New tools and insights to assist with the molecular identification of Simulium guianense s.l., main Onchocerca volvulus vector within the highland areas of the Amazonia onchocerciasis focus

•First evidence that Simulium guianense DNA escapes the effects of concerted evolution.•New ribosomal DNA barcoding tool for the onchocerciasis vector Simulium guianense.•New ribosomal mitochondrial barcoding tool for Simulium guianense.•Important Insights into the biology and evolution of Simulium guianense.•Important Insights for development of further subspecies Simulium guianense identification tools. Following the success of the Onchocerciasis Elimination Programme for the Americas (OEPA), there is now just one Latin American onchocerciasis focus where onchocerciasis transmission is described as ‘on-going:’ the Amazonia Onchocerciasis focus. In the hyperendemic highland areas of the Amazonia focus, Simulium guianense s.l. Wise are the most important vectors of the disease. Populations of S. guianense s.l. are, however, known to vary in their cytogenetics and in a range of behaviours, including in their biting habits. In the hypoendemic lowland areas of the Amazonia focus, for example, S. guianense s.l. are generally regarded as zoophilic and consequently unimportant to disease transmission. Robust tools, to discriminate among various populations of S. guianense s.l. have, however, not yet been developed. In the work reported here, we have assessed the utility of a ribosomal DNA sequence fragment spanning the nuclear ribosomal ITS-1, ITS-2 and 5.8S sequence regions and a ∼850 nucleotide portion of the mitochondrial cytochrome oxidase gene (CO1) for species-level identification and for resolving the within species substructuring. We report here how we have generated 78 CO1 sequences from a rich set of both zoophilic and anthropophilic populations of S. guianense s.l. that were collected from eight sites that are broadly distributed across Brazil. Consistent with previous findings, our analysis supports the genetic isolation of Simulium litobranchium from S. guianense s.l. In contrast with previous findings, however, our results did not provide support for the divergence of the two species prior to the radiation of S. guianense s.l. In our analysis of the S. guianense s.l. ribosomal DNA sequence trace files we generated, we provide clear evidence of multiple within-specimen single nucleotide polymorphisms and indels suggesting that S. guianense s.l. ribosomal DNA is not a good target for conventional DNA barcoding. This is the first report of S. guianense s.l. within individual ribosomal DNA variation and thus the first evidence that the species is not s