Recurrent introgression and geographical stratification shape Saccharomyces cerevisiae in the Neotropics

Domain: chemistry
Published: 2026-02-21
Editorial outcome: major_revision

Abstract
From yeasts to humans, introgressive hybridization significantly influences the evolutionary history of living organisms by introducing new genetic diversity. Strains of Saccharomyces cerevisiae worldwide exhibit introgressions from the sister species S. paradoxus, despite the average sequence identity between these species being lower than 90%. While S. cerevisiae isolates from the Neotropics are known for their high levels of introgression, the hybridization events originating them remain unclear. Here, we sequence 216 S. cerevisiae isolates from open, spontaneous agave fermentation across Mexico. The genomes of these strains reveal considerable genetic diversity and population structure linked to geographic distribution, which had been overlooked due to undersampling of this megadiverse region. These strains, along with those from French Guiana, Ecuador, and Brazil, form a broader Neotropical phylogenetic cluster that is notably enriched in introgressions. Surprisingly, their origins and the observed conservation patterns of these introgressions indicate multiple hybridization events, suggesting flexible species barriers in this region. Our findings underscore concurrent evolutionary processes—geographical stratification and multiple introgressions—that shape the genomes of a diverse lineage of S. cerevisiae. Neotropical yeasts thus provide a natural laboratory for exploring the mechanisms and adaptive significance of introgressive hybridization in eukaryotic genome evolution.

Introduction
Beyond its long-established status as the most extensively studied eukaryotic-cell model in the laboratory, the budding yeast Saccharomyces cerevisiae (S. cerevisiae) has emerged as an outstanding model for studies in ecology, population genomics, and evolution1–5. Genomic analyses of thousands of isolates from natural and human-related environments around the world reveal that the species is structured in clades that broadly correlate with geography and isolation source6–8. These global genomic studies have also shown that most strains harbor genetic material from other Saccharomyces species, a phenomenon referred to as genetic introgression. For instance, most S. cerevisiae isolates from around the world harbor genes introgressed from its sister species Saccharomyces paradoxus (S. paradoxus)6,9–12, even though these species diverged 4–6 MYA and have an average nucleotide divergence of ~14%13,14.

The neotropical region, which extends from northern Mexico to South America, is biogeographically relevant due to its rich biodiversity15. Some of the S. cerevisiae strains from this area group in a monophyletic branch referred to as a complex clade by Pontes et al.7. This branch aggregates four groups of strains: Mexican agave (MA), French Guiana (FG), Wild Brazil 3 (WB3) and South American mix 2 (SAM2). While WB3 strains were isolated from natural areas, most of the strains from this Neotropical cluster were isolated from anthropogenic environments. Considering the vast area encompassed by this region, the few available sequenced isolates likely provide an underestimate of genetic diversity, thus limiting our understanding of the species in this part of the world.

The handful of sequenced genomes available indicated that the S. cerevisiae Neotropical cluster harbors an unusually high number of introgressions from S. paradoxus6,7,12,16,17. This could explain the high divergence of the cluster when compared to other clades. A fraction of the introgressions from the Neotropical cluster has been associated with populations of S. paradoxus from the Americas6,12,16, which contrast with the origin of the introgressions in the European lineages6,18. It has even been suggested that the introgressions in Brazilian strains derive from two different S. paradoxus populations, raising the possibility of multiple hybridization events16. However, the precise origins of introgressions remained unclear in part due to the limited number of sequenced S. cerevisiae and S. paradoxus genomes available from the region. In Mexico, there are reports of S. paradoxus consistently at low frequency in agave fermentations19–23, including a lineage that coexists with S. cerevisiae in the agave distillery environment and that has not been considered by previous studies23. This lineage could be a potential donor for introgressions in the tropical Americas. Comprehensive understanding of both the vertical evolution of S. cerevisiae and the origins of its introgressions is essential to accurately determine the dynamics of interspecies gene flow in the region.

To explore the genomic diversity of S. cerevisiae in the tropical Americas—particularly in light of its strong signatures of introgression—we sequence 216 strains primarily isolated from traditional agave fermentations in Mexico. These spontaneous fermentations are anthropogenic, open systems that rely on microorganisms from the surrounding, often natural, environments22,23. Previous studies have highlighted the megadiversity in this region24 as well as the high diversity of S. cerevisiae in agave fermentation25–28, but whole-genome sequencing efforts have only included nine strains, mostly from a single agave fermenting region6. We show that most of the newly sequenced isolates belong to the Neotropical cluster, revealing new clades where diversity correlates with the geographic origin of the strains. Moreover, by analyzing the patterns and origins of the introgressions in the context of the relatedness of these lineages, we identify multiple introgression events in their evolutionary history. Notably, some of these introgression pulses are unique to strains isolated from agave fermentations. Our refined phylogeny allows us to illustrate how recurrent episodes of gene flow between two sister Saccharomyces species have unfolded in a megadiverse region of the world.

Results
To provide a comprehensive view of S. cerevisiae diversity in one of the most biodiverse regions in the world15,24, we sequenced the genomes of 216 isolates primarily isolated from traditional agave distilleries across Mexico (Supplementary Fig. 1 and Supplementary Data 1). This anthropogenic fermentation environment provides a good starting point to study yeast diversity, as spontaneous fermentations have taken place for over 3500 years in a wide variety of ecosystems, latitudes, and cultural contexts22,29,30. Specifically, 211 of the sequenced strains were isolated from agave fermentations collected through various sampling efforts conducted across the country between the years 1988 and 202120,23,25,26,28,31–33. Four of the five remaining isolates were from traditional fermentations of other substrates—pulque (fermented raw agave sap), apple cider, cactus pears, and cooked stems of sotol plants (Dasylirion spp.)—while the fifth was an uncharacterized commercial yeast strain used in one of the sampled distilleries; all of them were isolated in Mexico (Supplementary Data 1). With the 216 genomes (median depth coverage = 163X), we assessed the phylogenetic position and population structure of the strains in the context of the known diversity of the species, and we also analyzed the dynamics and origins of the prevalent S. paradoxus introgressions within these populations.

Agave fermentation strains belong to a genetically diverse Neotropical cluster
To assess the relatedness of the agave S. cerevisiae strains with other isolates of the species, we first performed multidimensional scaling analysis (MDS) with 1262 genomes, including the 216 newly sequenced strains. Most isolates from Mexico clustered next to other strains from the tropical Americas, such as those from the FG, the WB3, the SAM2, and the Mexican agave 1 (MA1) (formerly named “MA”) clades (Fig. 1a). The majority of the strains isolated from the tropical Americas form a consolidated group, separated from other clades of the world (Fig. 1a), a pattern that persisted even when strains from agave fermentation are not overrepresented in the MDS analysis (Supplementary Fig. 2). Only eleven of the sequenced strains here reported grouped with clades outside the Neotropical cluster. Meanwhile, all four isolates that we sequenced from non-agave substrates clustered with the agave-fermentation strains from the Neotropics.

To establish the phylogenetic relationships of the newly sequenced isolates, we compared them to a reference panel of strains (see Supplementary Data 2). Consistent with the MDS analysis, the phylogeny showed that 205 of the 216 newly sequenced strains (94.9%) were part of a monophyletic group that included two newly described clades, Mexican Agave 2 (MA2) and Tequila Distillery, along with the previously defined clades MA1, FG, WB3, and SAM2 (Supplementary Fig. 2). The genetic differentiation of MA1, MA2, WB3, and FG was supported by weighted Fst analyses (Supplementary Fig. 2). We refer to this higher hierarchy clade as the Neotropical cluster of S. cerevisiae, as nearly all the strains in this group come from the tropical Americas (Fig. 1b). Among the remaining sequenced isolates, five grouped with the North American Oak clade, which are commonly classified as wild strains6,8,34. Another five isolates—including the commercial strain—were closely related to the Mixed Origin clade and only one clustered with Wine strains.

The Neotropical cluster consistently exhibited a relatively long branch in the phylogeny, encompassing all strains from six clades: MA1, MA2, FG, WB3, Tequila Distillery, and SAM2. The long branch was supported by the high number of single nucleotide variants (SNVs) present in the strains of these clades (Fig. 2). In some of the phylogenetic reconstructions, the inclusion of the mosaic strains of Tequila Distillery and SAM2 changed the relative placement of MA1, MA2, WB3, and FG (Supplementary Fig. 2). These and other mosaic strains were excluded from the main phylogeny (Fig. 1b) to avoid artefactual topological shifts driven by admixture. To determine whether the extended branch lengths observed in the Neotropical cluster were driven by introgressed regions, we generated the phylogeny excluding introgressions (Methods). Removal of these regions had no impact on the overall topology of the phylogeny and exhibited only a modest effect on the branch lengths of the Neotropical cluster (Supplementary Fig. 2). The number of SNVs of the neotropical groups slightly diminished when introgressed regions were removed (Fig. 2). In contrast, removal of introgression markedly reduced branch lengths and SNV counts in the Alpechin clade, a group of European strains that harbors a high number of S. paradoxus introgressions. To assess whether the observed long branches were attributable to sampling bias of strains, we constructed the phylogenies using random downsampling to a maximum of five strains per clade (Supplementary Fig. 2). Resulting phylogenetic topologies consistently showed the same position and relatively long branches of the Neotropical cluster, although the more balanced dataset did slightly reduce the relative branch length (Supplementary Fig. 2), consistent with a previous report34. Collectively, these results highlight the robustness of the clades within the Neotropical cluster and its characteristically long branch, consistent with previously reported global phylogenies of S. cerevisiae6,8.

As mentioned above, three clades could be distinguished among the newly sequenced isolates from agave fermentations in neotropical S. cerevisiae. First, we identified a group of 20 isolates from northeastern Mexico, which included the seven strains previously reported as “Mexican Agave”6, herein referred to as the MA1 clade (Fig. 1b). It is worth mentioning that the monophyletic MA1 clade comprises so far only strains isolated northeast of the Sierra Madre Oriental, a mountain range which runs parallel to the coast of the Gulf of Mexico and acts as a natural barrier, exerting strong influence on the climate and biodiversity on either side of the range35,36. As a result of the comprehensive sampling of the isolates reported here, we identified a novel MA2 clade that is closely related to MA1 (Fig. 1b). Most of the newly sequenced strains in this study (81.9%) belonged to MA2 clade. Two previously sequenced strains isolated in Mexico6 formerly assigned to the SAM2 clade12, were reclassified after these analyses within the new MA2 clade. The third group of agave isolates was composed of 15 strains, most of them with admixed genotypes, all sequenced in this study (Fig. 1b and Supplementary Fig. 2). We refer to this group as “Tequila Distillery”, since eleven of the 15 strains were isolated from the tequila industry.

Our results show that most strains from spontaneous agave fermentations are part of the Neotropical cluster, revealing that the group previously described as “Mexican Agave” represents a fraction of the genetic diversity present in agave fermentations. Furthermore, the clades within this cluster, including the newly identified groups, rank among the highest in terms of SNVs when compared to the reference genome, surpassed only by the wild Asian clades (Fig. 2 and Supplementary Fig. 3). This trend persisted when pairwise identity by sequence was quantified (Supplementary Fig. 3), indicating that it is independent of the specific reference genome used. The high genetic divergence of the neotropical strains was recently supported by a pairwise comparison of 3334 S. cerevisiae genomes, which revealed that the greatest pairwise genetic distance within the species occurs between a wild Taiwanese isolate and a Mexican strain from agave8. Together, our phylogenomic and population analyses of S. cerevisiae from the tropical Americas bridge the gap of genetic knowledge for the species in this region.

