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. 2019 Mar 15;7:347. Originally published 2018 Mar 21. [Version 2] doi: 10.12688/f1000research.13807.2

Mitochondrial genomes of Anopheles arabiensis, An. gambiae and An. coluzzii show no clear species division

Mark J Hanemaaijer 1, Parker D Houston 1, Travis C Collier 1, Laura C Norris 1, Abdrahamane Fofana 2, Gregory C Lanzaro 1, Anthony J Cornel 3, Yoosook Lee 1,a
PMCID: PMC6489993  PMID: 31069048

Version Changes

Revised. Amendments from Version 1

The main difference between this version and the previous one is the analysis we performed to construct the phylogenetic tree. The newly created tree is shown in Figure 1. This approach is more in line of what previous studies that looked at mitogenomes in Anopheles specimens have done. This did not change the conclusion of the paper. We also added a new table (Table 1) where we list the chromosomal inversion of each specimen, as was suggested by one of the reviewers. Furthermore, we added Supplementary Table S1 with all the detected SNPs on the mitogenome for the different Anopheles species and chromosomal forms. We also addressed most of the comments the reviewers had and clarified where needed.

Abstract

Here we report the complete mitochondrial sequences of 70 individual field collected mosquito specimens from throughout Sub-Saharan Africa. We generated this dataset to identify species specific markers for the following Anopheles species and chromosomal forms: An. arabiensis, An. coluzzii (The Forest and Mopti chromosomal forms) and An. gambiae (The Bamako and Savannah chromosomal forms).  The raw Illumina sequencing reads were mapped to the NC_002084 reference mitogenome sequence. A total of 783 single nucleotide polymorphisms (SNPs) were detected on the mitochondrial genome, of which 460 are singletons (58.7%). None of these SNPs are suitable as molecular markers to distinguish among An. arabiensis, An. coluzzii and An. gambiae or any of the chromosomal forms. The lack of species or chromosomal form specific markers is also reflected in the constructed phylogenetic tree, which shows no clear division among the operational taxonomic units considered here.

Keywords: Mitogenome, species identification, Africa, malaria vector, mosquitoes, Anopheles, single nucleotide polymorphisms, phylogenomics

Introduction

Historically, mtDNA sequence has been used in taxonomy as a source of species diagnostic markers ( Cronin et al. (1991); De Barba et al. (2014); Pegg et al. (2006)) or in population genetics and evolutionary studies ( Fu et al. (2013); Harrison (1989); Llamas et al. (2016)). One advantage of using mitochondrial over nuclear DNA for such studies is that the mutation rate of mtDNA is about 10 times faster than nuclear DNA ( Brown et al. (1979); Haag-Liautard et al. (2008)), hence amplifying the evolutionary trajectory of populations and species. In addition, mtDNA is easy to amplify, because there are more copies of mitochondrial DNA relative to nuclear DNA. Also, universal primers can be applied to a wide range of species. Widely used universal primers target the cytochrome b and cytochrome oxidase 1 genes ( Tahir et al. (2016)), because both have conserved and highly variable regions. In addition to these, other genes as described in De Mandal et al. (2014), can also be used as markers. However, phylogenetic trees based on mtDNA can deviate from the ones that are derived from nuclear DNA ( Phillips et al. (2013); Shaw (2002); Sota & Vogler, 2001).

The Anopheles gambiae species complex consists of eight morphologically identical species that can only be distinguished with molecular markers ( Scott et al. (1993); Coetzee et al., 2013) or, for some of the species, by cytological examination of polytene chromosomes ( Green, 1972; Pombi et al., 2008). The currently used molecular markers to distinguish between An. coluzzii and An. gambiae ( Lee et al., 2014) are located within genomic islands of divergence located proximal to the centromeres ( Turner et al. (2005)). Monitoring additional species-specific markers on mitochondrial DNA (mtDNA) could increase the ease of application and accuracy of species detection assays. In addition, mtDNA markers could enhance our understanding of divergence times among taxa within the complex.

