The Heliconiini genera Agraulis and Dryas are widely distributed throughout the Neotropics and into adjacent temperate regions, and although they are currently treated as monotypic, both show significant geographic phenotypic variation. In this work, we employ six genetic markers (4199 bp), two mitochondrial and four nuclear, to perform coalescent species delimitation analyses in Bayesian Phylogenetics and Phylogeography (BPP) and in integrated BPP (iBPP), the latter also includes linear measurements and wings landmarks. We also analyze cytochrome c oxidase I (COI) barcode sequences for each genus using genetic distances, haplotype networks and a character-based approach. Based on the model testing results, complemented with data from previous studies, we performed morphometric analyses to compare fore and hindwing size, aspect ratio and shape among the new species. In addition, we compared the forewing spot pattern of hypothesizes species using the r package patternize and, for Dryas, compared the colour patterns of mature larvae. Model testing of the molecular species delimitation outputs favoured a seven species hypothesis for Agraulis and a four species hypothesis for Dryas. Average distances among COI barcode sequences of these groups were from 1.09 to 5.81% in Agraulis and from 1.09 to 3.44% in Dryas. Within-group distances ranged from 0 to 1.11% and between 0 and 2.43%, respectively. NeighborNet haplotype networks showed that all but one of the species are monophyletic, and the character-based approach found exclusive diagnostic positions for most species, while the rest can be recognized by unique combinations of the 44 informative nucleotide positions analysed. Morphometric analysis supported all species of Agraulis and Dryas based on wing shape, and also in several cases on wing sizes and aspect ratio (hindwing length–forewing length), including A. v. galapagensis, which was absent from the molecular study. The analysis of the forewing spot pattern also revealed differences among most species hypothesis. The colour pattern of the last instar larva is also diagnostic for each Dryas species hypothesis. Locality data for species in both genera show that most of them are allopatric albeit a few have contact zones being parapatric at some locations. Based on the observed genetic differences, which covary with morphology and geographical distribution, we recommend the recognition of eight species of Agraulis: A. incarnata, A. vanillae, A. forbesi new status, A. insularis reinstated status, A. maculosa new status, A. lucina reinstated status, A. galapagensis new status and one undescribed species, and four species of Dryas: D. iulia, D. dominicana revised status, D. lucia revised status and D. alcionea reinstated status. Further work is needed to investigate, which selective forces have led to the current configurations of venation and wing shape, the probable gene flow among species with a focus on Agraulis and gather more data on species ecology. LSIDurn: lsid: zoobank.org: pub: 9E8C9F6E-91C3-4FB2-804D-1A9C4BD2EABB.
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We especially thank Francis Deknuydt, Daniel Romé, Eddy Dumbardon‐Martial and Gwénaël David who kindly sent us specimens from Martinique, Guadeloupe, Saint Lucia and French Guiana and shared information and pictures of both adults and immature stages. We also thank colleagues where the field work was carried out, especially Douglas M. Fernández and Feliberto Bermúdez in Cuba, Carlos Suriel and Ruth Bastardo in the Dominican Republic and Jason Hall in Ecuador. We also thank Carlos de Soto Molinari, Dominican Republic ( https://www.flickr.com/people/cdesoto/ ) and Alfredo Colón, Puerto Rico ( http://alfredocolon.zenfolio.com/ ), for allowing us the use of their pictures of . mature larvae. We acknowledge curators and technicians at the visited collections and the distribution data provided by them, including Fernando Guerra, José Luis Aramayo, Santiago Villamarín and Yuvinka Gareca. We are also grateful to Jérôme Morinière and Isabella Stoeger for their support during the work at the ZSM molecular lab. We acknowledge Pável F. Matos–Maraví and Ledis Regalado for their suggestions during the molecular analyses. Funding for this project was made possible by Víctor González (San Juan, Puerto Rico), a Systematic Research Foundation grant (2015) and Georg Foster Research Fellowship from the Alexander von Humboldt Foundation (1162549 – CUB –‐ GFHERMES‐E). KRW thanks Santiago Villamarín, Sofía Nogales, the INABIO and Ecuadorian Ministerio del Ambiente for arranging the necessary permits for research in Ecuador, most recently under the project ‘Diversity and Biology of Lepidoptera in Ecuador’ (No. 006‐19 IC‐FLO‐FAU‐DNB/MA). Field work in Ecuador was supported in part by the Darwin Initiative, the National Geographic Society (Research and Exploration Grant # 5751‐96) and NSF (# 0103746, #0639977, #0639861, #0847582, #1256742). The authors declare that there are no conflicts of interest. Dryas D iulia
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