Additions, Losses, and Rearrangements on the Evolutionary Route from a Reconstructed Ancestor to the Modern Saccharomyces cerevisiae Genome
[['Inferring the genome organization and gene content of an extinct species has the potential to provide detailed information about the recent evolution of species descended from it .', 'If we know what was present in the genome of an ancestor , we can deduce how a current-day descendant differs from it .', 'We can then ask questions about how it came to be different .', 'The most recent changes in a genome are often the most interesting ones , because they reflect the most recent ( or even current ) evolutionary pressures acting on that genome [1] , [2] .', 'Yeast species offer the potential for the precise reconstruction of ancestral genomes , because many genomes have been sequenced and they show extensive colinearity of gene order among species [3]–[6] .', 'As the number of sequenced genomes from related species rises , so does the precision with which we can reconstruct their history .', 'In this study we compare the genomes of a group of species in the subphylum Saccharomycotina , spanning an evolutionary time-depth that is comparable to that of the vertebrates [7] .', 'A whole-genome duplication ( WGD ) event occurred during the evolution of this subphylum [8] , and we can compare the genomes of several species ( including S . cerevisiae ) that are descended from this event to the genomes of several species that branched off before the WGD occurred .', 'We focus on an ancestor that existed approximately 100–200 Mya , at the point immediately before the WGD occurred .', 'The evolutionary period beginning with this ancestor corresponds to a time during which the S . cerevisiae lineage became increasingly adapted to rapid fermentative growth [9] , [10] and extensive rearrangement of the genome occurred ( including the deletion of thousands of redundant copies of duplicated genes ) [11] .', 'Previous studies in other systems have employed both manual and computational approaches to reconstructing ancestral genomes .', 'One of the most successful applications of computational methods has been the estimation of the ancestral order of orthologous genes in the common ancestor of 12 Drosophila species [12] , [13] .', 'Ancestral reconstruction is more difficult when ancient polyploidizations are present [14] .', 'In studies of the 2R duplications in vertebrates , for example , the emphasis has been on establishing the ancestral gene content of paralogous chromosomal regions rather than on their precise gene order [15] , [16] .', 'We chose to use a manual , parsimony-based , approach to reconstructing the yeast ancestor at the point of WGD .', 'The manual approach has the attractions of being tractable ( whereas computational methods are still under development [17] , [18] ) , of providing an independent result to which computational results can be compared , and of forcing us to examine every rearrangement event without prejudice as to what mechanism might have caused it .', 'Sankoff and colleagues [14] , [17] , [18] have developed computational methods that aim to reconstruct ancestral gene order in datasets that include polyploidizations .', 'In recent work [18] , they evaluated their ‘guided genome halving’ ( GGH ) algorithm by comparing its results to ours , using a preliminary version of the manually-derived ancestral yeast gene order that we report here as a ‘gold standard’ .', 'As currently implemented , the GGH algorithm can only consider input from a single post-WGD genome and 1–2 non-WGD outgroups , and only considers genes that are duplicated in the post-WGD genome .', 'Inferring the set of genes that existed in a yeast ancestor , and the order of those genes along the chromosomes , is of interest from both genome-evolutionary and organismal-evolutionary standpoints .', 'Knowing the ancestral gene order enables us to trace all the inter- and intra-chromosomal rearrangements that occurred en route from this ancestor to the current S . cerevisiae genome , which is informative about the molecular mechanisms of evolutionary genome rearrangement and is also phylogenetically informative .', 'Knowing the ancestral gene content allows us to identify genes that have been added to , or lost from , the S . cerevisiae genome during the past 100 Myr .', 'Previous studies have shown that changes in gene content can provide a strong indication of changing evolutionary circumstances , either in cases of gene loss ( such as the losses of GAL , DAL and BNA genes in Candida glabrata [1] , [19] , [20] ) or