Scrutinising an inscrutable bark-nesting ant: Exploring cryptic diversity in the Rhopalomastix javana (Hymenoptera: Formicidae) complex using DNA barcodes, genome-wide MIG-seq and geometric morphometrics

Overlooking cryptic species diversity has grave implications on assessments of climate change impacts on biodiversity, ecosystems and organismal populations. Discriminating between cryptic species has long been challenging even for seasoned taxonomists, as interspecies morphological differences are often indiscernible by visual observation. Multi-disciplinary methods involving genetic analyses in conjunction with quantitative morphological data, should therefore be used to investigate boundaries between cryptic species. We adopted an integrated approach combining analyses of mitochondrial COI barcodes, a genome-wide dataset obtained via multiplexed inter-simple sequence repeats (ISSRs) genotyping by sequencing (MIG-seq), and geometric morphometrics to investigate species divergences in the inscrutable Rhopalomastix javana species complex. Objective clustering of COI suggested five putative molecular species units divergent from each other by thresholds within 4.2–10.6% uncorrected pairwise distance. Phylogenetic analyses based on concatenated MIG-seq data also recovered and strongly supported the monophyly of five major lineages in agreement with COI clusters. Co-ancestry analyses based on MIG-seq data using fineRADstructure resolved variable patterns of admixture linked to geography, and potential genetic drift within some putative species. Geometric morphometric analyses of specimen images further detected statistically significant differences in at least one of three anatomical aspects (Head, Meso, Profile) between all pairs of putative species. Head shape (full-face view) was determined to be the most informative character for species diagnosis, with relatively high classification accuracy. Thin-plate spline deformation grids highlighted areas of high variation between species in each shape for deeper taxonomic scrutiny. The presence of species from multiple distinct lineages existing in near-sympatry firmly demonstrates that R. javana comprises more than one closely-related species, but exact species boundaries are difficult to ascertain. Differences in elevation and its associated abiotic effects on ant adaptations and reproductive phenology may contribute to restricting gene flow and maintaining species boundaries between sympatric populations of the R. javana complex. We further assess the advantages and limitations of geometric morphometrics as a taxonomic tool. Despite its drawbacks, our combined approach has helped draw important insights on cryptic diversity in R. javana, and also identified gaps of knowledge that await address. Results from this study will inform and prime future in-depth taxonomic investigation on the R. javana complex, including formal descriptions and establishment of the five putative species.