We generated a high quality genome assembly for S. arboricolus using a combination of high-throughput sequencing platforms and strategies (Table  1). First, we generated single-end reads using the Roche 454 pyrosequencing platform. This gave long reads that facilitated assembly. Second, we used Roche 454 paired-end reads, with ~8 kb insert size, to join contigs into chromosome size scaffolds (combined Roche 454 sequence coverage ~49X). We anticipated that the large insert size of the paired-end library would be sufficient to span any repeat elements (e.g. full length single Ty elements). Finally, we used 50 bp reads from SOLiD (Life Technologies) sequencing (~100X sequence coverage) to correct homopolymer errors present in the Roche 454 sequence. This combination strategy resulted in high quality sequence with chromosome-sized scaffolds.

De novo assembly of the Roche 454 (fragment and pair-end) reads was performed using the Newbler algorithm (see Methods). This resulted in 290 contigs (≥500 bp; N50 117,280 bp) that were joined using the Roche 454 paired-end reads to give 35 scaffolds. There are 17 scaffolds that are comprised of a single contig (2024 - 5644 bp) and one scaffold comprising of two contigs (9948 bp; Additional file 1: Table S1). The remaining 17 scaffolds account for >99% of the assembly and are between 72 and 1246 kb long. The total base count of the assembly, 11.6 Mb, is comparable to the physical genome size predicted from the karyotype 11 and is similar to the completed S. cerevisiae genome sequence (12.1 Mb) and genome sequence of other sensu stricto yeasts (11.6 - 11.9 Mb) 3 13 .

Pyrosequencing suffers from an inherent difficulty in determining the number of incorporated nucleotides in homopolymer regions, due to the non-linear signal from the incorporation of >5 identical nucleotides 14 . Comparing our S. arboricolus genome sequence to S. cerevisiae, identified >700 open reading frames (ORFs) with putative frame-shifts (Figure 1A). These putative frame-shifts are predominantly in homopolymer runs and are therefore likely due to errors in the pyrosequencing (Figure 1). Indeed the S. paradoxus assembly 15 , which was based on Sanger sequence reads (that do not suffer from homopolymer errors), shows less than half the number of frame-shifts than S. arboricolus (Figure 1A). We further analyzed the homopolymeric runs that cause frame-shifts and found that they tended to be longer and more A-biased than the corresponding frame-shifts in S. paradoxus (Figure 1B and C). To overcome this problem we used SOLiD sequencing that relies on a different chemistry and is not subject to the same error. We used the Roche 454 de novo assembly to map the SOLiD reads, identify errors and then correct the assembly. This mapping, errors calling and correction process was then repeated a further 4 times. In total we corrected 121 single base substitutions and 1682 indels. This resulted in a dramatic reduction in the number of putative frame-shifts to levels comparable to that seen with conventional Sanger sequencing (as represented by the S. paradoxus genome, Figure 1A). The corrected assembly also improved the distribution of frame-shifts such that the over-abundance of long homopolymeric runs and the A-bias were greatly reduced (Figure 1B and C).

The coverage of mapped SOLiD reads gives a measure of sequence copy number and can be used to reveal repeat regions that have collapsed during assembly. Overall we find a scarcity of high-coverage regions (Additional file 2: Figure S1), implying that there has been very little collapse of repeat regions during assembly. Short (<100 bp) regions of elevated copy number frequently correspond to highly repetitive tandem repeats and/or homopolymer tracks. Longer regions of elevated copy number are predominantly subtelomeric with the noteworthy exception of the rDNA repeats on chromosome XII.

The resulting S. arboricolus genome assembly comprises of whole chromosome scaffolds with only 186 gaps. These gaps have an average size of 1206 bp, the smallest two are just 1 bp, and the largest is 5846 bp. These regions consist of complex or repetitive sequences resulting in poor mapping of the SOLiD data. By comparison to recently improved assemblies of other sensu stricto yeasts 3 , our S. arboricolus genome sequence has a higher proportion of the sequence (>99% compared with 96-98%) in a smaller number of scaffolds (35 compared with 147-226). Therefore, after the ‘gold standard’ S. cerevisiae, our S. arboricolus genome sequence represents the next most complete assembly.

