Integrative Taxonomy of Cryptic Amphibian Species Using Mitochondrial and Nuclear Markers

Abstract

Cryptic amphibian species — morphologically indistinguishable yet genetically divergent lineages — represent a
pervasive challenge for biodiversity assessment and conservation planning. This study applied an integrative taxonomic
framework combining three mitochondrial loci (16S rRNA, COI, ND2) and three nuclear markers (RAG1, POMC, NCX1)
to resolve species boundaries within three previously ambiguous amphibian complexes distributed across the
Mediterranean basin and Western Balkans. A total of 312 tissue samples from 47 localities were analysed.
Maximum-likelihood and Bayesian phylogenetic reconstructions, calibrated with five fossil constraints, recovered 11
well-supported lineages (posterior probability ≥ 0.95; bootstrap ≥ 80). Species delimitation analyses using bPTP and
GMYC confirmed nine independently evolving entities, of which two are formally described here as new species. Mean
interspecific K2P divergence across the mitochondrial 16S locus was 6.8 ± 1.2%, substantially exceeding the
intraspecific threshold of 0.9 ± 0.3%. Nuclear concordance factors corroborated mitochondrial topologies at 87% of
nodes. Morphometric analysis of 18 characters revealed significant multivariate separation (MANOVA: Wilks’ λ = 0.21, F
= 14.7, p < 0.001) among confirmed species. Divergence dating placed primary cladogenic events in the
Miocene–Pliocene transition (5.8–3.2 Ma), coinciding with major palaeogeographic reorganisation of the Western
Mediterranean. These findings underscore the extent to which morphological conservatism masks true amphibian
diversity and emphasise the urgent need for integrative approaches in threatened taxa assessments.