Experimental setup to study the long-term evolution of L. plantarum in Drosophila simulans populations.

Pangenome of L. plantarum genomes isolated from the Florida experiment (Table S1), sorted by phylogeny.

Left panel: Maximum likelihood tree built based on the core genome alignment. Tree leaves correspond to the name of the isolate and are coloured based on isolation origin: ancestral population (grey), hot-evolved population (red) or cold-evolved population (blue). Numbers at nodes represent bootstrap support values based on 1000 replicates. Middle panel: Pattern of genes presence (black)/absence (white) in each genome. Right panel: pairwise average nucleotide identity between the isolates. The colour ranges between 98.5% (purple) and 100% (yellow).

Maximum likelihood tree of 92 L. plantarum genomes.

Tree leaves are coloured by source of isolation. The genomes from our lab are coloured in according to experimental treatment: unevolved in grey, hot-evolved in red, and cold-evolved in blue. Numbers at nodes represent bootstrap support values based on 1000 replicates.

Clade composition over time in each temperature regime depicted as the relative abundance of each L. plantarum clade (top: cold, bottom: hot).

Clade C in light blue, clade H in red and clade U in grey. For each temperature regime we summed the reads from the ten replicate populations.

Clade-specific growth dynamics.

(A) L. plantarum growth curves overlapped. Lines are coloured by isolate’s clade. Background colour represents the growth temperature in the fluctuating environment. (B-D) Boxplots depicting the carrying capacity, growth rate, and inflection time of each clade. Each dot corresponds to a technical replicate. Data is grouped and coloured by clade. Statistical significance was determined using Dunn’s test with Holm-adjusted p-values. Only significant comparisons are indicated. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05.

Fitness effect of L. plantarum inoculation in axenic D. melanogaster.

(A, B) Total number of F1 flies eclosed normalised by day and female under the cold (A) and hot (B) regime. (C, D) Developmental time, estimated as the number of days it takes 50% of the offspring to eclose. Measurements were grouped by inoculation treatment and transfer. Each dot corresponds to a biological replicate (n = 10) Statistical significance was determined using Dunn’s test with Holm-adjusted p-values. Only significant comparisons are indicated. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05.

Pangenome of all L. plantarum genomes (Table S1), sorted by phylogeny.

Left panel: Maximum likelihood tree built based on the core genome alignment. Tree leaves correspond to the name of the isolate and are coloured based on isolation origin: unevolved populations (grey), hot-evolved populations (red) or cold-evolved populations (blue). Tree tips are coloured based on the experiment from which the isolate was obtained: Florida (black), Portugal (pink) or South Africa (green). Numbers at nodes represent bootstrap support values based on 1000 replicates. Middle panel: Pattern of genes presence (black)/absence (white) in each genome. Right panel: heat map depicting the pairwise average nucleotide identity between the isolates. The colour ranges between 98.5% (purple) and 100% (yellow).

Hierarchical clustering of 92 L. plantarum genomes based on the presence-absence of accessory genes.

Tree leaves are coloured by source of isolation. The genomes from our lab are coloured in according to experimental treatment: unevolved in grey, hot-evolved in red, and cold-evolved in blue. Red values in each branch indicate the p-value for each cluster’s robustness based on 1000 bootstrap iterations.

Clade composition over time per replicate population and temperature regime depicted as the relative abundance of each L. plantarum clade.

Clade C in light blue, clade H in red and clade U in grey.

Relative abundance of each L. plantarum clade across different reads depths in simulated reads sets with known clade composition.

Dashed vertical lines show the values of log(100), log(500), and log(1000) reads. Dashed horizontal lines show the true simulated relative abundances of each clade in the reads set. The top row titles reference to the ratio of reads from each clade following descending order: C:H:U. The bottom plots depict scenarios in which all the reads belong to a single taxon.

Boxplots depicting the carrying capacity (A), growth rate (B) and inflection time (C) of each isolate.

The boxplots were coloured by clade. Measurements were obtained from three independent technical replicates for each isolate.

Fitness effect of L. plantarum inoculation in conventionally reared D. simulans.

(A, B) Total number of F1 flies eclosed normalized by day and female under the cold (A) and hot (B) regime. (C, D) Developmental time, estimated as the number of days it takes 50% of the offspring to eclose. Measurements were grouped by inoculation treatment and transfer. Each dot corresponds to a biological replicate (n = 10) Statistical significance was determined using Dunn’s test with Holm-adjusted p-values. Only significant comparisons are indicated. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05.

Venn diagram depicting the overlap in KEGG Orthologs between the three clades.

Segments were coloured by number of KOs.

Graphical representation of the KEGG pathway map02060 (Phosphotransferase system).

Each rectangle represents a KEGG Ortholog (KO). Orthologs that are enriched in each clade are coloured in grey (present in clade U), blue (present in clade C) and/or red (present in clade H). The three clades differ in the set of sugar-related PTS transporters. Clades C and H have the capacity to internalize sorbitol and galactitol, whereas clades H and U have the ability to import galactosamine.

Graphical representation of the KEGG pathway map00740 (Riboflavin metabolism).

Each rectangle represents a KEGG Ortholog (KO). Orthologs that are enriched in each clade are coloured in grey (present in clade U), blue (present in clade C) and/or red (present in clade H). Clade U lacks the capacity to produce de novo FAD from GTP, since it is lacking several genes from the pathway.

Graphical representation of the KEGG pathway map00790 (Folate biosynthesis).

Each rectangle represents a KEGG Ortholog (KO). Orthologs that are enriched in each clade are coloured in grey (present in clade U), blue (present in clade C) and/or red (present in clade H). Clade U has the unique capacity to synthesize de novo guanylyl molybdenum cofactor.

Subset of the KEGG pathways map02020 (Two-component system; A), map00910 (Nitrogen metabolism; B), and map00920 (Sulfur metabolism; C).

Each rectangle represents a KEGG Ortholog (KO). Orthologs that are enriched in each clade are coloured in grey (present in clade U), blue (present in clade C) and/or red (present in clade H). Clade U encodes a unique alternative respiratory system. The operons nreABC and narGHIJ encode for genes that sense anoxic conditions and use nitrate as terminal electron donor instead of oxygen (A, B). The enzyme sulfur oxidoreductase (Nsr) is highly enriched in the clade C, which suggests that it can potentially use sulfur as terminal electron acceptor in absence of oxygen (C).