Introduction
Anopheles gambiae is a complex of six morphologically indistinguishable species of mosquitoes in the genus Anopheles (Besansky et al., 2003; Hunt et al., 1998). Major malaria vectors of the A. gambiae (Culicidae) complex includes (A. arabiensis, A. gambiae), with minor vectors (A. bwambae, A. melas, A. merus) and non-vectors (A. quadriannulatus). There is an overlap in the presence of A. gambiae and A. arabiensis in many regions spread across many areas in sub-Saharan Africa (Thelwell et al., 2000). In a small area around the geothermal springs located in the Semuliki National Park, Uganda there is a distinctive presence of Anopheles bwambae, where also A. gambiae and A. arabiensis species of the genus may be found (Harbach et al., 2007). A. merus and A. melas breed in saltwater (Coluzzi et al., 1979). A. quadriannulatus is a fresh water breeder, and although susceptible to Plasmodium infections, this species is not a natural vector of human malaria mainly because of its zoophilic behavior (Takken et al., 1999; Habtewold et al., 2008).

The ability to transmit Plasmodium efficiently makes members of the A. gambiae complex highly important vectors for public health and the scientific community. Malaria is the most important disease transmitted by the A. gambiae complex. This disease poses a great threat to half of the world’s population, and there are 300 million to 500 million clinical cases annually, resulting in approximately 1.5 million to 2.7 million deaths (WHO, 2010; WHO, 2011). Mosquitoes from the genus Anopheles are the only genus that transmits Plasmodium, the malaria-causing parasite. This genus is one of the most important of the three genera in the Culicidae subfamily Anophelinae. Of these families, Anopheles is the largest, with 427 species making up the family having an almost complete global presence (Harbach et al., 1997).

Phylogenetic reconstruction is a neglected area in mosquito vector systematics (Munstermann and Conn, 1997). Despite the fact that A. gambiae and A. arabiensis are highly efficient vectors, with an almost worldwide presence and the availability of the genome sequence (also molecular markers) for A. gambiae complex available. The phylogenetic relationships among the members are not fully delineated (Foley et al., 1998). The difficulty of determining the direction of evolution of the genus is made arduous because of the recent origin of the complex, the considerable level of sequence similarity, shared genetic elements, and shared molecular ancestral polymorphisms (Besansky et al., 1994; Mohanty et al., 2009). Phylogenic analysis as a tool for taxonomic studies has proved useful in mosquito sytematics (Mohanty et al., 2009).

Taxonomic classification of organisms have improved in light of advances made in DNA sequencing, which has provided DNA sequence data useful for reconstructing evolutionary relationships among several organisms. However, these new genetic classification often conflict with traditional taxonomy (Jobst et al., 1998). Thus, a good ideal on the molecular evolution and phylogenetic of the A. gambiae complex will help to understand the origins, evolution, classification and epidemiology (Foley et al., 1998; Thelwell et al., 2000). This has important implication in the control of malaria (Besansky et al., 2003).

Previous classification of the genus has not been tested using modern phylogenetics methods (Harbach et al., 1997), but instead, relies on intuitive taxonomic interpretations of a limited number of morphological similarities. Mohanty et al (2009) and Morgan et al (2009) provided the analysis of the phylogenetic relationship of Anopheles species, however the phylogenetics A. gambiae complex was not thoroughly investigated. Worked by Besansky et al (2003), after constructing a molecular phylogenetic tree of the A. gambiae complex with the A. gambiae and A. arabiensis, determined both species evolved from the same ancestor. The tree they constructed was contrary to the general accepted phylogenetics, which places the two principal vectors on distant branches. Coluzzi et al (1979) in their work noted that the A. gambiae complex represents one of the most recently diverged groups of sibling species studied to date. Thus the ability to infer evolutionary relationships in this species complex poses a challenge to all available phylogenetic techniques. Hence delineating the evolutionary position of Anopheles subfamily requires additional data.

In the present study, we explored cytochrome oxidase subunits I (COI) of mitochondrial DNA of the six member of the A. gambiae complex to infer the evolution and phylogeny of this group. Recently molecular markers have been used for a variety of genomic-based taxonomic, phylogenetic, population and evolutionary investigations in animal species (Hillis, 1996; Wilkerson et al., 2005; Khan et al., 2008). One of these DNA markers is the Mitochondrial DNA (mtDNA) sequence. Although mtDNA-sequence data have proved valuable in phylogenetic analysis, the selection of the appropriate gene for analysis is important. Among the coding genes in the mitochondrion genome, subunit I of the cytochrome oxidase (COI) gene possesses features suitable for evolutionary studies (Thompson et al., 1997). MtDNA posses a relatively fast mutation rate, which results in significant variation in mtDNA sequences between species providing an ample within species variance useful for phylogenetic investigations (Tamura and Nei, 1993; Mohanty et al., 2009). Our aim was to test the utility of COI genes of the species of A. gambiae complex to resolve the relationships among these species.

