ISSN (online): 1802-8829http://www.eje.cz free of null alleles and conforming to Hardy-Weinberg expectations) has proven challenging in E. diurnus. Indeed, a set of 16 loci had been reported for this species before our study (Hockham et al., 1999;Hamill et al., 2006). According to the authors themselves, these loci displayed strong heterozygote defi cit, and the incidence of null alleles was considerable for some of them. In a preliminary trial we tested 13 of these available 16 loci on samples from highly divergent populations previously characterized for mitochondrial DNA COI variation (Party et al., 2015). Most loci failed to amplify and/or presented complex allelic patterns impeding their scoring. This situation signifi cantly reduced the number of available markers to only fi ve, which is a minimum value for population genetic analyses. We therefore applied highthroughput (pyrosequencing) technology to a partial genomic library enriched in microsatellite motifs in order to increase the number of loci and fi lter out those of low quality according to criteria detailed below.
MATERIAL AND METHODS
Sample collection and DNA extractionFifty-one specimens of E. diurnus were collected from nine localities in southern France between 2011 and 2014 (Fig. 1) ). Five multiplex and one simplex PCR protocols were optimized, and the polymorphism at the 16 loci was assessed in two natural populations from southern France. The mean allele number and (expected mean heterozygosity) were 8.94 (0.71) and 6.57 (0.70), respectively, in each population. Several loci were at Hardy-Weinberg disequilibrium (HWD), possibly due to the incidence of null alleles. The occurrence of null alleles has been previously reported for this species, and it is a common feature of microsatellite loci in Orthoptera. Cross-amplifi cation tests demonstrated the transferability of some of these loci to other ephippigerine species. The microsatellite loci reported here substantially increase the number of available loci for this species and will afford an accurate picture of E. diurnus phylogeography, the genetic structure of its populations, and an improved understanding of the evolution of male song and other sexually-selected traits in this highly variable species.