Neotropical yeast populations are stratified by geography
Yeasts from the tropical Americas come from a diverse variety of environments across a broad range of geographical distances. We thus asked to what extent geographic parameters influence the population structure of S. cerevisiae in the Neotropical cluster. To this end, we carried out an ADMIXTURE analysis, selecting the number of populations that best explained the genotypes based on the lowest cross-validation error (Fig. 3a and Supplementary Fig. 4). The population structure analysis revealed eleven ancestral components within the Neotropical cluster. Specifically, strains from the MA1, Tequila Distillery, FG, and WB3 clades each formed distinct populations, while the remaining seven populations were all from the newly identified MA2 phylogenetic clade.

Given the stratification observed in the Neotropical cluster, we analyzed the geographical distribution of these isolates according to their population structure. The eleven colors in the ADMIXTURE plot representing ancestral components within the neotropical cluster showed good agreement with their geographic distribution (Fig. 3a). This geographic stratification was observed at two levels of resolution. First, each of the five clades of the Neotropical cluster are constrained to specific regions in the map. Second, at the resolution within the MA2 clade, the seven colors in the ADMIXTURE were also stratified by geography. In general, strains from the same agave-spirit producing region grouped together in the phylogeny and had the same ancestral population component. This was also demonstrated by a correlation between geographic and genetic distances in this set of strains (r=0.18, significance 0.0001 with 9999 permutations, Spearman Mantel test, one-sided, including MA1 and MA2 strains). We also observed increasing genetic diversity and heterozygosity from north to south within the MA2 clade (Supplementary Fig. 5). Together, these findings suggest that the genomic diversity in the Neotropical cluster is influenced by geography.

Despite the overall agreement between geography and population structure in the strains from the tropical Americas, there were exceptions suggesting gene flow within and even beyond the Neotropical lineage. For instance, a strain isolated from a spontaneous agave fermentation shared close ancestry with wild isolates of the North American Oak clade, while other four isolates closely related to the North American Oak clade showed signs of admixture with different genetic components of the neotropical cluster (Fig. 3a). The 15 WB3 showed little admixture, while the SAM2 strains had genetic components of MA2, FG, and WB3 populations, which is not surprising since they are closer relatives. Moreover, ten strains from the Tequila Distillery group showed admixture, mostly between different neotropical populations, but also with other clades, such as Wine. Taken together, these findings suggest that S. cerevisiae populations in the tropical Americas have diverged, at least partially, through geographic isolation, while other ecological processes or human-related factors have promoted gene flow, also shaping the genetic structure of the population.

We used the genomic data to further explore the patterns of genetic diversity in the neotropical strains. Each clade differed significantly in nucleotide diversity (π), Tajima’s D, and heterozygosity (Fig. 3b–d and Supplementary Fig. 6), with some exhibiting high genetic diversity despite most of their strains being isolated from anthropogenic environments. For instance, the MA1, MA2, and Tequila Distillery clades all exhibit higher nucleotide diversity than the Wine clade and even than the highly introgressed Alpechin clade (Supplementary Fig. 6). These findings suggest that the elevated genetic diversity observed in the agave-related clades cannot be attributed to introgressions alone. In contrast, the FG clade showed low genetic diversity and a homogenous degree of heterozygosity. The Tequila clade displayed the highest levels of heterozygosity and nucleotide diversity, partly due to the admixed genotypes of its strains, and consistent with trends observed in domesticated beer isolates6. Taken together, our population-structure and genetic-diversity analyses indicate that yeasts in the tropical Americas constitute diverse, structured populations stratified by geography.

Differences in the distribution of introgressions reveal recurrent interspecies gene flow
The genomes of S. cerevisiae strains within the Neotropical cluster are characterized by an atypically high number of introgressed regions from S. paradoxus12,16. To gain insight into the dynamics of gene flow, we identified the set of introgressed genes with a strategy that uses competitive mapping of sequencing reads against both parental genomes and phylogenetic analyses (Methods). Importantly, most of the introgressions that we identified were also found using other strategies, including: (i) genome assembly followed by a phylogenetic approach6, (ii) calling bona fide single-nucleotide diagnostic markers of introgressions12 or (iii) filtering orthogroups after competitive mapping16 (Supplementary Fig. 7).

All Neotropical clades showed more introgressed genes than the other groups worldwide, except for the Alpechin clade (Fig. 4a). The three clades from South America (WB3, SAM2, and FG) displayed fewer and shorter introgressions with a lower fraction of heterozygous introgressed genes, compared to the three agave clades (Tequila Distillery, MA1, and MA2) (Fig. 4a and Supplementary Fig. 8). Here, we call an introgressed gene heterozygous when both S. cerevisiae and S. paradoxus alleles are present within the same genome (Fig. 4a; Supplementary Figs. 8 and 9).

Among the Neotropical groups, we found that MA1 isolates exhibited both the highest number and the widest range in the number of introgressed genes, with counts going from 94 to 320 per strain. MA2 isolates featured between 64 and 122 introgressed genes (Fig. 4a). Moreover, we identified a subset of strains from the MA1 clade with more than 150 introgressed genes exhibiting longer and more heterozygous introgressions than most strains of the clade (Fig. 4 and Supplementary Fig. 8).

To infer the history of introgressed genes, we analyzed their retention patterns both within and between clades. We observed that the presence-absence patterns clustered the three agave associated clades together and clearly separated them from a second cluster comprising the three South American groups (SAM2, WB3, and FG. Fig. 4b; Supplementary Data 3 and Supplementary Fig. 9). Despite the differences between these two clusters, we identified significant overlap among most of the comparisons between subsets of the Neotropical clades (Supplementary Data 3), including five genes that are common to all six Neotropical clades. This number of shared introgressed genes exceeded the number expected by chance (hypergeometric test, one-sided, p=0.00234; Supplementary Data 3). This is likely explained by an introgression pulse in the common ancestor of all Neotropical strains although strong selection could also account for the presence of this shared set of introgressed genes. Yet, no significant GO enrichment was detected among the genes present in at least 75% of strains in each clade. As expected, analysis of the presence–absence patterns of introgressed genes in the Alpechin clade, used as a reference, revealed no significant overlap with the neotropical groups, confirming the previously reported independent hybridization event underlying this clade12,18.

Given the complex introgression patterns observed in the MA1 clade—multimodal distribution of number of introgressed genes (Fig. 4a), the presence of large heterozygous introgressed segments (Supplementary Fig. 8) and an unusually high number of genes exclusive to this clade but present at low frequency within it (Fig. 4b)—we examined the retention patterns alongside the phylogenetic relationships of the MA1 strains (Supplementary Fig. 9). While most strains shared the majority of their introgressed regions with closely related isolates, seven MA1 strains exhibited heterozygous introgressed segments that were exclusively found within restricted phylogenetic groups. This pattern could be explained by multiple convergent losses of the same introgressed genes in other branches of MA1 and in MA2, or more likely, by at least one additional interspecies gene flow event from S. paradoxus that occurred specifically in a subset of MA1 strains.

Introgressions in the Neotropical cluster trace to different lineages of S. paradoxus
To trace the origins of the observed introgressed regions, we inferred the most likely S. paradoxus lineage associated with each gene. To do so, we assessed the identity by sequence of each introgressed block against a panel of S. paradoxus genomes with representative strains from the Americas. This panel includes a diverse set of strains representing the previously defined populations from North America (SpA, SpB, SpC, and SpD), as well as a Brazilian SpB strain (SpB_Bra), along with representative strains from other established clades from China, Far East Asia, Hawaii and the reference strain from the European lineage (here grouped with SpA). Moreover, we included all sequenced strains from Mexico, encompassing three lineages exclusive to this region: SpB_MxAgave, SpB_Mx1 and SpB_Mx223. Our results indicate that most introgressed genes in the MA clades originated from SpB_MxAgave (Fig. 5a), a population found exclusively in agave distilleries in Mexico. A smaller fraction of introgressed genes in these strains appear to derive from the SpB_Mx2 and the broader SpB lineage. In contrast, the majority of introgressed genes in the South American strains were traced to the SpB lineage, which is commonly found in natural environments in the Neotropics but has not been reported in agave fermentations23. As expected, introgressed genes in the Alpechin group most closely resembled those from the European lineage, previously identified as the donor population in this clade (Supplementary Data 4)12,18,37. These findings indicate that at least two lineages of S. paradoxus have hybridized with ancestors of the extant neotropical strains, highlighting the role of interspecies interactions in shaping the evolutionary trajectory of S. cerevisiae in the region.

Methods
Genomes sequencing and variant calling
DNA was purified with the MasterPure DNA purification kit as recommended by the manufacturer, and was sequenced using the DNBSeq (2×150 bp; BGI, China). Raw reads were quality assessed with fastp V0.20.046. For each isolate, the filtered reads were aligned with BWA-MEM v0.7.447 to a concatenated species reference genome that includes S. cerevisiae S288C, S. paradoxus YPS138, S. mikatae IFO1815, S. kudriavzevii Cr85, S. jurei M1, S. arboricola H6, S. uvarum CBS7001, S. eubayanus FM1318, K. marxianus DMKU31042, and P. kudriavzevii CBS573. Duplicated reads were marked with Picard 2.6.048, and local realignment around indels and variant calling were performed with GATK v4.1.1.049. Allele balance information was incorporated in the VCF files with the GATK Variant Annotator. The called genotypes were filtered to keep only biallelic SNPs with a minimum depth of 5, a QUAL score of at least 30 and positions called in at least 90% of the isolates. Additionally, SNPs were also obtained by mapping the filtered reads only to the S. cerevisiae reference genome to call variants taking into account introgressed segments (Supplementary Figs. 3 and 4).

Multidimension scaling analysis
Genome-wide SNP data were used for multidimensional scaling (MDS) analysis with PLINK v1.950. A total of 1262 isolates were selected to provide a global overview at the species level, including 1011 from Peter et al.6, 216 newly sequenced in this study, 33 strains from previous sequencing efforts in the Neotropics (Barbosa et al.16, Barbosa et al.40) and four additional sequences from South America-associated lineages (SAM, Tellini et al.12). SRA Toolkit v3.0.0 was used to retrieve FASTQ files corresponding to samples other than those sequenced in this manuscript. Two sequences were excluded from Fig. 1a due to redundancy with other entries detected with PLINK50. Only nuclear SNPs were considered, and low-frequency or rare variants were excluded from this analysis using a threshold of missing call rate > 1%. After the application of this filter, isolates with missing genotypes > 10% were discarded. The quantitative indices (components) of the genetic variation for each isolate were calculated on the genome-wide average proportion of alleles shared between any pair of individuals within the sample. To perform the MDS analysis with equal representation across clades (Supplementary Fig. 2), we followed the same steps described above, but using only up to five randomly selected isolates from each phylogenetic clade described in the following section.

Phylogenetic analysis
Maximum-likelihood trees were constructed using genomes sequenced in this study along with global reference strains, with particular emphasis on the American clades. Specifically, in the supplementary complete phylogenetic analyses (Supplementary Fig. 1), we included 487 strains distributed as follows: all 216 newly sequenced strains generated in this study, 23 sequences from Barbosa et al.16, 10 isolates from Barbosa et al.40, four South American isolates (SAM) from Tellini et al.12 and at least one representative of each clade defined by Peter et al.6 (n=213). To improve resolution within lineages containing agave-associated isolates, we deliberately overrepresented strains from the Mixed Origin, Wine, and North American Oak clades from Peter et al.6. Additionally, we incorporated 21 Alpechin isolates from Pontes et al.37, as a reference to determine the effects of extensive introgression content. The 487 strains are indicated in the column Used_in_Supplementary_Complete_Phylogenies of Supplementary Data 2. For the primary phylogeny depicted in Fig. 1b, we used the same strain set but excluded admixed individuals. Only 332 strains exhibiting >85% of a predominant ancestral component as inferred from the ADMIXTURE analysis were retained for this tree. The 332 strains are indicated in the column Used_in_Main_Phylogeny of Supplementary Data 2.