Previous studies showed that there is a high amount of interspecific gene flow in mtDNA between An. coluzzii, An. gambiae and An. arabiensis specimens ( Besansky et al., 2003; Besansky et al., 1997; Donnelly et al., 2004). Although these data suggested no evidence for clear species division among the various species, the studies only focused on the ND5 loci ( Besansky et al., 2003; Donnelly et al., 2004) or included also cytochrome b and ND1 loci ( Besansky et al., 1997). In our study we use the complete mitogenome for comparison, which would make the analysis more robust. In addition, we specifically included the different chromosomal forms in our analysis. These chromosomal forms are genetically diverged from each other and display strong assortative mating in the An. gambiae chromosomal forms ( Touré et al., 1998). The An. coluzzii chromosomal forms differ from each other in their ecology: An. coluzzii-Mopti is found in dry areas whereas the An. coluzzii-Forest restrtict themselves to a wet climate ( Lee et al., 2009).

In this study we wished to identify species-specific markers within the mtDNA for Anopheles arabiensis, An. coluzzii and An. gambiae, including among the chromosomal forms currently subsumed under the designations An. gambiae and An. coluzzii, with the goal of adding these to our existing Anopheles species detection assay ( Lee et al. (2014)). We sequenced the whole mitogenomes of 70 individual mosquito specimens collected throughout Sub-Saharan Africa. The raw Illumina sequencing reads were mapped to the AgamP4 reference sequence, which included both nuclear and mitochondrial sequences. We explore the relationship among An. arabiensis, An. coluzzii, An. gambiae and four of the sub-specific chromosomal form mitogenome sequences.

Methods

Sample collection

Anopheles arabiensis raw Illumina sequencing reads were obtained from our previous study ( Marsden et al. (2014)). These included 20 female An. arabiensis mosquitoes which were collected indoors in houses using mouth aspirators from three villages in Tanzania in 2012 (Lupiro ((-8.38000°N, 36.66912°W), Sagamaganga (-8.06781°N, 36.80207°W), and Minepa (-8.25700°N, 36.68163°W) in the Kilombero Valley) and 4 samples from Cameroon collected in 2005 (9.09957°N, 13.72292°W). The DNA was extracted from the head and thorax of each mosquito species and An. arabiensis mosquitoes were identified using Scott primers ( Scott et al., 1993)). The adult An. gambiae and An. coluzzii samples were collected indoors using mouth aspirators in Kela, Mali (11.88683°N, -8.44744°W) in 2012 and Mutengene, Cameroon (4.0994°N, 9.3081°W) in 2011. We subdivided the An. coluzzii specimen into the Forest and Mopti chromosomal forms. Similarly, we did this for the An. gambiae Savannah and Bamako chromosomal forms. We examined the polytene chromosome to characterize the chromosomal forms as in Lanzaro & Lee, 2013 and used the same definitions. The results of chromosome determination are listed in Table 1. The An. quadriannulatus mosquito, used as an outgroup for the phylogenetic analysis, was collected as larvae in the Shingwidzi area (23.1160°S 31.3752°E) in South Africa in 2015 and was reared to adult.

Table 1. List of detected chromosomal inversions to detect chromosomal forms of An. coluzzii and An. gambiae according Toure and co-workers ( Touré et al., 1998). ‘2’ represents homozygous for the inversion, ‘1’ heterozygous for the inversion and ‘-‘ for homozygous for the standard arrangement.

