Results

We produced panels of 15,412 SNPs (7.6 % missing data) for 90 individuals of H. molleri and 33,140 SNPs (5.2 % missing data) for 83 individuals of P. cultripes .

Genetic structure

STRUCTURE runs converged well for low K values but not for larger K values (Table S2; Figures S3.1 and S3.2). The best-supported number of genetic clusters (K) identified using STRUCTURE varied according to the metric used (PI or ΔK) and marker type. In most cases, we found the best support for two genetic lineages (K = 2), but some metrics identified further substructure, with up to six genetic clusters (K = 6) when using PI (Table S3; Figures S4.1 and S4.2).
Ancestries derived from both markers were spatially coherent at different K values. That is, individuals from the same or nearby localities shared similar ancestries and more admixed individuals coincided with geographical shifts in cluster assignment (Figure 1). For K = 2, both marker types were congruent in identifiying major subdividions in each species: a northern and a southern lineage forH. molleri , and a central-western and a northeastern lineage forP. cultripes . From K = 3 to K = 8, the spatial patterns of genetic structure for both species were largely congruent between marker types in terms of admixture levels and ancestry group assignment (Figures 1 and S5). Both markers generally agreed on the genetic ancestry of localities or group of localities as sharing a singular genetic ancestry, although the K value at which for a given assignment to a cluster could differ between markers. For instance, for H. molleri , the western-coastal populations from Portugal (dark purple, Figure 1) formed a well-differentiated cluster at K = 3 with SNPs and at K = 4 with microsatellites. Another example is the locality Ojos de Villaverde, at the southeastern-most corner of the distribution ofH. molleri. This locality appeared well differentiated at K = 4 for SNPs (green), but at K = 5 in microsatellites (magenta) (Figure 1). In P. cultripes , we observed the same phenomenon. For instance, the localities from northwestern Portugal were very differentiated at K = 4 with SNPs (green), but at K = 5 with microsatellites (green, Figure 1). Both markers agreed in localities within the northern half of the Iberian Peninsula with nearly “pure” ancestries and no further clustering after K = 4, and yielded very admixed localities in the southern half of Iberia from K = 4 to K = 8, although the levels of admixture and the ancestry assignments differed notably between markers. In P. cultripes , for K = 7 and K = 8, microsatellites yielded more admixed individual ancestries compared to SNPs (Figure S5), driven by the more admixed southern localities (Figure 1). For H. molleri , we could not quantify reliably these differences in admixture levels between markers because the individuals analyzed for each dataset were not all the same.
Genetic structure based on STRUCTURE analyses was highly congruent with that inferred by model-free hierarchical clustering (Supplementary File S1), which yielded well-supported clades for SNPs but less so in microsatellite-based topologies.

Congruence in individual/locality ancestries between microsatellites and SNPs

Both species showed higher intra-marker similarity (H. molleri , SSCs = 0.27 - 1.00; P. cultripes , SSCs = 0.77 - 1.00) than inter-marker similarity (H. molleri , SSCs = -0.03 - 0.42;P. cultripes , SSCs = 0.55 – 0.89) (Figure 2). For microsatellites, ancestries were very similar (SSCs close to 1) from K = 2 to K = 8 (except K = 7) for H. molleri and from K = 2 to K = 4 for P. cultripes . For SNPs, STRUCTURE results were almost identical only from K = 2 to K = 4 for H. molleri , but up to K = 6 for P. cultripes . Larger K values were in all cases associated with less consistent results across STRUCTURE runs. For most K values, pairwise SSC values in microsatellite runs had a larger spread (i.e. a greater range of values), especially at larger K values. This spread was minimum for STRUCTURE results derived from SNPs, though at larger K values (K = 4 to K = 8 for H. molleri ; K = 6 to K = 8 forP. cultripes ) they tended to converge into 2 or even 3 regions of the parameter space (Figure 2). The similarity between SNP-microsatellite runs did not follow a clear pattern along increasing K. For H. molleri , SSCs were homogenously lower across all K values than for P. cultripes , highlighting the distinct solutions obtained between datasets. For this species, SSCs were maximum at K = 2 (0.89), and minimum at K = 4 (0.55). From K = 5 to K = 8, SSCs had a small increase in the 0.58 – 0.68 range.
Microsatellites yielded more admixed ancestries at larger values of K (i.e. K = 7 and K = 8; Figure S5) which seem to be driven by the more complex patterns of genetic structure in the southern localities (Figure 1).

Genetic diversity

Correlation of genetic diversity between microsatellites and SNPs-based measures was weak in both species (P. cultripes , Pearson’sr = 0.39, P < 0.001). Genetic diversity (sMLH) from SNPs in H. molleri was highest in southwest Iberia and decreased towards northern (β = -0.08; P < 0.001) and eastern localities (β = -0.04; P = 0.02) (Figure 3; Table S4). We did not detect a significant correlation of microsatellite diversity with latitude (P = 0.63) or longitude (P = 0.10).
For P. cultripes , genetic diversity decreased with latitude for SNPs (β = -0.07; P < 0.001) and microsatellites (β = -0.09; P < 0.001). Longitude had a marginal effect on diversity from SNPs (β = -0.02; P = 0.06) but not from microsatellites (P = 0.93). Both markers agreed in diversity being (1) extremely low in the north-eastern localities, in costal France, both on the Atlantic and Mediterranean sides, (2) moderately low in the Northern Plateau and along the Mediterranean coast and interior, and (3) greatest in the central south-western localities (Figure 3; Table S4). These south-western localities also showed the largest complexity in genetic structure and patterns of admixture across K (Figure 1).