To generate the phylogenies, haplotype sequences were obtained with vcf2phylip v2.351 from the nuclear genomic variants that mapped to the S. cerevisiae subgenome from the alignment to the concatenated species genome or, when indicated, directly to the S. cerevisiae reference genome to include variants from introgressions. The phylogenetic trees were inferred using IQTree 2.3.6 with the best model calculated with -m MFP and adding the +ASC parameter with 1000 ultrafast bootstraps and aLRT (aLRT shown for key branchings in Supplementary Fig. 2). Phylogenies were also reconstructed using RAxML v8.2.1253 (raxmlHPC-PTHREADS-AVX2) with the GTR+GAMMA model and 100 bootstrap replicates. Since the results were similar with the two methods, only IQTree trees were used for the figures of the manuscript. The visualization of the trees was performed using Microreact v.28254. To generate the trees with equivalent number of strains per clade (Supplementary Fig. 2), we randomly selected up to five isolates from the phylogenetic clades included in the set of 332 genomes and built the phylogeny with IQTree 2.3.6 with the ASC parameter.

ADMIXTURE
For the ADMIXTURE analysis, we analyzed a dataset comprising 466 strains centered on the newly sequenced agave-associated isolates, as indicated in the column Used_in_ADMIXTURE of Supplementary Data 2. This included all strains sequenced in the present study (n=216), along with 213 representative genomes of strains from Peter et al.6 ensuring coverage of all clades and inclusion of 20 strains without clade assignment. In addition, to capture phylogenetic and introgression diversity, we incorporated 23 strains from Barbosa et al.16, 10 strains from Barbosa et al.40 and 4 strains reported by Tellini et al.12 that belong to the SAM lineages. To enhance resolution within clades where agave-associated strains clustered, we intentionally overrepresented the Mixed Origin, Wine, and North American Oak lineages in the selection from Peter et al.6. To run ADMIXTURE 1.3.055, the VCF with 466 genomes was curated using PLINK 1.950 to remove linkage disequilibrium with parameters previously used for S. cerevisiae56 (50 window size, sliding every 5 nucleotides and correlation of 0.5). Curation led to a final set of 618,253 SNVs. Ten seeds were used to run ADMIXTURE. The cross-validation (CV) error was obtained for models with K=2 to K=35. The lowest median CV error K was 24, and therefore it was used for Fig. 3a. Visualization was made in Pong 1.4.957. The R package vegan (2.7-2)58 was used to perform the Spearman Mantel test.

Estimation of genetic diversity
To calculate genetic diversity (π) and Tajima’s D, we used VCFtools 0.1.1459 and the VCF with variants that mapped to S. cerevisiae after mapping to the concatenated reference and filtering high-quality biallelic SNPs with less than 10% of missing data. Per variant heterozygosity was estimated using BCFtools 1.960 on individual strain VCFs to calculate the ratio of the number of heterozygous variants by the number of variants.

Analysis of introgressions
To identify introgressed genes, we obtained the regions that mapped to the S. paradoxus genome when the alignment was performed to the concatenated reference. A gene was considered introgressed if over half of it had a coverage depth exceeding 25% of the median depth of the entire reference. Additionally, it had to be the ortholog with the highest depth or, if not, its depth had to be at least 25% of that of its ortholog in the S. cerevisiae subgenome. A minimum 5% nucleotide-level difference between the orthologs of S. cerevisiae and S. paradoxus was also a requisite. An introgressed gene was considered heterozygous if the ratio of the depth coverage between the two subgenomes in the competitive mapping was between 0.25 and 4.

After passing these mapping thresholds at the gene level, we grouped consecutive genes in blocks according to the annotation61 and assessed the phylogenetic position of each block with respect to a panel of two S. cerevisiae (Wine ERS1082532 and Taiwanese ERS1082750) and five S. paradoxus (SpA SRR4074385, SpC SRR7500262, SpB SRR4074411, SpB_Bra SRR4074412, SpB_MxAgave YMX005537) genomes with IQtree. Using ape 5.062, we only kept as true introgressions those blocks that grouped with S. paradoxus in the phylogeny.

Gene Ontology enrichment analysis was performed using the GO term finder tool from the Saccharomyces Genome Database. Introgressed genes present in at least 75% of strains within each clade were used as the query set, with no background set specified. Using the default parameters, no enriched terms were found or were too broad to provide any meaningful biological insights.

Once the introgressed blocks were determined, to infer the origin of introgressions, we mapped the full panel of S. paradoxus genomes from López-Gallegos et al.23 to the concatenated reference genome that includes both S. cerevisiae and S. paradoxus. We retained only the S. paradoxus subgenome. We filtered the resulting VCF to retain only regions previously identified as introgressed from S. paradoxus into S. cerevisiae strains. Using this VCF containing introgressed regions in S. cerevisiae and the corresponding S. paradoxus variants, we calculated pairwise identity-by-state (IBS) values using PLINK v1.950. For each introgressed region, we assigned its likely origin by identifying the S. paradoxus clade containing the strain with the highest IBS. When introgressed regions showed equally high similarity (IBS) to strains from multiple S. paradoxus lineages, no origin was assigned. We report the origin of introgressed regions as determined by the closest clade based on IBS similarity, as this approach leveraged the most extensive dataset, including all S. paradoxus strains from López-Gallegos et al.23, and provides improved resolution for assigning the origin of a larger number of introgressions. This assignment is concordant with results obtained using alternative metrics, including identification of phylogenetic siblings and assignment to the clade of the strain with the shortest phylogenetic distance (Supplementary Data 4, columns Closest_Clade_by_IBS_Similarity, Siblings_in_Phylogeny, and Strain_with_Lowest_Distance_in_Phylogeny). All the genes within a given block were considered to be from the same origin.

