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The present study explored the phylogenetic utility of mt-ND5 gene sequences for mosquitoes in genus Anopheles. Phylogenetic tree employing the Maximum Parsimony methods in PAUP strongly supported the traditional taxonomy of genus. Subgenus Anopheles represented by a single species i.e. Anopheles nigerrimus initially separated out from the rest with a strong bootstrap value of 100. Subgenus Cellia representing bulk of Pakistani mosquitoes showed a monophyletic lineage (bootstrap value: 97). Using two fossil calibration points (both at 34 Mya) mtDNA divergence times were estimated within a sympatric group of Anophelines of Pakistan. The divergence time between Subgenera Anopheles and Cellia was estimated at approximately 6 million years ago i.e. between mid to late Pliocene. Series Neocellia (Anopheles splendidus Anopheles stephensi Anopheles annularis Anopheles pulcherrimus) diverged some 5.7 million years ago from Myzomyia (Anopheles culicifacies and Anopheles fluviatilis)

while speciation within the respective series took place during the Pleistocene about 0.7 million years ago. The present study shows Pliocene as the starting point for mosquito evolution while Pleistocene as the point of diversification. The ongoing climate change uplifting of Himalaya evolution of a monsoon system rising and falling of the sea level are perhaps those features of Pleistocene that might have led to the process of speciation in genus Anopheles of Pakistan.

Keywords: malaria Pakistan Anopheles Pleistocene.


Anopheline systematics has reached its zenith in classical morphology some years ago and is now dominated by molecular genetics research (Harbach 2004). DNA based studies of this genus have mainly focused on the intra/interspecific identification phylogenies and divergence dates for various species and complexes (Collins and Paskewitz 1996; Foley et al. 1998; Krzywinski et al. 2001 a and b; Chen et al. 2004; Donnelly et al. 2004; Foley et al. 2007; Morgan et al. 2009). Despite the global advances in molecular systematics of the group; such studies are still lagging behind in Pakistan.

Pakistan (mainly Khyber Pakhtunkhwa and Baluchistan) has hosted millions of Afghans during the past few decades. The admittance and repatriation of these refugees has been linked to the changing disease patterns especially malaria (Kazmi and Pandit 2001). It is a disease that flourishes in conditions of crisis and population displacement for many reasons: the breakdown of health services and control pro-grammes; the displacement or concentration of non-immune refugees in malaria-risk areas; malnourishment within displaced populations; the siting of camps on marginal land prone to vector breeding; problems in gaining access or supplying medicine to displaced populations; and the lack of protective shelter and other man vector barriers (Rowland 2001). Malaria is regionally listed as a high priority health problem by the World Health Organization. In Pakistan two species are recognized as vectors i.e. Anopheles stephensi and Anopheles culicifacies (Mahmood et al. 1984)

while many others are suspected vectors (Suleman 1993). In this perspective genetic studies of Anopheline mosquitoes greatly deserve attention in Pakistan. Here we present for the first time baseline data on molecular systematics of seven Anopheline mosquitoes from Pakistan. The objectives are to determine (1) whether significant sequence heterogeneity exists within the mt-ND5 mtDNA sequences for biological species identification (2) to construct an Anopheline phylogeny for Pakistani mosquitoes (3) and to assess rate of speciation and divergence of a sympatric group of species from Pakistan. Fossil constraints are used to estimate divergence of taxa based on molecular data.


Sample species: The present study analyzed mitochondrial sequences of seven Anopheline species from Pakistan. The species were exclusively eusynanthropic and came from yearlong collections (2007) in three villages of district Charssada (34-03' and 34-28'N and 71-28' and 71-53'E) Khyber Pakhtunkhwa Pakistan. The study sites within the district were selected for their high prevalence of malaria. The specimens all adults and preserved in silica gel were kept at the department of Zoology (University of Peshawar Pakistan). About 22 mosquito specimens belonging to seven species were selected and sequenced for the present study.

Each PCR amplicon had a total volume of 50 l. For amplification 39 cycles consisting of 1 minute at 94C for denaturation 1 minute at 51C for annealing and 2 minutes at 72 C for extension were performed followed by 3 minutes at 72 C for final extension. Sequencing was performed using external services (Macrogen Inc. South Korea). Sequences generated during the present study were deposited in the Genbank with Accession Nos. JX985561-JX985583 as listed in Table 2.

Chromas Pro 34-Version 1.33 was used for manual edition of sequences while Clustal X was used for alignment and comparison of sequences.

Phylogenetic Analysis: Each mt-ND5 sequence constituted 500 nucleotide base pairs. Gaps were treated as missing data in all analysis. Parsimony analyses (MP) were implemented in PAUP4.0b10 (Swofford 2002) using the heuristic search option with Tree Bisection- Reconnection (TBR) branch-swapping. MP topologies were evaluated by bootstrap method with 1000 replicates (TBR) of 100 random stepwise addition replicates each. Parsimony-uninformative characters were excluded. A Majority rule consensus tree was thus obtained.

