An Integrative Approach Using Phylogenomics and High-Resolution X-Ray Computed Tomography for Species Delimitation in Cryptic Taxa

Morphologically cryptic taxa have proved to be a long-standing challenge for taxonomists. Lineages that show strong genomic structuring across the landscape but are phenotypically similar pose a conundrum, with traditional morphological analyses of these cryptic lineages struggling to keep up with species delimitation advances. Micro X-ray computed tomography (CT) combined with geometric morphometric analyses provides a promising avenue for identification of morphologically cryptic taxa, given its ability to detect subtle differences in anatomical structures. However, this approach has yet to be used in combination with genomic data in a comparative analytical framework to distinguish cryptic taxa. We present an integrative approach incorporating genomic and geometric morphometric evidence to assess the species delimitation of grassland earless dragons (Tympanocryptis spp.) in north-eastern Australia. Using mitochondrial and nuclear genes (ND2 and RAG1, respectively), along with >8500 SNPs (nuclear single nucleotide polymorphisms), we assess the evolutionary independence of target lineages and several closely related species. We then integrate phylogenomic data with osteological cranial variation between lineages using landmark-based analyses of three-dimensional CT models. High levels of genomic differentiation between the three target lineages were uncovered, also supported by significant osteological differences. By incorporating multiple lines of evidence, we provide strong support for three undescribed cryptic lineages of Tympanocryptis in north-eastern Australia that warrant taxonomic review. Our approach demonstrates the successful application of CT with integrative taxonomic approaches for cryptic species delimitation, which is broadly applicable across vertebrates containing morphologically similar yet genetically distinct lineages. Additionally, we provide a review of recent integrative taxonomic approaches for cryptic species delimitation and an assessment of how our approach can value-add to taxonomic research.