginosus , An. crypticus , An. namibiensis and An. symesi [ 11 ], our study faced challenges in determining the phylogenetic placement and species identification for one clade of specimens, which we designated as An. coustani s.l. These mosquito specimens were collected primarily in Nchelenge District on the border with the Democratic Republic of the Congo (DRC) where An. caliginosus has been reported [ 11 , 62 ], suggesting this species or perhaps other members of the An. coustani group may be more widely distributed in Zambia. Another caveat is the indistinguishable morphological features of adult female An. crypticus and An. coustani s.s. mosquitoes [ 11 , 30 ]. It is possible that the An. coustani s.s. specimens sequenced in this study, or alternatively the GenBank references, represent An. crypticus. This was implied by a study that identified ‘ An. coustani clade 2’ as putative An. crypticus [ 61 ]. Furthermore, pairwise distance estimates between representatives from these two groups suggest the potential prescence of An. crypticus circulating in Zambia. However, with the lack of reference specimens and the documented species range limited to South Africa [ 11 , 30 ], it is problematic to verify the presence of this species or correlate molecular and cytogenetic data to morphological identifications across different species and studies. Genetic distance matrices may provide definition of species boundaries [ 63 ], and the calculations derived from this study reinforce the complexity of relatedness among species such as An. coustani and An. ziemanni , further implying that cryptic speciation may be due to behavioral and ecological preferences [ 64 ]. Although studies for African anophelines have been biased towards well-recognized vectors such as An. funestus and An. gambiae [ 43 , 45 , 65 ], divergence estimations and phylogenies are also reported to be unresolved due to complexities such as introgression [ 25 , 58 , 66 ]. Our molecular divergence calculations suggest the An. coustani group diverged from the An. gambiae species complex ~ 110 MYA. This is consistent with inferences made by previous studies which reported the last common ancestor of Anopheles ~ 100 MYA [ 67 ] and the African distribution of the Anopheles subgenus ~ 113 MYA [ 68 ]. Molecular dating based on this phylogenetic analysis shows An. paludis splitting ~ 34 MYA from closely related species group members. This divergence time is older than that estimated between the other clades and like that for An. gambiae and An. funestus , suggests that reproductive or opportunistic behavioral adaptions may have occurred to explain why some species group members may be more involved in the transsion of Plasmodium falciparum . Conclusions This is the first publication using a genome skimming strategy to generate 17 mitochondrial genomes for representatives of the An. coustani group. We were able to estimate divergence times for members of the group for which there is data and this study emphasizes the importance of actively pursuing accurately identified morphological voucher specimens for molecular characterization collected from other African regions. This is required for the clear delineation of species boundaries as well as for the taxonomic rectification among An. coustani members which have been shown to be closely related in this study. These findings also highlight the need for study of the basic biology of this group, inlcuding reproductive compatibility between members of the group which may resolve some of the taxonomic mysteries and most critically, their biological capacity to vector human pathogens is largely unknown. With changes in land use, climate and the decrease or shifts in primary malaria vector populations, research should focus on the ecological and behavioral characteristics of species in this and similarly understudied anopheline groups, as their importance in malaria transmission becomes more prominent. Abbreviations IRS: Indoor residual spraying LLINs: Long lasting insecticidal nets COI: Cytochrome oxidase I ITS2: Internal transcribed spacer 2 PCGs: Protein coding genes tRNA: transfer RNA rRNA: ribosomal RNA ICEMR: International Centers of Excellence for Malaria Research CDC: Centers for Disease Control and Prevention MRT: Macha Research Trust TDRC: Tropical Diseases Research Centre BIC: Bayesian information criterion AIC: Akaike information criterion BEAST: Bayesian Evolutionary Analysis by Sampling Trees MYA: Million years ago MRCA: Most recent common ancestor DRC: Democratic Republic of Congo Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The dataset supporting the conclusions of this article are available in the GenBank repository under the BioProject PRJNA1061161. The mitochondrial genomes are available with accession numbers PP375116, PP385940-PP385945, PP392958-PP392959, PP413756, PQ585798 and PQ587036-PQ587041. Competing interests The authors declare that they have no competing interests. Funding This study was supported in part by by funds from the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (U19AI089680),T32 support to M.E.G. (T32AI138953) and A.C.M. (T32AI138953), a Johns Hopkins Malaria Research Institute Postdoctoral Award to R.L.M.N.A., the Bloomberg Philanthropies, and NSF-Accelerator Project D-688: Computing the Biome (2134862). Authors contributions S.U., R.L.M.N.A. and D.E.N. conceived and designed the study. M.E.G., L.S., K.S., W.H., H.C. and M.M. performed field collections and morphological identification of mosquito specimens. S.U. and R.L.M.N.A worked on laboratory extractions and bioinformatic pipelines for generated datasets. 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Skimming for barcodes: rapid production of mitochondrial genome and nuclear ribosomal repeat reference markers through shallow shotgun sequencing. PeerJ. 2022;10:e13790. da Silva FS, do Nascimento BLS, Cruz ACR, da Silva SP, Aragão A, de Dias O. Sequencing and description of the complete mitochondrial genome of Limatus durhamii (Diptera: Culicidae). Acta Trop. 2023;239:106805. Kneubehl AR, Muñoz-Leal S, Filatov S, de Klerk DG, Pienaar R, Lohmeyer KH, et al. Amplification and sequencing of entire tick mitochondrial genomes for a phylogenomic analysis. Scientifc Rep. 2022;12:19310. Dong Z, Wang Y, Li C, Li L, Men X. Mitochondrial DNA as a Molecular Marker in Insect Ecology: Current Status and Future Prospects. Ann Entomol Soc Am. 2021;114:470–6. Behura SK, Lobo NF, Haas B, deBruyn B, Lovin DD, Shumway MF, et al. Complete sequences of mitochondria genomes of Aedes aegypti and Culex quinquefasciatus and comparative analysis of mitochondrial DNA fragments inserted in the nuclear genomes. Insect Biochem Mol Biol. 2011;41:770–7. Ryazansky SS, Chen C, Potters M, Naumenko AN, Lukyanchikova V, Masri RA, et al. The chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus uncovers patterns of genome evolution in mosquitoes. BMC Biol. 2024;22:16. Miles A, Harding NJ, Bottà G, Clarkson CS, Antão T, Kozak K, et al. Genetic diversity of the African malaria vector Anopheles gambiae . Nature. 