in cases of gene gain ( such as the ADH2 and URA1 genes of S . cerevisiae [9] , [21] , [22] ) .', 'Even though it may not be possible to conclude that any particular gene gain was adaptive , the clear links between the functions of the gained genes ADH2 and URA1 and the adaptation of S . cerevisiae to a fermentative lifestyle [23] suggested to us that a systematic search for all the genes that were gained by S . cerevisiae since WGD would be worthwhile .'], ['We used a manual parsimony approach to reconstruct the gene order and gene content of the yeast ancestor that existed immediately prior to WGD ( Figure 1 ) .', 'The reconstruction was made by visually comparing the local gene orders in every region of the genome , stepping through the genome in overlapping 25-gene windows using the Yeast Gene Order Browser [YGOB; 6] .', 'Initially , during 2007–08 , we compared data from five post-WGD species ( S . cerevisiae , S . bayanus , C . glabrata , Naumovia castellii and Vanderwaltozyma polyspora ) and three non-WGD species ( E . gossypii , Kluyveromyces lactis and Lachancea waltii ) and inferred an ancestral genome based on these data .', 'Later , in 2009 , we added the genomes of three more non-WGD species ( Zygosaccharomyces rouxii , L . thermotolerans and L . kluyveri [24] ) and re-examined the whole genome window-by-window using YGOB .', 'This process confirmed that our initial ancestral reconstruction was largely correct , but identified a few places where the gene content or local gene order in the ancestor needed to be revised .', 'In particular , by adding data from more non-WGD species we were able in some cases to detect non-WGD orthologs of S . cerevisiae genes that are short and rapidly-evolving , which previously appeared to be unique to S . cerevisiae ( for example , YLR146W-A ) .', 'The gene order and content of the ancestor were inferred as shown in Figure 1B , C .', 'We first established the gene content , and then examined the adjacency relationships among these genes .', 'Within any post-WGD species such as S . cerevisiae , most of the genome can be sorted into pairs of sister regions that have a double-conserved synteny ( DCS ) relationship with any non-WGD species such as L . waltii [25] , [26] .', 'Breaks in the DCS pattern correspond to two types of event , called single-breaks and double-breaks of synteny [26] .', 'For each single-break of synteny ( Figure 1B ) , because we have genome sequences from multiple post-WGD species , and because the endpoints of the chromosomal rearrangements in different species generally do not coincide , we can infer the species and chromosomal track on which the break happened .', 'This inference also tells us the ancestral gene order across the site of breakage: in general , for a single break , the ancestral order has been disrupted in one track in one post-WGD species , but it is still conserved in the same track from the other post-WGD species , in the sister track from all the post-WGD species , and in the non-WGD species .', 'Similarly for each double-break of synteny ( Figure 1C ) , because we have multiple genome sequences from non-WGD species we can in general identify the break as having occurred in one particular non-WGD species .', 'A small number of double-breaks of synteny are caused by situations where all the non-WGD species show one gene order but both of the tracks from all the post-WGD species show a different order .', 'These breaks correspond to rearrangements that occurred on the branch between the Z . rouxii divergence and the common ancestor of the post-WGD species ( before the WGD happened ) .', 'We do not include these breaks in our analysis because we are only interested in events that occurred after the WGD .', 'Manual reconstruction by this method resulted in an inferred ancestral genome with eight chromosomes , containing 4703 protein-coding genes .', 'The ancestral gene set represents the intersections of orthologous genes between non-WGD and post-WGD species , and between ohnologs ( paralogs formed by WGD ) across the post-WGD species .', 'The ancestral genome is listed in Table S1 and can be browsed using YGOB ( http://wolfe . gen . tcd . ie/ygob ) .', 'Genes in this genome were given names such as Anc_1 . 