Map generation
Maps were generated in R using the ggplot2 4.0.063. World coastline and political boundary data were obtained from the Natural Earth dataset via the rnaturalearth package 1.1.064.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Avelar-Rivas et al present a study into the genomic characteristics of Saccharomyces cerevisiae strains isolated from the\nNeotropics via re-sequencing of 216 isolates. They define a new clade \"Mexican Agave 2\" and map numerous\nintrogressions from Sacharomyces paradoxus across the genomes of many of neotropic strains.\nThe expanded repertoire of isolates has expanded the catalogue of strains from this region. However the focus on the\nwidespread introgressions from S. paradoxus largely recapitulates findings from previous large-scale de novo sequencing\nefforts, such as those of Peter et al 2018 and D’Angiolo et al 2020, which defined the Alpecin, Mexican Agave and French\nGuiana lineages as displaying high degree of introgression from this sister species.\nGiven that the authors have used a mapping-based approach to defining introgression, more detailed explanation of the\nmethodology (with figures where appropriate) would be useful for determining the robustness of the predictions relative to\nthe a gene-prediction + phylogenomics approach. Direct comparison to data from papers such as Peter et al 2018 could be\nused to ground-truth the mapping-based methodology.\nLikewise, the application of introgressed gene blocks to specific S. paradoxus strain origin should be supported by a gene-\nwise phylogenetic approach rather than the distance-based methodology applied.", "reviewer_2": "This relevant study is based on the investigation of circa 200 isolates of S. cerevisiae collected from agave fermentations in\nMexico and is centered of the characterization of multiple and independent introgressions originating in the sister species S.\nparadoxus. I find this investigation well conduced and significant as the natural history and domestication of S. cerevisiae in\nthe Neotropics is poorly understood. Moreover, the pervasiveness of S. paradoxus introgressions in S. cerevisiae found in\nthe Central and South America, either in wild or in domesticated populations, is unique and understanding its causes is of\nrelevance as wild populations in other regions lack such introgressions. Although this study does not solve the question of\nwhy are introgressions found in Neotropics but not in other regions, it is a significant contribution to the topic. Below I list my\ncomments and questions that I hope the authors find pertinent.\nMain\n1. I wonder why the authors characterize the agave fermentations as “a habitat at the interface of natural and industrial\nenvironments” (lines 8-9). What are exactly the characteristics of this fermentation that separate it from other fermentations?\nFor example, are spontaneous wine fermentations also at this interface? If so, is wine must a natural substrate? I believe that\nthe distinction between wild and artificial environments should be made very clear from the onset of the paper, and I see no\nreason not to consider agave fermentations as a typical artificial environment created by humans. Also, why using “habitat”\nin this context (see also line 24)? In order to demarcate clearly wild and artificial substrates, perhaps “habitat” could be\nrestricted to the context of natural settings and a more neutral expression like “environment” could be used in situations\nrelating to artificial man-made environments.\n2. The authors use the expression “neotropical cluster” (line 37) for designate the three S. cerevisiae lineages from this\nregion. I agree that treating these lineages like this makes sense, but I miss a more thorough dissection of this group. I\nbelieve it would be important to emphasize (or at least mention) that two of these lineages are domesticated and one (Wild\nBrazil 3) is natural. That is relevant for several reasons, most importantly for discussing the occurrence of introgressions both\nin wild and in domesticated lineages. To add to this, the authors even state (line 94) “…distinguishing strains from the\nNeotropics (blue) from wild (green) and domesticated (red)”, implying that the strains from the Neotropics are neither wild or\ndomesticated, which is odd.\n3. The species “evolutionary history” needs to be studied on wild isolates, which is not the case in this study. I recommend\nreframing the way the study is presented as discussed. The following sentence (line 38) exemplifies my point “Considering\nthe vast area encompassed by this region, the few available sequenced isolates likely provide an underestimate of genetic\ndiversity, thus limiting our understanding of the species evolutionary history in this part of the world”.\n4. In lines 34-38 (see also line 86) the authors introduce three previously recognized neotropical lineages, that they refer to\nas Mexican Agave (MA), French Guiana (FG), and South American Mix 2 (SAM2). This last designation corresponds to an\nearlier designation, Wild Brazil 3 (see reference 17). I wonder why the authors preferred to use a designation that is not only\nsuperfluous but also confusing. In fact, “Mix” is normally used in the context of S. cerevisiae populations to label admixed\nlineages (see reference 6 for several examples). It is therefore unfortunate that the authors choose to associate a name that\nimplies admixture to a non-admixed population. This confusion is even exposed by the authors in the construction and\nlegend of Fig 1C in which the Wild Brazil 3 (SAM2) population is treated as admixed (see also lines 116, 122), which is\nfalse.\n5. I understand the interest in linking S. paradoxus with agave fermentations, but when checking reference 18 it is difficult to\nfind evidence for this. The reality is that for more than 1500 S. cerevisiae isolates one a single S. paradoxus isolate was\nfound. I think it is difficult to argue that S. paradoxus is present is agave fermentations and I think this part need to be turned\ndown. Moreover, is reference 19 adequate in this context?\n6. Fig.1A reports on 1264 genomes but table S1 lists only the 261 agave fermentation genomes. Moreover, Fig 1C reports\non 464 genomes. If they are included in table S2 this should be made clear by identifying which genomes were used in each\nanalysis. Moreover, how were these different subsets chosen? Was there a preoccupation in representing all known\npopulations? Why so many wine strains? Care much be applied because population structure analyses can be distorted by\nover-representing / neglecting individuals from different populations. The separation seen in figure 1A appears exacerbated\nand in fact cannot be confirmed. I would suggest that it oversimplifies the problem by showing a continuum from wine to wild\n(China?) isolates, with agave isolates as the only ones escaping this continuum. Moreover, the text in line 88 “The rich\ndiversity of the newly sequenced strains is evident, as strains from the tropical Americas encompass nearly half of the global\ndiversity” appears to ignore the fact that most diversity resides in wild oak-associated strains and that the agave strains\n(either those already known or the new ones, are domesticated and therefore are not expected to be considerably diverse). I\nam convinced that a less spectacular but probably more truly representation would not diminish the current study (and this is\ntrue for the entire perspective of the paper).\n7. Fig. 1B. This representation might the consequence of the sampling with a limited number of wild strains (again, no\ninformation on the origin of the strains used), a single known domesticated group (wine but not beer1 and 2, bread, sake,\nAsian beverages, etc) and an over-representation of agave strains.\n8. Fig 1C. Again, I wonder if the phylogeny is representative of the known phylogeny of the species, i.e. includes the relevant\nlineages. My point is that only the MA2 is a new addition, and previous phylogenies did not show the MA1 and FG in such a\ndistant position from the rest of the species lineages.\n9. Line 293. This new population or strain SpB_Mx needs to be presented. The reader also needs to be introduced to the A,\nB and B* populations.\n!0. The Wild Brazil 3 population is missing in the study of Fig. 4.\nMinor\n1. In this sentence (line 36-37) “Notoriously, the handful of sequenced genomes available to date indicate that this\nNeotropical cluster harbors an unusually high and heterogeneous number of introgressions from S. paradoxus”, the first\nstudy revealing these introgressions (ref 17) is not mentioned.\n2. Populations should be designated in a consistent manner throughout the paper – line 91 wild North American”; line (119)\n“wild North America Oak clade”;\n3. In Figure 1C and in the text what is the relevance in separating the Mexican agave strains in two subgroups? Wouldn’t it\nbe preferable presenting the strains from this fermentation as a single group, given its technological utilization, etc? For\nfuture studies having a single population regognized as originating form Agave fermentations might be advantageous. Think\nof th “wine” population and of what it represents for the understanding of S. cerevisiae biology and of the irrelevance of\nsubdividing it (currently being attempted by several authors).\n4. In line 148 “…revealing that what was previously described as “Mexican Agave” represents only a 148 minor fraction of\nthe great genetic diversity found in agave fermentations” this statement appears to be too strong (see comments above) as\nthe new agave strains are numerous but not vastly different from those already known.\n5. Figure 2. Why K=24?\n6. Line 194 “Likewise, there were SAM2 strains showing genetic components of the MA2 and French Guiana clades”. This is\nnot surprising since SAM2 (Wild Brazil 3) is the closest wild relative of the domesticated MA2 and French Guiana clades.\nThis could be added to the sentence.\n7. Line 204-205. Same for WB3.\n8. Line 233-235. I could not follow the reasoning as it appears to contradict what was stated in the lines before. Please clarify\nwhy this cannot be a recent introgression event.\n9. Line 279. The alpechin clade has a totally distinct introgression history that occurred in Europe and involved European S.\nparadoxus. Thus, it appears not relevant for this comparison.\n10. Line 294. “not commonly found” or not found at all?\n11. Line 320. With respect to anthropogenic environments the authors appear to have missed this study\nhttps://doi.org/10.1093/gbe/evy132.\n12. Line 327. Is it possible to conceive an alternative scenario in which there was a single pulse but MA1 did not lose as\nmany paradoxus genes as MA2?\n13. Line 331. Was any attempt made to investigate the pool of introgressed genes with respect to function?\n14. Line 342. I think it is important to focus on sampling natural environments rather than anthropic ones (see previous\ncomments on how to address the “true” natural history, rather than the recent domestication history.\n15. Line 349. Was the actual genetic divergence calculated?\n16. Line 397-398. Why not use IQ-TREE with the ‘ASC’ parameter associated with the best model inference? This takes into\naccount that the dataset is SNPs and not coding sequences/proteins.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "ITEMIZED RESPONSE TO REVIEWERS\nManuscript ID NCOMMS-24-72197-T\nTitle: \"Recurrent introgression and geographical stratification shape Saccharomyces cerevisiae\nin the Neotropics\" by Avelar-Rivas et al.\nWe thank the editor and the three reviewers for their thorough evaluation and constructive\nfeedback. We were encouraged to learn that our findings were found relevant and that a revised\nversion of our manuscript may be suitable for publication. While the main findings of the study\nremain unchanged, specifically the identification of recurrent introgression events from S.\nparadoxus in distinct S. cerevisiae lineages, these conclusions are now more strongly supported\nthrough expanded analyses, clearer methodological descriptions, more cautious interpretation of\nresults, and improved contextualization. These analyses required the contributions of two new\nmembers of our groups that are now included as co-authors. Overall, the integration of the\nreviewers’ suggestions has resulted in a more rigorous and clearly presented manuscript.\nThe revised manuscript includes the following major changes:\n1. Multiple down sampling analyses to address potential sampling biases in MDS and\nphylogenetic reconstructions, consistently confirming the phylogenetic position of the Neotropical\ncluster. These results are now included in the revised manuscript and new panels in Figure S2.\n2. Incorporation of new analysis showing SNV counts relative to the reference genome across\nstrains, to more accurately report the divergence and genetic diversity observed in the Neotropical\ncluster, which highlights that Neotropical strains rank among the most divergent after wild isolates\nfrom Taiwan and China (new Figure 2 and new Figure S3).\n3. Better description of the mapping-based pipeline and incorporated a phylogenetic validation\nstep. To identify the origin of introgressed segments, we used identity-by-sequence (IBS) to\ndetermine the most similar potential donor, leveraging the full panel of S. paradoxus strains to\nmaximize resolution (Figure 5A, Data S1).\n4. A more detailed characterization of the set of introgressed genes among the different clades,\nemphasizing the patterns that support an independent introgression pulse in MA1 (Table S3 and\nFigure S9).\n5. A more thorough description of the composition of strain sets used in each analysis (Figure\n1, Table S1, Table S2).\n6. Reviewer #2 raised relevant concerns about the reference genomes used to trace the origins\nof S. paradoxus introgressions. We note that, during the time of this revision, the results and\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\ngenome data from a separate project by our group (focused on the ecology of Saccharomyces\nstrains in wild and fermentative environments, including 58 sequenced strains of S. paradoxus\nfrom Mexico) are now publicly available as a preprint on bioRxiv. Reference to this work provides\na stronger and clearer context for our revised manuscript.\nSee doi: https://doi.org/10.1101/2025.05.31.656962.\n7. All remarks were carefully addressed and, in most cases, incorporated into the revised\nmanuscript. In the few instances where changes were not made, we provide a clear rationale for\nwhy the original version was kept.\nBelow is a point-by-point response to the reviewers’ comments. The page and line numbers in\nthe responses refer to the version of the manuscript with tracked changes\n(AgaveYeast_ReviewTrackedChanges.pdf) to facilitate identification of the revisions. Minor\ncorrections made during the revision, but not specifically requested by reviewers, were not\nmarked using track changes.\nEditor\nDear Dr Morales,\nThank you again for submitting your manuscript \"Recurrent introgression and geographical\nstratification shape Saccharomyces cerevisiae in the Neotropics\" to Nature Communications. We\nhave now received reports from 2 reviewers (one with an Early Career co-reviewer) and, after\ncareful consideration, we have decided to invite a major revision of the manuscript.