2017;552:96–100. Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab R, Nusskern DR et al. The Genome Sequence of the Malaria Mosquito Anopheles gambiae . Science 298(5591):129–49. Consortium TA, gambiae 1000 G, Clarkson CS, Miles A, Harding NJ, Lucas ER, Battey CJ, et al. Genome variation and population structure among 1142 mosquitoes of the African malaria vector species Anopheles gambiae and Anopheles coluzzii . Genome Res. 2020;30:1533–46. Ghurye J, Koren S, Small ST, Redmond S, Howell P, Phillippy AM, et al. A chromosome-scale assembly of the major African malaria vector Anopheles funestus . GigaScience. 2019;8:giz063. Jones CM, Lee Y, Kitchen A, Collier T, Pringle JC, Muleba M, et al. Complete Anopheles funestus mitogenomes reveal an ancient history of mitochondrial lineages and their distribution in southern and central Africa. Sci Rep. 2018;8:9054. Bouafou LBA, Ayala D, Makanga BK, Rahola N, Johnson HF, Heaton H et al. Chromosomal reference genome sequences for the malaria mosquito, Anopheles coustani , Laveran, 1900. Wellcome Open Research. 2024;9:551. Campos M, Crepeau M, Lee Y, Gripkey H, Rompão H, Cornel AJ, et al. Complete mitogenome sequence of Anopheles coustani from São Tomé island. Mitochondrial DNA Part B. 2020;5(3):3376–8. Wellcome Sanger Institute. Anopheles ziemanni genome assembly - BioProject - NCBI [Internet]. [cited 2024 Nov 27]. Available from: https://www.ncbi.nlm.nih.gov/bioproject/PRJEB53272/ Wellcome Sanger Tree of Life Programme. Anopheles ziemanni mitochondrion, complete genome- NCBI [Internet]. [cited 2024 Nov 27]. Available from: https://www.ncbi.nlm.nih.gov/nucleotide/NC_064609.1 Soghigian J, Sither C, Justi SA, Morinaga G, Cassel BK, Vitek CJ, et al. Phylogenomics reveals the history of host use in mosquitoes. Nat Commun. 2023;14:6252. Reidenbach KR, Cook S, Bertone MA, Harbach RE, Wiegmann BM, Besansky NJ. Phylogenetic analysis and temporal diversification of mosquitoes (Diptera: Culicidae) based on nuclear genes and morphology. BMC Evol Biol. 2009;9:298. Coetzee M. Key to the females of Afrotropical Anopheles mosquitoes (Diptera: Culicidae). Malar J. 2020;19:70. Chen T-Y, Vorsino AE, Kosinski KJ, Romero-Weaver AL, Buckner EA, Chiu JC et al. A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing. J Visualized Experiments. 2021; 15;170. Dierckxsens N, Mardulyn P, Smits G. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 2017;45:e18. Bernt M, Donath A, Jühling F, Externbrink F, Florentz C, Fritzsch G, et al. MITOS: improved de novo metazoan mitochondrial genome annotation. Mol Phylogenet Evol. 2013;69:313–9. Posada D, jModelTest. Phylogenetic Model Averaging. Mol Biol Evol. 2008;25:1253–6. Bouckaert R, Heled J, Kühnert D, Vaughan T, Wu C-H, Xie D, et al. BEAST 2: A Software Platform for Bayesian Evolutionary Analysis. PLoS Comput Biol. 2014;10:e1003537. Martinez-Villegas L, Assis-Geraldo J, Koerich LB, Collier TC, Lee Y, Main BJ, et al. Characterization of the complete mitogenome of Anopheles aquasalis , and phylogenetic divergences among Anopheles from diverse geographic zones. PLoS ONE. 2019;14:e0219523. Krzywinski J, Grushko OG, Besansky NJ. Analysis of the complete mitochondrial DNA from Anopheles funestus : An improved dipteran mitochondrial genome annotation and a temporal dimension of mosquito evolution. Mol Phylogenet Evol. 2006;39:417–23. Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol. 2021;38:3022–7. Cross DE, Healey AJE, McKeown NJ, Thomas CJ, Macarie NA, Siaziyu V, et al. Temporally consistent predominance and distribution of secondary malaria vectors in the Anopheles community of the upper Zambezi floodplain. Sci Rep. 2022;12:240. Hendershot AL. Understanding the Role of Anopheles coustani Complex Members as Malaria Vector Species in the Democratic Republic of Congo [Internet] [thesis]. University of Notre Dame; 2021 [cited 2024 Dec 13]. Available from: https://curate.nd.edu/articles/thesis/Understanding_the_Role_of_i_An_coustani_C_i_omplex_Members_as_Malaria_Vector_Species_in_the_Democratic_Republic_of_Congo/24851787/1 Marshall JWS Jr. Operational Criteria for Delimiting Species. Annu Rev Ecol Evol Syst. 2004;35:199–227. Hending D. Cryptic species conservation: a review. Biol Rev. 2025;100:258–74. Ditter RE, Campos M, Crepeau MW, Pinto J, Toilibou A, Amina Y, et al. Anopheles gambiae on remote islands in the Indian Ocean: origins and prospects for malaria elimination by genetic modification of extant populations. Sci Rep. 2023;13:20830. Fontaine MC, Pease JB, Steele A, Waterhouse RM, Neafsey DE, Sharakhov IV, et al. Extensive introgression in a malaria vector species complex revealed by phylogenomics. Science. 2015;347:1258524. Highly evolvable malaria vectors. The genomes of 16 Anopheles mosquitoes | Science. 2;347(6217):1258522. Freitas LA, Russo CAM, Voloch CM, Mutaquiha OCF, Marques LP, Schrago CG. Diversification of the Genus Anopheles and a Neotropical Clade from the Late Cretaceous. PLoS ONE. 2015;10:e0134462. Additional Declarations No competing interests reported. Supplementary Files SupplementaryforCoustani.docx Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Malaria Journal → Version 1 posted Editorial decision: Revision requested 24 Apr, 2025 Reviews received at journal 25 Mar, 2025 Reviewers agreed at journal 03 Mar, 2025 Reviewers invited by journal 01 Mar, 2025 Editor assigned by journal 06 Feb, 2025 Submission checks completed at journal 06 Feb, 2025 First submitted to journal 06 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Soha","middleName":"","lastName":"Usmani","suffix":""},{"id":433945387,"identity":"d6f3d4fb-b92e-432c-96b2-01ff85ea2bbd","order_by":1,"name":"Mary E. Gebhardt","email":"","orcid":"","institution":"The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Mary","middleName":"E.","lastName":"Gebhardt","suffix":""},{"id":433945389,"identity":"1a329735-0cad-4777-9c1d-fc5fd0321b43","order_by":2,"name":"Limonty Simubali","email":"","orcid":"","institution":"Macha Research Trust","correspondingAuthor":false,"prefix":"","firstName":"Limonty","middleName":"","lastName":"Simubali","suffix":""},{"id":433945390,"identity":"a276e59b-4c11-4d7b-b5c6-66399d4455e7","order_by":3,"name":"Kochelani Saili","email":"","orcid":"","institution":"Macha Research Trust","correspondingAuthor":false,"prefix":"","firstName":"Kochelani","middleName":"","lastName":"Saili","suffix":""},{"id":433945391,"identity":"ad046382-9a66-4463-b65a-0d0a90038675","order_by":4,"name":"Westone