Banked ID Chromosomal Form 2La 2Rb 2Rc 2Rd 2Rj 2Ru
11MUTE470 An. coluzzii-Forest - - - - - -
11MUTE472 An. coluzzii-Forest - - - - - -
11MUTE476 An. coluzzii-Forest - - - - - -
11MUTE477 An. coluzzii-Forest - - - - - -
11MUTE479 An. coluzzii-Forest - - - - - -
11MUTE480 An. coluzzii-Forest - - - - - -
11MUTE483 An. coluzzii-Forest - - - - - -
11MUTE487 An. coluzzii-Forest - - - - - -
11MUTE490 An. coluzzii-Forest - - - - - -
11MUTE491 An. coluzzii-Forest - - - - - -
11MUTE493 An. coluzzii-Forest - - - - - -
2012KELA022 An. coluzzii-Mopti 1 1 1 - - -
2012KELA024 An. coluzzii-Mopti 2 1 1 - - -
2012KELA046 An. coluzzii-Mopti 2 1 1 - - -
2012KELA085 An. coluzzii-Mopti 2 2 2 - - -
2012KELA087 An. coluzzii-Mopti 1 2 2 - - -
2012KELA088 An. coluzzii-Mopti 2 - - - - 1
2012KELA099 An. coluzzii-Mopti 2 - - - - 1
2012KELA112 An. coluzzii-Mopti 2 2 2 - - -
2012KELA161 An. coluzzii-Mopti 2 - - - - 1
2012KELA210 An. gambiae-Savannah 2 2 - - - -
2012KELA214 An. gambiae-Bamako 2 - 2 - 2 2
2012KELA219 An. gambiae-Bamako 2 - 2 - 2 2
2012KELA228 An. gambiae-Savannah 2 2 - - - -
2012KELA233 An. gambiae-Savannah 2 2 - - - -
2012KELA234 An. gambiae-Savannah 1 2 - - - -
2012KELA239 An. gambiae-Bamako 2 1 2 - 2 2
2012KELA240 An. gambiae-Bamako 2 1 2 - 2 2
2012KELA244 An. gambiae-Bamako 2 - 2 - 2 2
2012KELA285 An. gambiae-Savannah 2 2 - - - -
2012KELA321 An. gambiae-Savannah 2 2 - - - -
2012KELA334 An. gambiae-Savannah 2 2 - - - -
2012KELA348 An. gambiae-Savannah 2 2 - - - -
2012KELA367 An. gambiae-Bamako 2 1 2 - 2 2
2012KELA400 An. coluzzii-Mopti 2 - - - - 2
2012KELA406 An. gambiae-Bamako 2 - 2 - 2 2
2012KELA409 An. gambiae-Savannah 2 2 - - - -
2012KELA420 An. coluzzii-Mopti 2 - - - - 2
2012KELA423 An. coluzzii-Mopti 2 2 2 - - -
2012KELA443 An. gambiae-Bamako 2 1 2 - 2 2
2012KELA457 An. gambiae-Bamako 2 - 2 - 2 2
2012KELA458 An. coluzzii-Mopti 2 - - - - 2
2012KELA467 An. gambiae-Bamako 2 - 2 - 2 2
2012KELA468 An. gambiae-Savannah 2 1 - - - -
2012KELA481 An. gambiae-Bamako 2 2 2 - 2 2
2012KELA496 An. coluzzii-Mopti 2 1 - - - -
2012KELA651 An. gambiae-Bamako 2 2 2 - 2 2
2012KELA812 An. gambiae-Savannah 2 1 - - - -
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Genome sequencing

Sequencing methods for An. arabiensis samples are as described in Marsden et al. (2014). In short, individually barcoded Illumina paired-end sequencing libraries, with insert sizes of 320-400 basepairs (bp) using NEXTflex Sequencing kits (NOVA-5144) and barcodes (NOVA-514102)(Bio Scientific, Austin, TX, USA), were sequenced on an Illumina HiSeq2000 (Illumina, San Diego, CA, USA) with 100-bp paired-end reads using twelve samples per lane. For the An. coluzzii and An. gambiae samples we used the same methods as described in Norris et al. (2015) and Main et al. (2015). For the latter species, libraries were created using the Nextera DNA Sample Preparation Kit (FC-121-1031) and TruSeq dual indexing barcodes (FC-121-103)(Illumina) and the samples were sequenced on an Illumina HiSeq2500 with 100-bp paired end reads. We sequenced the whole genome, but only mapped the raw sequences to the NC_002084 reference mitogenome sequence.