Materials and Methods
We retrieved the nucleotide sequences of individual mtDNA gene sequences of COI, of 6 members of the A. gambiae complex from the GenBank database (www.ncbi.nim.nih.gov). The details of these sequences are given in Table 1. Sequence alignment was performed by using CLUSTAL-X software (http://www.clustal.org), version 2.1 (Saitou and Nei, 1987) through elimination of all positional gaps and missing data. The sequences were then trimmed to get their equal lengths for all the species. As a result, a total of COI was 524 bp used in the final dataset.
 

Table 1 COI Sequences of the A. gambiae complex

The Maximum Likelihood method based on the Tamura-Nei model (Tamura et al., 2011), Neighbor-Joining method (Roe and Sperling, 2007), Minimum Evolution method (Tamura et al., 2004). All the phylogenetic analyses were conducted in MEGA software (http://www.megasoftware.net), version 5.0 (Kamali et al., 2012). Ancestral sequences were inferred using web server: www.fastml.tau.ac.il. All the positions containing gaps or missing data were eliminated (complete deletion) from the dataset prior to analysis. However, MEGA software also provides alternatives to retain all such sites initially and excluding them as necessary in the pair-wise distance estimation (pairwise deletion option) or to use the partial deletion (site coverage) as a percentage. Evolutionary divergence between and over all Sequences pairs of the A. gambiae complex were using the Maximum Composite Likelihood model (Nei and Kumar, 2000). Evolutionary analyses were conducted in MEGA 5 (Kamali et al., 2012).

Results and Discussion
The estimates of evolutionary divergence between sequences of the A. gambiae complex are presented in Table 2. The evolutionary divergence is smallest between A. arabiensis and A. gambiae (0.004) and A. melas and A. quadriannulatus (0.02). The divergence between other pairs of species was much greater. From this result, it can be inferred that A. Arabiensis and A. gambiae are the most closely related taxa. This is in agreement with the morphological, behavioural, and ecological similarity between the two species described by the work of Besansky et al (2003).
 

Table 2 Estimates of evolutionary divergence between sequences

The nucleotide composition of each taxon in the A. gambiae complex is presented in Figure 1. All the members of this complex were all AT rich. A. gambiae and A. arabiensis had the highest AT composition with both having 39.0% thymine (T) and 30.4% adenine (A), while A. merus had the least among the complex with 28.8% thymine (T) and 30.6% adenine (A). The nucleotide frequencies are 29.68% (A), 36.77% (T), 16.92% (C), and 16.63% (G). There is more of thymine in the sequences and less of cytosine. The frequency of transversion rate is higher than that of transition (Table 3). Transition rate occur more between guanine and adenine while less between thymine and cytosine. The transition/transversion rate ratios are k₁ = 1000 (purines) and k₂ = 268.531 (pyrimidines). The overall transition/transversion bias is R = 267.796, where R = [A×G×k1 + T×C×k2]/ [(A+G)×(T+C)].

Figure 1 Nucleotide composition of the taxa of A. gambiae complex

Table 3 Maximum composite likelihood estimate of the pattern of nucleotide substitution

Evolutionary history was inferred using the Maximum Likelihood method (Kamali et al., 2012) and is presented in Figure 2. A. arabiensis and A. gambiae are the most recently evolved taxa among the A. gambiae complex with an estimated divergence of less than a million years. The ancestral node from which the A. gambiae and A. arabiensis evolved from was inferred to be A. stephensi, which shared the same ancestral with A. merus over 293.93 million years ago. A. merus evolve from A. albitarsis over 285 million years ago. A. melas and A. quadriannulatus are also recently evolved with an estimated divergence of less than a million year ago. Their ancestry was inferred to be from A. deaneorum, which shared the same ancestor with A. bwambae.