We compared our S. arboricolus genome assembly to the S. cerevisiae reference genome using LASTZ. We found that the 17 long scaffolds are each syntenic with a single S. cerevisiae chromosome or the mitochondrial genome with the exception of one predicted reciprocal translocation (Figure 2A) between the right arms of chromosome IV and XIII. The breakpoints are intergenic regions between ORFs MRPL1 and TMA64 on chromosome IV and YKU80 and SPG4 on chromosome XIII (Figure 2B). Interestingly, the breakpoint on chromosome XIII is adjacent to a tRNA gene, a feature previously reported to be associated with reciprocal translocations 7 . We used diagnostic PCR to experimentally confirm this reciprocal translocation (Figure 2C). The reciprocal translocation is unique to S. arboricolus, it is not present in S. bayanus or other sensu stricto assemblies 3 and therefore occurred after the S. arboricolus radiation.

The gene content of sensu stricto budding yeast species is thought to be similar 18 19 , therefore we used comparative gene-annotation methods based on the well-annotated S. cerevisiae proteome to identify and annotate the ORFs in the S. arboricolus genome. Using exonerate 20 , we aligned each S. cerevisiae protein to the S. arboricolus genome (see Methods). We assigned the top matching S. arboricolus ORF (based on the exonerate score) as a putative ortholog to each S. cerevisiae protein. We then compared the neighbouring genes of each S. cerevisiae gene with the neighbours of the putative orthologous S. arboricolus ORF to define a first set of 4798 orthologous gene pairs where the gene order has been conserved, which we refer to as “syntenic orthologs”. Because this method uses the best sequence match, missing assignments of syntenic orthologous ORFs may occur when the match with greatest sequence similarity is not the syntenic ortholog. To overcome this problem, we again used exonerate but emphasized the position of the predicted ORF, allowing the score to be slightly below the best scoring match (see Methods). An additional 519 S. cerevisiae genes had a high-scoring, but not top match with the expected syntenic gene pair. We considered these to be syntenic orthologs as sequence similarity together with gene order conservation is thought to be a more reliable indicator of orthology than sequence similarity alone 21 . To identify genes that may be found in S. arboricolus, but not in S. cerevisiae, we used Genemark 22 , which is a de novo gene prediction method, and does not rely on sequence similarity (see Methods) and identified 106 genes that were not predicted using exonerate. These Genemark predictions contain novel genes and ORFs that were missed by exonerate as only the best hit from exonerate was considered in our gene prediction.

We explored the possibility that our annotation of the S. arboricolus genome contained novel genes. As was observed with the S. bayanus genome 19 , the vast majority (96%) of the genes in S. arboricolus have conserved gene order with S. cerevisiae. The remaining “non-syntenic” genes include 104 that have similarity to another S. cerevisiae gene but are not syntenic (by our definition) and the 106 the genes predicted de novo (within the 16 assembled chromosomes). Analysis of the non-syntenic genes allowed the detection of at least two small local rearrangements relative to S. cerevisiae due to inversion of a large portion of DNA. The first one occurs on chromosome VI between ORFs FAR7 and YFR017C (Figure 3A) and the second one on chromosome XIV between YNL034W and COG6 (data not shown). To determine whether these were specific to the S. arboricolus genome, we compared these regions to the other sensu stricto genomes, and found that the S. cerevisiae gene order is likely to be the derived state, as S. bayanus and S. kudriavzevii shows the same gene order as S. arboricolus (Figure 4). Other synteny breaks occur predominantly in the subtelomeric regions: there is a significant enrichment of non-syntenic and novel genes predicted in the first and last 10% of the chromosomes (P-value = 5×10^-37, Figure 3B) 23 . We also considered the genes predicted in S. arboricolus that were not syntenic with S. cerevisiae. Of these 210 genes, 44 had no BLAST hits within the S. cerevisiae genome (e-value cutoff 1e-10). Interestingly, 3 of these 44 genes are likely to be S. cerevisiae specific gene losses, rather than new genes arising in S. arboricolus, as they are found in S. bayanus (Figure 3C). Two of the non-telomeric S. cerevisiae gene losses are SIR1 genes as previously reported 24 . Of the remaining 41 genes, 20 have no blast hit within Uniref90 (e-value cutoff 1e-10) and we considered the possibility that these were truly novel genes. After manual inspection based on presence of stop codons within the predicted peptide, protein sequence lengths, Pfam analysis andadditional blast searches, we concluded that 4 of these genes are likely to represent novel genes in S. arboricolus (Additional file 3).

