A widespread application of molecular markers across species is often hindered by a low cross-species amplification success, a high frequency of null alleles or a reduced level of polymorphism (Primmer et al., 2005). This is particularly true for hyper-variable markers, like microsatellites, where high mutation rates are both advantageous (because their polymorphism allows very fine-scale temporal and spatial resolution) and problematic (because mutations in flanking regions reduce cross-amplification success) (Primmer and Merilä, 2002). Thus, optimizing sets of applicable, reliable and informative markers, which are key to address questions about patterns of genetic diversity and structure, hybridization and speciation, involves selecting highly polymorphic markers that can nonetheless be consistently amplified and scored in long diverged taxa.
Recent molecular studies have resolved the phylogeny of the Bufo bufo (Linnaeus, 1758) species group, revealing the existence of four species and delimiting their respective ranges (Litvinchuk et al., 2008; Garcia Porta et al., 2012; Recuero et al., 2012; Arntzen et al., 2013a). As a result of these studies, B. spinosus Daudin, 1803 is defined as an Ibero-Maghrebian endemism, with populations in North Africa from Morocco to Tunisia, the Iberian Peninsula, the southern fringe of the British Isles (i.e., Jersey) and north of the Pyrenees across most of France, where it contacts B. bufo along a NW-SE line, roughly from Caen to Lyon (Arntzen et al., 2013b, 2014). Bufo bufo is also present in the Apennine and Balkan peninsulas and extends as far east as northern Kazakhstan and eastern Siberia (Agasyan et al., 2009).
Previous studies have used morphological characters, mtDNA and slowly evolving nuclear DNA markers to broadly delineate the contact zone between B. bufo and B. spinosus in France (Arntzen et al., 2013a, b, 2014). The results show an overall correspondence between morphology and molecules and congruence across molecular markers, with occasional discordance interpreted as resulting from incomplete lineage sorting, although hybridization-mediated gene flow cannot be ruled out.
Addressing questions about reproductive isolation versus hybridization and introgression in the two species requires characterization of molecular markers that amplify in both B. bufo and B. spinosus and the quantification of gene flow in areas of secondary contact. Bufo bufo has been the subject of a number of conservation genetics studies, especially in the UK and a number of polymorphic microsatellite loci are available (Scribner et al., 1994, 1997, 2001; Hitchings and Beebee, 1998; Brede and Beebee, 2004, 2006; Wilkinson et al., 2007). On the contrary, there are very few studies on B. spinosus and one of the reasons is low cross-amplification success of markers originally isolated from B. bufo (Brede et al., 2001; Martínez-Solano and González, 2008; Arntzen et al., 2014).
Here we describe a morphologically intermediate Bufo population from France with the help of a set of new polymorphic microsatellite markers isolated from a B. spinosus genomic library. We describe the variability of the newly developed markers in five populations of B. spinosus (two Iberian and three from western France), evaluate the cross-amplification success in two French populations of B. bufo and describe hybridization between the two species in a morphologically intermediate population in their contact zone.
Fig. 1. Sampling localities of Bufo bufo (blue) and B. spinosus (red) in France: 1) Saumur (n=12); 2) Perré (n=8); 3) Durtal (n=8); 4) Moyaux (n=22); 5) Audresselles (n=14); 6) Erloy (n=20). The population at Moyaux (open circle) is identified as a B. bufo x B. spinosus hybrid population (see results). Black dots represent populations studied in Arntzen et al. (2013b), whereas the dashed line represents the contact zone as approximated in that study.