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. 2023 Aug 16;18(8):e0290178.
doi: 10.1371/journal.pone.0290178. eCollection 2023.

Genetic diversity, phylogenetic and phylogeographic analysis of Anopheles culicifacies species complex using ITS2 and COI sequences

R A S Rathnayake  1 , W M M Wedage  2 , L S Muthukumarana  1 , B G D N K De Silva  1   2   3
Affiliations

Genetic diversity, phylogenetic and phylogeographic analysis of Anopheles culicifacies species complex using ITS2 and COI sequences

R A S Rathnayake et al. PLoS One. .

Abstract

Anopheles culicifacies is the major vector of malaria in Sri Lanka and the Indian subcontinent which is characterized as a species complex with five sibling species provisionally designated as A, B, C, D and E. The current study was carried out to understand the phylogenetic and phylogeographic relationships between the sibling species of the species complex while observing their genetic diversity and genetic differentiation. Thirty-five ITS2 and seventy-seven COI sequences of An. culicifacies species complex reported from different geographical locations of Asia and China at the NCBI public database were used for the analysis. Bayesian likelihood trees were generated for the phylogenetic analysis. The divergence of the species complex was obtained from the Bayesian phylogeographic model in BEAST. There were two clades of the sibling species of An. culicifacies species complex as A, D and B, C and E in both phylogenetic and phylogeographic analysis using ITS2 sequences. Based on the highly divergent COI sequences and the high mutation rate of the mitochondrial genome, there were four and three clades in both phylogenetic and phylogeographic analysis using COI sequences. The diversification of An. culicifacies species complex was obtained as ranging from 20.25 to 24.12 Mya and 22.37 to 26.22 Mya based on ITS2 and COI phylogeographic analysis respectively. There was a recent diversification of the sibling species A and D than the sibling species B, C and E. Low haplotype diversity was observed in the sequences reported from Sri Lanka in both ITS2 and COI analysis that can be due to bottlenecks resulting from the intense malaria control efforts. A high genetic differentiation was achieved for some populations due to the large geographical distance. The high genetic diversity based on the five sibling species implies the possibility of maintaining a relatively high effective population size despite the vector control efforts.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Bayesian likelihood tree generated by MrBayes-3.2.5_WIN32_x86 software using ITS2 sequences of An. culicifacies.
35 sequences, 439 characters, 460 000 generations, 2 mcmc runs. Percentages on the node labels are Bayesian posterior probabilities (PP). Ae. aegypti was used as the out-group. GenBank accession numbers of ITS2 sequences included in clade 1, 2 and outgroup were listed in S4 Table.
Fig 2
Fig 2. Bayesian likelihood tree generated by MrBayes-3.2.5_WIN32_x86 software using COI sequences of An. culicifacies.
77 sequences, 450 characters, 1 640 000 generations, 2 mcmc runs. Percentages on the node labels are Bayesian posterior probabilities (PP). Ae. aegypti was used as the out-group. GenBank accession numbers of COI sequences included in Clade 1 to 4 and outgroup were listed in S5 Table.
Fig 3
Fig 3. ITS2 haplotype network of An. culicifacies species complex (20 haplotypes) generated using DnaSP v5 software and Network 10.2 software.
The size of a circle indicates the relative frequency of sequences belonging to a certain sequence type. Each color indicates a different geographic area. The distance between two haplotypes includes no. of mutations. Red colored dots indicate median vectors.
Fig 4
Fig 4. COI haplotype network of An. culicifacies species complex (55 haplotypes) generated using DnaSP v5 software and Network 10.2 software.
The size of a circle indicates the relative frequency of sequences belonging to a certain sequence type. Each color indicates a different geographic area. The distance between two haplotypes includes no. of mutations. Red colored dots indicate median vectors.
Fig 5
Fig 5. Evolutionary timescale for An. culicifacies species complex generated by BEAST v1.8.2 software using ITS2 sequences.
35 sequences, 435 characters. An. subpictus was used as the out-group. Numbers near the nodes designate the average divergence time estimated (Million Years, Mya). Geological time scale includes Cretaceous, Pala(eocene), Eoce(ne), Olig(ocene) and Mioc(ene) respectively. GenBank accession numbers of ITS2 sequences included in clades 1, 2 and outgroup were listed in S8 Table. Posterior probability values, Mean values of diversification times and 95% highest posterior density (HPD) of each nodes of the ITS2 phylogeographic tree is listed in S9 Table.
Fig 6
Fig 6. Evolutionary timescale of An. culicifacies species complex generated by BEAST v1.8.2 software using COI sequences.
Sequences were retrieved from NCBI GenBank, 77 sequences, 450 characters. An. subpictus was used as the out-group. Numbers near the nodes designate the average divergence time estimated (Million Years, Mya). Geological time scale includes Cretaceous, Pala(eocene), Eoce(ne), Olig(ocene) and Mioc(ene) respectively. GenBank accession numbers of COI sequences included in clades 1 to 3 and outgroup were listed in S10 Table. Posterior probability values, Mean values of diversification times and 95% highest posterior density (HPD) of each nodes of the COI phylogeographic tree is listed in S11 Table.
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