125 , meaning the 125th gene on chromosome 1 of the ancestor .', 'The ancestral gene set accounts for 5158 ( 92% ) of the 5601 genes currently present in S . cerevisiae ( 1088 ohnologs and 4070 single copy genes ) , which covers all genomic regions in S . cerevisiae except for the subtelomeric regions ( discussed below ) .', 'The S . cerevisiae genome can be mapped onto the inferred ancestral genome in 182 DCS blocks that tile together in an unambiguous 2∶1 fashion across the ancestral genome ( Figure 2 ) .', 'Similarly , the other post-WGD species and non-WGD species can be mapped onto the ancestral genome , with 2∶1 and 1∶1 mappings , respectively , by the numbers of blocks shown in Figure 1A .', 'The C . glabrata genome is much more rearranged ( 582 blocks ) than S . cerevisiae as previously noted [19] , [27] .', 'The L . kluyveri genome is remarkably unrearranged , with the whole genome mapping into just 57 blocks relative to the ancestor .', 'Our inferred ancestral genome is incomplete in some regards: Using the breakpoints between S . cerevisiae synteny blocks in the ancestral genome , we inferred the large scale chromosomal rearrangements that have occurred in the S . cerevisiae lineage since the WGD .', 'Most rearrangement events could be classified as either reciprocal translocations ( Figure 3 ) or inversions .', 'Note that it is impossible to count inversions and reciprocal translocations with absolute precision , because if a genomic region that contains one endpoint of a reciprocal translocation subsequently undergoes inversion , the result is identical to one that could be produced by two successive reciprocal translocations ( Figure S1 ) .', 'We counted these situations as two reciprocal translocations , so we have probably misclassified some inversions as reciprocal translocations .', 'Inversions were defined as events where the two endpoints of the rearrangement were on the same ancestral chromosome and on the same post-WGD track .', 'In total we inferred 73 inversion events and 66 reciprocal translocations events on the evolutionary path from the ancestor to S . cerevisiae ( Table 1 ) .', 'Five of the inversions have endpoints at telomeres .', 'There were also five non-reciprocal translocations , which we call ‘telomeric translocations’ because they involved an exchange between a telomere and an internal region of another chromosome , which moved the end of an arm from one chromosome to another ( one of these events occurs at a shared inversion/translocation breakpoint ) .', 'The data indicate that some intergenic regions were re-used as breakpoints in more than one rearrangement event .', 'We classified the rearrangements as consisting of 34 simple inversion events ( not overlapping other inversions or reusing breakpoints ) , 39 complex inversion events ( overlapping other rearrangements and/or reusing breakpoints ) , 44 simple reciprocal translocation events , and 22 reciprocal translocation events involving breakpoint reuse ( Figure 4 ) .', 'These results are in reasonable agreement with our estimate from a decade ago of 70–100 rearrangement events , based only on S . cerevisiae data [33] .', 'If some post-WGD species share a rearrangement relative to the ancestor but others retain the ancestral gene order , the rearrangement event is a phylogenetically informative character [34] , [35] .', 'We searched for rearrangements shared by any pair of post-WGD species .', 'As described below , we found many that support the branching order of the post-WGD species shown in Figure 1A ( Table S2 ) .', 'We did not find any shared rearrangements supporting alternative topologies .', 'This result supports our previous conclusion , based on shared patterns of gene losses , that N . castellii is an outgroup to a clade containing C . glabrata and S . cerevisiae [11] , [36] .', 'In contrast , phylogenies based on sequence analysis tend to place C . glabrata outside N . castellii and S . cerevisiae [1] , [37] , [38] , a result that we believe is an artifact .', 'Given this phylogeny , the post-WGD species define four temporal intervals for rearrangements ( Figure 1A ) :', '( i ) no rearrangements are shared by all the post-WGD species relative to the ancestor;', '( ii ) 8 rearrangement events are shared by N . castellii , C . glabrata and S . cerevisiae ( 6 inversions , 1 reciprocal translocation , 1 telomeric translocation ) ;', '( iii ) 19 rearrangements are shared only by C . glabrata and S . cerevisiae , with N . castellii and V . polyspora retaining the ancestral organization ( 13 inversions , 6 reciprocal translocations ) ; and', '( iv ) 117 rearrangements are unique to S . cerevisiae or shared by this species and S . bayanus ( 54 inversions , 59 reciprocal translocations , 4 telomeric translocations ) .', 'Most of the rearrangements that are specific to S . cerevisiae are temporally ambiguous relative to each other .', 'We did not subdivide