We also searched for tRNA coding sequences within the 16 chromosomes using tRNAscan-SE 25 and annotated whether they were syntenic using a similar strategy to that described above (see Methods). In total, 257 tRNAs were found, 252 of which are syntenic with S. cerevisiae tRNAs. Next we used BLAST to search for the presence of repetitive elements in the genome such as subtelomeric genes and Ty elements. We detected the most distal subtelomeric element, Y’, in the genome sequence. This element is therefore present in all the sensu stricto species except S. bayanus 26 27 . We also detected Ty2 element sequences using as a query the region that does not share similarity with the Ty1 element (1.7-kb ClaI Ty2- specific sequences 26 ).

We tested five possible placements of S. arboricolus within the sensu stricto complex (Additional file 4: Figure S2), by sampling 100 sets of 50 random proteins for which we have data for all 6 sensu stricto species, as well as S. castellii as outgroup. These protein sequences were concatenated, and we computed the likelihood of the five phylogenetic trees using PAML. All 100 trees supported the grouping of S. arboricolus as diverging after the common ancestor with S. bayanus and before S. kudriavzevii, and all but 1 of these trees obtained bootstrap scores >0.9. Originally S. arboricolus was placed, based on a limited amount of ribosomal DNA sequence, between S. mikatae and S. kudriavzevii 11 , however our genome-scale phylogenetic analysis has much greater power and unambiguously supports the new tree structure (Figure 5).

We next set out to estimate the evolutionary distances between the species in the sensu stricto clade. To do so, we used the phylogeny as determined above, and computed codon-based maximum likelihood estimates (see Methods) of evolutionary distance based on alignments of 3899 genes for which we had 1 to 1 orthologs in all of the sensu stricto species, and that were syntenic between S. arbicolus and S. cerevisiae. We computed the median branch lengths (in substitutions per codon) for these, and they are shown in Figure 5.

To make the S. arboricolus genome sequence available and to facilitate analysis, we have made available a number of web resources (http://www.moseslab.csb.utoronto.ca/sarb). These include genome sequence, annotation and datasets for genes and proteins. The gene and protein sets are annotated based on the S. cerevisiae ortholog systematic name. Novel putative ORFs identified by Genemark are also reported. A BLAST server and a genome browser (Gbrowse 28 and GBrowse_syn 29 ) are available. The S. arboricolus genome browser offers the opportunity to view and compare the genome structure of S. cerevisiae, S. arboricolus and S. bayanus. Using S. arboricolus as the central reference species, gene order conservation or chromosomal rearrangements between the three species can easily be observed. Genes in S. arboricolus are coloured differently based on their annotation (e.g. syntenic orthologs, Genemark predictions, etc.). Finally, to facilitate experimental analysis of S. arboricolus the HO gene was disrupted (in the type strain H-6) and stable haploids were generated.