\nAs you will see from the reports copied below, the reviewers raise important concerns. We find\nthat these concerns limit the strength of the study, and therefore we ask you to address them with\nadditional work. Without substantial revisions, we will be unlikely to send the paper back to review.\nIf you feel that you are able to comprehensively address the reviewers’ concerns, please provide\na point-by-point response to these comments along with your revision. Please show all changes\nin the manuscript text file with track changes or colour highlighting. If you are unable to address\nspecific reviewer requests or find any points invalid, please explain why in the point-by-point\nresponse.\nImportant: In addition to the above, you must comply with the following editorial requests; we will\nnot be able to proceed with your revised manuscript otherwise. Please also see the Nature\nCommunications formatting instructions, which you may find useful while preparing your revised\nmanuscript.\nReviewer #1 (Remarks to the Author):\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nAvelar-Rivas et al present a study into the genomic characteristics of Saccharomyces cerevisiae\nstrains isolated from the Neotropics via re-sequencing of 216 isolates. They define a new clade\n\"Mexican Agave 2\" and map numerous introgressions from Sacharomyces paradoxus across the\ngenomes of many of neotropic strains.\nComment 1.1 The expanded repertoire of isolates has expanded the catalogue of strains from\nthis region. However the focus on the widespread introgressions from S. paradoxus largely\nrecapitulates findings from previous large-scale de novo sequencing efforts, such as those of\nPeter et al 2018 and D’Angiolo et al 2020, which defined the Alpecin, Mexican Agave and French\nGuiana lineages as displaying high degree of introgression from this sister species.\nWe agree with the reviewer that prior studies have reported introgressions from S. paradoxus in\ncertain S. cerevisiae lineages. However, we argue that our study is clearly distinct in showing\nmultiple, independent introgression events within a geographically structured population. To our\nknowledge, this pattern has not been previously documented, likely because it is not common\nand it required high-resolution genomic data from numerous strains of both admixing species,\nsampled within the same geographic region. In contrast to the work of Peter et al. (2018) and\nD’Angiolo et al. (2020), which identified the presence and broad evolutionary origins of\nintrogressed lineages in Europe (Alpechin) and the Americas, our study provides a novel\ncontribution by analyzing in detail the genomic population structure of such introgressed lineages.\nSpecifically, with these analyses we show that, in the Neotropical clade, introgressions are not\nthe result of a single hybridization event—such as that proposed for the origin of the highly\nintrogressed Alpechin lineage—but instead originate from multiple, recurrent hybridization events\nwith S. paradoxus.\nTo clarify this point, we have revised the Introduction (lines 45–58 in the tracked-changes file\nprovided) better explaining previous work and highlighting the novel contributions of our study\nregarding introgressions and geographical structure. This revised section includes references to\nthe studies of Peter et al. (2018) and D’Angiolo et al. (2020), as well as other studies that have\ninvestigated introgressions in S. cerevisiae (Barbosa 2016, Barbosa 2018, Tellini 2024).\nComment 1.2 Given that the authors have used a mapping-based approach to defining\nintrogression, more detailed explanation of the methodology (with figures where appropriate)\nwould be useful for determining the robustness of the predictions relative to the a gene-prediction\n+ phylogenomics approach. Direct comparison to data from papers such as Peter et al 2018 could\nbe used to ground-truth the mapping-based methodology.\nAs requested by the reviewer, our mapping-based pipeline is now better described in the “Analysis\nof Introgressions” section of the Methods (lines 692–698). Mapping-based strategies have been\npreviously used for identifying introgressed fragments in (Barbosa et al 2016). The parameters\nspecific to our workflow are reported in the Methods section.\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nIn addition, we now define introgression by complementing the mapping-based approach with a\nphylogenetic analysis of the 13,711 identified regions. This phylogenetic analysis was applied to\nintrogression blocks rather than to single genes, as the former provides greater size and variant\ndensity, and therefore higher resolution for the analysis. We think the integration of the\nphylogenetic analysis increases the robustness of our approach for identifying\nintrogressions.\nAlso based on the reviewer’s comment, we compared the introgressed genes identified in our\nanalyses with those reported by the gene prediction and phylogenomics approach in Peter et al.\n(2018), as well as with genes identified by using bona fide diagnostic SNP markers that\ndiscriminate S. cerevisiae from S. paradoxus, as reported in Tellini et al. (2024). In our revised\nmanuscript, we now show the overlap between our strategy for defining introgressions (based on\nthe mapping approach combined with phylogenetic analysis of introgressed blocks), and the two\nsignaled studies (lines 298–306, new Figure S7). Using 46 strains for which introgressions were\ndefined by all three studies, we complied a reference set of 3,662 genes (“Universe”), of which\n1,582 were common to all three studies, representing 63.68%, 54.60%, and 74.13% of the total\ngenes reported in this work, Tellini et al., and Peter et al., respectively. The new Figure S7 also\nshows a comparison in which we computed the overlap between our method and the strains\nreported in Barbosa et al. (2016), using the three strains shared between both datasets.\nSupplementary Figure S7. Comparison of the method used in this\nstudy for detecting introgressed genes with alternative approaches.\nVenn diagrams show the number of genes identified as introgressed\nacross 46 strains (left) and 3 strains (right) using three different\nidentification approaches.\nComment 1.3 Likewise, the application of introgressed gene blocks to specific S. paradoxus\nstrain origin should be supported by a gene-wise phylogenetic approach rather than the distance-\nbased methodology applied.\nTo perform the phylogenetic approach suggested by the reviewer, we took advantage of the\n13,711 phylogenies used to define introgressions (see response 1.2) and assigned the\nphylogenetic origin to each introgressed block when the sister sequence in the phylogeny was\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nalso the one with the smallest phylogenetic distance. This analysis showed good correspondence\nwith our previously reported results (see figures below, where (A) is our original result, and (B) is\nthe result from the phylogenetic approach). In the phylogenetic approach, we identified the donor\nstrain as the one with the lowest number of differences (i.e., smallest distance or greatest\nsimilarity) in a phylogeny of reconstructed sequences from a VCF file with 5 representative S.\nparadoxus strains, two S. cerevisiae strains as outgroups and the introgressed block. The use of\nlacked resolution, as many introgressed blocks could not be confidently assigned to a specific\nlineage. In such cases, we labeled the origin as “SpB_undefined” when the sister clade included\ntwo SpB strains or when the strain with the lowest phylogenetic distance was not the sister clade.\nBlocks that were sister to both SpB and SpC were assigned the label “Sp_American.”\nFor the main analysis in the revised article, we decided to keep a sequence similarity-based\napproach, taking advantage of the full panel of S. paradoxus donor strains. However, we only\nincluded introgressions that were independently validated by the phylogenetic approach (see\nresponse 1.2). In addition, to improve donor assignment, we calculated the identity for each\nintrogressed block using PLINK, comparing them to the entire S. paradoxus panel. The results\nshown in the revised manuscript are consistent with those from both strategies described above,\nthe original distance-based methodology and the phylogenetic approach proposed by the\nreviewer (new Figure 5A). In the main article we present only the improved similarity-based\nmethod, as it yields more precise assignments while supporting the same overall conclusions.\nWe note that we also incorporated uncertainty in donor lineage assignment into the model, by\nhighlighting introgressed blocks shared across all Neotropical cluster clades prior to full\ndivergence of the SpB subgroups (modified Figure 5B).\nA) Original results\nB) New results using the phylogenetic approach (discussed but not included in the final\nmanuscript)\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nReviewer #2 (Remarks to the Author):\nfrom agave fermentations in Mexico and is centered of the characterization of multiple and\nindependent introgressions originating in the sister species S. paradoxus. I find this investigation\nwell conduced and significant as the natural history and domestication of S. cerevisiae in the\nNeotropics is poorly understood. Moreover, the pervasiveness of S. paradoxus introgressions in\nS. cerevisiae found in the Central and South America, either in wild or in domesticated\npopulations, is unique and understanding its causes is of relevance as wild populations in other\nregions lack such introgressions. Although this study does not solve the question of why are\nintrogressions found in Neotropics but not in other regions, it is a significant contribution to the\ntopic. Below I list my comments and questions that I hope the authors find pertinent.\nMain\nComment 2.1. I wonder why the authors characterize the agave fermentations as “a habitat at\nthe interface of natural and industrial environments” (lines 8-9). What are exactly the\ncharacteristics of this fermentation that separate it from other fermentations? For example, are\nspontaneous fermentations also at this interface? If so, is wine must a natural substrate? I believe\nthat the distinction between wild and artificial environments should be made very clear from the\nonset of the paper, and I see no reason not to consider agave fermentations as a typical artificial\nenvironment created by humans. Also, why using “habitat” in this context (see also line 24)? In\norder to demarcate clearly wild and artificial substrates, perhaps “habitat” could be restricted to\nthe context of natural settings and a more neutral expression like “environment” could be used in\nsituations relating to artificial man-made environments.\nWe agree with the reviewer that agave fermentations are similar to other “spontaneous”\nfermentations and have revised the manuscript to avoid terminology that could be misleading in\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nthis regard (e.g., line 9 in the tracked-changes file provided). Given that agave spirit producers\ndo not intentionally inoculate these fermentations, the microorganisms involved likely originate\nfrom nearby natural environments. This is why we initially referred to them as occurring at the\n“interface of natural and industrial environments.” To clarify this point, we now explicitly state\nthroughout the Introduction, Results, and Discussion that agave fermentations are an\nanthropogenic environment (lines 83 and 490). For instance: “In this study, we sequenced 216\nstrains from open agave fermentations, providing an extensive population genomics dataset for\nisolates of anthropogenic environments in the Neotropics.” (line 490). We also added a\nparagraph to the Discussion (lines 556-569) to emphasize the anthropogenic nature of the\nenvironment and to acknowledge the overlapping zones between anthropogenic and natural\nenvironments.\nLikewise, we revised and modified the use of the term “habitat” in the few instances where it\npreviously appeared (lines 9, 25–26).\nComment 2.2. The authors use the expression “neotropical cluster” (line 37) for designate the\nthree S. cerevisiae lineages from this region. I agree that treating these lineages like this makes\nsense, but I miss a more thorough dissection of this group. I believe it would be important to\nemphasize (or at least mention) that two of these lineages are domesticated and one (Wild Brazil\n3) is natural. That is relevant for several reasons, most importantly for discussing the occurrence\nof introgressions both in wild and in domesticated lineages. To add to this, the authors even state\n(line 94) “…distinguishing strains from the Neotropics (blue) from wild (green) and domesticated\n(red)”, implying that the strains from the Neotropics are neither wild nor domesticated, which is\nodd.\nAs suggested by the reviewer, we have included a more thorough dissection of the cluster by\nseparating previously sequenced neotropical strains in four groups that now include the Wild\nBrazil 3 (WB3, line 36). We also now clarify which of these isolates come from anthropogenic\nenvironments and which are from natural areas (line 38). Importantly, the WB3 is now considered\nas a separate group in the new version of the phylogenetic analysis (see modified Figure 1) and,\nthroughout the text, we acknowledge and discuss that introgressions occur in both wild and\nhuman-associated lineages.\nThis reviewer’s comment helped us recognize that our initial description of the Neotropical\ncluster—contrasting it with \"wild\" and \"domesticated\"—was inaccurate. These terms were poorly\nchosen and have now been removed, along with the ADMIXTURE panel originally shown in\nFigure 1. Our intention was to convey that, in the admixture analysis with k = 3, the Neotropical\ncluster represents one of the genetic components, while the other two correspond predominantly\nto wild strains (e.g., Wild Asian and North American Oak, as broadly defined in Loegler et al.,\n2024 and Han et al., 2021) and human-associated strains (e.g., Wine, Dairy). However, this\nframing was an oversimplification, as all three components include a mix of strains from wild and\nanthropogenic origins. The use of the terms “wild” and “domesticated” was therefore misleading\nand now has been thoroughly revised. Additionally, the overrepresentation of Neotropical strains\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nin the ADMIXTURE analysis may have biased the inferred separation at this scale (see response\nto comment 2.6). These issues have been addressed in the revised version of Figure 1.\nComment 2.3. The species “evolutionary history” needs to be studied on wild isolates, which is\nnot the case in this study. I recommend reframing the way the study is presented as discussed.\nThe following sentence (line 38) exemplifies my point “Considering the vast area encompassed\nby this region, the few available sequenced isolates likely provide an underestimate of genetic\ndiversity, thus limiting our understanding of the species evolutionary history in this part of the\nworld”.\nWe acknowledge that the species’ evolutionary history is a subject of ongoing efforts and that wild\nisolates are essential to ultimately address this question. While our study focuses on\nanthropogenic environments, we believe the results contribute to understanding the broader\nnatural history of S. cerevisiae in the Neotropics, consistent with perspectives in previous studies\n(e.g. Duan et al., 2018 and De Chiara et al., 2022).