Species Delimitation in Morphologically Conserved Reptiles

Abstract

Morphological conservatism — the retention of ancestral phenotypes across deeply diverged lineages — is a persistent challenge for reptile taxonomy, where convergent evolution of scalation patterns, body proportions, and colouration frequently obscures species boundaries that are apparent only in molecular datasets. This study applied an integrative taxonomic framework combining mitochondrial (16S rRNA, ND4) and nuclear (PRLR, EXPH5) markers with geometric morphometrics of head shape and scale count data to resolve species boundaries in three morphologically conservative European lizard and snake complexes: the Lacerta viridis species group (Lacertidae), the Natrix natrix species group (Colubridae), and the Vipera aspis species group (Viperidae). A total of 486 specimens from 108 localities across 14 countries were sampled. Multi-locus Bayesian phylogenies recovered 12 strongly supported lineages across the three complexes (posterior probability ‡ 0.95 for all major nodes). Species delimitation under bPTP and GMYC confirmed 11 independently evolving entities (congruence between methods: 91.7%). Mean interspecific K2P divergence at 16S was 7.4 ± 1.8%, substantially exceeding intraspecific divergence of 0.8 ± 0.3%. Geometric morphometric analysis of head shape (22 landmarks) revealed significant multivariate differentiation between molecularly delimited species in 9 of 11 cases (MANOVA: Wilks’ l < 0.58, all p < 0.01), with DFA achieving 86.4 ± 5.2% correct classification despite overlap in traditional scalation characters. Scale count PCAs showed limited diagnostic power (species overlap in 8 of 11 comparisons), confirming that geometric morphometrics outperforms traditional meristic characters for cryptic reptile species identification. Divergence dating placed primary cladogenic events in the Miocene–Pliocene (8–2 Ma), consistent with Mediterranean palaeogeographic diversification. One new species is formally described from the Vipera aspis complex