Data analysis

De-multiplexed raw reads were trimmed using Trimmomatic ( Bolger et al. (2014)) version 0.36 and mapped to the mitogenome reference sequence of An. gambiae (Genbank accession number = NC_002084 ( Beard et al. (1993))). Freebayes (v1.0.1) ( Garrison & Marth, 2013) was used for mitochondrial variant calling assuming single ploidy and without population prior. Mapping statistics were calculated using qualimap version 2.2 ( Okonechnikov et al. (2016)) and the data is represented in Table 2. Following the recommendation of Crawford and Lazarro ( Crawford & Lazzaro, 2012), we used a minimum depth of 8 to call variants for each individual. Between positions 1-13,470bp of the mitogenome, we obtained consistently high quality reads for all samples, which were used for further analysis. An AT-rich region located between 13,471 and 15,388 suffers from low or zero coverage for sequences generated with the Nextera library preparation kit. Therefore, we excluded these regions from further analysis. The Vcf2fasta program ( Danecek et al. (2011)) was used to extract mitogenome sequences from vcf file to fasta format. Geneious version 10.1.3 was used for mitogenome alignments. The phylogenetic tree was generated using PhyloBayes MPI ( Lartillot et al., 2013) using the CAT-GTR model on the genomic sequences, which is shown to give similar results compared to amino acid sequences ( Foster et al., 2017). We ran the program twice for over 30000 iterations. Max difference between the two runs was 0.045 and minimum effective size was > 100 and created a consensus tree that we visualized in Geneious version 10.1.3. We used scikit-allel (v1.1.9), a software package for Python ( Miles & Harding (2017)), to identify species specific markers.

Table 2. List of samples that are used for the study.

Mapped reads indicates the reads that are mapped to the reference genome. Mean coverage indicates the average depth of reads on the mitochondrial DNA and standard deviation indicates the coverage deviation across the mitochondrial DNA.