the group of 117 events into those that occurred before and after the S . bayanus divergence because the S . bayanus genome assembly is quite fragmented .', 'The above analysis does not include gene transpositions , which we find to be relatively rare in yeast genomes but which are difficult to count precisely because to identify a transposed gene in a particular species , we need to be certain that it is orthologous to a gene at a non-syntenic location in the ancestral genome .', 'The lack of rearrangements in the first time interval is notable because it indicates that V . polyspora separated from the other lineages soon after the WGD .', 'We also found that no rearrangements occurred on one genomic track of all the post-WGD species , relative to the other track and the ancestor , prior to this speciation .', 'This observation argues against the possibility that the WGD event was an allopolyploidization rather than an autopolyploidization ( see ref . [11] for discussion ) : if it was an allopolyploidization , then the two hybridizing genomes must have been completely colinear .', 'It was necessary to infer breakpoint reuse at the ends of some synteny blocks .', 'Reused breakpoints appear as cycles in the map of breakpoint pairs ( Figure 4 ) .', 'The evolutionary re-use of breakpoints has previously been identified in studies on mammals and Drosophila [13] , [39] .', 'We find that for both reciprocal translocations and inversions , there are fewer breakpoints than expected if every event had unique ends ( Table 1 ) .', 'The average number of breaks per used site is 1 . 12 for reciprocal translocations and 1 . 16 for inversions .', 'Some sites were used as endpoints of both an inversion and a reciprocal translocation , and if we pool these two categories there are only 228 unique breakpoints instead of the expected 278 , implying an average of 1 . 22 breaks per site ( Table 1 ) .', 'We identified 96 sites in the ancestral genome at which genes are inferred to have been gained subsequently in the lineage leading to S . cerevisiae .', 'The total number of gained genes is 124 , because some sites contain groups of consecutive gained genes ( Figure 5 ) .', 'We were surprised to find that 33 ( 34% ) of these ‘gene gain’ sites are beside tRNA genes .', 'tRNA genes have previously been linked to sites of genomic rearrangement between E . gossypii and S . cerevisiae [26] .', 'Furthermore , it is known that origins of replication in yeast are often located near tRNA genes [40] , and it seems plausible that origins might be fragile sites for evolutionary breakage and/or integration of new DNA .', 'We used computer simulation to test the significance of the associations among tRNA genes , origins of replication , evolutionary breakpoints , and sites of gene gain ( see Methods ) .', 'tRNA genes are present at breakpoints and gain sites about three times more often than expected by chance ( Table 2 , rows 2 and 3 ) , and origins are present about twice as often ( Table 2 , rows 4 and 5 ) .', 'It should be noted however that the locations of all the tRNA genes are known whereas it is probable that many origins have not yet been identified [41] .', 'There are several plausible mechanisms by which tRNA genes could precipitate genomic rearrangements .', 'tRNA genes exist in multiple near-identical copies in the genome , so illegitimate recombination between these sequences could result in reciprocal translocations [42] , [43] .', 'Ty retroelements tend to integrate beside tRNA genes and provide long sections of near-identical sequence scattered around the genome that could be substrates for ectopic recombination , as seen in S . cerevisiae irradiation experiments [44] .', 'Ty LTRs , tRNA genes , and origins of replications have also all been associated with the endpoints of spontaneous segmental DNA duplications in S . cerevisiae [45] .', 'Replication forks tend to stall near highly-expressed genes ( such as tRNA genes ) , and sites of replication fork collapse are hotspots for chromosomal rearrangements [46] , [47] .', 'It is also possible that the Ty-encoded reverse transcriptase has played a direct role in the integration of new genes into sites beside tRNA genes , similar to the way that cDNA fragments of transcribed genes are sometimes captured at sites of double-strand break repair in S . cerevisiae experiments [48] .', 'We identified 124 genes , excluding those in subtelomeric regions , that are inferred to have been gained on the lineage leading to S . cerevisiae during the time since WGD ( Figure 5 ) .', 'The S . cerevisiae gene set