From black lights to taxonomic insights: Integrative taxonomy in the North American Enicospilus undulatus species-group of widespread, nocturnal parasitoid wasps uncovers species paraphyly and undiscovered species

Enicopsilus is a large genus of nocturnal koinobiont parasitoid wasps that attack various moth families. Most Enicospilus species have unusually wide distributions, sometimes encompassing the majority of multiple biogeographic regions. Particularly notable in North America (north of Mexico) are the species belonging to the E. undulatus species-group, distinguished by the absence of alar sclerites and larger size in some species. Although the seven species in eastern North America were revised in 1988, significant taxonomic and evolutionary questions remain, specifically: 1) Is the E. undulatus species-group monophyletic? 2) Do various lines of evidence, such as morphology, Ultra Conserved Elements (UCEs), and COI, support congruent species concepts? 3) Are there cryptic or otherwise undescribed species in North America? To resolve these questions, the morphology of over 1,000 specimens was examined and UCEs were sequenced for 121 specimens, both within and from outside the E. undulatus species-group. COI sequences were then mined from the UCE sequence data to evaluate COI diversity within and between each species using a pairwise distance analysis. Separate phylogenies using UCEs and COI concatenated alignments were then inferred using maximum likelihood. The resulting phylogenies were highly supported, showed a monophyletic E. undulatus species-group, and mostly aligned topologically with the current species concepts. Intriguingly, Enicospilus peigleri was found to be derived within the more widespread Enicospilus americanus, despite clear morphological differences, and four undescribed species were discovered. In summary, complementary morphological and genetic approaches were instrumental in clarifying the species boundaries in the E. undulatus species-group in North America.