Post on 09-Apr-2020
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Genomic diversity and global distribution of Saccharomyces eubayanus, the wild ancestor of 1
hybrid lager-brewing yeasts 2
3
Quinn K. Langdon1, David Peris1,2,3, Juan I. Eizaguirre4, Dana A. Opulente1,2, Kelly V. Buh1, 4
Kayla Sylvester1,2, Martin Jarzyna1,2, María E. Rodríguez5, Christian A. Lopes5, Diego 5
Libkind4,@, Chris Todd Hittinger1,2,@ 6
7
1Laboratory of Genetics, J. F. Crow Institute for the Study of Evolution, Wisconsin Energy 8
Institute, Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, WI 53706, 9
USA 10
2DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 11
53706, USA 12
3Department of Food Biotechnology, Institute of Agrochemistry and Food Technology (IATA), 13
CSIC, Valencia, Spain 14
4Laboratorio de Microbiología Aplicada, Biotecnología y Bioinformática de Levaduras, Instituto 15
Andino Patagónico de Tecnologías Biológicas y Geoambientales (IPATEC), Consejo Nacional 16
de Investigaciones, Científicas y Técnicas (CONICET)-Universidad Nacional del Comahue, 17
8400 Bariloche, Argentina 18
5Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías 19
Alternativas (PROBIEN, CONICET-UNCo), Neuquén, Argentina 20
@Corresponding authors: CTH cthittinger@wisc.edu & DL diego.libkind@gmail.com 21
22
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Abstract: 24
S. eubayanus, the wild, cold-tolerant parent of hybrid lager-brewing yeasts, has a 25
complex and understudied natural history. The exploration of this diversity can be used both to 26
develop new brewing applications and to enlighten our understanding of the dynamics of yeast 27
evolution in the wild. Here, we integrate whole genome sequence and phenotypic data of 200 S. 28
eubayanus strains, the largest collection to date. S. eubayanus has a multilayered population 29
structure, consisting of two major populations that are further structured into six subpopulations. 30
Four of these subpopulations are found exclusively in the Patagonian region of South America; 31
one is found predominantly in Patagonia and sparsely in Oceania and North America; and one is 32
specific to the Holarctic ecozone. S. eubayanus is most abundant and genetically diverse in 33
Patagonia, where some locations harbor more genetic diversity than is found outside of South 34
America. All but one subpopulation shows isolation-by-distance, and gene flow between 35
subpopulations is low. However, there are strong signals of ancient and recent outcrossing, 36
including two admixed lineages, one that is sympatric with and one that is mostly isolated from 37
its parental populations. Despite S. eubayanus’ extensive genetic diversity, it has relatively little 38
phenotypic diversity, and all subpopulations performed similarly under most conditions tested. 39
Using our extensive biogeographical data, we constructed a robust model that predicted all 40
known and a handful of additional regions of the globe that are climatically suitable for S. 41
eubayanus, including Europe. We conclude that this industrially relevant species has rich wild 42
diversity with many factors contributing to its complex distribution and biology. 43
44
45
46
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Introduction: 47
In microbial population genomics, the interplay of human association and natural 48
variation is still poorly understood. The genus Saccharomyces is an optimal model to address 49
these questions for eukaryotic microbes, as it contains both partly human-associated species (i.e. 50
Saccharomyces cerevisiae) and mostly wild species (e.g. Saccharomyces paradoxus). These two 51
examples also illustrate the complexity of studying yeast population genomics. Much of S. 52
cerevisiae population structure is admixed, and several lineages show signatures of 53
domestication (Liti et al. 2009; Schacherer et al. 2009; Gallone et al. 2016; Gonçalves et al. 54
2016). In contrast, S. paradoxus is almost exclusively found in the wild and has a population 55
structure that is correlated with geography (Leducq et al. 2014; Eberlein et al. 2019). Pure 56
