Molecular delimitation of morphologically cryptic species in globetrotting Nylanderia (Hymenoptera: Formicidae) species complexes

The ant genus Nylanderia Emery has a cosmopolitan distribution and includes 150 described species and subspecies, with hundreds more undescribed. Global taxonomic revision is stalled by strong intra- and interspecific morphological variation, limited numbers of diagnostic characters, and dependence on infrequently collected male specimens for species description and identification. Taxonomy is further complicated by widespread distributions of at least 15 globetrotting species that have expanded their ranges through human transport. Nylanderia is one of the most frequently intercepted ant genera at ports of entry worldwide and species-level diagnoses are often difficult. Three morphologically difficult species groups contain six of these problematic species: (1) the N. bourbonica complex, (2) the N. fulva complex, and (3) the N. guatemalensis complex. We used target enrichment of ultraconserved elements (UCEs) from 165 specimens representing approximately 98 Nylanderia species worldwide to elucidate the phylogenetic positions of these three complexes, delimit species boundaries within them, and estimate the number of species they contain. For each complex, allelic phasing was used to separate heterozygous sites within samples into two haplotypes, effectively doubling sample size and increasing population-level sampling. Each complex was found to be monophyletic with strong support. We also extracted mitochondrial COI (cytochrome C oxidase I) barcodes from the UCE data and found that single-gene delimitation using barcodes potentially over-splits each globetrotting species into multiple putative species when compared with genome-scale results. This work emphasizes the importance of integrative taxonomy for invasive species management, as globetrotting occurs independently across at least nine different lineages across Nylanderia.