Taking the phylogeny and gene content of S. arboricolus (described above) into account, we revisited recently generated data on its phenotypic diversity. Although included for completeness in our publication on the phenotypic landscape of Saccharomyces sensu stricto species 30 , the S. arboricolus phenotypes were not specifically analyzed or commented on. The sequenced strain (CBS 10644^TT) and two additional, genetically distinct lineages isolated from similar habitats in Southern China were subjected to high resolution phenotyping of proliferative capability across >120 environments selected to represent variations in common yeast habitats, such as carbon and nitrogen source variations, tolerance to metabolites and toxins produced by plants and bacteria, and variations in vitamins and mineral availability (Additional file 5: Table S2). The fitness components lag, rate (population doubling time) and efficiency of reproduction (population density change) were extracted from high density growth curves and normalized to those of the S. cerevisiae reference strain, providing >360 precise measures of organism-environment interactions (Figure 6A). In the absence of stress, S. arboricolus proliferated slightly slower than S. cerevisiae and one strain (CBS 10644^TT) also showed reduced efficiency (Figure 6A). However, these S. arboricolus growth aberrations in conditions with no stress were marginal compared to the dramatic proliferation deviations observed in a vast range of stress-inducing niche environments (Figure 6B). Remarkably, almost all of these aberrations constituted grave defects, many corresponding to more than 10-fold reductions in mitotic performance. Thus, S. arboricolus showed drastically reduced tolerance to fruit organic acids such as citric, tartaric and oxalic acid and to high temperatures and very poor utilization of adenine, serine and threonine as nitrogen sources. Notably, S. arboricolus failed to proliferate during conditions of elevated Li⁺ and Cu²⁺, traits likely explained by absence of the amplifications of the lithium exporter (ENA1) and the copper metallothionein (CUP1) that determine these traits in S. cerevisiae 30 (Figure 6C). The many niche specific proliferation deficiencies of S. arboricolus may explain its limited geographical and ecological distribution compared to S. cerevisiae.

Among the rare examples of superior S. arboricolus performance were better utilization of the sugar alcohol mannitol, one of the most abundant energy storage molecules in nature 31 and tolerance to biotin depletion, rarely observed in S. cerevisiae due to ancestral loss of the biotin synthesis genes BIO1 and BIO6 32 . Both BIO1 and BIO6 are present as conserved cistrons in S. arboricolus strain CBS 10644^TT (Figure 6B), explaining the biotin auxotrophy. S. arboricolus also featured consistently good utilization of the monosaccharide galactose (Figure 6C), a highly variable trait in both S. cerevisiae 30 and S. kudriavzevii 33 due to frequent loss-of-function mutations emerging in different lineages of these species that impair growth on galactose (e.g. the reference strain S288C). The coding sequences of the GAL pathway genes are also fully conserved in S. arboricolus strain CBS 10644^TT. Interestingly, S. arboricolus strain CBS 10644^TT has also retained an intact melibiase encoding MEL1, which is lost in most S. cerevisiae lineages 30 ; all three S. arboricolus isolates also utilized the disaccharide melibiose, a less common plant energy storage compound, with a vastly superior rate and efficiency.

Overall, the three S. arboricolus isolates showed virtually identical trait profiles (Pearson correlation, r=0.73-0.91). Moreover, using data from Warringer et al. 30 , we found S. arboricolus traits to closely mimic those of its relatives, S. bayanus and S. kudriavzevii (Figure 6D). These remarkable trait similarities, encompassing mitotic performance in a wide variety of environments, imply that these three species, despite billions of generations of separation, are adapted to rather similar ecological conditions. The few cases of S. arboricolus deviations from S. bayanus and S. kudriavzevii primarily included nitrogen utilization traits, such as superior S. arboricolus utilization of ammonium, glycine and ornithine, but inferior utilization of methionine, serine and citrulline (Figure 6E). Presumably, this reflects differences in nitrogen storage compounds among plant species that dominate the main habitats of these species and hint at ecological factors that may have driven speciation of the ancestral lineages. Further indications as to the nature of these factors may also be found in the reduced tolerance of S. arboricolus to malic acid, concentrated in e.g. apples, and to the toxin geneticin, produced by bacteria of the Micromonospora genus (Figure 6E).

