Population structure
The two populations ofA. alalia comprise one genetic cluster isolated from all populations of A. mantiqueira in the DAPC by location (Fig. 3). All individuals from Serra da Bocaina (‘mBoc’) have a high probability of belonging to one unique cluster. Similarly, all individuals collected in Poços de Caldas (‘mPC’) constituted a second cluster. In addition, two populations have some structure, ‘mI’ and ‘mPG’, although some individuals from these locations have mixed probabilities between four or more locations. Besides these locations, all individuals from Serra da Mantiqueira have mixed probabilities between locations, showing no geographic structure.
The best number of clusters indicated by the DAPC analyses based on BIC values was K = 2 (BIC = 978.6421), which perfectly defines one cluster for each species. In the sNMf analyses, the lowest cross-entropy is also K = 2 and the two ancestral populations correspond to the two species as well. Additionally, the cross-validation criterion did not show a minimum value or a plateau in the Tess3r analysis (Supplementary Material). For this reason, the K=2 value was investigated, and the same clustering result as the other analyses was obtained (Fig. 4).
Exploring other K values obtained in the DAPC analyses associated with close values of BIC, as K = 3 (BIC =9 80.9029), an additional cluster was obtained within A. mantiqueira , consisting of individuals from ‘mBoc’ (which is the only population sampled from the Serra do Mar mountain range). Using K = 4 (BIC = 983.8178), the clusters detected with values of K = 2 and K = 3 were also observed, and in addition to them, a fourth group subdivides the individuals of A. mantiqueira . This group contains all individuals sampled at location ‘mPC’, which is one of the two populations obtained from the Poços de Caldas plateau. For K = 5 (BIC = 987.0982), the full correlation between the sampled location and cluster membership is no longer observed. The four groups obtained with the lower K values (2, 3, and 4) were recovered, but the fifth group is composed of four individuals from the ‘mI’ location (of the 13 individuals sampled there) (Supplementary Material). To investigate the population structure of A. mantiqueira populations, the value of K=4 was applied in the sNMF and Tess3r analyses. The four clusters obtained in the DAPC analyses were equally recovered (Fig. 4).
Overall genetic structure inferred by a nonhierarchical AMOVA resulted in a significant ΦST value of 0.065 (P < 0.001). The hierarchical AMOVA considering the two species showed a ΦST value of 0.3243 (P < 0.0001), with 30.46% of the variation between species, 1.96% of the variation among populations within species, and 67.57% within populations. The hierarchical AMOVA considering the three clusters in A. mantiqueira (corresponding to Serra da Mantiqueira, Serra do Mar, and Poços de Caldas plateau) showed a ΦST value of 0.036 (P < 0.001), with 1.57% of the variation among mountain ranges, 2.04% of the variation among populations within groups, and 96.39% within populations.