The software BEAST 1.4 (Drummond and Rambaut 2007) was used to calculate a time line for Anopheline evolution. Modeltestv 3.7 (Posada and Crandall 1998) suggested the general time reversible (GTR) + I + G model of evolution. Culex pipiens was used as outgroup. The root age prior between Genus Anopheles and outgroup C. pipiens was set according to fossil-based minimum ages: Culex winchesteri 34 Ma; Anopheles dominicanus 34 Ma (Reidenbach et al. 2009) respectively. One tree was saved every 100 cycle for a total of 1.1 A- 10 cycles. The first 1000 cycles were discarded as burn-in. The final trees are displayed in results.


Molecular systematics of Pakistani Anophelines

Species identification: Mitochondrially encoded mt- ND5 gene segment was used during the present study. We also amplified COI and COII for some species though were unsuccessful in the case of others. Thus for the phylogenetic analysis of Anopheles species only mt- ND5 was used. For this purpose two year old specimens preserved in silica gel were successfully amplified and sequenced. Sequence variation accommodated identification of Pakistani taxa (Figure 1).

Phylogenetic relationships: The 50% majority rule consensus tree employing the Maximum Parsimony methods in PAUP strongly supported the traditional taxonomy of genus Anopheles (Figure 1).

Subgenus Anopheles represented by a single species i.e. Anopheles nigerrimus initially separated out from the rest with a strong bootstrap value of 100. Subgenus Cellia representing bulk of Pakistani mosquitoes showed a monophyletic lineage (bootstrap value: 97).

Within Subgenus Cellia the series Myzomyia showed paraphyly by branching A. culicifacies along with series Neocellia (bootstrap value: 78.7) though the clade culicifacies branched distinctly from those taxa representing Neocellia (89.7).

At a bootstrap value of 89.6 the Pakistani Neocellia formed a monophyletic clade showing four distinct lineages i.e. An. Pulcherrimus An. Annularis An. Stephensi and An. Splendidus.

Divergence estimates and speciation: Using two fossil calibration points (both at 34 Mya) we estimated mtDNA divergence times within a sympatric group of Anophelines from Pakistan. The divergence time between Subgenera Anopheles and Cellia was estimated at approximately 6 million years ago i.e. between mid to late Pliocene. Series Neocellia (An splendidus An stephensi An annularis An pulcherrimus) diverged from Myzomyia (An. Culicifacies and An fluviatilis) some 5.7 million years ago while Species diversification took place during the Pleistocene about 0.7 million years ago for almost all species (Figure 2). The complex annularis diverged about 4 million years ago.

Table 1. Brief taxonomy of species investigated analyzed during the present study.###

Subgenus###Subgenus Cellia###


Series###Series Myzomyia###Series Neocellia


(Edwards 1932)###1924)###


nigerrimus Giles###culicifacies Giles###annularis van

1900###1901###der Wulp 1884


###fluviatilis James###pulcherrimus

###1902###Theobald 1902



###Koidzumi 1920


###stephensi Liston


Table 2. Genebank accession numbers for the ND5 Anophelinesequencesreportedfrom Pakistan.

Species###Accession Numbers

Anopheles annularis 1###JX985561

Anopheles culicifacies1###JX985562

Anopheles culicifacies2###JX985563

Anopheles fluviatilis1###JX985564

Anopheles fluviatilis2###JX985565

Anopheles fluviatilis3###JX985566

Anopheles nigerrimus1###JX985567

Anopheles nigerrimus2###JX985568

Anopheles nigerrimus3###JX985569

Anopheles nigerrimus4###JX985570

Anopheles pulcherrimus 1###JX985571

Anopheles pulcherrimus 2###JX985573

Anopheles pulcherrimus 3###JX985574

Anopheles splendidus1###JX985575

Anopheles splendidus2###JX985576

Anopheles splendidus3###JX985577

Anopheles stephensi1###JX985578

Anopheles stephensi2###JX985579

Anopheles stephensi3###JX985580

Anopheles stephensi4###JX985581

Anopheles stephensi5###JX985582

Anopheles stephensi6###JX985583


Anopheline systematic: Anopheline mosquitoes (Culicidae Anophelinae) are of prime medical importance as human malaria vectors yet their phylogeny is poorly known (Krzywinski et al. 2001b). Anopheles with 437 species worldwide is classified into six subgenera: the cosmopolitan Anopheles the Old World Cellia and the Neotropical Kerteszia Nyssorhynchus Lophopodornyia and Stethornyia (Krzywinski et al. 2001b Harbach 2004). In Pakistan there are 24 anophelines species (Mehmood et al. 1984) with 11 in Khyber Pakhtunkhwa province i.e. A. stephensi and A. culicifacies A. splendidus A. pulcherrimus A. annularis A. maculatus A. nigerrimus A. subpictus A. superpictus A. fluviatilis and A. tarkhudi (Suleman 1993). Most of the species belong to subgenus Cellia. The present study investigated molecular systematics of seven of these species (Table 1). Mt-ND5 gene segment was employed for this purpose. Mt-ND5 has previously been successfully utilized for resolving taxonomic problems within genus Anopheles as it provides adequate variation for identification and phylogenetic reconstruction (Besansky et al. 1997; Lehman et al. 1997; Krzywinski et al. 2001a; Donnelly et al. 2004; Morgan et al. 2009). Subgenus Cellia representing bulk of Pakistani mosquitoes showed a monophyletic lineage (bootstrap value: 97). The results are in accordance to current phylogenetic scheme for Anopheline relationships as presented by Harbach 2004 and other studies (Sallum et al. 2000). Series Myzomyia showed paraphyly for our results according to which A. culicifacies (Myzomyia; funestus group) originated from the same ancestral stock as rest of Neocellia (Figure 1). In classical morphological taxonomy phenotypic overlap is known to exist between members of the funestus group of Myzomyia and the annularis group of the Neocellia series making it difficult to identify them as groups (Swain et al. 2010).

