Mitochondrial genome sequences reveal deep divergences among Anopheles punctulatus sibling species in Papua New Guinea
© Logue et al.; licensee BioMed Central Ltd. 2013
Received: 2 November 2012
Accepted: 6 February 2013
Published: 14 February 2013
Members of the Anopheles punctulatus group (AP group) are the primary vectors of human malaria in Papua New Guinea. The AP group includes 13 sibling species, most of them morphologically indistinguishable. Understanding why only certain species are able to transmit malaria requires a better comprehension of their evolutionary history. In particular, understanding relationships and divergence times among Anopheles species may enable assessing how malaria-related traits (e.g. blood feeding behaviours, vector competence) have evolved.
DNA sequences of 14 mitochondrial (mt) genomes from five AP sibling species and two species of the Anopheles dirus complex of Southeast Asia were sequenced. DNA sequences from all concatenated protein coding genes (10,770 bp) were then analysed using a Bayesian approach to reconstruct phylogenetic relationships and date the divergence of the AP sibling species.
Phylogenetic reconstruction using the concatenated DNA sequence of all mitochondrial protein coding genes indicates that the ancestors of the AP group arrived in Papua New Guinea 25 to 54 million years ago and rapidly diverged to form the current sibling species.
Through evaluation of newly described mt genome sequences, this study has revealed a divergence among members of the AP group in Papua New Guinea that would significantly predate the arrival of humans in this region, 50 thousand years ago. The divergence observed among the mtDNA sequences studied here may have resulted from reproductive isolation during historical changes in sea-level through glacial minima and maxima. This leads to a hypothesis that the AP sibling species have evolved independently for potentially thousands of generations. This suggests that the evolution of many phenotypes, such as insecticide resistance will arise independently in each of the AP sibling species studied here.
KeywordsAnopheles Anopheles punctulatus sibling species Molecular evolution Molecular dating
Anopheles mosquitoes are distributed worldwide, with the exception of Antarctica, and feed on a variety of hosts from birds to mammals ( and references therein). Within the Anopheles genus, 70 of over 500 species are able to transmit human malaria. These include the well-known species Anopheles gambiae, Anopheles arabiensis and Anopheles funestus that are the main vectors of malaria in Africa, Anopheles darlingi and Anopheles albitarsis in South America, Anopheles dirus and Anopheles minimus in Southeast Asia (SEA) and Anopheles punctulatus in Southwest Pacific (SW Pacific).
Despite their medical importance, the current understanding of the Anopheles phylogeny – the relationship among species as well as the times they diverged from each other – remains limited. Studies of this nature are complicated in Anopheles by the existence of species complexes (including morphologically identical sibling species)[3–6] and incipient species[7–9], as well as by the paucity of genetic data for most species besides An. gambiae. The current hypothesis of Anopheles evolution, mostly based on the extant geographic distribution of Anopheles mosquitoes, proposes that Anopheles originated in western Gondwana during the Cretaceous. They subsequently migrated across the world aided by land connections and radiated into multiple species adapted to unique habitats and climatic conditions[10, 11]. However, the timing and routes of these dispersions are unknown and the relationships among current species remain poorly understood. Determining the evolutionary relationships among Anopheles species has important clinical and vector control implications as it could clarify whether traits required for transmission of human blood-borne pathogens, avoidance of long-lasting insecticide-treated nets or insecticide resistance evolved only once in an ancestral population or, alternatively, whether different species acquired these traits independently.
The focus of this study is on members of the Anopheles punctulatus (AP) group, the principal vectors of malaria in Papua New Guinea (PNG), the Solomon Islands, Vanuatu, and northern Australia[12, 13]. Historically, the AP group was separated into four species based on morphological differences in the proboscis and wings: An. punctulatus, Anopheles koliensis, Anopheles farauti, and Anopheles clowi. Later studies involving cross-mating[14–16], allozyme analyses and DNA sequence analysis[18–22] provided evidence suggesting further differentiation of the AP group into 13 species, most of them morphologically indistinguishable. At least five of these species - An. punctulatus s.s, An. koliensis, An. farauti s.s (previously An. farauti 1), Anopheles hinesorum (previously An. farauti 2), and An. farauti 4 - have been described as competent vectors of malaria[23, 24]. Phylogenetic studies of this group have focused on DNA sequences of ribosomal RNAs[25, 26], mitochondrial genes[27, 28], ribosomal ITS2, and voltage-gated sodium channel gene. However, the genetic information generated in these studies has not allowed robust determination of the AP group phylogeny and has often yielded conflicting results[19, 27]. In addition, no study has yet evaluated the relationships between AP sibling species and other Anopheles species from neighbouring regions, such as species from SEA. As Beebe and Cooper have described this group of Anopheles sibling species as ‘unspecialized’, there is some reason to hypothesize that AP group members have evolved to acquire a broad range of feeding strategies to ensure survival.