We extracted DNA from the type strain of S. arboricolus H-6^T (CBS 10644^TT) isolated in China from the bark of Quercus fabri 11 . For the Roche 454 library construction and sequencing, 5 μg of high molecular weight genomic DNA was used to make standard shotgun DNA library as described in the Roche GS FLX Titanium General Library Preparation Method Manual with the exception of DNA fragmentation, which was done with Covaris S2 sonicator (fragmentation parameters: Duty cycle - 5%, Intensity - 1, cycles/burst - 200, time - 85 s, bath temperature - 5°C). 15 μg of high molecular weight genomic DNA was used to make the 8 Kb paired end library as stated in the Roche GS FLX Titanium 8 Kb Span Paired end library preparation method manual. Exceptions include: DNA extraction was done using QIAquick Gel extraction kit (Qiagen, Cat no. 28760) instead of Electroelution as stated in the manual and fragmentation of circularised DNA was done using Covaris S2 sonicator (Duty cycle −5%, Intensity - 3, cycles/burst - 200, time - 120 s, bath temperature - 5°C). Sequencing of standard shotgun fragment library was carried out on ¾ of a PTP and the 8 Kb paired end library was sequenced on a full PTP using Roche 454 Titanium sequencing chemistry. For SOLiD library construction and sequencing, 500 ng of high molecular weight genomic DNA was used to make a barcoded DNA fragment library as stated in the SOLiD 4 library preparation guide. Enzymes and reagents were used from NEBNext DNA sample prep Master mix set 3 (NEB, Cat no. E6060L). The barcoded DNA fragment library was quantified using Kapa Library Quantification kit (Kapa Biosystems, Cat no. KK4823). 200-300 bp library size selection was carried out using 2% SizeSelect E-Gel (Life Technologies, Cat no. G6610-02). SOLiD EZ Bead System was used according to manufacturer’s guide to prepare ePCR and templated bead enrichment. Sequencing was performed on a SOLiD 4 analyser according to the manufacturer’s instructions to generate 50 bp reads in colour space.

We assembled the genome of S. arboricolus using the Newbler algorithm (v2.3, Roche) for de novo assembly of reads generated by the 454 pryosequencing platform. Combinations of read datasets, reads added in assembly iterations, and assembler parameters were explored before selecting the optimal combination according to assembly metrics (number of scaffold sequences and contigs, the average and longest contig length and N50 value). All reads were trimmed against a dataset of adapter and vector sequences in the initial step of the assembly process.

The selected assembly parameters used an expected coverage value of 40X with all other settings remaining at default values. Two assembler iterations were employed; the first iteration included all 734,353 single fragment reads and one set of 583,674 paired-end reads. The second iteration incorporated an additional set of 518,434 paired-end reads. A third set of paired-end reads was excluded from the assembly due to decreased performance with their inclusion.

The resulting genome assembly comprised of 32 scaffold sequences with a total length of 11,465,281 bp. The scaffolds were comprised of 266 contigs (≥500 bp) with an average length of 43,102 bp (538,482 bp max.) and an N50 value of 136,945 bp. The mapped read coverage of the assembly was 49X.

In order to resolve small errors in the assembly arising from pyrosequencing artifacts, such as homopolymer sequence regions 43 44 , we acquired deep sequence coverage (~100X) from short reads. We generated a total of 31,316,59 short (50 bp) reads from a SOLiD 4 single fragment library. Subsequent gapped read alignment and variant calling was achieved using Bioscope 1.3.1 (Life Technologies).