that we used in this study consists only of genes that are conserved between S . cerevisiae and at least one of the other Saccharomyces sensu stricto species ( dN/dS ratio<1 in the analysis of Kellis et al . [49] ) , or that are duplicates of other genes in S . cerevisiae ( again with dN/dS<1 ) , so we can be confident that the all the gains we identity are real genes and not annotation artifacts .', 'Some of the gained genes are unique to S . cerevisiae and sensu stricto species , while others are shared by the other post-WGD species ( Figure 5 ) .', 'The 124 gained genes range from those with high similarity to another gene in the S . cerevisiae genome to those with no similarity to any known gene from any organism .', 'We classified the gained genes into nine groups as described in Figure 5 , and then into three larger categories according to their apparent mechanism of formation .', 'The three large categories are: Analysis of the functions of the gained genes should provide insight into the evolutionary pressures that have acted on S . cerevisiae in the period since WGD but , remarkably , there is no functional information in the Saccharomyces Genome Database ( SGD ) for almost half of the recently gained genes .', 'None of the 124 genes is essential when deleted , according to SGD .', 'The non-essentiality of gained genes is not surprising because they were gained by an organism that was already fully functional in its environment before they were gained .', 'It is particularly notable that only 16 of the 51 orphans in Figure 5 have been assigned genetic names , which would indicate that something is known about their function .', 'In the sections below , we discuss some of the functional groups of gained genes .', 'The gene information in these sections is derived primarily from summaries in the SGD and YPD databases [52] , [53] , and from a MIPS ( Munich Information Centre for Protein Sequences ) catalog analysis .', 'Reconstructing the content and gene order of the ancestral yeast genome just prior to WGD has provided a mechanism for studying the structural rearrangements that occurred subsequent to WGD .', 'Our reconstruction is dependant on the set of extant genomes available for comparison , so it is likely that our list of candidate gene gains includes some false positives that will turn out to have been present at the time of WGD .', 'As more genome sequences become available the ancestral gene set will become progressively more complete and the list of gains may shrink .', 'From a biological perspective , the main shortcoming of our work is that we were unable to reconstruct the telomeric regions of the genome , corresponding to the last ∼10 genes on each arm of each chromosome in S . cerevisiae .', 'These regions turn over so dynamically that synteny breaks down almost completely between the species considered here .', 'This is unfortunate because many of the most interesting evolutionary events such as the gain of genes by horizontal gene transfer ( HGT ) from other species [22] , seem to occur preferentially near telomeres .', 'Our set of candidate gene gains in S . cerevisiae contains only two possible cases of gene gain by HGT at internal chromosomal sites ( YLR011W/LOT6 and YLR012W; we did not study these in detail ) , whereas Hall et al . [22] found eight examples of apparent transfer of bacterial genes into telomeric sites .', 'A second shortcoming is that we relied on sequence conservation ( dN/dS<1 ) among the sensu stricto species as a way of distinguishing between genuine S . cerevisiae genes and annotation artifacts , which had the inadvertent effect that we overlooked any genes that may have been gained by S . cerevisiae in the time since it diverged from the other sensu stricto species; one such case is BSC4 , which appears to have been formed de novo in S . cerevisiae [87] .', 'The set of genes inferred to have been gained on the S . cerevisiae lineage is relatively small ( 2% of the gene set ) and their functions point squarely towards increasing adaptation to the ‘fermentative lifestyle’ [23] .', 'They indicate increasing throughput of the glycolysis and fermentation pathways , and adaptation towards growth in conditions with little oxygen , including modifications to the cell wall and the bypass of biochemical pathways that require molecular oxygen by importing substances from outside the cell .', 'There are also many gained genes in our set that we have not discussed in detail here because they did not fall into larger functional groups .', 'Further analysis of these gains on an individual