Species Banked_id Year Country Village Mapped bases Mean
coverage		 Standard
deviation
An. coluzzii-Forest 11MUTE470 2011 Cameroon Mutengene 4265836 277.7 144.5
An. coluzzii-Forest 11MUTE472 2011 Cameroon Mutengene 1862892 121.3 23
An. coluzzii-Forest 11MUTE476 2011 Cameroon Mutengene 2130531 138.7 50.5
An. coluzzii-Forest 11MUTE477 2011 Cameroon Mutengene 806611 52.5 16.7
An. coluzzii-Forest 11MUTE480 2011 Cameroon Mutengene 804015 52.3 21
An. coluzzii-Forest 11MUTE483 2011 Cameroon Mutengene 1702247 110.8 42.9
An. coluzzii-Forest 11MUTE487 2011 Cameroon Mutengene 812839 52.9 21.2
An. coluzzii-Forest 11MUTE490 2011 Cameroon Mutengene 1882088 122.5 52.4
An. coluzzii-Forest 11MUTE491 2011 Cameroon Mutengene 1422997 92.6 46.6
An. coluzzii-Forest 11MUTE493 2011 Cameroon Mutengene 627590 40.9 17.3
An. coluzzii-Mopti 12KELA022 2012 Mali Kela 3695920 240.6 64.4
An. coluzzii-Mopti 12KELA024 2012 Mali Kela 574282 37.4 30.8
An. coluzzii-Mopti 12KELA046 2012 Mali Kela 4152520 270.3 87.2
An. coluzzii-Mopti 12KELA085 2012 Mali Kela 10883282 708.4 345
An. coluzzii-Mopti 12KELA087 2012 Mali Kela 3351158 218.1 79.8
An. coluzzii-Mopti 12KELA088 2012 Mali Kela 1704283 110.9 91.3
An. coluzzii-Mopti 12KELA099 2012 Mali Kela 349531 22.8 11
An. coluzzii-Mopti 12KELA112 2012 Mali Kela 8550102 556.5 198.2
An. coluzzii-Mopti 12KELA161 2012 Mali Kela 33794208 2199.7 629.3
An. gambiae-Savannah 12KELA210 2012 Mali Kela 3007375 195.8 53.3
An. gambiae-Bamako 12KELA214 2012 Mali Kela 26441050 1721.1 566.4
An. gambiae-Bamako 12KELA219 2012 Mali Kela 3617355 235.5 130.2
An. gambiae-Savannah 12KELA228 2012 Mali Kela 7783776 506.7 262.8
An. gambiae-Savannah 12KELA233 2012 Mali Kela 7827363 509.5 138.6
An. gambiae-Savannah 12KELA234 2012 Mali Kela 6721204 437.5 205.9
An. gambiae-Bamako 12KELA239 2012 Mali Kela 6683521 435 126.4
An. gambiae-Bamako 12KELA240 2012 Mali Kela 15131480 984.9 270.8
An. gambiae-Bamako 12KELA244 2012 Mali Kela 12851754 836.5 306.5
An. gambiae-Savannah 12KELA285 2012 Mali Kela 407888 26.6 119.8
An. gambiae-Savannah 12KELA321 2012 Mali Kela 1034014 67.3 43.8
An. gambiae-Savannah 12KELA334 2012 Mali Kela 20949015 1363.6 400.4
An. gambiae-Savannah 12KELA348 2012 Mali Kela 12053890 784.6 280.9
An. gambiae-Bamako 12KELA367 2012 Mali Kela 12109235 788.2 240.1
An. coluzzii-Mopti 12KELA400 2012 Mali Kela 13707820 892.3 398.2
An. gambiae-Bamako 12KELA406 2012 Mali Kela 17605437 1146 463.2
An. gambiae-Savannah 12KELA409 2012 Mali Kela 10526480 685.2 259.1
An. coluzzii-Mopti 12KELA420 2012 Mali Kela 31785953 2069 845.5
An. gambiae-Bamako 12KELA443 2012 Mali Kela 25740781 1675.5 669.1
An. gambiae-Bamako 12KELA457 2012 Mali Kela 1360654 88.6 36.6
An. coluzzii-Mopti 12KELA458 2012 Mali Kela 153686 10 10.4
An. gambiae-Bamako 12KELA467 2012 Mali Kela 10499093 683.4 249.1
An. gambiae-Savannah 12KELA468 2012 Mali Kela 10315033 671.4 197.1
An. gambiae-Bamako 12KELA481 2012 Mali Kela 20308589 1321.9 307.6
An. coluzzii-Mopti 12KELA496 2012 Mali Kela 2975297 193.7 162.9
An. gambiae-Bamako 12KELA651 2012 Mali Kela 376689 24.5 11.3
An. gambiae-Savannah 12KELA812 2012 Mali Kela 799071 52 29.3
An. arabiensis 12LUPI001 2012 Tanzania Lupiro 2843317 185.1 34.9
An. arabiensis 12LUPI007 2012 Tanzania Lupiro 6288802 409.3 40
An. arabiensis 12LUPI024 2012 Tanzania Lupiro 6328898 412 78.5
An. arabiensis 12LUPI056 2012 Tanzania Lupiro 5440256 354.1 39.2
An. arabiensis 12LUPI059 2012 Tanzania Lupiro 39721262 2585.5 801.8
An. arabiensis 12LUPI071 2012 Tanzania Lupiro 3433158 223.5 59.2
An. arabiensis 12LUPI074 2012 Tanzania Lupiro 10096062 657.2 100.5
An. arabiensis 12LUPI082 2012 Tanzania Lupiro 5732773 373.2 69.6
An. arabiensis 12MINE001 2012 Tanzania Minepa 7768923 505.7 66.9
An. arabiensis 12MINE040 2012 Tanzania Minepa 2784428 181.2 54.9
An. arabiensis 12MINE100 2012 Tanzania Minepa 10753877 700 93.9
An. arabiensis 12MINE101 2012 Tanzania Minepa 5684230 370 41.9
An. arabiensis 12MINE105 2012 Tanzania Minepa 1526829 99.4 32.8
An. arabiensis 12MINE111 2012 Tanzania Minepa 5578562 363.1 76.3
An. arabiensis 12SAGA066 2012 Tanzania Sagamaganga 12745079 829.6 142.3
An. arabiensis 12SAGA107 2012 Tanzania Sagamaganga 14460217 941.2 259.2
An. arabiensis 12SAGA131 2012 Tanzania Sagamaganga 15333239 998.1 282.9
An. arabiensis 12SAGA133 2012 Tanzania Sagamaganga 3792945 246.9 62.5
An. arabiensis 12SAGA134 2012 Tanzania Sagamaganga 2439101 158.8 34.5
An. arabiensis 12SAGA141 2012 Tanzania Sagamaganga 3130504 203.8 33.3
An. arabiensis 05OKJ017 2005 Cameroon Ourodoukoudje 9041052 588.5 78.8
An. arabiensis 05OKJ042 2005 Cameroon Ourodoukoudje 148752684 9682.5 785.7
An. arabiensis 05OKJ045 2005 Cameroon Ourodoukoudje 35514980 2311.7 262.8
An. arabiensis 05OKJ070 2005 Cameroon Ourodoukoudje 22847478 1487.2 400.5
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Aligned FASTA file of mitogenome samples

