Molecular Evolution and Phylogenomics of the Anopheles gambiae Complex  

Benson otarigho , Mofolusho O. Falade
Cellular Parasitology Unit, Department of Zoology, University of Ibadan, Nigeria
Author    Correspondence author
Journal of Mosquito Research, 2013, Vol. 3, No. 9   doi: 10.5376/jmr.2013.03.0009
Received: 26 Mar., 2013    Accepted: 03 Apr., 2013    Published: 26 Apr., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Otarigho and Falade, 2013, Molecular Evolution and Phylogenomics of the Anopheles gambiae Complex, Journal of Mosquito Research, Vol.3, No.9 65-70 (doi: 10.5376/jmr.2013.03.00009)

Abstract

Malaria vectors of the Anopheles gambiae complex are made up six species of mosquitoes that are stable and are highly efficient vectors. This group comprises of major and minor vectors, some of which are responsible for transmission of the most deadly strain of malaria parasite, Plasmodium falciparum. The evolutionary history of this species group was inferred using publically available DNA sequence data. The retrieved sequences were aligned using CLUSTAL W; evolutionary history was inferred using Maximum Likelihood, Neighbor-Joining, and Minimum Evolution methods. Ancestral sequences were also inferred using the FASTML Server-based program for computing ancestral sequences. Based on morphology, ecology and behaviour, Anopheles gambiae and Anopheles arabiensis (major vectors) were found to evolve from a common ancestor. All the members of this complex were all AT rich. A. gambiae and A. arabiensis had the highest AT composition, while A. merus was the least AT rich among the complex. Evolutionary divergence estimates show that these two major vectors are genetically similar. A. quadriannulatus (non vector) and A. melas (minor vector) were also found to evolve from the same ancestor. Overall, this study gives an understanding of the ancestral lineage of the A. gambiae complex, which will be essential for understanding the origins, evolution, classification and epidemiology of this important disease vector. This may have important implications for the control of malaria.

Keywords
Anopheles gambiae complex; Vectorial capacity; Evolution; Phylogenetics; Cytochrome c oxidase (COI); Mitochoindria DNA sequence; Evolutionary history; Evolutionary divergence

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.

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