Abstract
Anopheles bellator is a primary malaria vector in the Atlantic Forest. Partial sequences of timeless and Clock genes were used to assess the genetic differentiation of five Brazilian populations, which showed strong population structure (e.g. high F ST values and fixed differences) in all pairwise comparisons between Bahia sample and the others from Paraná, São Paulo and Rio de Janeiro states. Also, the resulting phylogenetic trees clearly grouped the sequences from Bahia in a different cluster with high bootstrap values. Among southern and southeastern populations low levels of genetic differentiation were found suggesting a general stability of the genetic structure.
Highlights
DNA sequence analysis - Anopheles bellator DNA sequences were aligned with MAFFT[19] and phylogenetic trees were constructed for each gene under the Maximum Likelihood method using the ape and phangorn packages[20,21] in R version 3.3.2.(18) The best-fit substitution models HKY+G (Clock) and TIM3e+G+I were selected following the AIC criterion using the modelTest function from phangorn.[21]. An additional timeless phylogenetic tree using the coding regions from An. bellator, An. laneanus and An. cruzii s.s. were constructed (K80+G model) as described above
Polymorphism data from a variety of measures — e.g. nucleotide diversity (θ and π values) — showed that in general the timeless gene has lower genetic diversity compared with Clock [Supplementary data (Tabela II)], the first has a bigger fragment size
Fig. 2: maximum likelihood trees of Anopheles bellator timeless and Clock sequences (TIM3e+G+I and HKY+G models, respectively). These phylogenies show that two structured groups were identified in the An. bellator populations from Brazil: An. bellator B occurs in the NorthEast Atlantic Rainforest (Camacan) and An. bellator A is found in the other more Southern Brazilian regions
Summary
Novel molecular evidence of population structure in Anopheles (Kerteszia) bellator from Brazilian Atlantic Forest. Partial sequences of timeless and Clock genes were used to assess the genetic differentiation of five Brazilian populations, which showed strong population structure (e.g. high FST values and fixed differences) in all pairwise comparisons between Bahia sample and the others from Paraná, São Paulo and Rio de Janeiro states. From the south of Brazil to the north-east, An. bellator, An. homunculus and An. cruzii are sympatric in the Atlantic Forest.[1,8] while An. homunculus populations from these regions showed no clear genetic separation ― they were considered a single species ― An. cruzii was shown to be a complex of at least two siblings: one major group with broad range dispersion from south and south-east Brazil and a distinct sibling species in BA.[8,9,10,11] This knowledge raises an interesting question: are An. bellator populations highly structured like those from An. cruzii; or, like An homunculus, is it a species without significant genetic differentiation in its wide range distribution? Clock and timeless loci are involved in the circadian rhythms of insects,(12) and have previously been shown to be good molecular markers to define different species in the An. cruzii complex.[10,11,13,14]
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