Copyright: © 2019 Hanemaaijer MJ et al.

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Click here for additional data file. (60.3KB, tgz)

Results and Discussion

We identified a total of 783 single nucleotide polymorphisms (SNPs) over the entire mitogenome. The majority of these (58.7%) were singletons (found on one of the 70 mitogenomes). We did not identify any SNPs unique to the species or chromosomal forms ( Supplementary Table S1) and therefore conclude that mtDNA is not suitable for Anopheles gambiae complex species identification.

The lack of species-specific markers is also reflected in the phylogenetic tree ( Figure 1). An. arabiensis, An. coluzzii and An. gambiae did not cluster separately, which is consistent with previous reports that compared mitochondrial genome sequence data from specimens originating from Kenya, Senegal and South Africa ( Besansky et al. (1997)) and Burkina Faso, Cameroon, Kenya, Mali, South Africa, Tanzania and Zimbabwe ( Fontaine et al. (2015), Supplemental material).

Figure 1. Phylogenetic tree inferred from mtDNA genome sequence data.

Figure 1.

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The phylogenetic tree fails to reveal a clear division of the operational taxonomic units included in this analysis. Colors indicate the species or chromosomal form and numbers at the branches indicate the accuracy of the inferred branches on a scale of 0–1, where 1 represents the highest confidence. The three An. arabiensis lineages are previously reported by Maliti and co-workers ( Maliti et al., 2016).

Our data may indicate that there is no divergent selection in mitogenome among An. gambiae complex. Since mitochondrial genomes have a higher (1–10 times) substitution rate than nuclear genomes ( Havird & Sloan, 2016; Lynch & Walsh, 2007), one might expect some level of divergence in the mitogenome in the absence of selection if the taxa have been separated by reproductive barrier even if they are in sympatry just as people have observed in nuclear genome. Therefore, our data showing lack of any species-specific markers on the mitogenome may due to the results of episodic hybridizations occurred between two species. Of note, 36 of the samples that we used in our study originated from Kela (Mali). Kela is located near the village of Selinkenyi, where previous studies have shown a history of hybridization and introgression between An. gambiae and An. coluzzii ( Lee et al. (2013); Main et al. (2015); Norris et al. (2015)), which may have resulted in shared polymorphisms in their mitochondrial genomes. Shared polymorphisms in their mitochondrial genomes, where history has not been reported, also appeared to have occurred in Mutengene (Cameroon), where both An. gambiae and An. coluzzii occur sympatrically. Hybridization between either An. coluzzii or An. gambiae with An. arabiensis yields sterile males ( Slotman et al. (2004)), but phylogenomic analysis of these species show patterns of introgression between all of them ( Fontaine et al. (2015)), which could be the reason that we do not find any species-specific markers on the mitogenome. Our mitochondrial genome study does not provide conclusive evidence for hybridization and introgression among the taxa under study. However, our data suggest that this is a possibility and it would be consistent with results reported by ( Fontaine et al., 2015) and ( Besansky et al., 1997). Future modeling work may illuminate the likely contribution of different evoluationary forces that shapes mitogenome and nuclear genome evolution.

