AimsTo characterize the genetic and morphological diversification of the endangered Balearic lizard Podarcis lilfordi and to assess the relevance of this diversity, and how it is described, to conservation measures.LocationThis study covers all the populations of the Balearic lizard, Podarcis lilfordi, present in its range of distribution at coastal islets of Menorca, Mallorca and Cabrera Archipelago.MethodsWe analysed genetic and morphological variation across the 43 known extant populations of the Balearic lizard, using mitochondrial and nuclear markers. We examined morphometric and scalation characters using, in some cases, phylogenetically independent contrasts. We also incorporated the study of dorsal coloration and dorsal colour pattern including the analysis of melanism in several populations.ResultsWe detected clear genetic divergence between Menorcan populations and populations from Mallorca and Cabrera, in both nuclear and mtDNA markers, but genetic divergence is relatively low among different insular populations within these groups. In contrast, morphological divergence was substantial both between Menorcan and remaining populations and within these groups. Morphological traits, such as dorsal coloration, body size and the number and size of scales, seemed to be linked with differences in climatic conditions between populations. In addition, some traits, as melanism, showed a strong phylogenetic signal.Main conclusionsThe morphological and genetic diversity of the Balearic lizard is incongruent with the subspecies described in the classical taxonomic literature. Moreover, current populations differ not only in some genetic and morphological features, but also in several ecological and ethological characteristics, in many cases unique to one population. Based on our results, we propose abandoning the use of subspecies to describe the extraordinary morphological diversity of the Balearic lizard and its replacement with the concept of evolutionary significant units (ESUs). ESUs are particularly suitable to describe and recognize such diversity and, especially, to ensure the continuity of the evolutionary process.

Gut microbial communities provide essential functions to their hosts and are known to influence both their ecology and evolution. However, our knowledge of these complex associations is still very limited in reptiles. Here we report the 16S rRNA gene faecal microbiota profiles of two lizard species endemic to the Balearic archipelago (Podarcis lilfordi and P. pityusensis), encompassing their allopatric range of distribution through a noninvasive sampling, as an alternative to previous studies that implied killing specimens of these IUCN endangered and near-threatened species, respectively. Both lizard species showed a faecal microbiome composition consistent with their omnivorous trophic ecology, with a high representation of cellulolytic bacteria taxa. We also identified species-specific core microbiota signatures and retrieved lizard species, islet ascription, and seasonality as the main factors in explaining bacterial community composition. The different Balearic Podarcis populations are characterised by harbouring a high proportion of unique bacterial taxa, thus reinforcing their view as unique and divergent evolutionary entities.

The diets of insular lizards are extremely varied, depending on the different environmental characteristics of each island population. This is particularly evident in the case of the populations of small coastal islets of the Balearic Islands, where the Balearic lizard, Podarcis lilfordi, is found. The study of trophic ecology carried out by means of traditional tools, such as morphological analysis of feces, has made it possible to detect numerous prey and nutritional elements. However, these methods are clearly insufficient, as some rare groups are not detected. It is also difficult to identify remains of marine subsidies or of foods contributed to these small islands by other predators, such as seabirds. The current study demonstrates the advantages of combining morphological diet analysis with the molecular study of individual feces samples obtained from the same populations. We obtained a greater diversity of prey groups using the combined methodologies, with each method identifying prey items that were not detected using the other method. Particularly, the study of diets at the molecular level identified plant species consumed by lizards that were, occasionally, not identified in morphological analyses. Conversely, the traditional morphological study of an equivalent number of fecal samples allowed for the identification of several prey groups that had not been detected in the molecular study. From this viewpoint, the advantages and disadvantages of each methodology are discussed.

Dietary studies are essential to unravel the functioning of ecosystems and ultimately to understand biodiversity. This task, which at first may seem simple, becomes especially complex in those cases of omnivorous species with highly variable diets. In this regard, the emergence of next-generation DNA sequencing methodologies represents a powerful tool to address the problem. Here we implement a high-throughput metabarcoding strategy based on the analysis of four molecular markers aimed at sequencing both mitochondrial (animal prey) and chloroplast (diet plants) genome fragments from fecal samples of two lizard species endemic to the Balearic Archipelago (Podarcis lilfordi and P. pityusensis) obtained through non-invasive methods. The results allowed for the characterization of their diets with a high degree of taxonomic detail and have contributed a large number of new trophic records. The reported diets are based mainly on the consumption of arthropods, mollusks and plants from a diversity of taxonomic orders, as well as carrion and marine subsidies. Our analyses also reveal inter- and intra-specific differences both in terms of seasonality and geographical distribution of the sampled lizard populations. These molecular findings provide new insights into the trophic interactions of these threatened endemic lizards in their unique and isolated ecosystems.

The Columbretes archipelago consists of a group of small volcanic islets located in the western Mediterranean near the east of the Iberian Peninsula. Four of its islands are inhabited by the wall lizard Podarcis liolepis, whose populations have been considered vulnerable. The purpose of this study was to assess the level of genetic diversity across the archipelago and the possible evolutionary origin of the Columbretes lizard populations. Additionally, we investigated the evolutionary ecology of these populations using a DNA-based metabarcoding approach to characterise both their microbiota and trophic interactions. The genetic results reported very low genetic diversity and corroborated the conspecificity between insular populations and P. liolepis from the mainland (Peñagolosa region). The results of the metabarcoding analyses based on faecal samples were in accordance with an omnivorous ecology, suggesting that specific microbiota communities in the insular populations might be correlated with differences in host ecology and phylogeny. These results are a valuable contribution to the current understanding of the evolution of Columbretes’ lizards and provide important information for conservation management.