Figure 2 Molecular phylogenetic anaylsis by Maximum Likelihood method

 
Evolutionary history inferred using the Neighbor-Joining method is presented in Figure 3. The topology of the tree is similar to the tree built using the Maximum Likelihood method. Notably, A. arabiensis and A. gambiae are on the same clade in both trees. These species are the most recently evolved, together with A. melas and A. quadriannulatus, which also have an estimated divergence time less than one million years. A. melas and A. quadriannulatus appear to have evolved from A. deaneorum, which shared the same ancestor with A. bwambae. The species with the longest estimated divergence times were A. bwambae (187 million years ago) and A. merus (307 million year ago). In agreement with the work of Besansky et al (2003), Foley et al (1998), Morgan et al (2009), A. gambiae and A. arabiensis were supported by our analysis as monophyletic taxa.

Figure 3 Evolutionary relationships of taxa Neighbor-Joining method

Proper understanding of the ancestral lineage of A. gambiae complex species is essential for defining proper taxonomy. Until recently, there has not been detail molecular phylogenetic analysis of this complex. Using phylogenetics tools, the present study provides evidence of the ancestry, evolutionary history and divergence of members of the complex. Furthermore, it is also noted that A. gambiae and A. arabiensis shared the same ancestry, which may probably be A. stephensi. A. quadriannulatus (non vector) and A. melas (minor vector) were also found to have evolved from a common ancestor. Our findings provide clues that will aid understanding of phylogenetic relationships in this complex, which would prove useful in vector control.

References
Besansky N.J., Krzywinski J., Lehmann T., Simard F., Kern M., Mukabayire O., Fontenille D., Toure Y., and Sagnon N., 2003, Semipermeable species boundaries between Anopheles gambiae and Anopheles arabiensis: evidence from multilocus DNA sequence variation, Proc. Natl. Acad. Sci. USA, 100(19): 10818–10823
http://dx.doi.org/10.1073/pnas.1434337100 PMid:12947038 PMCid:196886

Besansky N.J., Powell J.R., Caccone A., Hamm D.M., Scott J.A., and Collins F.H., 1994, Molecular phylogeny of the Anopheles gambiae complex suggests genetic introgression between principal malaria vectors, Proc. Natl. Acad. Sci. 91(15): 6885-6888
http://dx.doi.org/10.1073/pnas.91.15.6885 PMid:8041714 PMCid:44302

Coluzzi M., Sabatini A., Petrarca V., and Di Deco M.A., 1979, Chromosomal differentiation and adaptation to human environments in the Anopheles gambiae complex, Trans. R. Soc. Trop. Med. Hyg., 73(5): 483–497
http://dx.doi.org/10.1016/0035-9203(79)90036-1

Foley D.H., Bryan J.H., Yeates D., and Saul A., 1998, Evolution and Systematics of Anopheles: Insights from a Molecular Phylogeny of Australasian Mosquitoes, Molecular Phylogenetics And Evolution, 9(2): 262–275
http://dx.doi.org/10.1006/mpev.1997.0457 PMid:9562985

Habtewold T., Povelones M., Blagborough A.M., and Christophides G.K., 2008, Transmission blocking immunity in the malaria non-vector mosquito Anopheles quadriannulatus species A, PLoS Pathog., 4(5): e1000070
http://dx.doi.org/10.1371/journal.ppat.1000070 PMid:18497855 PMCid:2374904

Harbach R.E., Garros C., Manh N.D., and Manguin S., 2007, Formal taxonomy of species C of the Anopheles minimus sibling species complex (Diptera: Culicidae), Zootaxa 1654: 41–54

Harbach R.E., Townson H., Mukwaya L.G., and Adeniran T., 1997, Use of rDNA-PCR to investigate the ecological distribution of Anopheles bwambae in relation to other members of the An. gambiae complex of mosquitoes in Bwamba County, Uganda, Med. Vet. Ent., 11(4): 329-334
http://dx.doi.org/10.1111/j.1365-2915.1997.tb00418.x

Hillis D.M., 1996, Inferring complex phylogenies, Nature 383(6596): 130–131
http://dx.doi.org/10.1038/383130a0 PMid:8774876

Hunt R.H., Coetzee M., and Fettene M., 1998, The Anopheles gambiae complex: a new species from Ethiopia, Trans. R. Soc. Trop. Med. Hyg., 92(2): 231–235
http://dx.doi.org/10.1016/S0035-9203(98)90761-1

Jobst J., King K., and Hemleben V., 1998, Molecular Evolution of the Internal Transcribed Spacers (ITS1 and ITS2) and Phylogenetic Relationships among Species of the Family Cucurbitaceae, Molecular phylogenetics and evolution, 9(2): 204–219
http://dx.doi.org/10.1006/mpev.1997.0465 PMid:9562980

