Phenotypic diversity across L. cidri populations
To estimate the impact of genetic diversity upon phenotypic diversity, we assessed the phenotypic diversity of L. cidri in the complete set of 56 isolates. For this, we performed microcultures and estimated growth rates (µmax, corresponding to the maximum specific growth rate) from curves obtained under 12 conditions comprising different carbon sources, stresses, and environmental parameters. A clustered heat map of the phenotypic correlations between strains showed a separation of the isolates according to their geographic origin, either from SoAm or from Aus, except for three Australian isolates that clustered together with strains from Patagonia (Fig. 4a, Table S11). In this sense, hierarchical clustering of the phenotypic data distributed the strains across four main clades (Fig. 4a). Cluster A contained two isolates (one from SoAm and one from Aus), showing the lowest growth rates for most conditions (Fig. 4a). The different SoAm isolates distributed in Clusters B and C. Cluster B showed the greatest growth rates compared to the other three clusters, while Cluster C showed intermediate values and exhibited low growth rates in fructose and ethanol. Interestingly, all isolates in Cluster C belong to localities close to the Pacific Ocean (low altitude, Valdivian Coastal Reserve, and Chiloé National Park) (Fig. 4a). Most strains from Australia distributed in Cluster D (n =20). However, three Australian strains grouped in cluster B, likely because of their greater growth rates using maltose and fructose as carbon sources. The main difference between clusters is the ability of the strains to grow in ethanol-containing media. In this case, we observed significant differences when we compared the growth rate between all clusters (p -value < 0.001, t-test). For example, in Clusters B and D we found isolates with the highest growth rates under 6% ethanol. Cluster B contains SoAm isolates from high altitude localities together with strain CBS2950 and three strains of the Aus lineage. On the other hand, Cluster D consists entirely of Aus lineage isolates.
We observed a clear separation between the SoAm and Aus populations (Fig. S4) using PCA, allowing us to further dissect the phenotypic data (Fig. S4). When analyzing the distribution of each isolate, we also observed a separation based on locality (Fig. S4b). Nevertheless, the SoAm populations exhibited the greatest growth rates across the different conditions compared to the Aus isolates (p -value < 0.001, t-test) (Fig. S5a). The SoAm populations exhibited phenotypic differences based on longitude. We found two phenotypic groups, termed high and low altitude locations (HA and LA, respectively), depending on whether they were collected near the Andes or the Pacific Coast, respectively (Fig. 4a). In general, strains isolated from HA locations show greater growth rates than those isolated from LA locations (p -value < 0.001, t-test) (Fig. S4b). Interestingly, LA strains showed a higher growth rate than HA only in 2% maltose (p -value < 0.05, t-test) (Fig. S5c). The longitude comparison showed significant differences for 8 of the 12 conditions evaluated (p -value < 0.05, t-test) (Fig. S5c), with the most notable differences (p -value < 0.001, t-test) in ethanol (6 and 8% v/v), glucose (20% w/v), sorbitol (20% w/v) and NaCl (1.25 mM) (Fig. S5c). Overall, the phenotypic data demonstrate that the ecological niche is determinant in the differences observed, highlighting the different conditions in which L. cidrican grow from localities near to the Pacific Coast up to high altitudes in the Andes.