An iterative correction process was devised in which errors in the assembled sequence were identified from the SOLiD read alignment data as either a single nucleotide polymorphisms (SNP) for single base errors, or as small InDels (insertion/deletion) for homopolymer pyrosequencing errors. Each iteration of the assembly correction process involved the initial mapping of SOLiD reads against the 454 assembly, followed by SNP calling. Selected putative SNPs were then integrated into the assembly sequence and SOLiD reads were remapped to allow InDels to be called and integrated. This process was repeated until no additional variants were detected. In subsequent iterations additional reads were mapped allowing the identification and correction of a small number of further errors. Both SNPs and InDels were calculated from alignment data using Bioscope 'high stringency' variant parameter settings. Additionally, integrated variants were required to represent a minimum of 60% of the alignment data.

S. cerevisiae was used as the reference proteome for the program exonerate, which uses comparative approaches for gene finding based on protein sequence similarity. An initial pass with the protein2dna model and a refine boundary of 2000 was used to find the best orthologous candidate of each S. cerevisiae gene. For intronic genes, the max intron size was limited to 1500 bp and the model used was protein2genome.

To annotate genes within the S. arboricolus genome, we first identified gene orthologs with conserved synteny. To do so, we analysed the top hit by exonerate for each S. cerevisiae gene. When three neighbouring genes within S. cerevisiae all identified three neighbouring genes within S. arboricolus, we assigned the S. arboricolus gene in the middle (flanked by its two neighbours) as a syntenic ortholog. This initial step discovered most of the syntenic genes within S. arboricolus. Other genes within S. cerevisiae that had not been assigned an ortholog were further analysed with the hypothesis that these may have exonerate hits within the expected positions but were not the most similar sequence within S. arboricolus. We looked at the top 10 exonerate hits of the remaining S. cerevisiae genes for matches in S. arboricolus between the initially assigned syntenic ortholog. When only one hit was found between these syntenic orthologs, we used this hit as a newly discovered syntenic ortholog. This process was repeated until no more syntenic orthologs could be found. Finally, we assigned the top exonerate hit of few remaining S. cerevisiae genes that were still not assigned a syntenic ortholog as the non-syntenic ortholog provided that they did not overlap with another gene prediction.

De novo gene prediction on the S. arboricolus genome was performed using GeneMark-ES, version 2 45 . The total number of the predicted genes was 5005 within the 16 assembled chromosomes (5038 in total). Of these, 95 genes had non-overlapping coordinates with the genes predicted by Exonerate within the 16 assembled chromosomes (106 in total when including the 19 scaffolds that did not assemble into the chromosomes).

A significant issue when using a comparative-based method, such as exonerate, for gene prediction is that gene boundaries are often incorrectly predicted if there is a lack of homology at these ends. Initially, a large number of predicted genes did not contain a start or stop codon (637 genes and 1121 genes respectively). We have attempted to rectify these starts and ends by extending or truncating the predicted CDS. First, CDSs were extended if a stop codon could be found within 9 codons from the end of our gene prediction. This corrected 857 cases of missing stop codons and further extension only slightly improved the annotation. Second, for start codons, the methionine can be on either side of the predicted gene start. We therefore extended the predicted gene until a methionine was found, but only when a methionine could be found within 9 codons and without any intervening stop codons. In the cases where a stop codon occurred before a suitable methionine was identified, we truncated the CDS to a downstream methionine if it occurred within 9 codons. This corrected 348 cases of missing start codons. Finally, intron-containing genes were left untouched as missing starts and ends for these genes could be due to a missing exon. We note that for intron-containing genes, we specifically use the protein2genome model in exonerate that explicitly attempts to predict all exons found in S. cerevisiae genes. This assumes that the presence of introns is conserved between S. cerevisiae and S. arboricolus.

We aligned the protein sequence orthologs for the sensu stricto using MAFFT 46 with default settings, either with or without S. castellii orthologs as an outgroup. For the coding sequence analysis we inserted the gaps back into the DNA sequences. Phylogenetic analysis was performed using PAML 47 , either with the codon model for the DNA sequence analysis or with empirical model for the amino acid analysis. Because we are only concerned with the placement of S. arboricolus within the established sensu stricto yeast phylogeny, we compared the likelihood of several putative tree topologies that differ only in the position of S. arboricolus (Figure S2).