Our results however showed that despite the paraphyly of Myzomyia series; A. culicifacies (funestus) was itself a monophyletic lineage and mt-ND5 facilitated the identification of funestus and annularis groups (Figure 1). As for Series Neocellia monophyly was obvious in the results which are similar to a study carried out in neighboring India (Swain et al. 2010). On contrary the phenomenon of paraphyly has been well documented in Neocellia throughout the globe (Pape 1992; Foley et al. 1998; Ma et al. 2011).

The refuge model of Anopheline speciation: The abounding biodiversity of Orient has been attributed to habitat fragmentation and creation of refuges over time (Haffer 1969). The refuge model thus states that the repeated expansion and contraction of tropical forests in response to climatic fluctuation resulted in allopatric divergence and eventual speciation within many taxa including mosquitoes (Morgan et al. 2009). The question is when did it happen Many studies have indicated Pliocene to be the time when major Anopheline speciation events took place in the world (Donnelly et al. 2004; Morgan et al. 2009). The present study however shows Pliocene as the starting point while Pleistocene as the point of diversification for the genus Anopheles.

It has previously been asserted that Pleistocene with its changing climate might have favored radiation of species yet evolutionary role of Pleistocene has remained doubtful (Morgan et al. 2009). Quite the opposite Pleistocene in Pakistan was a suitable candidate for mosquito evolution. Following factors might have played an important role in this regard.

Changing Climate: The changing climate of Pleistocene favored mosquito evolution (Morgan et al. 2009). Alternation of cool glacial periods with the warm interglacial periods (Hills and Hills 2005) perhaps gave way to refuges that acted as hotspot zones where speciation ran rampant.

Rising and Falling Sea levels: Pleistocene was the time when sea levels were continuously rising and falling in South East Asia (Heaney 1991). The phenomenon perhaps further assisted in the creation of refuges on the Indian Subcontinent.

Evolution of Monsoon and phased uplift of Himalaya: Since antiquity the climates of Asia are affected significantly by the extent and height of the Himalaya (Kutzbach et al. 1993; Kutzbach et al. 1997). Uplift of this region began about 50Myr ago. Significant increases in altitude occurred in the mid Pliocene i.e. some 10-8 million years ago (Molnar et al. 1993 Harrison et al. 1992). The formation of Himalaya is correlated with evolution of the Monsoon system. Three stages are recognized in this perspective; the enhanced aridity and onset of the Indian and east Asian monsoons in Pliocene; continued intensification of Summer and Winter Monsoons during early Pleistocene; and increased variability and possible weakening of the Indian and east Asian summer monsoons and continued strengthening of the east Asian winter monsoon since about 2.6Myr ago Zhisheng et al. 2001).

Today Monsoons are known to have a profound impact on natality/mortality of Anopheline species. In the arid Punjab for example rainfall facilitates increased breeding and lifespan of the malarial vectors (Bouma and Kaay 1996).

The historical development of a Monsoon system since the late Pliocene through the Pleistocene supports the refuge model of Anopheles speciation in Pakistan.

Changing Habitats: Reconstruction of Pleistocene environment in the last glacial maximum suggests major habitat replacements in South Asia (Morgan et al. 2009). Thus moist" thick forests continuously interchanged with arid" grasslands and Savannahs (Heaney 1991; Hope et al. 2004; White et al. 2004; Morgan et al. 2009). Similar changes were occurring in vegetation i.e. from C3 (forests) to C4 (grasses) in Pakistan beginning about 8Myr ago (from mid Pliocene onwards) (Cerling et al. 1997). As mosquitoes are very much sensitive to habitat nature such events might have inevitably led to spatial and temporal isolation of mosquito populations which ultimately gave rise to new species.

Conclusions: The present study demonstrated the utility of mitochondrial mt-ND5 sequences in phylogenetic inferences of Anopheline species. It also showed a recent speciation for Pakistani mosquitoes about 0.7 million years ago which might be due to the major climatic and geological changes that occurred in Pakistan during the Plesitocene. These events such as development of monsoon system uplifting of himalaya glaciation etc might had played an important role in evolution of various taxa including mosquitoes.

Acknowledgements: We are heartly thankful to Entomological museum of Department of zoology for providing us mosquito specimens for our research work.


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Publication:Journal of Animal and Plant Sciences
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Date:Aug 31, 2014

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