Integrating phylogeography and high-resolution X-ray CT reveals five new cryptic species and multiple hybrid zones among Australian earless dragons.

ABSTRACT

Cryptic lineages, comprising species complexes with deep genetic structuring across the landscape but without distinct morphological differences, impose substantial difficulties for systematists and taxonomists in determining true species diversity. Here, we present an integrative approach that combines data from phylogeography and geometric morphometric analyses of three-dimensional cranial models to revisit the uncertain taxonomy of earless dragons from southern and central Australia that at one time or another have been included under the name Tympanocryptis lineata. Our approach finds strong support for seven previously described species, and more importantly, five undescribed Tympanocryptis taxa for which we provide a taxonomic treatment. We also find evidence of introgression and hybridization in three discrete contact zones between lineages, supported by mitochondrial and nuclear genes, as well as morphological analyses. With a sampling design that includes at least five individuals for each genetic lineage with corresponding X-ray microcomputed tomography scans, we perform comparative evolutionary analyses to show that there is a significant phylogenetic signal in Tympanocryptis cranial shape. Our results demonstrate the importance of using multiple specimens in each genetic lineage, particularly in cases of potential hybridization, and that geometric morphometrics, when used in an integrative framework, is a powerful tool in species delimitation across cryptic lineages. Our results lay the groundwork for future evolutionary studies in this widespread group across multiple environmental types and identify several species of immediate conservation concern with a focus on T. petersi sp. nov. We suggest that this species has undergone significant population declines and warrants a full conservation assessment.