Abstract
BackgroundSkipper butterflies (Hesperiidae) are a relatively well-studied family of Lepidoptera. However, a combination of DNA barcodes, morphology, and natural history data has revealed several cryptic species complexes within them. Here, we investigate three DNA barcode lineages of what has been identified as Urbanus belli (Hesperiidae, Eudaminae) in Área de Conservación Guanacaste (ACG), northwestern Costa Rica.ResultsAlthough no morphological traits appear to distinguish among the three, congruent nuclear and mitochondrial lineage patterns show that “Urbanus belli” in ACG is a complex of three sympatric species. A single strain of Wolbachia present in two of the three cryptic species indicates that Urbanus segnestami Burns (formerly Urbanus belliDHJ01), Urbanus bernikerni Burns (formerly Urbanus belliDHJ02), and Urbanus ehakernae Burns (formerly Urbanus belliDHJ03) may be biologically separated by Wolbachia, as well as by their genetics. Use of parallel sequencing through 454-pyrosequencing improved the utility of ITS2 as a phylogenetic marker and permitted examination of the intra- and interlineage relationships of ITS2 variants within the species complex. Interlineage, intralineage and intragenomic compensatory base pair changes were discovered in the secondary structure of ITS2.ConclusionThese findings corroborate the existence of three cryptic species. Our confirmation of a novel cryptic species complex, initially suggested by DNA barcode lineages, argues for using a multi-marker approach coupled with next-generation sequencing for exploration of other suspected species complexes.
Highlights
Skipper butterflies (Hesperiidae) are a relatively well-studied family of Lepidoptera
Given three cryptic species in northwestern Costa Rica, the different geologic history of Meso- and South America, and the degree to which we are finding that supposedly single species occurring in both continents comprise two or more related species that are much more limited in distribution, we believe that extending the application of the name Urbanus belli to ACG lowland Asteraceae-eating Urbanus is unwarranted
Were alva an applicable epithet, it would pertain to a full species because it appears to be broadly sympatric with Urbanus viterboana viterboana, which Evans likewise recorded as pan-neotropical (Mexico to Bolivia)
Summary
Skipper butterflies (Hesperiidae) are a relatively well-studied family of Lepidoptera. We investigate three DNA barcode lineages of what has been identified as Urbanus belli (Hesperiidae, Eudaminae) in Área de Conservación Guanacaste (ACG), northwestern Costa Rica. Cryptic species—those species that lack anatomical differences—require additional lines of evidence for species identification beyond traditional measures, including but not limited to: ecological distinctiveness, behavioral differences, morphological dissimilarity in undescribed life stages, and molecular divergences e.g., [4,5,6]. This study focuses on the latter and, utilizes DNA barcode divergences as initial proxies for distinguishing cryptic species. Since 2003, DNA barcoding has been applied to a long-standing morphological and ecological inventory of Lepidoptera from the 140,000 ha terrestrial portion of Área de Conservación Guanacaste (ACG), northwestern Costa Rica (http://www.acguanacaste.ac.cr)
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