Hamwata","email":"","orcid":"","institution":"Tropical Diseases Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Westone","middleName":"","lastName":"Hamwata","suffix":""},{"id":433945392,"identity":"4b3ade69-4672-440a-9dff-eea56cf0032b","order_by":5,"name":"Hunter Chilusu","email":"","orcid":"","institution":"Tropical Diseases Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Hunter","middleName":"","lastName":"Chilusu","suffix":""},{"id":433945393,"identity":"240d92ab-8a5c-4018-abb4-2a56e326fe62","order_by":6,"name":"Mbanga Muleba","email":"","orcid":"","institution":"Tropical Diseases Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Mbanga","middleName":"","lastName":"Muleba","suffix":""},{"id":433945394,"identity":"023c5f3c-a75f-4982-b8bb-9a2bcd2c70ce","order_by":7,"name":"Conor J. McMeniman","email":"","orcid":"","institution":"The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Conor","middleName":"J.","lastName":"McMeniman","suffix":""},{"id":433945395,"identity":"72f37713-052d-4dea-a396-938e3adf921d","order_by":8,"name":"Anne C. Martin","email":"","orcid":"","institution":"Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health,","correspondingAuthor":false,"prefix":"","firstName":"Anne","middleName":"C.","lastName":"Martin","suffix":""},{"id":433945397,"identity":"60fe42f9-8583-4645-822f-65be1cf9c834","order_by":9,"name":"William J. Moss","email":"","orcid":"","institution":"Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health,","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"J.","lastName":"Moss","suffix":""},{"id":433945399,"identity":"5229b64d-daff-4807-998c-e6aa081c4e03","order_by":10,"name":"Douglas E. Norris","email":"","orcid":"","institution":"The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Douglas","middleName":"E.","lastName":"Norris","suffix":""},{"id":433945401,"identity":"65607a30-8196-4039-802c-86342b234136","order_by":11,"name":"Reneé L.M.N. Ali","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYFAC5oYDH37YMBsAmRJEamFsODizJ41ELcw8bIcZiNdi3n6w8QAPz3l2cwbmg7d5iNEicyax4YCExW1mywa2ZGuitEgwALUY8NxmNjjAYyZNnBb+hw0HEtjOAbXwfyNSiwTQlgNsB0C2sBGr5WHDwcaeZGaDw2zGlnOIc1jy4c9/ftglGxxvfnjjDTFaYCCZgZkU5SBgR6qGUTAKRsEoGEEAAINAMMSUBVsSAAAAAElFTkSuQmCC","orcid":"","institution":"The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":true,"prefix":"","firstName":"Reneé","middleName":"L.M.N.","lastName":"Ali","suffix":""}],"badges":[],"createdAt":"2025-02-06 22:23:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5976492/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5976492/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12936-025-05461-z","type":"published","date":"2025-07-01T15:57:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80057827,"identity":"e51a9fbd-858f-4cf0-b1a9-a8ea25a06dfe","added_by":"auto","created_at":"2025-04-07 11:49:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":198465,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing the four Districts used for mosquito collections in this study study.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5976492/v1/a751a03dcd8200f286027f9f.jpg"},{"id":80057829,"identity":"0f715692-b312-4e47-8486-f20164a10ca8","added_by":"auto","created_at":"2025-04-07 11:49:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":154723,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative mitochondrial genome of the \u003cem\u003eAn. coustani\u003c/em\u003e group comprising 37 genes: 13 PCGs, 22 tRNAs and 2 rRNAs.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5976492/v1/c7c8d3b0528086dbb4137b73.png"},{"id":80057832,"identity":"93d03cb7-5131-48b2-965d-c9754362cf52","added_by":"auto","created_at":"2025-04-07 11:49:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":213222,"visible":true,"origin":"","legend":"\u003cp\u003eBayesian tree showing phylogenetic relationships of 17 new mitogenomes (highlighted in blue) of the \u003cem\u003eAn. coustani\u003c/em\u003e group with other \u003cem\u003eAnopheles\u003c/em\u003e species. The tree was constructed using the concatenated PCGs using BEAST v 2.7.6 as described in the methods. The posterior probabilities supporting the tree topology are represented by the values at the nodes.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5976492/v1/e207a5be1fbb12aa835a887f.png"},{"id":80058800,"identity":"a575a9ff-3b2e-4292-ab0e-323a351602b8","added_by":"auto","created_at":"2025-04-07 11:57:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":296670,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree showing inferred molecular divergence estimates (MYA) for members of the \u003cem\u003eAn. coustani\u003c/em\u003e group (highlighted in blue) using the concatenated PCGs from mitogenomes generated in this study. The mean divergence time (MYA) predicted for each event is represented by the values at the tree nodes. The bars show the 95% confidence intervals.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5976492/v1/5f0744bd0b726cbb263adb63.png"},{"id":86179050,"identity":"e4d045d4-612e-4723-b692-f3df47ae68be","added_by":"auto","created_at":"2025-07-07 16:14:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1421313,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5976492/v1/914d2bd3-f397-453b-ade2-78470fc81e27.pdf"},{"id":80058798,"identity":"902d528c-f428-4363-bd59-89c38e9046a9","added_by":"auto","created_at":"2025-04-07 11:57:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":67578,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryforCoustani.docx","url":"https://assets-eu.researchsquare.com/files/rs-5976492/v1/b6c2bf90bb0efeb0ce8046c9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phylogenetic taxonomy of the Zambian Anopheles coustani group using a mitogenomics approach","fulltext":[{"header":"Background","content":"\u003cp\u003eVector control methods like indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) have been instrumental in progress toward malaria elimination [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Primary, well-studied vectors like \u003cem\u003eAnopheles gambiae\u003c/em\u003e and \u003cem\u003eAn. funestus\u003c/em\u003e, which typically engage in endophagic and endophilic behaviors by seeking human hosts indoors, are the focus of these key intervention measures [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, selection pressure driven by the broad deployment of IRS and LLINs have either reduced these populations, driven insecticide resistance, yielded shifts in vector species composition and/or resulted in changes in biting and resting behaviors [\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Shifts to outdoor biting or having a high plasticity in this behavior, and the existence of other exophagic malaria vectors have been identified as a significant barriers to malaria control and elimination [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Though frequently collected, exophagic anopheline mosquitoes such as members of the \u003cem\u003eAn. coustani\u003c/em\u003e group [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], \u003cem\u003eAn. squamosus\u003c/em\u003e, and \u003cem\u003eAn. rufipes\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] are understudied despite contributing to malaria transmission in sub-Sahran Africa.