Species Delimitation in Morphologically Conserved Lizards

Abstract

Morphological conservatism in lacertid lizards generates extensive cryptic species diversity that is systematically underestimated by traditional taxonomy, with conservation consequences for species classified as widespread and common that may in fact comprise multiple range-restricted endemics. This study applied an integrative taxonomic framework combining mitochondrial phylogenetics (cytochrome b, ND4; 2,840 bp), genome-wide SNP genotyping (RADseq; 8,420 loci from 284 individuals), multivariate morphometrics (38 scalation and biometric characters), and ecological niche modelling (MaxEnt) to delimit species within the Lacerta viridis complex across 42 sampling localities in Central and Southern Europe. Mitochondrial phylogenetic analysis recovered eight well-supported lineages (bootstrap support > 84% in all cases; mean uncorrected p-distance between lineages 4.8 +/- 0.8%), exceeding the 2% barcoding threshold for species-level divergence in all pairwise comparisons. STRUCTURE analysis of RADseq SNPs identified K = 7 genomic clusters with mean pairwise FST of 0.184 +/- 0.042. Discriminant analysis of morphometric characters correctly reclassified 82.4% of individuals to their molecular lineage group (cross-validated). Ecological niche models showed significant niche divergence between six of eight lineage pairs (Schoener's D < 0.40). Applying the general lineage concept of species delimitation, we recognise six species as diagnosable units supported by congruent molecular, morphological, and ecological evidence, including the formal description of two lineages as new species. These results increase the known species richness of European lacertids by two and identify three single-locality endemic lineages requiring immediate conservation status assessment.