PERMANENT GENETIC RESOURCES ARTICLE Strong correlation between cross-amplification success and genetic distance across all members of ‘True Salamanders’ (Amphibia: Salamandridae) revealed by Salamandra salamandra-specific microsatellite loci RALF HENDRIX, *† J. SUSANNE HAUSWALDT,‡ MICHAEL VEITH§ and SEBASTIAN STEINFARTZ* *Department of Animal Behaviour, University of Bielefeld, Morgenbreede 45, 33615 Bielefeld, Germany, †Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plo ¨n, Germany, ‡Department of Evolutionary Biology, Zoological Institute, Technische Universita ¨t Braunschweig, Spielmannstr. 8, 38106 Braunschweig, Germany, §Department of Biogeography, University of Trier, Am Wissenschaftspark 25 + 27, 54296 Trier, Germany Abstract The unpredictable and low cross-amplification success of microsatellite loci tested for conge- neric amphibian species has mainly been explained by the size and complexity of amphibian genomes, but also by taxonomy that is inconsistent with phylogenetic relationships among taxa. Here, we tested whether the cross-amplification success of nine new and 11 published microsatellite loci cloned for an amphibian source species, the fire salamander (Salamandra salamandra), correlated with the genetic distance across all members of True Salamanders (genera Chioglossa, Lyciasalamandra, Mertensiella and Salamandra that form a monophyletic clade within the family of Salamandridae) serving as target species. Cross-amplification suc- cess varied strongly among the species and showed a highly significant negative relationship with genetic distance and amplification success. Even though lineages of S. salamandra and Lyciasalamndra have separated more than 30 Ma, a within genus amplification success rate of 65% was achieved for species of Lyciasalamandra thus demonstrating that an efficient cross-species amplification of microsatellite loci in amphibians is feasible even across large evolutionary distances. A decrease in genome size, on the other hand, paralleled also a decrease in amplified loci and therefore contradicted previous results and expectations that amplification success should increase with a decrease in genome size. However, in line with other studies, our comprehensive dataset clearly shows that cross-amplification success of microsatellite loci is well explained by phylogenetic divergence between species. As taxo- nomic classifications on the species and genus level do not necessarily mirror phylogenetic divergence between species, the pure belonging of species to the same taxonomic units (i.e. species or genus) might be less useful to predict cross-amplification success of microsatellite loci between such species. Keywords: cross-amplification, genome size, microsatellite loci, neutral genetic distance, Salaman- dra salamandra, Salamandridae, True Salamanders Received 29 September 2009; revision received 24 January 2010; 11 March 2010; accepted 22 March 2010 Introduction Microsatellite loci are the most important and frequently used population genetic markers in amphibian research, addressing questions related to general biological issues, such as landscape ecology (e.g. Palo et al. 2003; Funk et al. 2005), fine-scale population structure (e.g. Palo et al. 2004; Jehle et al. 2005; Steinfartz et al. 2007a), mating systems (e.g. Jones et al. 2002; Steinfartz et al. 2006; Jehle et al. 2007), amphibian decline-linked disease infections (e.g. Pearman & Garner 2005; Teacher et al. 2009) and Correspondence: Sebastian Steinfartz, Fax: +49-521-106-2998; E-mail: sebastian.steinfartz@uni-bielefeld.de Ó 2010 Blackwell Publishing Ltd Molecular Ecology Resources (2010) doi: 10.1111/j.1755-0998.2010.02861.x