Results
SNPs were considered species-diagnostic for 55 out of 64 markers, with missing data amounting to 2.7% (Table S.2). Significant deviations from Hardy-Weinberg equilibrium (heterozygote deficit) were found four times in four populations, involving the loci BF, mrpl41 and sostdc1. Per population deviations were significant three times in populations 10, 13 and 21. No significant pairwise linkage disequilibrium was detected per locus pair or population. Admixture linkage disequilibrium was significant for population 13. The water bodies involved in significant instances of heterozygote deficit and admixture linkage disequilibrium were highly isolated and had small dimensions (< 3 m2).
Protein functions were described for the 47 markers that could be annotated (Table S.3). Interactions were uncovered among nine marker pairs; however, these markers do not appear to be co-expressed or involved in significant deviations from Hardy-Weinberg equilibrium. All markers were included in downstream analyses, as they appear to be physically and functionally unlinked.
Structure classified 20 individuals from two populations (nos. 1 and 2) as admixed and 334 individuals from 30 populations (nos. 3 - 32) asT. pygmaeus (Table S.4). For mtDNA, the SNP allele representing the T. marmoratus haplotype was found in populations 1 and 2, and the T. pygmaeus allele was found in all other populations. Allelic profiles consistent with enclave formation or genomic footprints were not observed (Table S.4). Accordingly, the genetic signature ofT. marmoratus was restricted to the previously documented enclave and displayed notable levels of introgression with T. pygmaeus(Figure 1).
The selected two-species distribution model is represented by the logistic equation Pm=(1/(1+exp(-0.156*Bio17+7.767), in which Pm is the probability for the presence of T. marmoratus at the locality investigated, on a zero to unity scale and bio17 is ‘precipitation of driest quarter’. Model fit is AUC=0.931±0.025. The model describes more arid summer conditions forT. pygmaeus (mean precipitation over 60 localities = 36.4 mm) than for Triturus marmoratus (mean precipitation over 48 localities = 67.4 mm). The spatial interpretation of the model is shown in Figure 2A. Temporal extrapolations of the model (or ‘hindcasts’) are in Figure 2B for climate conditions of the Mid Holocene and in Figure 2C for the Last Glacial Maximum.