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eAnopheles coustani\u003c/em\u003e group is widely distributed throughout sub-Saharan Africa and the Middle East, with members typically exhibiting zoophilic and outdoor foraging behaviors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Within the group, morphologically similar species including \u003cem\u003eAn. coustani, An. zeimanni\u003c/em\u003e, \u003cem\u003eAn. paludis\u003c/em\u003e, and \u003cem\u003eAn. tenebrosus\u003c/em\u003e, have demonstrated opportunistic foraging towards anthropophilic and endophilic feeding [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Little is known about the basic biology, ecology and behaviors of most of these species. This knowledge gap is particularly noteworty given members of the group have been implicated as established vectors with a key role in sustaining residual malaria transmission in Kenya, Madagascar, Ethiopia, Cameroon, Mozambique and Zambia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Members of this group present an imminent threat to malaria elimination efforts due to inherent plasticity in their foraging behaviors, which enable them to evade many of the existing vector control strategies that target endophagic and endophilic mosquitoes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMorphological and molecular techniques have proved to be challenging for identification of species in this group due to cryptic features, damaged specimens which obscures key morphological attributes [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and the paucity of reference molecular data for comparison in genomic repositories [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, the well-established cytochrome oxidase I gene (COI) and the internal transcribed spacer 2 (ITS2) molecular barcodes commonly used for species confirmation have limited power in delineating phylogenetic disparities in cryptic species groups [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Though limited in number, published genetic and molecular studies have highlighted cryptic members within the \u003cem\u003eAn. coustani\u003c/em\u003e group [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Early studies using chromosomal inversion analyses identified \u003cem\u003eAn. coustani\u003c/em\u003e and \u003cem\u003eAn. crypticus\u003c/em\u003e as separate species [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Genetic diversity analyses in Zambia and the Democratic Republic of the Congo also reported two distinct phylogenetic groups of \u003cem\u003eAn. coustani\u003c/em\u003e populations [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] in 2020, and definitive species identification remained unverified based on conventional barcoding methods in Mozambique in 2024 for \u003cem\u003eAn. tenebrosus\u003c/em\u003e and \u003cem\u003eAn. zeimanni\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMitochondrial genomes (mitogenomes) are circular, double stranded DNA molecules with high copy numbers, low incidence of recombination, absence of introns, and maternal inheritance [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These characteristics facilitate utility for inferring phylogenies, addressing species identification, and evolutionary studies in a range of organisms including metazoans [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The mitogenome encodes for 13 protein coding genes (PCGs), 22 transfer RNA (tRNA), 2 ribosomal RNA (rRNA) and a non-coding control region [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Developments in computational and sequencing technologies enable more datasets to include chromosomal and mitochondrial reference genomes for mosquito species, where both data are available [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, sequencing efforts to date have been biased toward well-studied and defined species groups such as \u003cem\u003eAn. gambiae\u003c/em\u003e [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and \u003cem\u003eAn. funestus\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAt the present time, there are five mitochondrial and two chromosomal genomes collectively available in the GenBank databse for \u003cem\u003eAn. coustani\u003c/em\u003e sensu stricto and \u003cem\u003eAn. ziemanni\u003c/em\u003e [\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Generating additional reference mitogenomes for members of the \u003cem\u003eAn. coustani\u003c/em\u003e group would prove beneficial for phylogenetic analyses and these data can inform taxonomic classification, mosquito diversity, and evolutionary history in relation to malaria transmission of this understudied group [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Although full genomes would be ideal for these tasks, mitochondrial genomes can be sequenced and assembled quickly and inexpensively compared to full nuclear genome sequencing and annotation.\u003c/p\u003e \u003cp\u003eGiven that accurate species identification is crucial for vector incrimination and the development and evaluation of vector control strategies, the taxonomic resolution of species in the \u003cem\u003eAn. coustani\u003c/em\u003e group is essential for malaria control efforts [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Additionally, it is not plausible to generate significant inferences regarding population and evolutionary histories or actual taxonomic species boundaries based on currently available evidence. This study aims to contribute complete reference mitochondrial genomes for members of the \u003cem\u003eAn. coustani\u003c/em\u003e group in Zambia and delineate the phylogenetic taxonomy for this epidemiologically important mosquito complex.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cem\u003eMosquito collection and morphological identification.