The isolation of Saccharomyces strains was conducted using a selective enrichment protocol previously described (Sampaio and Gonçalves, 2008). For the strains isolated in Slovenia, samples were directly used for yeast isolation without enrichment. Preliminary species-level identifications were performed by sequencing the D1/D2 region of the 26S rDNA. Crosses involved ascospore micro-manipulation. Positive crosses between two parental strains were confirmed by sequencing of GAL1 and confirmation of the expected heterozygous sites. For each cross, interspecific spore viability was determined by examining 200 ascospores.

Olives of the Oblica variety (approx. 200 g) collected in Évora, Portugal were used to prepare olive brine (200ml H₂O, 8% NaCl w/v) during 3 months at 17°C. After this period, the brine was sterilized by filtration and kept at 4°C. This brine was analyzed by HPLC to identify and quantify the sugars and sugar-like compounds present. Sugar concentrations were determined using a carbohydrate analysis column (250 mm × 4 mm + Aminotrap, Dionex Carbopac PA10; DIONEX ICS3000). The column was kept at 25°C and 0.1M NaOH was used as the mobile phase at 1 ml min^-1. For the phenolic compounds the concentrations (%) were determined using a Waters Novapack C18 15 mm column (DIONEX ICS3000). The column was kept at 30°C and 2% methanol was used as the mobile phase at 0.5 ml min^-1.

Two experiments were done independently for the group of strains selected. Pre-cultures (20 ml in 50 ml flasks) grown for 24 h in YNB (Yeast Nitrogen Base, Difco) supplemented with 1% (w/v) glucose (incubation at 25°C) were used to inoculate approximately 1 × 10⁵ cells/ml in a volume of 2 ml of olive brine in 2 ml micro-centrifuge tubes. Cells were grown in batch cultures for 70 days at 17°C without shaking, and cell viability was estimated by performing regular plate counts after preliminary counts in a hemocytometer. Statistical significance was tested using an unpaired t-test with Welch’s correction, implemented in GraphPad Prism v5 (p-value cut-off <0.01). At the end of the experiment, the supernatants of two cultures from the Olives population and two cultures from the Wine population were randomly chosen for HPLC analysis to determine the residual concentrations of sugars.

Strains were pre-grown overnight in liquid YPD medium [yeast extract 1% (w/v), peptone 2% (w/v), D-glucose 2% (w/v), at 25°C and were subsequently transferred to fresh medium (20 ml YPD or YPD supplemented with NaCl 6% or 8% w/v) and incubated with orbital shaking (180 r.p.m.) at 30°C in 50 ml flasks]. The initial OD_640nm was 0.1 – 0.2. Growth rates were calculated in the exponential phase using OD_640nm measurements.

Strains were pre-grown overnight at 25°C in YNB medium supplemented with 1% (w/v) glucose. Cells were then transferred to phosphate buffer at pH 5.8 (30 ml in 50 ml flasks) supplemented with 0.6% (w/v) glucose 0.1% (w/v), fructose, and 8% (w/v) NaCl, to mimic the conditions in olive brine and incubated at 20°C. A similar experiment was also conducted in the absence of NaCl. Sugar consumption was monitored for 10 days by HPLC. Extracellular concentrations of fructose and glucose were determined using a carbohydrate analysis column (300 mm × 7 mm, Thermo HyperREZ XP Carbohydrate Ca++; KNAUER Smartline) and a differential refractometer. The column was kept at 85°C and H₂O was used as a mobile phase at 0.6 ml min^-1.

DNA was extracted from overnight grown cultures of monosporic or single-cell derivatives and paired-end Illumina MiSeq 250 bp genomic reads were obtained after sequencing for 500 cycles. Genomic data for other strains was obtained from the NCBI-SRA archive and from the Saccharomyces Genome Resequencing Project v2 (SGRP2) (Bergström et al., 2014). When only finished genome sequences were available in public databases (NCBI), the corresponding error-free Illumina reads were simulated using dwgsim¹. Reads for each isolate were mapped to the S. cerevisiae reference genome (UCSC version sacCer3) using SMALT v0.7.5 aligner². The reference Index was built with a word length of 13 and a sampling step size of 2 (-k 13 -s 2). An intensive search for alignments (-x) was performed during the mapping step with the random assignment of ambiguous alignments switched off (-r -1) and the base quality threshold for the look-up of the hash index set to 10 (-q 10). With these settings, SMALT v0.7.5 only reports the best unique gapped alignment for each read. For the paired-end data the insert size distribution was inferred with the “sample” command of SMALT prior to mapping. Conversion of SAM format to BAM, sorting, indexing, several mapping statistics, and consensus genotype calling were performed using the tools available in the SAMtools package v1.18 (Li et al., 2009) and as described previously (Almeida et al., 2014). Multiple sequence alignments for each reference chromosome were generated from the resulting fasta files. For downstream analysis, all bases with Phred quality score below Q40 (equivalent to a 99.99% base call accuracy) or ambiguous base calls were converted to “N.” For obtaining the S. cerevisiae and S. paradoxus sub-genomes of the hybrid strains, reads for each strain were mapped to an extended Saccharomyces spp. reference with assembled sequences from the genomes for S. cerevisiae (UCSC version sacCer3), S. paradoxus, S. mikatae, S. kudriavzevii, S. uvarum (Scannell et al., 2011), and S. arboricolus (Liti et al., 2013).