Here, the evolutionary history of the AP group of PNG is investigated by sequencing the mitochondrial (mt) genome of 14 individual mosquitoes from the AP group and neighbouring An. dirus complex. Next generation sequencing technologies were used to generate mt genome sequences and de novo assemble each individual sequence. The concatenated sequence of all mitochondrial protein coding genes (10,770 bp) was used to reconstruct robust phylogenies and estimate divergence times among available Anopheles mt genomes. The implications of this study’s findings are discussed with regard to the evolutionary history of anophelines in general and the origin of the AP group.
Sampling and laboratory procedures
Sample collection and DNA extraction
AP mosquitoes were obtained from the Entomology Unit of the PNG Institute of Medical Research (PNGIMR). Anopheles dirus samples were obtained from the Faculty of Tropical Medicine, Mahidol University (Thailand). Genomic DNA from individual mosquitoes was extracted using the Qiagen DNeasy® blood and tissue kit according to the supplementary protocol for purification of insect DNA. The species of each AP mosquito was determined using a PCR-based assay targeting the ITS2 locus.
Whole genome shotgun sequencing and assembly
The whole genome of five mosquitoes (An. punctulatus s.s. (n = 1), An. koliensis (n = 1), An. farauti s.s. (n = 1), and An. farauti 4 (n = 2)) were sequenced. Genomic DNA was fragmented into ~300 base pairs and sequencing libraries were prepared using the New England Biolabs (NEB) NEBNext® kit protocol and standard Illumina paired-end adaptors. Each library was sequenced on one lane of an Illumina GAIIx or HiSeq2000 instrument to generate 37 to 150 million paired-end reads from each sample [see Additional file1].
To identify reads originating from the mt genome and separate them from reads originating from the nuclear genome, the program Bowtie was used to map all reads generated from one sample on the cytochrome oxidase I (COI), cytochrome oxidase II (CO2), and the voltage gated sodium channel (VGSC) gene sequences previously generated for each species. As expected based on the copy number, the sequence coverage of mt genes, COI and CO2, was 50–60 fold greater than the coverage of the nuclear gene, VGSC [see Additional file2]. The ~500 X coverage of mtDNA implied that multiple identical reads mapped to the exact same nucleotide position along the entire mt genome sequence, therefore, One hypothesis is that most reads occurring twice or more in each shotgun sequencing dataset were likely to originate from the mt genome. These reads were therefore selected (regardless of their DNA sequence) for reconstructing the complete mtDNA sequence of each sample. All assemblies were performed using ABySS with a k-mer size of 29 and C = 70. The assembled contigs were aligned to the mtDNA sequence of An. gambiae with MUMmer and any remaining gaps were filled by PCR and Sanger sequencing [see Additional file3]. To identify possible artefacts or assembly errors, all reads generated from a given sample were then mapped to the final mtDNA contig (assembled from a subset of these reads). If necessary, any base in the contig differing from the nucleotide carried by a majority of the reads was replaced by this nucleotide.
Multiplexed mt genome sequencing and assembly
For the remaining nine samples, a multiplex approach was used to simultaneously sequence the mt genome [see Additional file4]. First, primers were designed to amplify any Anopheles mt genome using seven overlapping long-range PCRs. An Anopheles consensus sequence was generated by aligning the mt genomes of An. punctulatus s.s., An. farauti s.s, An. gambiae, An. quadrimaculatus and An. darlingi with ClustalW and masking any variant site. Primers were then designed based on this consensus sequence using Primer3 following the Roche Expand Long Range dNTPack® kit recommendations. Primers were able to be designed at overlapping sites with two or less variants and without known variants in the last 3’ positions [see Additional file3].
Each amplicon was amplified using the Roche Expand Long Range dNTPack® kit protocol with 20–40 ng of gDNA per PCR reaction and 3% DMSO. Amplification conditions were as follows: 3 minute denaturation step at 94°C, 39 cycles of 94°C for 45 seconds, 50°C for 45 seconds, 60°C for 5 minutes followed by a 10 minute final elongation at 60°C. Product amplification was verified by electrophoresis on a 1% agarose gel.