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eOutdoor mosquito collections were carried out in Zambia as part of the Southern and Central Africa International Centers of Excellence for Malaria Research (ICEMR). Specimen collections were performed in 2023\u0026ndash;2024 using standard Centers for Disease Control and Prevention (CDC) miniature light traps in Choma and Nchelenge Districts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Larvae were collected in the Chilubi and Mbala Districts and were reared to adults at the Tropical Diseases Research Centre (TDRC), Ndola, Zambia. Mosquitoes were sorted and identified using a morphological key [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] by members of the ICEMR team. Specimens morphologically identified as \u003cem\u003eAn. coustani\u003c/em\u003e, \u003cem\u003eAn. ziemanni\u003c/em\u003e, \u003cem\u003eAn. tenebrosus\u003c/em\u003e, and \u003cem\u003eAn. paludis\u003c/em\u003e were stored in tubes containing silica gel and shipped to the Johns Hopkins Bloomberg School of Public Health (Maryland, USA) for molecular analysis. The specimens with intact morphological characteristics that allowed clear identification as \u003cem\u003eAn. coustani\u003c/em\u003e, \u003cem\u003eAn. tenebrosus, An. paludis\u003c/em\u003e and \u003cem\u003eAn. ziemanni\u003c/em\u003e, were molecularly confirmed and selected for sequencing and downstream analysis. Specimens that could not be further keyed to species type due to damage or cryptic features were labelled as \u003cem\u003eAn. coustani\u003c/em\u003e sensu lato (s.l.).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eDNA extraction, sequencing, mitogenome assembly and annotation.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eSingle mosquito specimens were homogenized in a mixture containing 98 \u0026micro;L of PK buffer (Applied Biosystems, Waltham, MA) and 2 \u0026micro;L of proteinase K (Applied Biosystems, Waltham, MA) followed by an incubation at 56\u003csup\u003eo\u003c/sup\u003eC for 2.5 hours [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. After incubation, DNA was extracted using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) as per the manufacturer\u0026rsquo;s instructions. Using the Qubit dsDNA assay kit (Thermo Fisher Scientific, Waltham, MA) the extracted DNA was quantified and stored at -20\u003csup\u003eo\u003c/sup\u003eC. Extracted DNA was shipped to SeqCenter (Pittsburgh, USA) for library construction and Illumina sequencing. Libraries were 150 bp paired end sequenced to a depth of 13.3\u0026nbsp;million reads.\u003c/p\u003e \u003cp\u003eUsing NOVOPlasty [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] (RRID:SCR_017335) version 4.3.5, the mitochondrial genomes were assembled with k-mer set at 39 and reference mitogenomes (MT_806097, NC_064609, NC_064611) as seed sequences. The generated contigs were automatically annotated using the MITOchondrial genome annotation (MITOS) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] galaxy tool under the invertebrate genetic code with default settings. Using reference \u003cem\u003eAn. coustani\u003c/em\u003e mitochondrial genomes as guides, start and stop codon positions were manually modified in Geneious Prime (RRID:SCR_010519) version 2023.2.1 (Biomatters, Auckland, Australia). Resulting sequences and their corresponding annotations were uploaded to the GenBank database.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis and divergence time estimation\u003c/h2\u003e \u003cp\u003eThe protein coding genes of the mitogenomes constructed in this study and those from \u003cem\u003eAn. coustani\u003c/em\u003e (MT_806097, NC_064611, OX_030899), \u003cem\u003eAn. ziemanni\u003c/em\u003e (NC_064609, OX_030922), \u003cem\u003eAn. gambiae\u003c/em\u003e (NC_083487), \u003cem\u003eAn. arabiensis\u003c/em\u003e (NC_028212), \u003cem\u003eAn. pharoensis\u003c/em\u003e (PP_068257), \u003cem\u003eAn. rufipes\u003c/em\u003e (PP_068269) and \u003cem\u003eAe. aegypti\u003c/em\u003e (NC_035159) reference sequences were imported from the GenBank repository, aligned, and exported in nexus format using the MAFFT amino acid alignment mode in Geneious Prime (RRID:SCR_010519) version 2023.2.1 (Biomatters, Auckland, Australia). Using jModelTest (v2.1.10) software [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] with default settings. The best fit base pair substitution model for the aligned sequence matrix was identified based on the Bayesian information criterion (BIC) and the Akaike information criterion (AIC). Bayesian inference analysis and node age calculations were performed in Bayesian Evolutionary Analysis by Sampling Trees (BEAST) version 2.7.6 [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] using the GTR\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;I substitution model with three independent runs as described [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. An application of 20% burn-in rate was implemented for tree building purposes and FigTree v.1.4.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree/\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/software/figtree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to visualize trees. Molecular dating time estimations were inferred alongside the previously mentioned parameters using \u003cem\u003eAedes-Anopheles\u003c/em\u003e divergence time as the calibration point. The \u003cem\u003eAedes-Anopheles\u003c/em\u003e divergence was set as a prior with normal distribution around 154.7\u0026nbsp;million years ago (MYA) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Pairwise genetic distances between representative groups were computed in the MEGA X 10.0.5 software [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] using the exported MAFTT amino acid alignment from Geneious Prime.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial genome characteristics\u003c/h2\u003e \u003cp\u003eReview of collections from 2023\u0026ndash;2024 provided 81 putative \u003cem\u003eAn. coustani\u003c/em\u003e group specimens. From these, 17 specimens passed morphological and molecular confirmation, and were sequenced and annotated. The 17 novel mitogenomes produced in this study were arranged similarly to the reference \u003cem\u003eAn. coustani\u003c/em\u003e and \u003cem\u003eAn. ziemanni\u003c/em\u003e mitochondrial genomes available in the GenBank database, with lengths ranging from 15,404 bp (\u003cem\u003eAn. tenebrosus\u003c/em\u003e) to 15,425 bp (\u003cem\u003eAn. paludis\u003c/em\u003e) and an average AT content of 78.3% (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The \u003cem\u003eAn. coustani\u003c/em\u003e group mitogenomes comprised of 13 PCGs, 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhylogenetic and Divergence time analysis\u003c/h3\u003e\n\u003cp\u003eThe aligned and concatenated protein coding sequences from the 25 mitogenomes (24 \u003cem\u003eAnopheles\u003c/em\u003e and 1 \u003cem\u003eAedes\u003c/em\u003e mosquito species as an outgroup) resulted in a matrix of 11,023 bp, which was included in the Bayesian analyses for the phylogenetic tree construction and molecular dating. Bayesian inferences resulted in well supported phylogenies with posterior probabilities close to or at one for the mitogenomes generated in this study. Six main clades were identified. Five clades represent four taxa (\u003cem\u003eAn. tenebrosus\u003c/em\u003e, \u003cem\u003eAn. coustani\u003c/em\u003e, \u003cem\u003eAn. ziemanni\u003c/em\u003e, \u003cem\u003eAn. paludis\u003c/em\u003e) from \u003cem\u003ethe An. coustani\u003c/em\u003e group and an \u0026lsquo;unspeciated\u0026rsquo; group comprised of specimens morphologically identified as \u003cem\u003eAn. coustani\u003c/em\u003e s.l. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The sixth clade is comprised of the GenBank reference sequences labeled as \u003cem\u003eAn. coustani\u003c/em\u003e and \u003cem\u003eAn. ziemanni\u003c/em\u003e as identified in GenBank.\u003c/p\u003e \u003cp\u003eThe most recent common ancestor (MRCA) of all \u003cem\u003eAnopheles\u003c/em\u003e was dated at 109.77 MYA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) with a 95% confidence interval spanning from 68.4 to 157.02 MYA (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), using the \u003cem\u003eAnopheles\u003c/em\u003e-\u003cem\u003eAedes\u003c/em\u003e divergence period set at 154.7 MYA [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The MRCA for \u003cem\u003eAn. coustani\u003c/em\u003e s.l. and \u003cem\u003eAn. ziemanni\u003c/em\u003e within the \u003cem\u003eAn. coustani\u003c/em\u003e group dates to 10.4 MYA, with a credibility interval that spans from 0.7 to 14.3 MYA. This MRCA is more recent than those determined for the unspeciated group and \u003cem\u003eAn. paludis\u003c/em\u003e from other members of the \u003cem\u003eAn. coustani\u003c/em\u003e group, estimated at 15.9 and 34.4 MYA respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The pairwise genetic distance matrix calculations (Table S2) between representatives of each group/clade ranged from 0.0008\u0026ndash;0.0217, except for \u003cem\u003eAn. paludis\u003c/em\u003e which resulted in a much wider genetic distance.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDivergence estimations output from BEAST v 2.7.6 for \u003cem\u003eAnopheles\u003c/em\u003e species including the mitochondrial genomes generated in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSelected nodes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean ages\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95% credibility interval\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAedes/Anopheles\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e155.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150.00 -158.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnopheles\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e109.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68.40\u0026ndash;157.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn.\u003c/em\u003e paludis/\u003cem\u003eAn. coustani\u003c/em\u003e group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.40\u0026ndash;57.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnspeciated group/\u003cem\u003eAn. coustani\u003c/em\u003e group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.20 \u0026minus;\u0026thinsp;24.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. coustani\u003c/em\u003e s.s./\u003cem\u003eAn. ziemanni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70 \u0026minus;\u0026thinsp;14.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. coustani\u003c/em\u003e s.s./\u003cem\u003eAn. tenebrosus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.00 -12.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study generated 17 new full-length mitochondrial genomes for members of the \u003cem\u003eAn. coustani\u003c/em\u003e group from Zambia that improve the resolution of within-group species taxonomy and provide insight into the species group\u0026rsquo;s complexity. Bayesian analyses using the concatenated PCGs from the mitogenomes generated in this study supported phylogenies and separated the specimens into distinct taxonomic groups including \u003cem\u003eAn. coustani\u003c/em\u003e s.s., \u003cem\u003eAn. tenebrosus\u003c/em\u003e, \u003cem\u003eAn. paludis\u003c/em\u003e and \u003cem\u003eAn. ziemanni\u003c/em\u003e. These new phylogenies have better taxonomic resolution and stronger branch support when compared to earlier studies in Zambia using the COI and ITS2 molecular barcodes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Those studies separated \u003cem\u003eAn. coustani\u003c/em\u003e s.l. specimens into two general groups, \u003cem\u003eAn. coustani\u003c/em\u003e clade 1 or 2 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], or undefined \u003cem\u003eAnopheles\u003c/em\u003e species groups [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Furthermore, a subset of the \u003cem\u003eAn. coustani\u003c/em\u003e s.s. specimens in this study formed a separate clade from the GenBank reference genome sequences identified as \u003cem\u003eAn. coustani\u003c/em\u003e and \u003cem\u003eAn. ziemanni\u003c/em\u003e, an indication of additional complexity within the \u003cem\u003eAn. coustani\u003c/em\u003e species group or perhaps, morphological misidentification prior to sequencing.\u003c/p\u003e \u003cp\u003eThis study highlights the significance of anopheline morphological data and molecular verification for identifying both known and unknown anopheline species, especially those implicated as malaria vectors. Though previous studies have shed light on mosquitoes in the \u003cem\u003eAn. coustani\u003c/em\u003e group and their association with malaria transmission [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], there remains a paucity of sequence data corresponding to well-curated specimens which can be used to accurately speciate members of this group. As a result, the majority of available COI and ITS2 sequences are categorized as \u0026lsquo;\u003cem\u003eAn. coustani\u003c/em\u003e s.l.