Chromosomal single nucleotide polymorphisms (SNPs) were extracted from multiple sequence alignments only if the evaluated site was represented by unambiguous high confidence alleles in at least 85% of the strains. SNPs were then concatenated to generate a whole-genome SNP alignment. The phylogeny was inferred using maximum likelihood as implemented in IQ-TREE v 1.6.7 (Nguyen et al., 2015) using an empirically determined substitution model, SH-like approximate likelihood ration test (1000 replications) (Guindon et al., 2010), and rooted with S. paradoxus. The phylogeny was visualized using ITOL, version 3.0 (Letunic and Bork, 2016). Whole-genome levels of divergence were estimated using Variscan v2.0 (Hutter et al., 2006). Divergence was calculated for each mapped strain in comparison with the reference genome of S. cerevisiae using RunMode 21. The results were processed using a 10 kb sliding window with 10 kb step increments.

Evidence of the presence of genes from other Saccharomyces species was investigated by mapping the reads to a combined reference that includes the annotated coding sequences of S. arboricola, S. cerevisiae, S kudriavzevii, S. mikatae, S. paradoxus, and S. uvarum (Scannell et al., 2011; Liti et al., 2013). Reads were mapped to this combined reference using BWA V0.6.2 (Li and Durbin, 2009) with default parameters, but setting the quality threshold to 10 (-q 10). SAMtools V1.1852 (Li et al., 2009) was then used for manipulation of the resulting BAM files. Only ORFS with orthologs unambiguously annotated in all the species were analyzed. An ORF was considered to have a foreign origin to S. cerevisiae if its coverage was higher at least one-fourth of the median whole genome coverage for a given strain. The ORF coverage was defined as the product of the total number of mapped reads to a given ORF by the read length, dividing by the sum of all the ORFS length (considering only ORFS that have at least 25% of reads mapped to, when comparing to the orthologous ORF with the highest number of reads). This measure was taken to control spurious alignment counts. The coverage threshold allowed for some heterogeneity in the read counts and for the eventual presence of a foreign ORF together with the native S. cerevisiae ORF. For some of the S. paradoxus genes detected in the hybrid genomes, their assignment to this species was confirmed with phylogenetic analyses involving homologous sequences from other Saccharomyces species.

Standard gene ontology (GO) term find was performed with the GO TERM FINDER tool v0.83, available at SGD, using a p-value cut-off of <0.01. We performed de novo genome assemblies using SPAdes v.3.11.1. Prior to assembly, reads were processed with Trimmomatic v.036 based on quality score threshold of 20 for windowed trimming, discarding reads less than 100 bp in length or harboring ambiguities. To retrieve genes of interest, a local BLAST database was set up for each genome. Copy number variation of the two CUP1 genes (CUP1-1 and CUP1-2) and the three ENA genes (ENA1, ENA2, and ENA5) was investigated using CNVNator (Abyzov et al., 2011) on mapped genomes and using ACT1 as control. The query sequences were defined by the coordinates in the reference sequence of S. cerevisiae for the coding regions of the genes of interest. The results obtained were manually validated by checking the chromosomal context of the hits using UGENE (Okonechnikov et al., 2012) and by analyzing the copy number of the genes flaking the genes of interest.

Genome sequence data have been deposited in the European Nucleotide Archive (ENA) database under the accession code PRJEB30431.

Given earlier reports on the occurrence of S. cerevisiae in association with table olives (Arroyo-López et al., 2008; Cromie et al., 2013; Bonatsou et al., 2018) and with alpechin (Santa María, 1958), we asked if the original source of these yeasts was the olive tree itself. For this reason we conducted an isolation program employing samples of olive tree bark, leaves, fruits and soil underneath the trees, and a selective enrichment protocol for yeasts of the genus Saccharomyces. In parallel, processed products such as olive oil and olive brine from table olives were also investigated. In total 163 samples from olive trees were investigated, together with 53 samples from olive oil and 7 samples from olive brine. Although the number of samples collected from olive trees was much higher, the frequency of isolation of Saccharomyces spp. was very low (3.7%), and only six strains were collected. The frequency of isolation in olive oil was higher (7.6%), and yielded four strains but was still markedly lower than that of olive brine (85.7%, six strains). Therefore, in total 16 new strains of Saccharomyces spp. were isolated from the olive niche.

