Microsatellite loci are still considered valuable tools for addressing basic questions in ecology, evolution, and behavior in nonmodel organisms, despite the fact that other molecular markers have become increasingly popular due to next-generation sequencing (NGS) approaches (e.g., genotyping or sequencing of single-nucleotide polymorphisms (SNPs)). Microsatellite loci are currently still the marker of choice for comprehensive analyses of population structure (e.g., Palo et al. 2004; Jehle et al. 2005, 2007; Dogaç et al. 2013), mating systems (e.g., Jones et al. 2002; Schmeller et al. 2005; Steinfartz et al. 2006; Jehle et al. 2007; Loyau and Schmeller 2012), landscape (Storfer et al. 2010) and conservation genetics (reviewed by Jehle and Arntzen 2002; Beebee 2005; Schmeller and Merilä 2007; Duong et al. 2013). Moreover, the current impact of microsatellite loci as genetic tools is also illustrated by more than 4000 scientific studies that have been published in the past two years matching the query “microsatellite loci” in the Web of Science and by recent publications reporting the development of new loci (i.e., Prunier et al. 2012; Castoe et al. 2012a,b; Dobeš and Scheffknecht 2012). Before the application of NGS in the process of developing microsatellite loci, the isolation and characterization of new loci was a costly and sometimes elaborate endeavor (e.g., Zane et al. 2002). However, by using NGS approaches on genomic libraries enriched for microsatellite motifs, the isolation process has become much simpler and more cost-effective (e.g., Abdelkrim et al. 2009). Normally, these approaches result in tens of thousands of sequence reads, which are expected to lead to a large amount of suitable microsatellite loci (e.g., Yang et al. 2012). However, the correlation between the initial number of sequence reads obtained and the number of usable polymorphic microsatellite loci may be low as the number of potentially amplifiable loci (PALs) is negatively influenced by many factors. These factors include sequence read quality (cut-off score values), motif length (type and number of repeat), and the presence, quality, and necessary length of the primer region, in addition to the amplification success and confirmed polymorphism of loci across the studied populations. Accordingly, systematic approaches to estimate success rates of microsatellite loci development for quite distinct taxa are important to finally obtain a sufficient number of applicable loci (e.g., Castoe et al. 2010, 2012a,b; Prunier et al. 2012). For example, in the copperhead snake (Agkistrodon contortrix), Castoe et al. (2010), isolated 4,564 PALs from 128,773 reads, but only found 80 tetra-nucleotide PALs (i.e., 0.062% of all reads) with more than 10 repeat units. In the alpine newt (Ichthyosaura alpestris), Prunier et al. (2012) obtained 1015 microsatellite motif-bearing sequence reads, with a final yield of 14 microsatellite loci from 61 tested primer pair combinations. Microsatellite development might be especially tedious in amphibians due to their large genome sizes and comparably low numbers of PALs, which have made development and isolation approaches in the past both cost- and time-intensive (e.g., Hendrix et al. 2010; Hauswaldt et al. 2012). Accordingly, NGS-based microsatellite loci development approaches should be efficient in obtaining a sufficient number of loci in such species (e.g., amphibians).
In this study, we used a 454-sequencing approach with enriched libraries to develop highly polymorphic tetra-nucleotide microsatellite loci for three distinct newt species within the family of Salamandridae (Calotriton asper, Lissotriton helveticus, and Triturus cristatus). We determined the success rate of our approach by estimating the number of PALs based on the number of usable polymorphic loci tested across several populations of each species and compared it with Illumina-based sequencing approaches of recently published studies. Furthermore, we tested the cross-amplification success rate of the developed loci for C. asper in the highly endemic and threatened species C. arnoldi, the Montseny brook newt (see Carranza and Amat 2005).