Data availability

The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2019 Hanemaaijer MJ et al.

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). http://creativecommons.org/publicdomain/zero/1.0/

Aligned sequences were submitted to the National Center for Biotechnology Information (NCBI) Accession number: MG930826 - MG930896

Dataset 1. Aligned FASTA file of mitogenome samples 10.5256/f1000research.13807.d192892 ( Hanemaaijer et al., 2018)

Acknowledgments

We thank Michelle Sanford for her assistance in the field collection in Cameroon in 2011. We thank Clare Marsden for providing the raw data of An. arabiensis samples.

Funding Statement

We thank University of California - Irvine, Malaria Initiatives (UCIMI) for their support.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[version 2; peer review: 2 approved]

Supplementary material

Supplementary Table S1. List of SNP variants in the different Anopheles species and chromosomal forms.
Click here for additional data file. (38.7KB, tgz)

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F1000Res. 2019 Apr 29. doi: 10.5256/f1000research.20094.r45747

Reviewer response for version 2

Maria Anice Mureb Sallum 1

The revised version is suitable for publication and previously mentioned concerns have been clarified. The mitogenome annotation was well done, and the phylogenetic analyses were adequate. The writing is clear and correct. This work is an important contribution to our knowledge of the mitochondrial genome of the Anophelinae species complexes.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

F1000Res. 2018 May 14. doi: 10.5256/f1000research.15009.r33371

Reviewer response for version 1

Maria Anice Mureb Sallum 1

General comment

Phylogenetic analysis need to be improved, and the choice for NJ methods and JC model, justified in the article. There are several programs that have been largely employed for phylogenetic analysis, including for mitogenome data. The paper authored by Foster et al. 1 contains useful information about analyses that have been carried out for inferring phylogenetic relationships within Anophelinae mosquitoes. I strongly suggest authors to verify how analyses were done.

Sample collection

Authors - “The An. gambiae and An. coluzzii samples were collected as resting adults using mouth aspirators in Kela, Mali (11.88683°N, -8.44744°W) in 2012 and Mutengene, Cameroon (4.0994°N, 9.3081°W) in 2011.”

Comment - Can you please give more details the micro environment where your specimens of An. gambiae and An. coluzzii were resting?

Authors - “Similarly, we did this for the An. gambiae Savannah and Bamako chromosomal forms. We used the same definitions and methods to characterize the chromosomal forms as in Lanzaro & Lee, 2013.”

Comment - It is not clear to me if you examined the polytene chromosome of each specimen you identified as the Savannah, Bamako, Forest and Mopti forms. Please clarify.

Genome sequencing

Authors - “For the An. coluzzii and An. gambiae samples we used the same methods as described in Norris et al. (2015) and Main et al. (2015). For the latter species, libraries were created using the Nextera DNA Sample Preparation Kit (FC-121-1031) and TruSeq dual indexing barcodes (FC-121-103) (Illumina) and the samples were sequenced on an Illumina HiSeq2500 with 100-bp paired end reads.”

Comment - Please add a short sentence to clarify if you sequenced the whole genome and from the full sequence data you obtained the positions 1-13,470 of the mitogenome.