isolates of their more distant relative Saccharomyces eubayanus have only ever been isolated 57
from wild environments; yet, hybridizations between S. cerevisiae and S. eubayanus were key 58
innovations that enabled cold fermentation and lager brewing (Libkind et al. 2011; Gibson and 59
Liti 2015; Hittinger et al. 2018; Baker et al. 2019). Other hybrids with contributions from S. 60
eubayanus have been isolated from industrial environments (Almeida et al. 2014; Nguyen and 61
Boekhout 2017), indicating that this species has long been playing a role in shaping many 62
fermented products. This association with both natural and domesticated environments makes S. 63
eubayanus an excellent model where both wild diversity and domestication can be investigated. 64
Since the discovery of S. eubayanus in Patagonia (Libkind et al. 2011), this species has 65
received much attention, both for brewing applications and understanding the evolution, ecology, 66
population genomics of the genus Saccharomyces (Sampaio 2018). In the years since its 67
discovery, many new globally distributed isolates have been found (Bing et al. 2014; Peris et al. 68
2014; Rodríguez et al. 2014; Gayevskiy and Goddard 2016; Peris et al. 2016; Eizaguirre et al. 69
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4
2018). Prior research has suggested that S. eubayanus is most abundant and diverse in the 70
Patagonian region of South America, where there are two major populations (Patagonia 71
A/Population A/PA and Patagonia B/Population B/PB) that recent multilocus data suggested are 72
further divided into five subpopulations (PA-1, PA-2, PB-1, PB-2, and PB-3) (Eizaguirre et al. 73
2018). There are two early-diverging lineages, West China and Sichuan, which were identified 74
through multilocus data (Bing et al. 2014) and whose sequence divergences relative to other 75
strains of S. eubayanus are nearly that of currently recognized species boundaries (Peris et al. 76
2016; Sampaio and Gonçalves 2017; Naseeb et al. 2018). A unique admixed lineage has been 77
found only in North America, which has approximately equal contributions from PA and PB 78
(Peris et al. 2014, 2016). Other isolates from outside Patagonia belong to PB, either the PB-1 79
subpopulation that is also found in Patagonia (Gayevskiy and Goddard 2016; Peris et al. 2016), 80
or a Holarctic-specific subpopulation that includes isolates from Tibet and from North Carolina, 81
USA (Bing et al. 2014; Peris et al. 2016). This Holarctic subpopulation includes the closest 82
known wild relatives of the S. eubayanus subgenomes of lager-brewing yeasts (Bing et al. 2014; 83
Peris et al. 2016). 84
To explore the geographic distribution, ecological niche, and genomic diversity of this 85
industrially relevant species, here, we present an analysis of whole genome sequencing data for 86
200 S. eubayanus strains. This dataset confirms the previously proposed population structure 87
(Peris et al. 2014, 2016; Eizaguirre et al. 2018) and extends the analysis to fully explore genomic 88
diversity. Even though S. eubayanus is genetically diverse and globally distributed, there are not 89
large phenotypic differences between subpopulations. This genomic dataset includes evidence of 90
gene flow and admixture in sympatry, as well as admixture in parapatry or allopatry. While S. 91
eubayanus has a well-differentiated population structure, isolation by distance occurs within 92
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subpopulations that are found globally, as well as within subpopulations restricted to a handful of 93
locations. Much of the genetic diversity is limited to northern Patagonia, but modeling suggests 94
that there are more geographic areas that are climatically suitable for this species, including 95
Europe. S. eubayanus maintains genetic diversity over several dimensions, including multiple 96
high-diversity sympatric populations and a low-diversity widespread invasive lineage. The 97
diversity and dispersal of this eukaryotic microbial species mirror observations in plants and 98
animals, including humans, which shows how biogeographical and evolutionary forces can be 99
shared across organismal sizes, big and small. 100