Next, we obtained draft genome sequences of the new isolates in order to ascertain if they belonged to S. cerevisiae and, if so, to determine to which population they belonged to. As shown in Figure 1A, the new isolates were all identified as members of S. cerevisiae, thus showing that S. paradoxus was not isolated during our survey. Interestingly, the S. cerevisiae strains were found to belong to different populations. The six strains isolated directly from olive tree bark or ripe olives did not belong to the same population (Figure 1A and Supplementary Table S1). A single strain had substantial genomic contributions from S. paradoxus and, accordingly, was assigned to the Olives clade. Two strains belonged to the Wine clade, one strain to the Sake clade and two additional strains occupied an isolated position in the phylogeny and subsequent analyses showed that they had “mosaic” genomes with major contributions from the Wine and North American – Japan clades. With respect to the four strains isolated from olive oil, three of them had S. paradoxus contributions and belonged to the Olives clade. The remaining strain belonged to the Wine clade. For the strains isolated from olive brine, more homogeneous results were obtained and all of them were found to belong to the Olives clade (Figure 1A). Our phylogenetic analysis included also other strains that belong to the Olives clade and that had been isolated as far back as 1957 from olives, olive brine, alpechin, and from the gut or feces of humans and pigs (van Uden and Assis-Lopes, 1957; Santa María, 1958, 1962). It is noteworthy that the 25 S. cerevisiae × S. paradoxus hybrid strains isolated during this study or in previous studies formed a monophyllum even if the phylogeny of Figure 1A was prepared only with S. cerevisiae ORFs, thus avoiding the strong bias that would be introduced if S. paradoxus ORFs were considered. This suggests that all hybrid strains share the same S. cerevisiae ancestor irrespective of the geographical origin and particular substrate from which they were collected, a possibility also supported by the divergence plots depicting the S. cerevisiae and S. paradoxus blocks along the genome, that were similar for all strains of the Olives clade (Figure 1B). Moreover it appears the S. cerevisiae ancestor of the hybrids was a member of the wine population.

In conclusion, the results from our ecological survey do not support the hypothesis that the members of the Olives clade reside in the olive tree environment. Although the possibility that such strains are associated with olive trees cannot be entirely ruled out, it appears more likely that the ecological niche of this clade are processed olives and their products like olive oil, alpechin, which is the corresponding waste product, and table olives /olive brine. Also, the occurrence of hybrid strains in the intestinal tract is of notice. Besides the two strains (YJM 248 and YJM 1078) already reported in Strope et al. (2015) we found three additional strains from this source (PYCC 2613, PYCC 2708, and PYCC 8033).

Most strains (70%) of the Olives clade were sexually competent (Supplementary Table S1). Spore viability for two strains in this clade (PYCC 4935 and YO652) ranged 95.5 – 96% and a cross between them was also fertile (90% spore viability), thus suggesting that sexual recombination within the clade can occur. Also, a cross between YO 652 and the wine strain EXF 6719 (97% spore viability) had an ascospore fertility of 87%, thus indicating that sexual contact between the Olives and Wine populations appears not to be significantly hampered.

In order to characterize the genomic nature of the hybrids, we analyzed in detail 23 strains (Supplementary Dataset S1). We detected a total of 540 S. paradoxus ORFs and between 193 and 314 S. paradoxus ORFs per strain, with 103 ORFS being shared among all the strains. The S. paradoxus ORFs originated in the European population of this species (Supplementary Figure S2), thus suggesting that the hybridization event occurred in this continent. The co-existence of S. cerevisiae and S. paradoxus alleles for a given ORF was not frequent. In total 148 ORFs (27.4% of the total number of S. paradoxus ORFs) were found to occur in that configuration in at least one strain. One strain was devoid of ORFs represented in the genome by alleles belonging to the two species and 16 strains had only two to four ORFs with S. cerevisiae and S. paradoxus alleles. Together, these strains represent 74% of the total number of strains analyzed. Three strains had between 12 and 25 ORFs with S. cerevisiae and S. paradoxus alleles and another three strains had between 34 and 54 ORFs with S. cerevisiae and S. paradoxus alleles. The distribution of strains having more heterozygous ORFs did not show any association with the isolation substrate or with the phylogeny.

