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Abstract
BackgroundWall lizards of genus Podarcis are abundant and conspicuous reptiles inhabiting Europe and North Africa. In recent years, they have become a popular lizard model for phylogeographical and evolutionary ecology studies. However a lack of suitable nuclear markers currently presents a limitation on analyses of molecular evolution within this genus. We address this limitation by developing twenty-one new primer pairs for polymerase chain reaction (PCR) amplification and sequencing of anonymous sequence markers in Podarcis vaucheri and performed an assay of their cross-amplification and polymorphism levels in two closely- (P. bocagei and P. liolepis) and two distantly-related (P. muralis and P. tiliguerta) congeners.FindingsCross-amplification and sequencing was straightforward among members of the Iberian and North-African group within genus Podarcis (which includes P. vaucheri), and somewhat less successful in species belonging to other groups (one and four loci out of 21 failed to amplify in P. muralis and P. tiliguerta, respectively, and overall success rates were lower). Nucleotide diversity for the five species examined ranged from 0.35% to 3.5%, with an average of 1.5% across all loci. Insertion and deletion polymorphisms were found in all but three loci.ConclusionsGiven the high cross-amplification rates, these markers constitute a valuable addition to set of genomic resources available for Podarcis, especially in studies dealing with phylogenetics, species delimitation, population genetics and phylogeography.
Highlights
Wall lizards of genus Podarcis are abundant and conspicuous reptiles inhabiting Europe and North Africa
Given the high cross-amplification rates, these markers constitute a valuable addition to set of genomic resources available for Podarcis, especially in studies dealing with phylogenetics, species delimitation, population genetics and phylogeography
A recent review [1] indicated that Podarcis, more than any other genus of lizard including Anolis, has been used extensively in studies pertaining to evolutionary ecology and phylogeography
Summary
Given the high cross-amplification rates, these markers constitute a valuable addition to set of genomic resources available for Podarcis, especially in studies dealing with phylogenetics, species delimitation, population genetics and phylogeography. Five replicate 25 μL reactions were carried out, each containing 1X PCR buffer (50 mM Tris–HCl, 50 mM NaCl, pH 8.5); 2 mM MgCl2; 1 mM each dNTP, 2U of GoTaq DNA polymerase (Promega), 1 μM of TSPADSHORT and 1,25 μL of the DNA-adapter mixture. The output of the ligation reaction was transformed into Escherichia coli competent cells and grown on standard LB medium with ampicillin/IPTG/X-Gal (details on this medium can be found on the pGEM-T Easy Vector Systems protocol); conventional blue/white screening was used to discriminate clones that contained inserts (white) from those that did not (blue) Using this protocol, a total of 72 positive colonies were obtained. Sequencing was performed in a Applied Biosystems 3130xl Genetic Analyzer According to this preliminary screening, 23 primer pairs showed consistent amplification and sequencing success.
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