101
Results: 102
Global and regional S. eubayanus population structure and ecology: 103
To expand on existing data (Libkind et al. 2011; Bing et al. 2014; Peris et al. 2014; 104
Rodríguez et al. 2014; Gayevskiy and Goddard 2016; Peris et al. 2016; Eizaguirre et al. 2018), 105
we sequenced the genomes of 174 additional strains of S. eubayanus, bringing our survey to 200 106
S. eubayanus genomes. This large collection provides the most comprehensive dataset to date for 107
S. eubayanus. We note that the dataset does not contain West China or Sichuan strains (Bing et 108
al. 2014), which were unavailable for study and may constitute a distinct species or subspecies. 109
These strains were globally distributed (Figure 1A), but the majority of our strains were from 110
South America (172 total, 155 newly sequenced here). The next most abundant continent was 111
North America with 26 strains (19 new to this publication). We also analyzed whole genome 112
sequence data for the single strain from New Zealand (Gayevskiy and Goddard 2016) and the 113
single Tibetan isolate with available whole genome sequence data (Bing et al. 2014; Brouwers et 114
al. 2019). The collection sites in South America span from northern Patagonia to Tierra del 115
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Fuego (Figure 1B), while the North American isolates have been sparsely found throughout the 116
continent, including the Canadian province of New Brunswick and the American states of 117
Washington, Wisconsin, North Carolina, and South Carolina (Figure 1C). 118
119
Figure 1. S. eubayanus distribution and population structure. 120 S. eubayanus has a global distribution and two major populations with six subpopulations. (A) Isolation 121 locations of S. eubayanus strains included in the dataset. For visibility, circle size is not scaled by the 122 number of strains. Subpopulation abundance is shown as pie charts. The Patagonian sampling sites have 123 been collapsed to two locations for clarity. Details of sites and subpopulations found in South America 124 (B) and North America (C) with circle size scaled by the number of strains. (D) Whole genome PCA of S. 125 eubayanus strains and five hybrids with large contributions from S. eubayanus. (E) PCA of just PA. (F) 126 PCA of just PB and hybrid S. eubayanus sub-genomes. Color legends in A and D apply to this and all 127 other figures. 128 129 To determine population structure, we took several approaches, including Principal 130
Component Analysis (PCA) (Jombart 2008), phylogenomic networks (Huson and Bryant 2006), 131
and STRUCTURE-like analyses (Lawson et al. 2012; Raj et al. 2014). All methods showed that 132
S. eubayanus has two large populations that can be further subdivided into a total of six non-133
25
30
35
40
45
50
−120 −100 −80 −60Longitude
Latitude
PB−1
PB−2
PB−3Holarctic
PA−1 PA−2
NoAm
SoAm
−150
−100
−50
0
50
−100 0 100 200PC1 = 40.06%
PC2
= 9.
95%
PA−1
PA−2
PB−1
PB−2
PB−3
Holarctic
Hybrid
NoAm
SoAm
PA−1PA−2
−75
−50
−25
0
25
50
−150 −100 −50 0 50PC1 = 41.56%
PC2
= 7.
81%
PA−1
PA−2
−50
0
50
−100 0 100 200Longitude
Latitude
PA−1PA−2
PB−1PB−2
PB−3Holarctic
NoAmSoAm
−55
−50
−45
−40
−75 −70 −65 −60Longitude
Latitude
B
C
E
D
A
PB1
PB−2
PB−3
Holarctic−50
0
50
−50 0 50 100PC1 = 19.03%
PC2
= 13
.96%
PB−1
PB−2
PB−3
Holarctic
Hybrid
F
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admixed subpopulations and one abundant North American admixed lineage (Figure 1D and 134
Figure S1). We previously described the two major populations, PA and PB-Holarctic (Peris et 135
al. 2014, 2016), as well as the subpopulations PA-1, PA-2, PB-1, PB-2, Holarctic, and the North 136
American admixed lineage (Peris et al. 2016). PB-3 had been suggested by multilocus data 137
(Eizaguirre et al. 2018), and our new analyses confirm this subpopulation with whole genome 138
sequence data. All of the strains isolated from outside of South America belonged to either the 139
previously described North American admixed lineage (NoAm) or one of two PB 140
subpopulations, PB-1 or Holarctic. This dataset included novel PB-1 isolates from the states of 141