Gene Ontology analysis of the 103 ORFs shared among all the strains revealed a significant enrichment in genes encoding for proteins of the fungal cell wall and plasma membrane, like TIP1, HLR1, DAN1, FCY21, and STL1. However, for several strains, when an individual analysis was performed, a significant result for an enrichment in hexose transporters was also observed (Figure 2 and Supplementary Table S2). Taken together, these results support the view that the hybrid strains have a similar core genomic composition, thus suggesting that they share a common (hybrid) ancestor and also that after hybridization have evolved adaptations to the processed olives niche.

Given that the hybrid strains descend from a S. cerevisiae wine strain (Figure 1A), we surveyed the hybrid genomes for the presence of typical domestication signatures of wine strains (Almeida et al., 2015; Barbosa et al., 2018). For regions A, B, and C, that encompass 39 genes potentially relevant for the winemaking process acquired by horizontal gene transfer from non- Saccharomyces species, at least one of these regions was present in 68.8% of the control group of 32 wine strains listed in Supplementary Table S1, whereas only 13% (3 out of 23) hybrid genomes shared the same characteristic. It thus appears that these regions are less prevalent in hybrid strains, either because their ancestor S. cerevisiae wine strain already lacked most of them and/or because there are not relevant in the olives niche and were therefore lost. With respect to the inactivation of aquaporin genes AQY1 and AQY2, associated with the domestication of wine strains and with the adaptive loss of those water channels, a trait that increases fitness in sugar-rich environments (Will et al., 2010), no differences were found between the two groups and all strains had at least one aquaporin gene coding for a non-functional protein. We also investigated the variation of the number of copies of CUP1, a gene involved in resistance to copper toxicity in S. cerevisiae, especially in wine strains, due probably to their expose to copper sulfate used in vineyards (Fay et al., 2004; Strope et al., 2015). Copy number variation (CNV) of the two paralogs of CUP1 among reference wine and wild (oak-associated) strains is shown in Supplementary Table S1. Whereas among wine strains CNV of CUP-1 could exceed 30 (in two cases), the Mediterranean oak (MO) strains did not show an enrichment in the number of CUP-1 copies. Some of the hybrid strains showed also elevated numbers of copies of CUP-1, with the two most enriched genomes having 33 and 35 copies. Statistically, wine and olives strains could not be distinguished in terms of presence and expansion of CUP-1.

In order to investigate whether strains of the Olive clade were adapted to thrive in the processed olives niche, we estimated absolute fitness of a set of six strains isolated from olive brine, olive oil, and alpechin, and compared it to six S. cerevisiae strains from the wine population. We measured absolute fitness as the number of viable cells maintained in a long-term batch culture of table olive brine, here used as a proxy for a habitat to which the S. cerevisiae hybrids are adapted to (Figure 3, Supplementary Figure S1 and Supplementary Table S3). Employing freshly collected ripe olives, we prepared a brine containing 8% (w/v) NaCl (see section “Materials and Methods” for details) which was used to test each strain separately. The absolute fitness of the olive and wine strain cohorts was inferred by measuring viable cell numbers for 70 days in two independent experiments (Figure 3, Supplementary Figure S1 and Supplementary Table S3). Although strain fitness varies within both groups, variation is much more pronounced in the wine group. In spite of the within-group differences among strains, a clear difference is observable between fitness of the olive group and the wine group (p < 0.0001, unpaired t-test with Welch’s correction), the former being able both to attain higher cell numbers and to sustain viability throughout the duration of the experiment (70 days). On the contrary, wine strains tended to start losing viability already during the first month of incubation.

We reasoned that one possible cause for the difference in fitness between the two groups might be related to their ability to use the nutrients available in olive brine. Contrary to what is typical of the initial stages of wine fermentation, olive brine has low concentrations of sugars. To analyze this in more detail, we identified and quantified the sugars and sugar-related compounds present in olive brine and measured their consumption by two representatives of the wine and two representatives of the olive cohorts (Table 1). While strains belonging to the Olives clade virtually exhausted the glucose and fructose present initially in the brine, the representatives of the Wine clade consume only about half of the available sugars. Mannitol was left untouched in both cases.

This finding was intriguing because some wine strains were previously found to have an impairment in high affinity hexose transport (the type of transporters expressed under the low sugar concentrations measured in olive brine), a trait that was subsequently associated to certain variants of the HXT hexose transporter genes (Luyten et al., 2002). Numerous HXT genes are present in the genomes of the species of the genus Saccharomyces, encoding transporters with different affinities for their substrates. Since hexose transporter genes encoding the main high affinity transporters (HXT6/7) were among those “replaced” in the hybrids by their S. paradoxus counterparts (Supplementary Dataset S1), we asked if these substitutions might have contributed to improve high affinity hexose transport in the hybrids.