\nIn response to the reviewer’s comment, we have made the following changes to the manuscript:\nFirst, we have revised wording in the Introduction section to avoid broad claims about\n“evolutionary history” (line 43, line 63). In addition, we now explicitly note the need for additional\nsampling from environmental sampling (Discussion, line 484-487, 532). In the Discussion\n(starting at lines 572-574), we clarify that the observed genetic diversity could reflects\nevolutionary processes in both natural and human-associated contexts. Finally, we added the\nfollowing sentence to the Discussion: “Comprehensive sequencing of S. cerevisiae genomes in\nmegadiverse regions is essential to understand the genomic diversity of this species, its natural\nevolutionary history, and its relationship with domestication.” (lines 484-487), explicitly separating\nboth concepts. These revisions provide a more cautious and precise framing of our study’s\ncontributions, in line with the reviewer’s comment.\nComment 2.4. In lines 34-38 (see also line 86) the authors introduce three previously recognized\nneotropical lineages, that they refer to as Mexican Agave (MA), French Guiana (FG), and South\nAmerican Mix 2 (SAM2). This last designation corresponds to an earlier designation, Wild Brazil\n3 (see reference 17). I wonder why the authors preferred to use a designation that is not only\nsuperfluous but also confusing. In fact, “Mix” is normally used in the context of S. cerevisiae\npopulations to label admixed lineages (see reference 6 [Peter et al. 2018] for several examples).\nIt is therefore unfortunate that the authors choose to associate a name that implies admixture to\na non-admixed population. This confusion is even exposed by the authors in the construction and\nlegend of Fig 1C in which the Wild Brazil 3 (SAM2) population is treated as admixed (see also\nlines 116, 122), which is false.\nWe thank the reviewer for this clarification. We acknowledge that our initial labeling of strains as\n“SAM2” and its interpretation as a previously recognized lineage oversimplified the original\ndefinitions. In particular, we did not fully account for the distinction between the Wild Brazil 3\npopulation described by Barbosa et al. (2016) and the admixed strains assembled as SAM2 in\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nTellini et al. (2024). We now address this distinction more carefully and in line with the reviewer’s\nobservations.\nAs described in the response to comment 2.2, we have separated the three Wild Brazil strains\nfrom Barbosa et al. (2016) into a distinct group labeled Wild Brazil 3 (WB3; see line 36). This\nimplied re-running most of the analyses accordingly. After this adjustment, the strains remaining\nin the SAM2 group (as defined by Tellini et al., 2024) display multiple genetic components at most\nK values in the ADMIXTURE analyses, consistent with the reviewer’s definition of a population\nlabeled \"Mix\" (see Figure S4 and Table S2, columns Main_Ancestral_pop, Main_Anc_Pop_Q,\nSecondary_Ancestral_Pop, Secondary_Anc_Pop_Q). In the revised manuscript, all the admixed\nstrains were removed for phylogenetic analyses, including three from SAM2, while the non-\nadmixed WB3 strains are shown (see modified Figure 1B). These changes are also now reflected\nin the text: Lines 36, 101-102, 131–136, 144–145, 156-157, 776, 793, and 10121.\nComment 2.5. I understand the interest in linking S. paradoxus with agave fermentations, but\nwhen checking reference 18 (Gallegos-Casillas, 2024) it is difficult to find evidence for this. The\nreality is that for more than 1500 S. cerevisiae isolates one a single S. paradoxus isolate was\nfound. I think it is difficult to argue that S. paradoxus is present in agave fermentations and I think\nthis part needs to be turned down. Moreover, is reference 19 (Peris et al, 2023) adequate in this\ncontext?\nBased on this comment, we have toned down the statement about the presence of S. paradoxus\nin agave fermentations, acknowledging that it is found at low frequency in this environment (lines\n55–58). However, we note that the presence of S. paradoxus in agave-spirit distilleries has been\nconsistently reported, and we now cite recent studies to support this claim in a much stronger\nmanner. Specifically, we now reference a recent preprint by our group, showing that 7.5% of over\nparadoxus as based on MALDI-TOF species identification (López-Gallegos et al., 2025). We also\nreference a recent review (Colon-Gonzalez, 2025) which compiles reports of yeast communities\nin the agave fermentation environment. These studies make it clearer that S. paradoxus has a\nlow but consistent presence in agave fermentations (see line 57).\nWe note that the reported isolation frequency of S. paradoxus in Gallegos-Casillas et al. (2024)\nis actually higher than one out of 1,500 isolates, but we fully recognize that this information is\ndifficult to extract from the Supplementary Material of that manuscript. Heading 3.3 of the Results\nsection of Gallegos-Casillas et al. (2024) (“Validation of MALDI‐TOF biotyping by ITS\nsequencing”) states: “Noteworthy, a small fraction (7.6%) of the putative S. cerevisiae isolates\nwas assigned to its sister species Saccharomyces paradoxus by ITS sequencing.”\nIn sum, to address the reviewer’s well-taken point, we have replaced these citations in the revised\nmanuscript with more recent studies (line 55–58), which we believe provide strong evidence that\nS. paradoxus is consistently present in agave fermentations, albeit at a much lower prevalence\nthan its sister species.\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nComment 2.6. Fig.1A reports on 1264 genomes but table S1 lists only the 261 agave fermentation\ngenomes. Moreover, Fig 1C reports on 464 genomes. If they are included in table S2 this should\nbe made clear by identifying which genomes were used in each analysis. Moreover, how were\nthese different subsets chosen? Was there a preoccupation in representing all known\npopulations? Why so many wine strains? Care much be applied because population structure\nanalyses can be distorted by over-representing / neglecting individuals from different populations.\nThe separation seen in figure 1A appears exacerbated and in fact cannot be confirmed. I would\nsuggest that it oversimplifies the problem by showing a continuum from wine to wild (China?)\nisolates, with agave isolates as the only ones escaping this continuum. Moreover, the text in line\n88 “The rich diversity of the newly sequenced strains is evident, as strains from the tropical\nAmericas encompass nearly half of the global diversity” appears to ignore the fact that most\ndiversity resides in wild oak-associated strains and that the agave strains (either those already\nknown or the new ones, are domesticated and therefore are not expected to be considerably\ndiverse). I am convinced that a less spectacular but probably more truly representation would not\ndiminish the current study (and this is true for the entire perspective of the paper).\nTo provide a more structured and detailed response, we have organized our responses\naddressing the concerns raised in this comment under the following points:\nComment 2.6.1. Fig.1A reports on 1264 genomes but table S1 lists only the 261 [216] agave\nfermentation genomes. Moreover, Fig 1C reports on 464 genomes. If they are included in table\nS2 this should be made clear by identifying which genomes were used in each analysis.\nWe have reformatted the two Supplementary Tables to present the information more clearly\nregarding which strains were used in each analysis. Table S1 now includes all 216 strains\nsequenced in this study, along with their associated metadata, while Table S2 lists a total of 1,284\nstrains:\n-The 216 strains sequenced in this study (Table S1)\n-1,010 strains from Peter et al. (2018)\n-37 strains from the Americas used in Tellini et al. 2024 (strains from Barbosa et al. 2016;\nBarbosa et al. 2018; Gallone et al. 2016; Legras et al. 2018)\n-21 strains of the Alpechin clade (Pontes et al. 2019).\nBinary indicator columns in Table S2 indicate which set of strains was used in each analysis:\nUsed_in_MDSA, Used_in_main_phylogeny, Used_in_ADMIXTURE, Used_in_complete_phylogenies, and\nUsed_in_downsamplMDSPhylogeny.\nThese designations are now presented in a “readme” sheet in Table S2 and are also described\nin the Materials and Methods section (lines 606-612, 623-637and 658-667).\nComment 2.6.2. Moreover, how were these different subsets chosen?\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nWe selected strain sets that were most relevant to each analysis, balancing informative value with\ncomputational feasibility. We have included explanation of the justification of the strain set in the\ncorresponding section of each analysis as explained below:\nLine 605: For the Multidimensional Scaling Analysis (Used_in_MDSA), we used 1,261 sequences\nto have a global overview of the species. These included the 1,010 strains from (Peter et al. 2018),\nthe Neotropical strains sequenced in this study (n=216) and the 37 strains from SAM clades\n(Tellini et al 2024) that include strains from Barbosa et al. (2016) (n=23) from Barbosa 2018 (n=10)\nLegras et al 2018 (n=1) and Gallone et al 2016 (n=3). Two sequences were excluded from the\nfinal image due to redundancy with other entries detected with PLINK. As a result, the MDSA plot\nprovides a comprehensive global overview of the species’ diversity.\nLine 623-637: For the phylogenetic analysis we focused on the newly sequenced agave-\nassociated strains and their closest relatives. In the supporting, complete phylogenetic analyses\n(Figure S1), we included all newly sequenced strains from this study, those from any SAM group\nfrom Tellini et al. (2024), at least one representative from each clade described in Peter et al.\n(2018) and 20 strains without clade designation (Used_in_complete_phylogenies). To improve\nresolution in the agave-associated strains, we intentionally overrepresented the Mixed Origin,\nWine, and North American Oak clades (Peter et al. 2018) since some of our isolates clustered\nwithin these groups. We also incorporated the 21 Alpechin strains from Pontes et al. (2019) given\nits high number of introgressions. For the main phylogeny shown in Figure 1, we used the same\nstrain set defined for the supporting trees but excluded admixed strains (Used_in_main_phylogeny).\nFor this figure, only strains with a predominant ancestral component, as determined by the\nADMIXTURE analysis, were retained (n=332). For the new Figure S2E, we downsampled\nrandomly the set of 332 strains selecting up to five sequences per clade ten times, the iteration\nthat is shown as a representative result is in the column Used_in_downsamplMDSPhylogeny (new\nFigure S2A).\nLine 658-667: For the ADMIXTURE analysis (Used_in_ADMIXTURE), we employed 466 strains and\nalso focused on the newly sequenced agave-associated strains. Accordingly, we included all\nstrains generated in this study (n=216), representative strains from each lineage described by\nPeter et al. (2018) (n=213) including at least one strain from each clade and 20 strains without\nclade designation. We also added strains that were of interest due to their previous classification\nas American strains that could inform the genetic components of our newly sequenced strains\nincluding strains from Tellini et al (2024, SAM clades), Barbosa et al. (2016), Barbosa et al (2018),\nGallone et al (2016) and Legras et al (2018). As in the phylogenetic analysis, to improve resolution\nof the groups where agave strains clustered, in the selection of the 213 strains from Peter et al.\n(2018) we overrepresented the Mixed Origin, Wine, and North American Oak clades, as a few of\nour newly sequenced isolates clustered within these groups.\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nComment 2.6.3. Was there a preoccupation in representing all known populations? Care much\nbe applied because population structure analyses can be distorted by over-representing /\nneglecting individuals from different populations.\nWe agree with the reviewer that population structure analyses can be biased by the\noverrepresentation or omission of specific populations. In response to this concern, we have\nremoved the k = 3 ADMIXTURE from Figure 1, along with the corresponding conclusions (line\n110-115), as it overstated the prominence of the Neotropical cluster and the groups containing\nour newly sequenced strains. The ADMIXTURE analysis now show in Figure 3A is focused\nspecifically on the Neotropical cluster. As detailed above, it was performed using all newly\nsequenced isolates, other strains from the Americas and representative strains from other clades\nof the world, as a reference. The conclusions drawn from this new analysis only concern the\npopulation structure of the Neotropical cluster (section starting on line 219). These changes have\nled to a more balanced and accurate interpretation of the population structure, particularly\nregarding the agave-associated and other strains from the Neotropics.\nComment 2.6.4. Why so many wine strains?\nThis comment likely arose from our ambiguous use of the “Wine” label in the original version of\nFigure 1 and from insufficient clarity about which strains were included in each analysis. The label\nwas intended merely as a reference point to indicate the location of selected wine strains, which\nrepresent the most abundant group in Peter et al. (2018). However, not all strains under that label\nwere actually wine strains. In the revised manuscript, we have improved Figure 1 by including the\nlabels of key clades mentioned in the text and by specifying in the figure legend that all relevant\nlineages of the species were included as shown in Table S2, thereby eliminating the source of\nthe confusion. In addition, the exact composition of the strains used in each analysis is now clearly\nprovided in the relevant sections of Material and Methods and in Table S2.\nComment 2.6.5: The separation seen in figure 1A appears exacerbated and in fact cannot be\nconfirmed. I would suggest that it oversimplifies the problem by showing a continuum from wine\nto wild (China?) isolates, with agave isolates as the only ones escaping this continuum.\nTo address the reviewer’s comment and test whether the separation shown in Figure 1A could\nresult from the overrepresentation of the neotropical strains, we repeated the MDS analysis ten\ntimes using a downsampled dataset limited to five strains per clade. As shown in a representative\niteration, the neotropical cluster still separated from the rest of the strains, although the distance\nwas more modest (new Figure S2A). It is also worthwhile noting that in the dataset that we used\nfor the MDS, the Neotropical cluster (n=250) was represented similarly as the Wine group\n(n=268), but only the former dispersed throughout the MDS plot.\nWe now restrain from drawing strong claims about the magnitude of the diversity based on the\nMDS analysis (line 104–108) and included our downsampling analysis in new Figure S2.\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nComment 2.6.6: Moreover, the text in line 88 “The rich diversity of the newly sequenced strains\nis evident, as strains from the tropical Americas encompass nearly half of the global diversity”\nappears to ignore the fact that most diversity resides in wild oak-associated strains and that the\nagave strains (either those already known or the new ones, are domesticated and therefore are\nnot expected to be considerably diverse). I am convinced that a less spectacular but probably\nmore truly representation would not diminish the current study (and this is true for the entire\nperspective of the paper).