Washington (yHRMV83) and North Carolina (yHKB35). Unexpectedly, from this same site in 142
North Carolina, we also obtained new isolates of the NoAm admixed lineage (Figure 1C and 143
Table S1), and we obtained additional new NoAm strains in South Carolina. Together, with the 144
North Carolina strains reported here and previously (Peris et al. 2016), this region near the Blue 145
Ridge Mountains harbors three subpopulations or lineages, PB-1, Holarctic, and NoAm. We 146
were also successful in re-isolating the NoAm lineage from the same Wisconsin site, sampling 147
two years later than what was first reported (Peris et al. 2014) (Table S1), indicating that the 148
NoAm admixed lineage is established, not ephemeral, in this location. Additionally, we found 149
one novel South American strain that was admixed between PA (~45%) and PB (~55%) (Figure 150
1D “SoAm”). This global distribution and the well-differentiated population structure of S. 151
eubayanus is similar to what has been observed in S. paradoxus (Leducq et al. 2014, 2016) and 152
Saccharomyces uvarum (Almeida et al. 2014). 153
S. eubayanus has been isolated from numerous substrates and hosts, and our large dataset 154
afforded us the power to analyze host and substrate association by subpopulation. We found that 155
PA-2 was associated with the seeds of Araucaria araucana (45.71% of isolates, p-val = 6.11E-156
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07, F-statistic = 15.29). Interestingly, while PB-1 was the most frequently isolated subpopulation 157
(34% of isolates), it has never been isolated from A. araucana seeds. Instead, PB-1 was 158
associated with Nothofagus antarctica (52.31% of isolates, p-val = 0.017, F-statistic = 3.10). PB-159
1 was also the subpopulation isolated the most from Nothofagus dombeyi (75% of isolates from 160
this tree species), which is a common host of S. uvarum (Libkind et al. 2011; Eizaguirre et al. 161
2018). PB-2 was positively associated with Nothofagus pumilio (36.59% of isolates, p-val = 9.60 162
E-04, F-statistic = 6.59), which could be an ecological factor keeping PB-2 partly isolated from 163
its sympatric subpopulations, PA-2 and PB-1 (Figure 1C). PB-3 was associated with the fungal 164
parasite Cyttaria darwinii (14.29% of isolates, p-val = 0.039, F-statistic = 25.34 ) and 165
Nothofagus betuloides (28.57% of isolates, p-val = 5.02E-06, F-statistic = 60.35), which is only 166
found in southern Patagonia and is vicariant with N. dombeyi, a host of PB-1. PB-3 was 167
frequently isolated in southern Patagonia (49% of southern isolates) (Eizaguirre et al. 2018), and 168
its association with a southern-distributed tree species could play a role in its geographic range 169
and genetic isolation from the northern subpopulations. Neither Nothofagus nor A. araucana are 170
native to North America, and we found that our North American isolates were from multiple 171
diverse plant hosts, including Juniperus virginiana, Diospyros virginiana, Cedrus sp., and Pinus 172
sp. (Table S1), as well as from both soil and bark samples. In Patagonia, S. eubayanus has been 173
isolated from exotic Quercus trees (Eizaguirre et al. 2018), so even though Nothofagus and A. 174
araucana are common hosts, S. eubayanus can be found on a variety of hosts and substrates. 175
These observed differences in host and substrate could be playing a role in the maintenance of its 176
population structure, especially in sympatric regions of Patagonia. 177
178
All subpopulations grow at freezing temperatures and on diverse carbon sources: 179
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S. eubayanus comes from a wide range of environments, so we tested if there were 180
phenotypic differences between these subpopulations. We measured growth rates on several 181
carbon sources and stress responses for a large subset of these strains (190) and 26 lager-brewing 182
strains (Figure 2 and Figure S2). Lager-brewing strains grew faster on maltotriose than all 183
subpopulations (p-val < 0.05, Figure 2A), which is consistent with this sugar being one of the 184
most abundant in brewing wort but rare in nature (Salema-Oom et al. 2005). The Holarctic 185