basis may reveal insights into the evolution of S . cerevisiae and the other species in the WGD clade .'], ['In this paper we have adopted the revised genus nomenclature proposed by Kurtzman [88]: Saccharomyces castellii becomes Naumovia castellii; Kluyveromyces polysporus becomes Vanderwaltozyma polyspora; Ashbya gossypii becomes Eremothecium gossypii; Kluyveromyces waltii becomes Lachancea waltii; Kluyveromyces thermotolerans becomes Lachancea thermotolerans; and Saccharomyces kluyveri becomes Lachancea kluyveri .', 'In this scheme each genus name refers to a monophyletic group , whereas previously Saccharomyces and Kluyveromyces were polyphyletic .', 'We did not change any gene names , even though in many species the gene names have a prefix that is an acronym of the obsolete species name .', 'The numbers of double-conserved synteny ( DCS ) and synteny blocks between the reconstructed ancestor and each other species ( Figure 1A ) were counted automatically using an algorithm that smoothes over small inversions and other interruptions in cases where endpoints are ≤20 genes apart in the ancestral genome .', 'For S . cerevisiae our manual analysis identified 228 breakpoints ( Table 1 and Figure 4 ) , which subdivide the 16 linear chromosomes into 244 segments .', 'The discrepancy in numbers between these 244 segments and the 182 DCS blocks in S . cerevisiae ( Figures 1A and 2 ) is due to the use of the smoothing algorithm .', 'We described the inferred ancestral gene order in terms of synteny blocks of current Saccharomyces cerevisiae genes .', 'We manually identified intrachromosomal rearrangements ( inversions ) between the ancestor and S . cerevisiae and reversed them , revising our synteny blocks , in order to more easily identify the endpoints of reciprocal translocations .', 'For each synteny block end not at a telomere , the location is at a position in the ancestral genome that underwent a reciprocal translocation in its transition towards the current S . cerevisiae genome .', 'Each synteny block end in the ancestral genome is bordered by another synteny block found elsewhere in the current S . cerevisiae genome .', 'The two breakpoints at the ends of two ancestral synteny blocks now adjacent in the current S . cerevisiae genome were created by a reciprocal translocation event that joined them together from different ancestral locations .', 'Concurrently the other synteny blocks that border each breakpoint in the ancestral genome were joined together by the same event .', 'We ordered the synteny blocks in the manner in which they are found along each chromosome in S . cerevisiae , thus inferring all the interchromosomal rearrangements between the ancestral polyploid genome and S . cerevisiae ( Figure 4 ) .', 'To confirm that the inferred reciprocal translocation events were correct , we found the location in S . cerevisiae of the other synteny block ends joined by each event .', 'In cases where these synteny blocks are not adjacent in the S . cerevisiae genome , we found the ancestral breakpoint locations of the blocks that are adjacent to each of these blocks in S . cerevisiae , inferring another reciprocal translocation event .', 'If the synteny blocks bordering each breakpoint were again not adjacent , this process was repeated .', 'To obtain a set of likely gene gains ( Figure 5 ) we subtracted the set of S . cerevisiae genes represented in the ancestral genome from the curated set of 5601 S . cerevisiae genes currently used in YGOB .', "YGOB's S . cerevisiae gene set is based on the SGD annotation ( ‘verified’ and ‘uncharacterized’ protein-coding loci only ) with some additional manual curation .", "It omits loci that failed Kellis et al . 's test of reading frame conservation among sensu stricto species [49] .", 'S . cerevisiae genes that are present in YGOB set but absent from the inferred ancestral set are candidates for having been gained in the S . cerevisiae lineage after the WGD .', 'We did not include subtelomeric genes from the YGOB set , as orthologous relationships across species break down at the telomeres [49] .', 'This candidate set of gains was then manually checked to ensure that there were no possible non-syntenic homologs that were ancestral but missing from our ancestral genome reconstruction due to a breakdown of synteny information .', 'Any cases where a good candidate non-syntenic homolog was found were removed from the gained set and flagged as a likely transposition event .', 'It is possible that the set of candidate gained genes may also contain ancestral genes that were lost in all of