Population structure and conservation units
After excluding the candidate adaptive loci and loci deviating from HWE, we kept 2,663 and 1,711 SNPs for the NIDGS and SIDGS, respectively. With these sets of neutral loci, we determined that there is a significant pattern of IBD for NIDGS (r = 0.136, p = 0.039) but not for SIDGS (r = 0.556, p = 0.090). IBD was not significant for the NIDGS adaptive dataset (r = 0.018, p = 0.396), or for the SIDGS adaptive dataset (r = -0.378, p = 0.950).
PCA results for the total and neutral datasets in both species were identical (Figure S8, Supporting information). From the NIDGS PCA analysis, there were four axes for the total and neutral datasets and three axes for the adaptive dataset explaining a significant proportion of the variance (Figures 5A, 5B and S8, Supporting information). Both the neutral and adaptive datasets identified similar populations as the most distinct, particularly Lower Butter (LB), Rocky Top (RT) and Tamarack (TA) (Figure S8, Supporting information). Mud Creek (MC) was only distinctive at the neutral level. Considering potential groups, at the first two PCs, Lower Butter (LB) was clearly separated from an eastern group (MC, TA, PV, LV) and a western group (all other populations). Based on these data, we defined three ESUs for NIDGS: ESU1 represented by the western group, ESU2 represented by Lower Butter, and ESU3 represented by the eastern group (Figure 6A). From the SIDGS PCA analysis, we considered as significant one axis for the total the neutral and the adaptive datasets, the first two identified Olds Ferry (OF) as the most distinctive populations, while the adaptive dataset showed a clear separation of Paddock (PA) (Figure 5C and 5D, and S8, Supporting information). Based on these data we defined two ESUs for SIDGS: ESU1 represented by Olds Ferry, and ESU2 represented by all other populations east of the Weiser River (Figure 6B).
The PCA neutral results for both species agree with the STRUCTURE analyses (Figure 6). For NIDGS, STRUCTURE suggested separation of NIDGS into two main groups corresponding to a western group (ESU1 and ESU2) and also an eastern group (ESU3) (Figure 6A, K = 2). Here, Lower Butter was included in the western group, although there was a high proportion of admixture with the eastern group as well. Lower Butter (ESU2) was differentiated from all other populations at all Kvalues starting from K = 3 (Figure 6A). The STRUCTURE analysis indicated a second peak in the marginal likelihood for K = 6 using the Evanno et al. (2005) method, suggesting the presence of fine-scale population structure within NIDGS (Figures 6A, and S9A, S9B and S10A, Supporting information). At K = 6, the eight populations of ESU1 split into three geographically distinct populations or MUs, and the four populations of ESU3 split into three MUs, with Lost Valley showing evidence of admixture (Figure 6A). In terms of adaptive variation, within ESU1 we identified two AUs from eight populations, corresponding to the same structure found for MUs, ESU2 (Lower Butter) was also a distinct AU, and for ESU3, only Tamarack was adaptively differentiated (Figure 6A). These results are also reflected in the pairwise F ST estimates, where there is no significant differentiation among 7 of the 8 populations of ESU1, even at the adaptive level (Figures S11A and S11B, Supporting information). Rocky Top (ESU1), Lower Butter (ESU2), and Mud Creek (ESU3) were the NIDGS populations with the highest neutral average pairwiseF ST, with 0.09, 0.08 and 0.08, respectively, considering an overall average of 0.06, while for the highest adaptive average pairwise F ST was found for Rocky Top (ESU1) and Lower Butter (ESU2), with 0.26 and 0.20, respectively, considering an average of 0.10. The SIDGS STRUCTURE results also mimicked those from the neutral PCA where individuals were found to be divided in two groups, with a separation of Olds Ferry from all other SIDGS localities at K = 2 (Figure 6B). This resulted in the definition of two MUs, according to our criteria. In terms of adaptive differentiation, Paddock was identified as a separate AU.
In terms of population differentiation, averageF ST between NIDGS and SIDGS was 0.143, compared to 0.05 and 0.100 within NIDGS and SIDGS for the same dataset, respectively. At the intraspecific level and considering the NIDGS and SIDGS datasets separately, average neutral pairwiseF ST was highest for Olds Ferry (0.13), considerably higher than the average value of 0.04 (Figure S11C, Supporting information). However, as seen in the SIDGS adaptive PCA, Paddock (PA) was the most distinct population and also had the highest average pairwise F ST (0.24), considerably higher than the average value of 0.15 (Figure S11D, Supporting information).
Considering the levels of genetic diversity, for the IDGS dataset, no NIDGS population showed significantly decreasedH O compared H E, but Lower Butter did show significantly higher H O thanH E (Table 4). Contrarily, for the same dataset, two out of four SIDGS populations showed significantly lowerH O than H E. SIDGS populations tended to have significantly lower H Ethan NIDGS populations (Table S1, Supporting information). At the intraspecific level, for NIDGS we observed an average of neutralH O of 0.237, which was significantly lower than the average H E of 0.280 (Table 4). Values of heterozygosity for the adaptive dataset were lower than the neutral dataset, and H O (0.199) was lower thanH E (0.190). Of the 10 populations for which we estimated H E, four had significantly lower neutral H O than H E, while only two showed lower H O thanH E for the adaptive dataset (Table 4). Considering the comparisons among populations, Steve’s Creek/Squirrel Valley (SS, H E = 0.305), Tamarack (TA,H E = 0.296) and Mud Creek (MC,H E = 0.286) showed significantly higher neutral HE (Table S1, Supporting information) than all other NIDGS populations. For the adaptive dataset, Rocky Top (RT,H E = 0.294), Tamarack (TA,H E = 0.270) and Lower Butter (LB,H E = 0.263) showed the highest HEvalues, also significantly higher than most other NIDGS populations (Table S1, Supporting information). Comparing the HEestimated for the neutral and adaptive datasets, Mud Creek (MC), Rocky Top (RT), and Steve’s Creek/Squirrel Valley (SS) had significantly lower adaptive than neutral H E (Table S1, Supporting information). For the SIDGS, we observed an average of neutralH O of 0.231, which was significantly lower than the average HE of 0.276. Values of heterozygosity for the adaptive dataset were similar to the neutral dataset, again withH O (0.232) lower than H E(0.275). Of the four populations for which we estimatedH E, three presented significantly lower neutralH O than H E, while none showed lower H O than H Efor the adaptive dataset (Table 4). Considering the comparison among populations, Olds Ferry (OF, H E = 0.287) and Paddock (PA, H E = 0.283) showed significantly higher neutral H E than the other three SIDGS populations (Table S1, Supporting information). For the adaptive dataset, Paddock (PA, H E = 0.410) showed significantly higher HE compared to all other SIDGS populations, as well as when compared to its neutral HEestimate (Table S1, Supporting information).