Population structure analyses reveal two main genetic groups inL. cidri.
To explore the connection between strains and localities, we performed an analysis of the distribution of the genetic variants found in the 55L. cidri strains (Fig. 2a and b). The principal component analysis (PCA) showed a clear separation of the strains depending on their geographical origin, where according to the first two components, PC1 (11.2 %) and PC2 (8.98%), the Aus group is completely separated from the other set of strains (Fig. 2a), illustrating again the genetic differentiation between Aus and SoAm L. cidri . Interestingly, SoAm exhibited a separation into two additional groups, one corresponding to AL (most divergent clade) located in the upper part of the PCA plot and the other in the lower part corresponding to the remaining SoAm strains (Fig. 2a). A second PCA considering just the latter group, clearly revealed that the strains distributed based on their geographic origin of isolation, including latitude and longitude. According to the first two principal components, PC1 (7.84 %) and PC2 (6.62 %), those strains collected near to the Pacific Ocean (Valdivian Coastal Reserve and Chiloé National Park) separated from those isolates obtained in mountainous regions (color chart, Fig. 2b).
To determine the population genetic structure of L. cidri , we performed several complementary approaches, including STRUCTURE, ADMIXTURE, and fineSTRUCTURE clustering (Fig. 2c and d). The three analyses revealed a high degree of differentiation between strains isolated from South America and Australia. STRUCTURE and ADMIXTURE analyses indicated an optimum K = 2 groups (ΔK2 = 301 for STRUCTURE, Fig. 2c, Table S8), showing the presence of two genetic groups, SoAm and Aus. Further analysis using the fineSTRUCTURE Co-ancestry matrix indicated a clear separation between the SoAm and Aus isolates (Fig. 2d), confirming the presence of two large populations. Additionally, we identified a series of subgroups within the SoAm clade, suggesting sub evolutionary units in central and southern Chile confirming the great differentiation and divergence of the group of isolates obtained from AL. To corroborate the results obtained, we calculated the genetic differentiation (F ST) between each of the localities from which L. cidri isolates were obtained (Fig. 2e, Table S9). In this case, we found low to highF ST values ranging from 0.04 (p -value < 0.001) between Villarrica National Park and the Huilo-Huilo Biological Reserve, up to 0.99 (p -value < 0.001) between Altos de Lircay National Park (AL) and Central Plateau of Tasmania (CP), Australia (Fig. 2e). Although the STRUCTURE and ADMIXTURE analyses did not suggest AL as a third lineage, we found the highestF ST values between AL and the other localities from South America, suggesting that this could represent a third population in our study (Fig. 2e, Table S9). Intermediate and lowF ST values were found between closer localities in southern Chile (Fig. 2e, Table S9). Under this scenario, with IBD we found a moderately significant correlation (R2 = 0.39,p -value = 0.05) (Fig. 2f) between genetic differentiation and geographic distance in the localities of southern Chile, suggesting that the genetic differences found in Patagonian L. cidri could result from the geographical distribution (distance in kilometers).
SoAm isolates exhibited higher genetic diversity compared to those from Australia. In this case, we calculated nucleotide diversity (π) (Fig. S3a, Table S10). In general, we observed a higher nucleotide diversity in the SoAm population, most likely due to the number of different locations from which the L. cidri strains were isolated (Fig. 3b). When analyzing the diversity at each locality, higher π values were observed in sampling sites from southern Chile (e.g., Coyhaique National Reserve) compared to central Chile (e.g., Altos de Lircay) or Australia, which exhibited the lowest π values (Fig. S3b), despite being the location with the highest number of isolates (n=25). To confirm the results obtained, we compared the nucleotide diversity of the Huilo-Huilo Biological Reserve (locality with the highest number of isolates in South America, n=10) with the same number of randomly-selected isolates from the Australian population. As in the previous case, we observed a great diversity in the strains of the Huilo-Huilo population compared to the Australian strains, confirming the greater genetic diversity found in Patagonia. Furthermore, the low number of SNPs between the French reference strain and the Australian population, also suggest a likely low genetic diversity in Australia. The low nucleotide diversity found in the Australian population could be due to a short divergence time, suggesting a recent introduction of the species in Australia or Europe (Fig. S3b, Table S10).