To assess this, we compared the ability of the same strains of the Wine and Olives clades used in the previous experiment (see Table 1) to consume the sugars present in olive brine in the course of the first 8 days after inoculation of brine (Figure 4A). Surprisingly, and although in this period olive strains grew on average in the brine fitness experiments shown in Figure 3 an order of magnitude more than wine strains, sugar consumption was very similar between wine and olive strains. Fructose consumption in particular was indistinguishable, while olive strains seemed to assimilate glucose slightly better, an observation that nevertheless does not explain the differences in growth in brine between the two sets of strains (Figure 3). A distinct experiment was subsequently performed in which brine was replaced by phosphate buffer supplemented with NaCl, glucose and fructose in concentrations identical to those found in brine. This time wine strains seemed to be slightly more proficient in fructose assimilation while in glucose no clear differences were observed (Figure 4B). Nevertheless, when this experiment was performed without NaCl, glucose and fructose were totally consumed after 2 days by wine and olives strains. Taken together these results suggest that no considerable differences in sugar uptake capacities exist between the two groups of strains that justify the better growth of olive strains. It seems therefore that the observed difference in growth is due to a better capacity to adapt to the harsh conditions of olive brine, of which high salt concentrations stand out, resulting in better growth for olive strains during the first 8 days while consuming the same amount of sugar in the same period as wine strains. The same experiment as shown in Figure 4B was subsequently performed but this time adding 0.1% yeast extract to the phosphate buffer and adding more strains to both cohorts to increase representativeness (Figure 4C). The sugar consumption profiles of both strain cohorts were different in these conditions, with olive strains exhausting the available sugars significantly faster (p < 0.05, unpaired t-test), which means that in the presence of the required nutrients olive strains are better equipped to adapt to the high salt medium. Interestingly, under these conditions wine strains were capable of consuming all the sugar available after 8–10 days while they only consumed about 50% of the available sugars during the fitness experiments in brine. This could mean that brine contains other inhibitors that affect the metabolism of wine strains more than that of olive strains, in addition to NaCl.

In summary, while S. paradoxus HXT genes may confer a slight advantage for glucose consumption in brine, this advantage does not explain the considerable difference in the ability of wine and olive strains to grow in brine. Instead, this difference seems to be derived from a better adaptation of olive strains to the particular conditions of brine of which the high NaCl concentration appears as a relevant factor.

The results of the experiments shown in Figure 3, 4 suggested that fitness and sugar consumption aptitude in brine might be related, at least partly, to salt resistance. To investigate this hypothesis, we started by determining the copy number of the three ENA genes found in the S. cerevisiae reference genome (ENA1, ENA2, and ENA5) in the hybrid strains of the Olives clade and compared their abundance using 10 representative strains of the Wine clade. The ENA proteins are sodium pumps that help the cells to cope with an excess of sodium ions in their environment (Ruiz and Ariño, 2007). ENA copy number variation is shown in Figure 5A,C. Interestingly, the highest ENA copy number (14–18 copies) was detected among strains isolated from olive brine (Figure 5C), although a marked dispersion in the number of ENA copies was observed in this group (Figure 5A). Overall, strains isolated from olive brine and the intestinal tract were more likely to have a higher number of ENA copies than strains isolated from olive oil, alpechin or wine (Figure 5A). It is possible that the strains found in the intestinal tract originate from the olive brine environment, having been subsequently ingested. This would explain their increased number of ENA copies. The differences between the number of ENA copies were found to be statistically significant between the Wine and Olive Brine populations (Figure 5C, p < 0.01, Dunn post hoc test with Bonferroni correction). The comparison of ENA copy numbers was also significantly distinct when all hybrid strains from the olives niche were compared with wine strains and for the comparison between the olive brine and alpechin groups (p < 0.05).

To investigate to which extent ENA gene copy numbers determined fitness of the strains under study in the presence of salt, the ability of the various strains to grow in the presence of 6% and 8% NaCl was also tested (Figure 5B). There was, as expected, a correlation between ENA gene copy numbers and the ability to grow in the presence of NaCl, but this correlation was not complete. For example, while all strains isolated from olive brine performed well in the growth tests even if they had only moderately high ENA copy numbers (e.g., AP 17.1; 4–5 copies), strains associated with the intestinal tract behaved heterogeneously, varying between an excellent performance (PYCC 2613; 9–13 copies) and a very poor performance (e.g., YJM 1078; 7–8 copies).