\nWe have revised this section of the Results to avoid drawing conclusions about the magnitude of\ndiversity based on the MDS analysis (line 104–106). While our data indicate that the Neotropical\ncluster represents a diverse S. cerevisiae lineage, it clearly does not account for half of the global\ndiversity, as we had inadvertently suggested.\nTo further address this point, we now provide additional evidence highlighting the unusually high\ndiversity of the Neotropical strains. In a new analysis, we report the number of SNVs per group\nrelative to the reference genome, based on 487 strains (new Figure 2). As the reviewer correctly\nnoted, wild strains from Taiwan and China clearly showed the highest median number of SNVs.\nHowever, notably, the next most divergent groups are those from the Neotropical cluster.\nIn addition, we used a reference free approach that showed that Neotropical cluster clades show\nless identity by sequence than most groups to clades such as CHNII, Asian Fermentation, etc.\nAgain, excluding Taiwanese and CHNI strains (Figure S3). We also note that Loegler et al. (2024)\nalso remarked the high divergence of Mexican Agave isolates (line 212-215).\nIn sum, the revised version offers a more descriptive picture of the relative divergence and\ndiversity observed in the Neotropical lineage, avoiding overstatements or exaggerated\nconclusions.\nComment 2.7. Fig. 1B. This representation might the consequence of the sampling with a limited\nnumber of wild strains (again, no information on the origin of the strains used), a single known\ndomesticated group (wine but not beer1 and 2, bread, sake, Asian beverages, etc) and an over-\nrepresentation of agave strains.\nIn response to this and related comments by the reviewer, we have removed the ADMIXTURE\nanalysis originally shown in Figure 1B. As noted in our response to point 2.6.4, the “Wine” label\nwas meant to indicate selected wine strains as a reference, though neighboring strains included\nBeer, Alpechin, among others. Likewise, strains from North American Oak, Asian Islands, Sake,\nand Asian beverages were present but appeared near the “Wild” label, which likely caused\nconfusion. We have clarified the composition of the strains sets in the corresponding section of\nthe Materials and Methods and in Table S2.\nComment 2.8. Fig 1C. Again, I wonder if the phylogeny is representative of the known phylogeny\nof the species, i.e. includes the relevant lineages. My point is that only the MA2 is a new addition,\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nand previous phylogenies did not show the MA1 and FG in such a distant position from the rest\nof the species lineages.\nThe previous phylogeny in Peter et al. (2018) did show that the lineage is well separated in\nrelatively long branches, even though it included a much smaller proportion of strains from the\nneotropics. The same was observed in the more recent study by Loeger et al. (2024), which\nincluded a larger number of neotropical strains, specifically from agave fermentations. These\nreported phylogenies are shown below. To make this point clear, we briefly describe it in the\nrevised manuscript (line 176).\nTo further explore the impact of strain selection in the phylogeny, we repeated the analysis ten\ntimes using random downsampling to a maximum of five strains per clade. An example including\nadmixed strains is shown below, and a version excluding admixed strains is now provided in the\nnew Figure S2F-G. The resulting topologies were consistent with that shown in Figure 1 whether\nor not introgressed regions were included: the Neotropical clade consistently occupied a distant\nposition. This analysis is now described in the revised manuscript (line 172) and the resulting\nphylogenies are included as Figure S2.\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nComment 2.9. Line 293. This new population or strain SpB_Mx needs to be presented. The\nreader also needs to be introduced to the A, B and B* populations.\nTo introduce the SpB_MxAgave population we now provide reference to our recent preprint about\nthe ecology of S. paradoxus isolates from agave fermentations and natural environments (López-\nGallegos et al., 2025). This study provides a better context to describe the origins of introgressions\nfrom this sister species (lines 55–58). Likewise, we now present the S. paradoxus lineages used\nto identify the origins of introgressions in the main text (lines 450-457).\nComment 2.10. The Wild Brazil 3 population is missing in the study of Fig. 4.\nAs described above, we now include the Wild Brazil 3 (WB3) as a standalone group throughout\nthe manuscript, which is also shown now in Figure 5 (formerly Figure 4).\nMinor\nComment 2.11. In this sentence (line 36-37) “Notoriously, the handful of sequenced genomes\navailable to date indicate that this Neotropical cluster harbors an unusually high and\nheterogeneous number of introgressions from S. paradoxus”, the first study revealing these\nintrogressions (ref 17 [Barbosa et al. 2016]) is not mentioned.\nThe reference has been added (line 45) and we have expanded the description of the findings in\nthis study as they are directly relevant for our work (sentences starting in line 50).\nComment 2.12. Populations should be designated in a consistent manner throughout the paper\n– line 91 wild North American”; line (119) “wild North America Oak clade”;\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nWe replaced the designation “Wild North American” to “North American Oak” (lines 148 and 260)\nand made sure we used population names consistently throughout the manuscript.\nComment 2.13. In Figure 1C and in the text what is the relevance in separating the Mexican\nagave strains in two subgroups? Wouldn’t it be preferable presenting the strains from this\nfermentation as a single group, given its technological utilization, etc? For future studies having a\nsingle population recognized as originating form Agave fermentations might be advantageous.\nThink of the “wine” population and of what it represents for the understanding of S. cerevisiae\nbiology and of the irrelevance of subdividing it (currently being attempted by several authors).\nWe appreciate the reviewer’s perspective. After careful consideration, we decided to retain the\nMA1 and MA2 designations, which is justified and appropriate given the scope and findings of\nthis study. The decision is based on the following arguments:\ni) Although MA1 and MA2 are phylogenetically related, they form two clearly distinct clades\nseparated by a mountain range known to act as a natural biogeographic barrier. This geographic\nstructuring is a key finding of our study.\nii) MA1 showed a recent pulse of introgression from S. paradoxus that is absent in MA2,\nhighlighting differences in hybridization history, which is another major result of our study.\niii) The two groups differ in patterns of admixture within species; for example, only MA1 has strong\nsignatures of admixture with the wild clade North American Oak.\niv) MA1 is only found in one of the seven agave-spirit producing regions, while MA2 is distributed\nacross six regions and represents the dominant group in traditional agave-spirit production. This\ndistinction is relevant for understanding and preserving regional diversity.\nv) We now show that MA1 and MA2 have an Fst=0.43, which is higher than all of the Fst values\ninvolving SAM2 and Tequila vs MA1, MA2, and WB3 (see response to comment 2.20).\nFurthermore, the IBS between MA1 and MA2 is 0.97, comparable to that between other well-\ndefined clades such as between Mediterranean Oak and Alpechin (Figure S3).\nWe agree with the reviewer that unnecessary subdivision of strain groups may be\ncounterproductive and anticipate that the community will likely consider Mexican Agave strains\nas a single group in future global-scale studies of S. cerevisiae populations. In the Discussion\nsection of the current manuscript, we do refer to them as “Mexican Agave clades” (line 524).\nHowever, for more detailed population-level analysis presented here, we find it useful to\ndistinguish between the two Mexican Agave groups.\nComment 2.14. In line 148 “…revealing that what was previously described as “Mexican Agave”\nrepresents only a 148 minor fraction of the great genetic diversity found in agave fermentations”\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nthis statement appears to be too strong (see comments above) as the new agave strains are\nnumerous but not vastly different from those already known.\nAgreed, we toned down this statement, as suggested (line 207).\nComment 2.15 Figure 2. Why K=24?\nWe used K=24 because it was the K value with the lowest cross-validation error (Figure S4). This\nhas been clarified in the main text (line 224) and explicitly stated in the figure legend.\nComment 2.16 Line 194 “Likewise, there were SAM2 strains showing genetic components of the\nMA2 and French Guiana clades”. This is not surprising since SAM2 (Wild Brazil 3) is the closest\nwild relative of the domesticated MA2 and French Guiana clades. This could be added to the\nsentence.\nAs mentioned in our previous responses, the WB3 strains are now distinguished from SAM2\nstrains. We have adapted the text accordingly (lines 227-229).\nComment 2.17. Line 204-205. Same for WB3. Lines 204-205: “For instance, the MA1, MA2, and\nTequila Distillery clades all show higher genetic diversity than yeasts from the Wine clade.”\nIn the revised version, population metrics are reported only for groups with more than ten isolates,\nas these metrics are highly sensitive to outlier and sampling biases (legend of Figure 3). This\napproach has also been adopted in previous studies analyzing large numbers of lineages and\nstrains (e.g. Peter et al., 2018).\nComment 2.18. Line 233-235: “The presence of both alleles in several introgressions of the\nTequila Distillery clade suggests admixture with a non-introgressed population, which may also\nexplain the lower number of introgressed genes”. I could not follow the reasoning as it appears to\ncontradict what was stated in the lines before. Please clarify why this cannot be a recent\nintrogression event.\nWe have substantially revised and expanded the explanation of the observed introgression\npatterns in these strains in the section beginning on line 294.\nWe removed focus from the patterns in the Tequila Distillery strains and instead we focused on\nthose that in our opinion provide stronger evidence for multiple introgression pulses, without the\npossible confounding effects of high admixture.\nComment 2.19. Line 279. [In terms of “private” introgressions—those present in a clade but\nabsent in all others—we scored 18.9% in FG, 32.2% in MA2, and 58.6% in MA1 (Figure 3E), while\nmost introgressed genes in the Alpechin reference clade (76.3%) were exclusively found in this\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nclade (Figure 3E)]. The alpechin clade has a totally distinct introgression history that occurred in\nEurope and involved European S. paradoxus. Thus, it appears not relevant for this comparison.\nAs suggested by the reviewer, we have removed the comparison with the Alpechin linage when\nit was not useful (e.g. Figure 5A). We also deleted previous Figure 3C-D. We retained the\nAlpechin group as a reference for comparison when useful.\nComment 2.20. Line 294: “In contrast, the majority of the introgressed genes in the FG strains\ntracked to SpB, a different lineage of S. paradoxus, which is not commonly found in agave\ndistillery contexts”. “not commonly found” or not found at all?\nThe SpB lineage has actually been recovered only from natural environments, not from agave\nfermentation samples. We have modified the sentence an included the reference to the recent\npreprint by López-Gallegos, et al. (2025) (lines 465-468).\nComment 2.21. Line 320. “In the Americas, several S. cerevisiae lineages feature high number\nof introgressions from S. paradoxus, both in wild and anthropogenic environments”. With respect\nto anthropogenic environments the authors appear to have missed this study\nhttps://doi.org/10.1093/gbe/evy132. https://academic.oup.com/gbe/article/10/8/1939/5047776\nWe have included the reference in the relevant sentence (line 497), as well as in other appropriate\nsections of the manuscript.\nComment 2.22. Line 327. “At least one additional, more recent pulse of introgression from the\nSpB_Mx lineage took place in the MA1 clade, which is supported by high numbers of large and\nheterozygous introgressions in some strains of this clade”. Is it possible to conceive an alternative\nscenario in which there was a single pulse but MA1 did not lose as many paradoxus genes as\nMA2?\nThis comment led to the inclusion of new Figure S9, which shows the patterns of persistently\nintrogressed genes in the Neotropical clades, with particular emphasis on the large heterozygous\nregions present only in a small subset of MA1 strains. Considering the phylogenetic history of\nthese strains, explaining the observed patterns without invoking a second introgression event in\nthis clade would require assuming a highly intricate history of introgression losses, as described\nin lines 381–445.\nWhile it is possible that a single introgression pulse followed by differential loss of introgressed\nsegments could explain the observed patterns, it would require unusually strong and localized\nselective pressures acting only on these strains to preserve large heterozygous blocks while\nallowing erosion and loss in others. Consistent with this, the 7 MA1 strains share a similar number\nof introgressed genes (from 60 to 96) with other neotropical lineages but have 2x to 6x times more\nprivate introgressions, suggesting an additional, lineage-specific event. The proportion of\nheterozygous private introgressions in these strains is also nearly twice that of the heterozygous\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nshared introgressions, consistent with a younger hybridization signal. Taken together, these\npatterns are much more consistent with a secondary introgression event than with differential loss\nfrom a single ancestral hybridization.\nStill, alternative scenarios suggested by the reviewer cannot be completely ruled out and they are\nnow mentioned in the following lines: Results, lines 389–392; Discussion, lines 508-510. We do\nunderscore, however, that multiple pulses of introgression are the most parsimonious\nevolutionary scenario\nComment 2.23. Line 331. Was any attempt made to investigate the pool of introgressed genes\nwith respect to function?\nYes, we performed a GO analysis enrichment of the genes that are present in the introgressed\nblocks. No significant enrichment was found, at least focusing on those shared among most\nisolates. We now include a sentence summarizing this analysis in the Results section of the\nrevised manuscript (lines 372-373).\nComment 2.24. Line 342. I think it is important to focus on sampling natural environments rather\nthan anthropic ones (see previous comments on how to address the “true” natural history, rather\nthan the recent domestication history.\nAs described above, we agree about these comments and now provide more carefully worded\nstatements in the revised manuscript (see lines 532-534).