Data analysis

Authors - “The phylogenetic tree was generated using the Jukes-Cantor genetic distance model and Neighbor-Joining tree methods available in Geneious version 10.1.3.”

Comment - Authors should clarify their choice for sequence analysis. The Geneious software has been developed for editing and aligning DNA / amino acid sequences. There are several softwares, which have been largely used to infer phylogenetic relationships. I suggest authors to refining and improving the phylogenetic analysis using appropriate programs and models that have been chosen for the mitogenome data you have at hand.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

References

  • 1. Foster PG, de Oliveira TMP, Bergo ES, Conn JE, Sant'Ana DC, Nagaki SS, Nihei S, Lamas CE, González C, Moreira CC, Sallum MAM: Phylogeny of Anophelinae using mitochondrial protein coding genes. R Soc Open Sci.2017;4(11) : 10.1098/rsos.170758 170758 10.1098/rsos.170758 [DOI] [PMC free article] [PubMed] [Google Scholar]
F1000Res. 2018 Apr 30. doi: 10.5256/f1000research.15009.r32266

Reviewer response for version 1

Beniamino Caputo 1, Verena Pichler 2

General comments

The present research note entitled:” Mitochondrial genomes of Anopheles arabiensis, An. gambiae and An. coluzzii show no clear species division” is well analysed, reported and written. As already reported in previous study the submitted manuscript suggested the absence of any species-specific differences in the mitogenome of the three species examined. Although the manuscript is not innovative and the research is not based on any previous evidence, the present note confirms previous suggestions by examining the whole mitogenome of 70 specimens from field specimens and find the lack of species or chromosomal form specific markers.

Title and abstract

Title and abstract are appropriate and summarize well the content of the article.

Introduction

The introduction gives a good description of the aims of the present study, although I would have added some references to previous studies performed on mtDNA of the examined species (for example Besansky 1997) and why you expected to obtain different results compared to previous studies.

Please revise also:

“morphologically identical species that can only be distinguished with molecular markers”  (Scott  et al., 1993; Coetzee et al., 2013)

The currently used molecular markers are located within genomic islands of divergence located proximal to the centromeres ( Lee et al. (2014);  Turner et al. (2005)) please rephrase the citation and refer it only to detect genomic differences between An.gambiae e and An.coluzzii.

Please insert a sentence about chromosomal forms of An.gambiae.

Methods

Please specified the method for collecting An. arabiensis as you already described for An.gambiae (e.g. indoor specimens, mouth aspirators, PSC collections).

Please insert a table with inversion polymorphism of chromosomal forms analyzed.

Please add the source of the An. quadriannulatus specimens you included in the phylogenetic analysis.

Results

Study design is well explained and results are given concisely.

Please add in Table 2 also the number of specimens you included for each species in the analysis.

Please add in Figure two an explanation of what “lineage” means for An. arabiensis specimens.

Please give results (also without table or figure) for each country separately.

Discussion

Discussion is very concise but deals with most major points of interest. We would just suggest to explain better the conclusion on possible introgression (the more plausible hypothesis) between taxa and to evaluate other possible explanations for the absence of fixed differences between species (e.g. absence for divergent selection, or evolutionary characherestic of mitogenomes).

We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Aligned FASTA file of mitogenome samples

    Copyright: © 2019 Hanemaaijer MJ et al.

    Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

    Click here for additional data file. (60.3KB, tgz)
    Click here for additional data file. (38.7KB, tgz)

    Data Availability Statement

    The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2019 Hanemaaijer MJ et al.

    Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). http://creativecommons.org/publicdomain/zero/1.0/

    Aligned sequences were submitted to the National Center for Biotechnology Information (NCBI) Accession number: MG930826 - MG930896

    Dataset 1. Aligned FASTA file of mitogenome samples 10.5256/f1000research.13807.d192892 ( Hanemaaijer et al., 2018)

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