\u0026rsquo;, rather than to specific species within the group [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the increased taxonomic power the data in this study provided, there were some limitations to identification of all specimens. In the absence of voucher specimens available for sequencing or genomic data for other members of the group such as \u003cem\u003eAn. caliginosus\u003c/em\u003e, \u003cem\u003eAn. crypticus\u003c/em\u003e, \u003cem\u003eAn. namibiensis\u003c/em\u003e and \u003cem\u003eAn. symesi\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], our study faced challenges in determining the phylogenetic placement and species identification for one clade of specimens, which we designated as \u003cem\u003eAn. coustani\u003c/em\u003e s.l. These mosquito specimens were collected primarily in Nchelenge District on the border with the Democratic Republic of the Congo (DRC) where \u003cem\u003eAn. caliginosus\u003c/em\u003e has been reported [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], suggesting this species or perhaps other members of the \u003cem\u003eAn. coustani\u003c/em\u003e group may be more widely distributed in Zambia. Another caveat is the indistinguishable morphological features of adult female \u003cem\u003eAn. crypticus\u003c/em\u003e and \u003cem\u003eAn. coustani\u003c/em\u003e s.s. mosquitoes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It is possible that the \u003cem\u003eAn. coustani\u003c/em\u003e s.s. specimens sequenced in this study, or alternatively the GenBank references, represent \u003cem\u003eAn. crypticus.\u003c/em\u003e This was implied by a study that identified \u0026lsquo;\u003cem\u003eAn. coustani\u003c/em\u003e clade 2\u0026rsquo; as putative \u003cem\u003eAn. crypticus\u003c/em\u003e [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Furthermore, pairwise distance estimates between representatives from these two groups suggest the potential prescence of \u003cem\u003eAn. crypticus\u003c/em\u003e circulating in Zambia. However, with the lack of reference specimens and the documented species range limited to South Africa [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], it is problematic to verify the presence of this species or correlate molecular and cytogenetic data to morphological identifications across different species and studies.\u003c/p\u003e \u003cp\u003eGenetic distance matrices may provide definition of species boundaries [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], and the calculations derived from this study reinforce the complexity of relatedness among species such as \u003cem\u003eAn. coustani\u003c/em\u003e and \u003cem\u003eAn. ziemanni\u003c/em\u003e, further implying that cryptic speciation may be due to behavioral and ecological preferences [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Although studies for African anophelines have been biased towards well-recognized vectors such as \u003cem\u003eAn. funestus\u003c/em\u003e and \u003cem\u003eAn. gambiae\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], divergence estimations and phylogenies are also reported to be unresolved due to complexities such as introgression [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Our molecular divergence calculations suggest the \u003cem\u003eAn. coustani\u003c/em\u003e group diverged from the \u003cem\u003eAn. gambiae\u003c/em\u003e species complex\u0026thinsp;~\u0026thinsp;110 MYA. This is consistent with inferences made by previous studies which reported the last common ancestor of \u003cem\u003eAnopheles\u003c/em\u003e\u0026thinsp;~\u0026thinsp;100 MYA [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] and the African distribution of the \u003cem\u003eAnopheles\u003c/em\u003e subgenus\u0026thinsp;~\u0026thinsp;113 MYA [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Molecular dating based on this phylogenetic analysis shows \u003cem\u003eAn. paludis\u003c/em\u003e splitting\u0026thinsp;~\u0026thinsp;34 MYA from closely related species group members. This divergence time is older than that estimated between the other clades and like that for \u003cem\u003eAn. gambiae\u003c/em\u003e and \u003cem\u003eAn. funestus\u003c/em\u003e, suggests that reproductive or opportunistic behavioral adaptions may have occurred to explain why some species group members may be more involved in the transsion of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis is the first publication using a genome skimming strategy to generate 17 mitochondrial genomes for representatives of the \u003cem\u003eAn. coustani\u003c/em\u003e group. We were able to estimate divergence times for members of the group for which there is data and this study emphasizes the importance of actively pursuing accurately identified morphological voucher specimens for molecular characterization collected from other African regions. This is required for the clear delineation of species boundaries as well as for the taxonomic rectification among \u003cem\u003eAn. coustani\u003c/em\u003e members which have been shown to be closely related in this study. These findings also highlight the need for study of the basic biology of this group, inlcuding reproductive compatibility between members of the group which may resolve some of the taxonomic mysteries and most critically, their biological capacity to vector human pathogens is largely unknown. With changes in land use, climate and the decrease or shifts in primary malaria vector populations, research should focus on the ecological and behavioral characteristics of species in this and similarly understudied anopheline groups, as their importance in malaria transmission becomes more prominent.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eIRS: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Indoor residual spraying\u003c/p\u003e\n\u003cp\u003eLLINs: \u0026nbsp; \u0026nbsp;Long lasting insecticidal nets\u003c/p\u003e\n\u003cp\u003eCOI: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cytochrome oxidase I\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eITS2: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Internal transcribed spacer 2\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCGs: \u0026nbsp; \u0026nbsp; Protein coding genes\u003c/p\u003e\n\u003cp\u003etRNA: \u0026nbsp; \u0026nbsp; \u0026nbsp;transfer RNA\u003c/p\u003e\n\u003cp\u003erRNA: \u0026nbsp; \u0026nbsp; \u0026nbsp;ribosomal RNA\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eICEMR: \u0026nbsp; International Centers of Excellence for Malaria Research\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCDC: \u0026nbsp; \u0026nbsp; \u0026nbsp; Centers for Disease Control and Prevention\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMRT: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Macha Research Trust\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTDRC: \u0026nbsp; \u0026nbsp; Tropical Diseases Research Centre\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBIC: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Bayesian information criterion\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAIC: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Akaike information criterion\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBEAST: \u0026nbsp; \u0026nbsp;Bayesian Evolutionary Analysis by Sampling Trees\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMYA: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Million years ago\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMRCA: \u0026nbsp; \u0026nbsp; Most recent common ancestor\u003c/p\u003e\n\u003cp\u003eDRC: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Democratic Republic of Congo\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe dataset supporting the conclusions of this article are available in the GenBank repository under the BioProject PRJNA1061161. The mitochondrial genomes are available with accession numbers PP375116, PP385940-PP385945, PP392958-PP392959, PP413756, PQ585798 and PQ587036-PQ587041.