\nComment 2.25. Line 349. “Finally, the genetic divergence between the MA1 and MA2 clades is\nassociated with the presence of the Sierra Madre Oriental Mountain range, which is found at the\nedge where the Neotropical and Nearctic biogeographic regions coincide”.\nWas the actual genetic divergence calculated?\nAs suggested, the Fst values between the Neotropical groups are now reported in Figure S2.\nNotably, the genetic divergence between these clades is higher than that observed between WB3\nand Tequila or between MA1 and SAM2, underscoring the significance of this biogeographic\nbarrier in shaping yeast population structure and diversity (see line 144–145).\nManuscript ID NCOMMS-24-72197-T\nITEMIZED RESPONSE TO REVIEWERS\nSupplemenary Figure S2C\nComment 2.26. Line 397-398. Why not use IQ-TREE with the ‘ASC’ parameter associated with\nthe best model inference? This takes into account that the dataset is SNPs and not coding\nsequences/proteins.\nAs suggested, we used IQ-TREE with the ‘ASC’ parameter and replaced all phylogenies in the\nmanuscript with the ones generated using method. We also highlight the consistency of the results\nregardless of the specific phylogeney-building strategy used (lines 642-649 and images below).\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank this reviewer for their collaborative review and constructive feedback.\nSeptember 5th, 2025\nITEMIZED RESPONSE TO REVIEWERS\nManuscript ID NCOMMS-24-72197-T\nTitle: \"Recurrent introgression and geographical stratification shape Saccharomyces cerevisiae\nin the Neotropics\" by Avelar-Rivas et al.\nWe were encouraged to learn that all reviewers found their original comments thoroughly\naddressed in our revised manuscript. We also thank the reviewers for their constructive\nfeedback and an outstanding review experience, including attention to the final details in this\nsecond round of comments. Below, we provide a point-by-point response to the reviewers’\ncomments.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have addressed my previous comments and have improved the manuscript\nsignificantly.\nWith the re-review of the material, I have a question regarding the models proposed by\nthe authors regarding the formation of the MA1 genotype(s). As pointed out by the authors,\nthe strains within the MA1 clade show far higher variation in the specific introgressed\ngenes than strains within other clades. How do the authors propose that this variation in\ngene content in the MA1 cluster occurred? Would a full hybridization cerevisiae x\nparadoxus event have occurred followed by differential gene loss? Are the various strains\nstill undergoing genome reduction? Could this be a continual process with hybridisation\ncommonly occurring?\nThese questions regarding the variation in gene content in the MA1 cluster, are important and\nstimulating areas we look forward to exploring in future work. The MA1 strains show considerable\nvariation in the introgressed genes that they retain, likely reflecting genome instability and\nbackcrossing. This suggests they are in an ongoing transition from hybridization to stable\nintrogression, though more data and testing are needed to clarify the process. To address this\npoint, we have expanded our discussion starting on line 391 (tracked changes file), with a\nparagraph that now opens with an explicit reference to the variation within MA1. It suggests\npossible scenarios, highlights open questions, and mentions potential future work such as\nexperimental and computational tests to estimate the timing of hybridization and backcrossing\nevents, and broader sampling to better capture the evolutionary dynamics of interspecies gene\nflow.\nReviewer #2 (Remarks to the Author):\nI was very pleased to see that the authors carried out a thorough and very careful revision\nof the manuscript. As far the concerns I raised in my first revision, I see that the authors\naddressed them, and I do not have are relevant request for change in the manuscript.\nThere is however one issue, that of the divergence of the Neotropical cluster (Fig. 1B),\nin which I tend to disagree with the authors. I do not think that further scrutiny will support\nthis divergent placing of the Neotropical cluster that is probably due to a unbalanced\nsampling of the Neotropical representatives, and perhaps of other groups like the wine\nclade, by comparison with the representatives of other populations. It is not the number,\ne.g. phylogenies with more than 1000 or 3000 genomes, but the fine balance of the\nnumbers of genomes of representatives of ALL the populations that is of importance.\nPlease check here https://doi.org/10.1016/j.cub.2025.04.056 how a probably more\nbalanced sampling (that of course misses the “Mexican Agave 2”) fails to show the\nneotropical cluster as the utterly divergent lineage seen in Fig. 1. However, I want to\nstress that I am making this comment “for the record” not as a request for change.\nWe thank the reviewer for sharing their concern and fully agree on the importance of achieving\na more balanced sampling across populations, as fine balance among representatives is indeed\ncritical and may still be partly missing in our dataset. In line with this observation, Fig. 2S of our\nmanuscript already shows that a more balanced sampling reduces the branch length of the\nNeotropical cluster. A more comprehensive and balanced sampling will be required to clarify the\nextent and causes of this pattern. To acknowledge this point, we have introduced new text in the\nresults (line 147, tracked changes file) and discussion (line 434, tracked changes file).\nI conclude by congratulating the authors for this very interesting and finely prepared\nmanuscript that I am sure will stand as a relevant contribution for S. cerevisiae population\nbiology.\nMinor notes\nLine 8, 102, 172, 224, 341, 401, 575 “open agave fermentation”. Why not employing\n“spontaneous fermentation” which provides the reader with an objective concept, clearer\nthan that of “open fermentation”?\nWe agree that “spontaneous fermentation” is a clearer and more objective term, and we have\nincorporated it where appropriate, as suggested. At the same time, we chose to retain “open\nfermentation” in places where we want to emphasize the physical context, namely, that these\nfermentations typically occur in open tanks and locations exposed to the natural environment,\nwhere microorganisms constantly colonize the agave must.\nLine 30 “The Neotropical region, which extendS from…”\nLine 97 Agave fermentation strains belong TO a genetically diverse Neotropical cluster\nWe corrected both errors in the manuscript in line 30 and in the updated line 88 of changes\ndocument.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my previous comments and have improved the manuscript significantly.\nWith the re-review of the material, I have a question regarding the models proposed by the authors regarding the formation of the MA1 genotype(s).\nAs pointed out by the authors, the strains within the MA1 clade show far higher variation in the specific introgressed genes than strains within other clades.\nHow do the authors propose that this variation in gene content in the MA1 cluster occurred? Would a full hybridisation cerevisiae x paradoxus event have occurred followed by differential gene loss? Are the various strains still undergoing genome reduction? Could this be a continual process with hybridisation commonly occurring?", "reviewer_2": "I was very pleased to see that the authors carried out a thorough and very careful revision of the manuscript.\nAs far the concerns I raised in my first revision, I see that the authors addressed them, and I do not have are relevant request for change in the manuscript.\nThere is however one issue, that of the divergence of the Neotropical cluster (Fig. 1B), in which I tend to disagree with the authors. I do not think that further scrutiny will support this divergent placing of the Neotropical cluster that is probably due to a unbalanced sampling of the Neotropical representatives, and perhaps of other groups like the wine clade, by comparison with the representatives of other populations. It is not the number, e.g. phylogenies with more than 1000 or 3000 genomes, but the fine balance of the numbers of genomes of representatives of ALL the populations that is of importance. Please check here https://doi.org/10.1016/j.cub.2025.04.056 how a probably more balanced sampling (that of course misses the “Mexican Agave 2”) fails to show the neotropical cluster as the utterly divergent lineage seen in Fig. 1. However, I want to stress that I am making this comment “for the record” not as a request for change.\nI conclude by congratulating the authors for this very interesting and finely prepared manuscript that I am sure will stand as a relevant contribution for S. cerevisiae population biology.\nMinor notes\nLine 8, 102, 172, 224, 341, 401, 575 “open agave fermentation”. Why not employing “spontaneous fermentation” which provides the reader with an objective concept, clearer than that of “open fermentation” ?\nLine 30 “The Neotropical region, which extendS from…”\nLine 97 Agave fermentation strains belong TO a genetically diverse Neotropical cluster", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "September 5th, 2025\nITEMIZED RESPONSE TO REVIEWERS\nManuscript ID NCOMMS-24-72197-T\nTitle: \"Recurrent introgression and geographical stratification shape Saccharomyces cerevisiae\nin the Neotropics\" by Avelar-Rivas et al.\nWe were encouraged to learn that all reviewers found their original comments thoroughly\naddressed in our revised manuscript. We also thank the reviewers for their constructive\nfeedback and an outstanding review experience, including attention to the final details in this\nsecond round of comments. Below, we provide a point-by-point response to the reviewers’\ncomments.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have addressed my previous comments and have improved the manuscript\nsignificantly.\nWith the re-review of the material, I have a question regarding the models proposed by\nthe authors regarding the formation of the MA1 genotype(s). As pointed out by the authors,\nthe strains within the MA1 clade show far higher variation in the specific introgressed\ngenes than strains within other clades. How do the authors propose that this variation in\ngene content in the MA1 cluster occurred? Would a full hybridization cerevisiae x\nparadoxus event have occurred followed by differential gene loss? Are the various strains\nstill undergoing genome reduction? Could this be a continual process with hybridisation\ncommonly occurring?\nThese questions regarding the variation in gene content in the MA1 cluster, are important and\nstimulating areas we look forward to exploring in future work. The MA1 strains show considerable\nvariation in the introgressed genes that they retain, likely reflecting genome instability and\nbackcrossing. This suggests they are in an ongoing transition from hybridization to stable\nintrogression, though more data and testing are needed to clarify the process. To address this\npoint, we have expanded our discussion starting on line 391 (tracked changes file), with a\nparagraph that now opens with an explicit reference to the variation within MA1. It suggests\npossible scenarios, highlights open questions, and mentions potential future work such as\nexperimental and computational tests to estimate the timing of hybridization and backcrossing\nevents, and broader sampling to better capture the evolutionary dynamics of interspecies gene\nflow.\nReviewer #2 (Remarks to the Author):\nI was very pleased to see that the authors carried out a thorough and very careful revision\nof the manuscript. As far the concerns I raised in my first revision, I see that the authors\naddressed them, and I do not have are relevant request for change in the manuscript.\nThere is however one issue, that of the divergence of the Neotropical cluster (Fig. 1B),\nin which I tend to disagree with the authors. I do not think that further scrutiny will support\nthis divergent placing of the Neotropical cluster that is probably due to a unbalanced\nsampling of the Neotropical representatives, and perhaps of other groups like the wine\nclade, by comparison with the representatives of other populations. It is not the number,\ne.g. phylogenies with more than 1000 or 3000 genomes, but the fine balance of the\nnumbers of genomes of representatives of ALL the populations that is of importance.\nPlease check here https://doi.org/10.1016/j.cub.2025.04.056 how a probably more\nbalanced sampling (that of course misses the “Mexican Agave 2”) fails to show the\nneotropical cluster as the utterly divergent lineage seen in Fig. 1. However, I want to\nstress that I am making this comment “for the record” not as a request for change.\nWe thank the reviewer for sharing their concern and fully agree on the importance of achieving\na more balanced sampling across populations, as fine balance among representatives is indeed\ncritical and may still be partly missing in our dataset. In line with this observation, Fig. 2S of our\nmanuscript already shows that a more balanced sampling reduces the branch length of the\nNeotropical cluster. A more comprehensive and balanced sampling will be required to clarify the\nextent and causes of this pattern. To acknowledge this point, we have introduced new text in the\nresults (line 147, tracked changes file) and discussion (line 434, tracked changes file).\nI conclude by congratulating the authors for this very interesting and finely prepared\nmanuscript that I am sure will stand as a relevant contribution for S. cerevisiae population\nbiology.\nMinor notes\nLine 8, 102, 172, 224, 341, 401, 575 “open agave fermentation”. Why not employing\n“spontaneous fermentation” which provides the reader with an objective concept, clearer\nthan that of “open fermentation”?\nWe agree that “spontaneous fermentation” is a clearer and more objective term, and we have\nincorporated it where appropriate, as suggested. At the same time, we chose to retain “open\nfermentation” in places where we want to emphasize the physical context, namely, that these\nfermentations typically occur in open tanks and locations exposed to the natural environment,\nwhere microorganisms constantly colonize the agave must.\nLine 30 “The Neotropical region, which extendS from…”\nLine 97 Agave fermentation strains belong TO a genetically diverse Neotropical cluster\nWe corrected both errors in the manuscript in line 30 and in the updated line 88 of changes\ndocument.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts."}]

Decision letter
Editor
Dear Dr Morales,
Thank you again for submitting your manuscript "Recurrent introgression and geographical stratification shape Saccharomyces cerevisiae
in the Neotropics" to Nature Communications. We
have now received reports from 2 reviewers (one with an Early Career co-reviewer) and, after
careful consideration, we have decided to invite a major revision of the manuscript.
As you will see from the reports copied below, the reviewers raise important concerns. We find
that these concerns limit the strength of the study, and therefore we ask you to address them with
additional work. Without substantial revisions, we will be unlikely to send the paper back to review.
If you feel that you are able to comprehensively address the reviewers’ concerns, please provide
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