the non-WGD species used here but have orthologs in more distantly related outgroups .', 'We compiled lists of the 245 S . cerevisiae intergenic regions that contain one or more tRNA genes [from SGD; 52] , the 228 intergenic regions that contain evolutionary breakpoints on the S . cerevisiae lineage , the 96 sites of gene gain in S . cerevisiae , and 267 intergenic regions that contain an origin of replication in S . cerevisiae ( from OriDB [41]; we included origins that overlap with genes ) .', 'We counted the numbers of intergenic regions that contain combinations of multiple types of site .', 'We then used computer simulation to estimate the significance of the observed numbers of coinciding sites ( Table 2 ) .', 'In each of 1 million replicates we simulated a genome with 5100 intergenic spacers ( the estimated number of intergenic spacers between S . cerevisiae genes that are at an ancestral locus ) .', 'We placed the same numbers of tRNA genes , origins , breakpoints , and gene gain sites as above into randomly chosen spacers in the simulated genome .', 'Each type of site was placed randomly and independently of the other types of site .', 'We then counted the numbers of spacers containing all possible combinations of types of site in the replicate .', 'Finally , we compared the observed numbers of coinciding sites in the real data to the distribution of results from the simulation ( Table 2 ) .', 'The proportion of simulated genomes in which the number of sites with a particular colocalization pattern matches or exceeds the observed number of such sites in the real genome is an empirical measure of the statistical significance of the observation , under the null hypothesis of a random distribution of sites .', 'We then applied a false discovery rate correction to these empirical P-values .']]
['Introduction', 'Results/Discussion', 'Methods']
['Comparative genomics can be used to infer the history of genomic rearrangements that occurred during the evolution of a species .', 'We used the principle of parsimony , applied to aligned synteny blocks from 11 yeast species , to infer the gene content and gene order that existed in the genome of an extinct ancestral yeast about 100 Mya , immediately before it underwent whole-genome duplication ( WGD ) .', 'The reconstructed ancestral genome contains 4 , 703 ordered loci on eight chromosomes .', 'The reconstruction is complete except for the subtelomeric regions .', 'We then inferred the series of rearrangement steps that led from this ancestor to the current Saccharomyces cerevisiae genome; relative to the ancestral genome we observe 73 inversions , 66 reciprocal translocations , and five translocations involving telomeres .', 'Some fragile chromosomal sites were reused as evolutionary breakpoints multiple times .', 'We identified 124 genes that have been gained by S . cerevisiae in the time since the WGD , including one that is derived from a hAT family transposon , and 88 ancestral loci at which S . cerevisiae did not retain either of the gene copies that were formed by WGD .', 'Sites of gene gain and evolutionary breakpoints both tend to be associated with tRNA genes and , to a lesser extent , with origins of replication .', 'Many of the gained genes in S . cerevisiae have functions associated with ethanol production , growth in hypoxic environments , or the uptake of alternative nutrient sources .']
['Genomes evolve in structure as well as in DNA sequence .', "We used data from 11 different yeast species to investigate the process of structural evolution of the genome on the evolutionary path leading to the bakers' yeast S . cerevisiae .", 'We focused on an ancestor that existed about 100 million years ago .', 'We were able to deduce almost the complete set of genes that existed in this ancestor and the order of these genes along its chromosomes .', 'We then identified the complete set of more than 100 structural rearrangements that occurred as this ancestor evolved into S . cerevisiae and found that some places in the genome seem to be fragile sites that have been broken repeatedly during evolution .', 'We also identified 124 genes that must be relatively recent additions into the S . cerevisiae genome because they were not present in this ancestor .', 'These genes include several that play roles in the unique lifestyle of this species , as regards the intensive production and consumption of alcohol .']
['genetics and genomics/microbial evolution and genomics', 'genetics and genomics/comparative genomics', 'computational biology/genomics', 'evolutionary biology/genomics']