An ancient lineage of slow worms, genus Anguis (Squamata: Anguidae), survived in the Italian Peninsula.

Four species of legless anguid lizard genus Anguis have been currently recognized: A. fragilis from western and central Europe, A. colchica from eastern Europe and western Asia, A. graeca from southern Balkans, and A. cephallonica from the Peloponnese. Slow worms from the Italian Peninsula have been considered conspecific with A. fragilis, despite the fact that the region served as an important speciation center for European flora and fauna, and included some Pleistocene glacial refugia. We used mitochondrial and nuclear DNA sequences to investigate the systematic and phylogenetic position of the Italian slow-worm populations and morphological analyses to test for phenotypic differentiation from A. fragilis from other parts of Europe. Our phylogenetic analyses revealed that Italian slow worms form a distinct deeply differentiated mtDNA clade, which presumably diverged during or shortly after the basal radiation within the genus Anguis. In addition, the specimens assigned to this clade bear distinct haplotypes in nuclear PRLR gene and show morphological differentiation from A. fragilis. Based on the differentiation in all three independent markers, we propose to assign the Italian clade species level under the name Anguis veronensisPollini, 1818. The newly recognized species is distributed throughout the Italian Peninsula to the Southern Alps and south-eastern France. We hypothesize that the Tertiary Alpine orogeny with subsequent vicariance might have played a role in differentiation of this species. The current genetic variability was later presumably shaped in multiple glacial refugia within the Italian Peninsula, with the first splitting event separating populations from the region of the Dolomite Mountains.

Phenotypic differentiation of the slow worm lizards (Squamata: Anguis) across their contact zone in Central Europe.

BACKGROUND: The application of molecular-phylogenetic approaches to taxonomy has had a dramatic effect on our understanding of the diversity of reptiles. These approaches have allowed researchers to reveal previously hidden lineages as well as taxonomic overestimation in morphologically plastic taxa. Slow worms, legless lizards of the genus Anguis (Squamata: Anguidae), were previously considered to comprise either one or two species, and morphology-based intraspecific taxonomy of Anguis fragilis remained controversial throughout the 20th century. After the discovery of deep genetic divergences within the genus, its taxonomy was reconsidered, and as a result, five extant species have been recognized. In order to better understand the patterns of their interspecific differentiation, here we studied phenotypic differences between the two most widespread of them-A. fragilis and A. colchica, and their putative hybrids across the contact zone of both species in Central Europe. METHODS: We used multivariate and univariate statistics and analyzed ten metric, eleven meristic, and six categorical phenotypic variables in material comprising a total of 326 individuals. We also genotyped individuals from the contact zone for one mitochondrial and two nuclear DNA fragments in order to delineate the distribution of individuals of hybrid and non-hybrid origin. The clines in morphological traits were studied using HZAR. RESULTS: We show that the two species are morphologically differentiated. Anguis fragilis has a less robust head, fewer scales covering the body, lower frequency of the external ear opening presence, lower frequency of separated prefrontal scales, higher frequency of prefrontal scales in contact with each other, and body coloration more similar to the juvenile coloration than A. colchica. Slow worms from the contact/hybrid zone are characterized by an intermediate morphology, with more similarities to A. fragilis than to A. colchica. DISCUSSION: None of the analyzed characters alone proved to be fully diagnostic, although more than 90% of all individuals could be successfully assigned to one or another species based on numbers of scales around the body. Our results indicate concordant, coincident, and steep clines in character states change. We present several hypotheses on the origin and evolutionary maintenance of the morphological divergence between both species and suggest that different evolutionary histories of the taxa rather than recently acting selection explain the observed morphological variation.