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported in part by by funds from the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (U19AI089680),T32 support to M.E.G. (T32AI138953) and A.C.M. (T32AI138953), a Johns Hopkins Malaria Research Institute Postdoctoral Award to R.L.M.N.A., the Bloomberg Philanthropies, and NSF-Accelerator Project D-688: Computing the Biome (2134862).\u003c/p\u003e\n\u003cp\u003eAuthors contributions\u003c/p\u003e\n\u003cp\u003eS.U., R.L.M.N.A. and D.E.N. conceived and designed the study. M.E.G., L.S., K.S., W.H., H.C. and M.M. performed field collections and morphological identification of mosquito specimens. S.U. and R.L.M.N.A worked on laboratory extractions and bioinformatic pipelines for generated datasets. S.U., D.E.N. and R.L.M.N.A. drafted the manuscript. C.J.M and A.C.M reviewed and approved the manuscript with all authors. W.J.M. and D.E.N. attained funding, read and approved the manuscript with all authors.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Zambian study teams at the Tropical Diseases Research Centre and Macha Research Trust. We are grateful to the communities of Nchelenge and Choma Districts for their cooperation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePryce J, Medley N, Choi L. Indoor residual spraying for preventing malaria in communities using insecticide-treated nets. 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PLoS ONE. 2015;10:e0134462.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Anopheles coustani, mitochondrial genome, phylogeny, Zambia, malaria","lastPublishedDoi":"10.21203/rs.3.rs-5976492/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5976492/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMosquito species belonging to the \u003cem\u003eAnopheles coustani\u003c/em\u003e group have been implicated in driving residual malaria transmission in sub-Saharan Africa and are regarded as an established primary vector in Madagascar. The morphological identification of mosquitoes in this group is challenging due to cryptic features and their molecular confirmation is difficult due to a paucity of reference sequence data representing all members of the group. Conventional molecular barcoding with the cytochrome oxidase I (COI) gene and the internal transcribed spacer 2 (ITS2) region targets is limited in their discrimination and conclusive identification of members of species complexes. In contrast, complete mitochondrial genomes (mitogenomes) have demonstrated much improved power over barcodes to be useful in rectifying taxonomic discrepancies in Culicidae.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe utilized a \u0026nbsp;genome skimming approach via shallow shotgun sequencing on individual mosquito specimens to generate sequence reads for mitogenome assembly. Bayesian inferred phylogenies and molecular dating estimations were perfomed on the concatenated protein coding genes using the Bayesian Evolutionary Analysis by Sampling Trees 2 (BEAST 2) platform. Divergence estimates were calibrated on published calucations for \u003cem\u003eAnopheles\u003c/em\u003e-\u003cem\u003eAedes\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study generated 17 new complete mitogenomes which were comprable to reference \u003cem\u003eAn. coustani\u003c/em\u003e mitogenomes in the GenBank repository by having 13 protein coding, 22 transfer RNA and 2 ribosomal RNA genes, with an average length of 15,400 bp and AT content of 78.3%. Bayesian inference using the concatenated protein coding genes from the generated and publicly available mitogenomes yielded six clades: one for each of the four taxa targeted in this study, the GenBank references, and a currently unknown species. Divergence times estimated that the \u003cem\u003eAn. coustani\u003c/em\u003e group separated from the \u003cem\u003eAn. gambiae \u003c/em\u003ecomplex approximately 110 million years ago (MYA), and members within the complex diverged at times points ranging from~34 MYA to as recent as ~7 MYA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese findings demonstrate the value of mitochondrial genomes in differentiating cryptic taxa and help to confirm morphological identities of \u003cem\u003eAn. coustani s.s.\u003c/em\u003e, \u003cem\u003eAn. paludis\u003c/em\u003e, \u003cem\u003eAn. zeimanni\u003c/em\u003e and \u003cem\u003eAn. tenebrosus\u003c/em\u003e. Divergence estimates with the \u003cem\u003eAn. coustani\u003c/em\u003e group are similar to those for well-studied anopheline vector groups. These analyses also highlight the likely prescence of other cryptic \u003cem\u003eAn. coustani\u003c/em\u003e group members circulating in Zambia.\u003c/p\u003e","manuscriptTitle":"Phylogenetic taxonomy of the Zambian Anopheles coustani group using a mitogenomics approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 11:49:51","doi":"10.21203/rs.3.rs-5976492/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-24T11:53:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-25T19:57:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"339735993393052969909735162864982880131","date":"2025-03-03T17:20:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-01T16:01:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-07T01:54:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-07T01:54:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2025-02-06T22:08:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9f8cff7b-bf3c-4410-bef4-a9b9edc33955","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:04:00+00:00","versionOfRecord":{"articleIdentity":"rs-5976492","link":"https://doi.org/10.1186/s12936-025-05461-z","journal":{"identity":"malaria-journal","isVorOnly":false,"title":"Malaria Journal"},"publishedOn":"2025-07-01 15:57:54","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-04-07 11:49:51","video":"","vorDoi":"10.1186/s12936-025-05461-z","vorDoiUrl":"https://doi.org/10.1186/s12936-025-05461-z","workflowStages":[]},"version":"v1","identity":"rs-5976492","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5976492","identity":"rs-5976492","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}