Following amplification, all seven PCR products from a given individual were pooled together and DNA molecules were sheared into 300 bp fragments [see Additional file4]. A sequencing library for each individual was prepared using Illumina adapters including a unique 6-nucleotide barcode. Finally, libraries were pooled from all mosquitoes in equal concentrations and the resulting pool was sequenced on one lane of an Illumina HiSeq 2000 instrument [Additional file4], generating an average of 28 million paired-end reads of 100 bp per sample [see Additional file1] [NCBI SRA: SRP013853].
List of the samples used in this study with their collection site or colony ID and corresponding NCBI accession numbers
An. punctulatus s.s.
An. punctulatus s.s.
An. punctulatus s.s.
An. punctulatus s.s.
An. punctulatus s.s.
An. farauti s.s.
An. farauti 4
An. farauti 4
An. dirus s.s.
An. dirus s.s.
An. albitarsis F
An. albitarsis G
An. darlingi North
An. darlingi South
Phylogenetic analysis and molecular dating Since relying on the gene annotations from one of the Anopheles (i.e. An. gambiae) may introduce systematic biases in the phylogenetic analyses, the DNA sequence of each gene for each mt genome (i.e. the sequences that were generated as well as those retrieved from NCBI) was determined with tBlastn using the Drosophila melanogaster protein annotations as references [Genbank:U37541.1]. Briefly, for each protein coding sequence, the DNA sequences were translated into amino acid sequences, aligned to each other and the amino acid sequence was reverse-translated back into nucleotide sequences with Translator X using the default parameters and the invertebrate mt genetic code. The aligned coding protein sequences from all 13 mt genes (resulting in 10,770 nucleotides) were concatenated and the best model of nucleotide substitutions was determined using the program jModeltest v0.1.1. According to the Akaike Information Criterion, the best nucleotide substitution model for this data set was the General Time Reversible with gamma distribution (GTR + G) model.
Bayesian phylogenies were reconstructed using BEAST v1.7.2 with the following program parameters: an uncorrelated lognormal relaxed clock model, allowing for rate heterogeneity among species, the GTR + G substitution model, the SRD06 model of partitioning, which allows estimation of nucleotide substitution parameters separately for the 1st + 2nd and 3rd codon positions, and a Yule model for tree reconstruction. Using the above parameters, three independent runs of 20 million generations were performed, with trees sampled every 1,000 generations. All runs were then combined after a burn-in of 10% using LogCombiner v1.7.2. Tracer v1.5 was used to verify adequate mixing of the Markov chains and ensure that each parameter had been appropriately sampled (i.e., effective sampling size >200). The maximum credibility tree was determined using TreeAnnotator v1.7.2 and visualized the phylogenic tree with FigTree v1.3.1.
The program BEAST was used to estimate divergence times using the Drosophila-Anopheles divergence using a prior distribution normally distributed around a mean of 260 million years ago (mya) and ranging from 243 to 276 mya as suggested in. For comparison, divergence times were also estimated using a mutation rate of 0.0115 mutations per nucleotide per million years, which was estimated from the divergence times and sequence divergence of several insect mt genomes.
Sequence data and assembly
This study focuses on analysis of the mt genome of 14 individual Anopheles mosquitoes from seven species. These include 11 individuals from the AP group in PNG (An. punctulatus s.s (n = 5), An. koliensis (n = 2), An. farauti s.s (n = 1, An. farauti 1), An. hinesorum (n = 1, An. farauti 2), An. farauti 4 (n = 2)) and three samples from the An. dirus complex in Thailand (An. dirus s.s (n = 2, An. dirus species A) and An. cracens (n = 1, An. dirus species B) (Table 1).
This study was initiated by sequencing the whole genome of five individual mosquitoes, generating ~37-150 million paired-end reads and resulting in an average 500 X coverage of mtDNA [see Additional file1 and Additional file2]. Each individual mt genome was then de novo assembled. For nine additional mosquitoes, the mt genome was amplified by overlapping long-range PCR products and all samples were simultaneously sequenced on one lane of an Illumina HiSeq 2000 [see Additional file4]. This pooled mtDNA sequencing strategy generated 392,983,105 million paired-end reads of 100 bp resulting in an average 188,000 X coverage of each individual mt genome. Each genome was de novo assembled separately. For each of the newly sequenced genomes, the genes and gene organization (i.e., orientation and order) were identical to that of previously sequenced Anopheles mt genomes[11, 35–37, 43].
The AP group from PNG clusters most closely with the An. dirus complex distributed across SEA (Figure 2). This tree also partially resolves the phylogeny within the AP group with An. farauti 4 being the most divergent while An. farauti s.s. and An. hinesorum being most closely related (Figure 1).
