A barcoding approach to phylogenetic classification of Aedini mosquitoes (Aedes, Ochlerotatus)

Abstract

14Traditionally, entomologists have used morphological characteristics for mosquito taxonomy 15 and systematics. However, this approach does not take into consideration the genetic 16 relatedness of species. In 2000, the Aedes genus of mosquitoes in the tribe Aedini was split into 17 two genera (Aedes and Ochlerotatus), thereby elevating Ochlerotatus from subgenus to genus 18 rank, strictly based on morphology of adults. Herein, we use the genetic barcoding marker COI 19 to generate a phylogeny of 65 species of Aedes, Ochlerotatus, and Anopheles outgroup from 20 almost 900 sequences downloaded from BOLD systems. Our results reveal evidence of non-21 random, but polyphyletic clustering of Aedes and Ochlerotatus species, with a monophyletic 22 outgroup. We do find support for the validity of Ochlerotatus as an evolutionary unit, although 23 we find insufficient evidence to support its retention as a genus. We suggest that mosquito 24 phylogenetic analyses incorporate a greater number of genetic markers to help clarify our 25 understanding of Aedini species classifications, but caution that recent assessments based 26 solely on morphology may be insufficient. 27 28 45 Ochlerotatus (Meigen 1818, as cited in Harbach 2016). Through a series of morphology-46 informed phylogenetic studies, Reinert et al. 47 Ochlerotatus to genus status. This decision was based on the analysis of morphological 48 characteristics of 119 Aedini species across all life stages. The resulting morphology-based 49 phylogeny proposed this change to the previous classification that was based solely on adult 50 4 mosquito morphology. These revisions to the genera created instant controversy among 51 researchers and led to many journals that focus on these medically important species to 52 suggest caution with adopting the new designations (Reisen 2016). Because many species 53 within the genus Aedes are of significant medical importance (e.g., Aedes aegypti), 54 redesignation of any species would pose challenges for public health officials in relation to 55 using long standing species names when communicating with the public. In addition, as it has 56 been nearly two decades since Reinert first proposed elevating Ochlerotatus to a genus (Reinert 57 2000), and using a molecular approach to resolving the phylogenetic relationships among these 58 species is long overdue. Indeed, in an editorial about Aedini mosquitoes, Reisen (2016) noted: 59 "As more mosquito sequencing data become available … genetic analyses should be done to 60 confirm these phenotypic groupings." 61 DNA barcoding has been promoted as a universal tool for reliable species identifications 62 (Hebert et al. 2003, Hebert et al. 2004), and also as a tool for helping to resolve phylogenetic 63 relationships among species (Hajibabaei et al. 2007, Erpenbeck et al. 2007). The 648 base-pair 64 mitochondrial cytochrome c oxidase 1 gene (COI) is regarded as the standardized barcode gene 65 for species identification (iBOL 2018). Thus far, there is a mixed record of success of using COI...