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).