Mean divergence times and 95% credibility intervals for selected nodes
95% Credibility (mya)
Drosophila / Anopheles (Calibration - 260 mya)a
Anophelinae / Culicinae
An. dirus complex / An. punctulatus group
South and Central American Anophelesb
Anopheles punctulatus group
Anopheles albitarsis complex
An. farauti s.s./ An. hinesorum
Within the AP group, a deep divergence among sibling species with the MRCA of the AP group was observed dating back to 25–54 mya, roughly half as old as the MRCA of all Anopheles (Table 2). Importantly, this ancient origin of the AP group does not appear to result from a single highly divergent sibling species: most species analysed here (An. punctulatus s.s., An. koliensis and An. farauti 4) seem to have diverged 25–54 mya, and only An. farauti s.s. and An. hinesorum share a recent common ancestor (5–17 mya) (Figure 2). This finding is especially striking when compared with the only other sequenced Anopheles group: An. albitarsis mosquitoes are distributed across South America (from southern Brazil to Columbia) over a much larger geographic range than the AP group in PNG but share an MRCA dating back to only 6–24 mya, significantly younger than the MRCA of the AP group (Figure 2).
Dating using the estimated insect mt mutation rate of 0.0115 mutations per nucleotide per million years instead of a calibration point, led, overall, to more recent divergence times [see Additional file5] (but see below).
The predominant hypothesis regarding the origin of Anopheles mosquitoes predicts that they originated in western Gondwana[10, 11] and that, by 95 mya, Anopheles had migrated into Africa. Ancestral Anopheles are predicted to have then colonized Europe and North America (via land bridges), and migrated through Asia into the SW Pacific. The topology of the tree in Figure 2 is globally consistent with this hypothesis. However, the position of North American mosquitoes (An. quadrimaculatus) relative to African and other non-American Anopheles remains unclear. In particular the lack of mt genomes from European Anopheles mt genomes preclude determining whether African Anopheles are ancestral to European and North-American Anopheles or, alternatively, whether North-American Anopheles derive directly from South-American Anopheles. Additional sampling of mt genomes would provide better resolution of Anopheles early dispersal routes.
Regarding SEA and the SW Pacific, it is generally believed that Anopheles from the SW Pacific derives from SEA mosquitoes[10, 26, 27]. The results of this study suggest that the AP group is most closely related to the An. dirus complex of SEA, consistent with an origin of the AP group in SEA. However, currently it cannot be determined whether SW Pacific and Australian Anopheles originate from an SEA ancestor as currently hypothesized or, alternatively, whether SW Pacific and SEA Anopheles have an Australian origin. The molecular dates in this study, suggest that the ancestor of the AP group was present in PNG between 25 and 54 mya but does not allow rejecting either of these scenarios. Plate tectonic models show that the Australia/PNG plate, that separated from Gondwana during the Cretaceous, moved from a southern position in the Eocene to its current position near SEA. While the upper limit of the age of the AP ancestor (54 mya) corresponds to a time when the distance between PNG and SEA would not have allowed migration between these regions, the lower limit (~25 mya) corresponds to a time when the Australian plate had moved close enough to the Asian plate to enable possible migration of species between the two regions. Inclusion of additional mt genome sequences, in particular from Anopheles complexes restricted to Australia (e.g., Anopheles annulipes) may allow better understanding of these early dispersal routes in SEA and SW Pacific.
The monophyly of AP mosquitoes (Figure 1) suggests that they colonized PNG through a single migration event followed by speciation (as opposed to multiple migrations of pre-established species). This study suggests that the different AP sibling species diverged from each other 25–54 mya, much earlier than proposed in previous studies of the AP group[26, 28]. This deep divergence among AP mosquitoes is unlikely to be caused by a single species that could have diverged from the other sibling species in SEA and colonized PNG later. In fact, most of the AP sibling species are equally divergent from each other, suggesting rapid radiations of AP sibling species upon arrival in the SW Pacific area. Since this analysis relies on a single non-recombining locus, it cannot rule out the possibility that the estimates are influenced by the action of natural selection. However, when only Anopheles mt genomes are analysed, there is little evidence for deviation from a clock-like model of evolution, which suggests that nucleotide substitutions occur at a similar rate on each lineage. Therefore, if natural selection is driving the evolution of the mt genome in Anopheles, it is likely to have acted in a similar manner on all lineages and consequently is unlikely to bias the molecular dates significantly. An additional possible complication is that the phylogenetic tree inferred from mt sequences differs from the actual species tree: since these analyses are based on a single locus, one cannot rule out that incomplete lineage sorting and introgression lead to a phylogenetic reconstruction that does not represent the true evolutionary pathways of the species studied[56, 57]. The long internal branches separating species coupled with the very short branches separating individuals from the same species indicate that incomplete sorting of ancestral polymorphisms is unlikely to affect this phylogeny. Ruling out introgression is difficult without genetic data on multiple unlinked loci (i.e., nuclear), which are complicated to generate due to the lack of a good reference genome sequence (An. gambiae being too divergent), the high divergence among AP species at the nuclear level and the high level of genetic diversity within species. However, there is little evidence of gene flow among AP mosquitoes (but see also Ambrose) and early forced mating studies suggested that most hybrids are non-viable or sterile. Overall, these observations suggest that, for this dataset, the mt gene tree likely recapitulates the actual species tree.