To annotate tRNA coding sequences, we predicted tRNAs using tRNAscan-SE 25 with default settings on the 16 assembled chromosomes. To determine whether or not these predicted tRNAs are syntenic with respect to S. cerevisiae we used an analogous strategy to that described above for gene annotations. tRNA coding sequences were annotated as syntenic orthologs if they were flanked by genes within S. arboricolus that were assigned as syntenic orthologs and if a tRNA was also found in S. cerevisiae between those genes. In all but one cases, the syntenic tRNAs code for the same amino acids.

Chromosome structure plots for the Saccharomyces sensu stricto species were constructed using Mauve 48 . Assembled chromosomes for S. paradoxus (strain CBS432) were obtained from 15 and for S. mikatae (IFO 1815 ^T), S. kudriavzevii (IFO 1802 ^T) and S. bayanus var. uvarum (strain CBS 7001) from 3 . As these chromosome assemblies have been constructed partly by using the S. cerevisiae genome to orient and order scaffolds, alignments were also made to the unordered scaffolds using MUMmer 49 to confirm the relative orientation of chromosomal segments inverted between species.

The bulk of the phenotypic data was taken from our recent publication 30 on sensu stricto phenotypes where it was included for completeness but where S. arboricolus phenotypes were not specifically analyzed or considered. The data displayed as growth curves in this study correspond to novel confirmatory runs performed to ensure the reliability of specific statements. Three diploid isolates of Saccharomyces arboricolus were collected as described previously 11 and long time stored in 20% glycerol at -80C. Isolates were subjected to high throughput phenotyping by micro-cultivation (n=2) in an array of environments (Additional file 5: Table S2) essentially as previously described 50 . For pre-cultivations, strains were inoculated in 350 μL of SD medium (0.14% yeast nitrogen base, 0.5% ammonium sulfate and 1% succinic acid; 2% (w/v) glucose; 0.077% Complete Supplement Mixture (CSM, ForMedium), pH set to 5.8 with NaOH or KOH) and incubated for 48 h at 30°C. For experiments where the removal of a specific media component was studied, the pre-culture was performed in absence of this component in order to completely deplete the component in question. For experiments where alternative nitrogen sources were used, two consecutive pre-cultures were performed, the first containing low amounts of ammonium sulphate (0.05%), the second replacing ammonium with the indicated nitrogen source in amounts corresponding to equivalent moles of N. For all experimental runs, strains were inoculated to an OD of 0.03 - 0.1 in 350 μL of SD medium and cultivated for 72 h in a Bioscreen analyser C (Growth curves Oy, Finland). Optical density was measured using a wide band (450-580 nm) filter. Incubation was at 30.0°C (±0.1°C) with ten minutes preheating time. Plates were subjected to shaking at highest shaking intensity with 60s of shaking every other minute. OD measurements were taken every 20 min. Strains were run in duplicates on separate plates with ten replicates of the universal S. cerevisiae reference strain BY4741 or its prototrophic mother S288C, in randomised (once) positions on each plate as a reference. The reproductive rate (population doubling time), lag (population adaptation time) and efficiency (population total change in density) were extracted from high density growth curves and put in relation to the corresponding fitness variables of the reference strain BY4741, or in conditions directly involving alterations of nitrogen content, its prototrophic mother S288C, as described previously 30 . The derived Log₂ ratios (Log₂ (BY4741/isolate) or, in case of efficiency, Log₂ (isolate/BY4741) were used for subsequent analysis.

Raw sequencing reads are available from the European Nucleotide Archive (EBI ENA) for the SOLiD reads [EMBL: ERP001702], Roche 454 single fragment reads [EMBL: ERP001703] and Roche 454 paired-end reads [EMBL: ERP001704]. The assembled genome is available from NCBI as Saccharomyces arboricola [GenBank: ALIE00000000].