Kamali M., Xia A., Tu Z., and Sharakhov I.V., 2012, A New Chromosomal Phylogeny Supports the Repeated Origin of Vectorial Capacity in Malaria Mosquitoes of the Anopheles gambiae Complex, PLoS Pathog., 8(10): E1002960
http://dx.doi.org/10.1371/journal.ppat.1002960 PMid:23055932 PMCid:3464210

Khan H.A., Arif I.A., Bahkali A.H., Al Farhan A.H., Al Homaidan A.A., 2008, Bayesian, maximum parsimony and UPGMA models for inferring the phylogenies of antelopes using mitochondrial markers, Evol. Bioinform., 4: 263–270

Mohanty A., Swain S., Kar S.K., and Hazra R.K., 2009, Analysis of the phylogenetic relationship of Anopheles species, subgenus Cellia (Diptera: Culicidae) and using it to define the relationship of morphologically similar species, Infection, Genetics and Evolution, 9(6): 1204–1224
http://dx.doi.org/10.1016/j.meegid.2009.06.021 PMid:19577013

Morgan K., O’Loughlin S.M., Mun-Yik F., Linton Y.M., Somboon P., Min S., Htun P.T., Nambanya S., Weerasinghe I., Sochantha T., Prakash A., and Walton C., 2009, Molecular phylogenetics and biogeography of the Neocellia Series of Anopheles mosquitoes in the Oriental Region, Molecular Phylogenetics and Evolution 52(3): 588–601
http://dx.doi.org/10.1016/j.ympev.2009.01.022 PMid:19603555

Munstermann L.E., and Conn J.E., 1997, Systematics of mosquito disease vectors (Diptera, Culicidae): Impact of molecular biology and cladistic analysis, Annu. Rev. Entomol., 42: 351–369
http://dx.doi.org/10.1146/annurev.ento.42.1.351 PMid:9017898

Nei M., and Kumar S., 2000, Molecular Evolution and Phylogenetics, Oxford University Press, New York
PMCid:27115

Roe A.D., and Sperling F.A., 2007, Patterns of evolution of mitochondrial cytochrome c oxidase I and II DNA and implications for DNA barcoding, Mol. Phylogenet. Evol., 44 (1): 325-345
http://dx.doi.org/10.1016/j.ympev.2006.12.005 PMid:17270468

Saitou N., and Nei M., 1987, The neighbor-joining method: A new method for reconstructing phylogenetic trees, Molecular Biology and Evolution, 4(4): 406-425
PMid:3447015

Takken W., Eling W., Hooghof J., Dekker T., Hunt R., and Coetzee M., 1999, Susceptibility of Anopheles quadriannulatus Theobald (Diptera: Culicidae) to Plasmodium falciparum, Trans. R. Soc. Trop. Med. Hyg., 93(6): 578–580
http://dx.doi.org/10.1016/S0035-9203(99)90054-8

Tamura K., and Nei M., 1993, Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees, Molecular Biology and Evolution, 10(3): 512-526
PMid:8336541

Tamura K., Nei M., and Kumar S., 2004, Prospects for inferring very large phylogenies by using the neighbor-joining method, Proceedings of the National Academy of Sciences (USA), 101(30): 11030-11035
http://dx.doi.org/10.1073/pnas.0404206101 PMid:15258291 PMCid:491989

Tamura K., Peterson D., Peterson N., Stecher G., Nei M., and Kumar S., 2011, MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods, Molecular Biology and Evolution, 28(10): 2731-2739
http://dx.doi.org/10.1093/molbev/msr121 PMid:21546353 PMCid:3203626

Thelwell N.J., Huisman R.A., Harbach R.E., and Butlin R.K., 2000, Evidence for mitochondrial introgression between Anopheles bwambae and Anopheles gambiae, Insect Molecular Biology, 9(2): 203-210
http://dx.doi.org/10.1046/j.1365-2583.2000.00178.x PMid:10762428

Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., and Higgins D.G., 1997, The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucl. Acids Res., 25(24): 4876–4882
http://dx.doi.org/10.1093/nar/25.24.4876 PMid:9396791 PMCid:147148

WHO, 2010, World Malaria Report 2010, World Health Organization

WHO, 2011, World Malaria Report 2011, World Health Organization

Wilkerson R.C., Foster P.G., Li C., and Sallum M.A., 2005, Molecular phylogeny of neotropical Anopheles (Nyssorhynchus) albitarsis species complex (Diptera: Culicidae), Ann. Entomol. Soc. Am., 98(6): 918–925
http://dx.doi.org/10.1603/0013-8746(2005)098[0918:MPONAN]2.0.CO;2