Given the old divergence times among most AP sibling species, one would expect that most AP species today are reproductively isolated and that hybridization is unlikely to occur in nature, with the possible exception of An. farauti s.s and An. hinesorum that only diverged 5 to 17 mya (see also). This potential reproductive isolation among AP sibling species is supported by early cross-mating experiments suggesting that F1 hybrids between any combination of An. farauti s.s, An. hinesorum, An. koliensis and An. punctulatus s.s are non-viable or sterile in laboratory. These results have important implications for vector control in the SW Pacific. For example, control of malaria by releasing genetically modified-, sterile-, male mosquitoes as was recently proposed would require, in PNG, independent engineering of mosquitoes from, at least, five highly divergent species to significantly impact the populations of the main malaria vectors. In addition, if AP sibling species are reproductively isolated from each other, insecticide resistance arising in one species is unlikely to spread quickly across all AP mosquitoes, but instead resistance mutations would have to occur independently in each species. Further investigations are required to definitively rule out the existence of gene flow among most AP sibling species, as well as to confirm the observation of putative mt genome introgression between An. farauti s.s and An. hinesorum recently described in Southern New Guinea.
The ancient divergences among Anopheles species, including among AP from SW Pacific, also raises questions for the evolution of traits related to human malaria transmission. Transmission of plasmodium parasites to humans is facilitated by the anthropophilic behaviour of some Anopheles species. Such behaviour includes usage of human-made habitats as breeding sites and preferential feeding on human blood. Some Anopheles mosquitoes, including most populations of An. gambiae, are highly specialized feeders and rely preferentially on humans for their blood meal. Others are considered more “opportunistic” or “generalist” and the source of their blood meal varies according to several factors including host density (for review see). There are limited data on the feeding preference and behaviour of AP mosquitoes. Early studies of AP mosquitoes seem to indicate a relatively unspecialized feeding behaviour[62, 63].
On the other hand, recent studies by Cooper and colleagues have shown that at least two populations of An. hinesorum show strong feeding preferences and specifically avoid biting humans[22, 28]. Frequency of utilization of human-made habitats for breeding also varies among the AP sibling species. The phylogenetic study described here suggests that the AP sibling species diverged from each other long before humans arrived in PNG, probably ~50,000 years ago. In fact, the molecular dates place the divergence time of AP mosquitoes before the emergence of the Hominidae (or great ape) family[65, 66]. This indicates that any adaptations to humans (for blood meal or larval habitats) would have occurred independently in each AP sibling species, rather than being inherited from a common ancestor, and suggests that co-occurrences of malaria-related traits in Anopheles are the results of convergent evolution[67, 68].
The ancient divergence among AP sibling species, coupled with the recent arrival of humans in PNG, indicates that AP mosquitoes were present in PNG long before humans colonized the island. This observation suggests that the AP mosquitoes have independently evolved to adapt their behaviour to humans. While further studies are needed to better characterize the behaviour of AP mosquitoes, these findings emphasize the potential of the AP group to serve as a model for studying the evolution of vector competence and potentially for identifying the genetic basis of the ability to transmit human malaria.
We thank the staff of the Entomology Unit of the Papua New Guinea Institute of Medical Research (PNGIMR) and of the Faculty of Tropical Medicine, Mahidol University (Thailand) for their help in collecting Anopheles samples. We also thank the sequencing group of the McGill University and Genome Quebec Innovation Center for their help with Illumina sequencing. This work was supported by Cleveland Clinic funds to DS and the Fogarty International Center (TW007872) and NIAID International Centers of Excellence in Malaria Research Program (5U19AI089686) to PAZ.
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