subpopulation grew slower on glucose and maltose compared to all other subpopulations (p-val < 186
0.05, Figure 2A, Table S2). Overall, the admixed NoAm lineage performed better than PB-1 (p-187
val = 0.038, Figure 2A), but there was no interaction with carbon source. Therefore, the admixed 188
lineage’s robustness in many conditions could play a role in its success in far-flung North 189
American sites where no pure PA or PB strains have ever been found. 190
191 Figure 2. Phenotypic differences. 192 (A) Heat map of mean of maximum growth rate (change in OD/hour) (GR) on different carbon sources by 193 subpopulation. Warmer colors designate faster growth. (B) Heat map of log10 normalized growth at 194 different temperatures by subpopulation. 195 196
Since S. eubayanus’ contribution to the cold-adaptation of hybrid brewing strains is well 197
established (Libkind et al. 2011; Gibson et al. 2013; Baker et al. 2019), we measured growth at 198
0°C, 4°C, 10°C, and 20°C. All subpopulations grew at temperatures as low as 0°C (Figure 2B 199
GR
Raffinose
Maltotriose
Glycerol
EthanolGlucose
Maltose
Galactose
PA−2NoAmPA−1PB−2PB−3PB−1LagerHolarctic
0.01
0.02
0.03
0.04
0.05
0.06
RaffinoseMaltotriose
Glycerol
EthanolGlucose
Maltose
Galactose
PA−2NoAmPA−1PB−2PB−3PB−1LagerHolarctic
0.01
0.02
0.03
0.04
0.05
0.06GR
20C10C0C 4C
LagerHolarcticPA−2NoAmPB−2PB−3PB−1PA−1 0.05
0.1
0.15
20C10C0C 4C
LagerHolarcticPA−2NoAmPB−2PB−3PB−1PA−1 0.05
0.1
0.15BA
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and Figure S2), and all S. eubayanus subpopulations outperformed lager-brewing yeasts (p < 200
0.05). Within pure S. eubayanus, there were no temperature by subpopulation interactions, 201
indicating that no subpopulation is more cryotolerant than any other subpopulation. In summary, 202
we found that all strains that we tested grew similarly in many environments, and despite the 203
large amount of genotypic diversity observed for this species, we observed much less phenotypic 204
diversity (Figure 2). 205
206
Subpopulations are well differentiated: 207
The mating strategies and life cycle of Saccharomyces, with intratetrad mating and 208
haploselfing, often lead to homozygous diploid individuals (Hittinger 2013). Nonetheless, in S. 209
cerevisiae, many industrial strains are highly heterozygous (Gallone et al. 2016; Gonçalves et al. 210
2016; Peter et al. 2018). Here, we analyzed genome-wide heterozygosity in our collection of 200 211
strains and found only one individual with more than 20,000 heterozygous SNPs (Figure S3). 212
When we phased highly heterozygous regions of its genome and analyzed the two phases 213
separately, we found that both phases grouped within PB-1 (Figure S3C). Thus, while this strain 214
is highly heterozygous, it has contributions from only one subpopulation. 215
This large collection of strains is a powerful resource to explore natural variation and 216
population demography in a wild microbe, so we analyzed several common population genomic 217
statistics in 50-kbp windows across the genome. We found that diversity was similar between 218
subpopulations (Figure S4A). We also calculated Tajima’s D and found that the genome-wide 219
mean was zero or negative for each subpopulation (Figure S4B), which could be indicative of 220
population expansions. In particular, the most numerous and widespread subpopulation, PB-1, 221
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had the most negative and consistent Tajima’s D, suggesting a recent population expansion is 222
especially likely in this case. 223
224 Figure 3. Population genomic parameters. 225 (A) Network built with pairwise FST values < 0.8 between each subpopulation. FST values are printed and 226 correspond to line thickness, where lower values are thicker. Circle sizes correspond to genetic diversity. 227 (B) LD decay for each subpopulation (colors) and the species in whole (black). 228 229 For the non-admixed lineages, genome-wide average FST was consistently high across the 230
genome (Figure S4C). In pairwise comparisons of FST, PB-1 had the lowest values of any 231
subpopulation (Figure 3A, Figure S4D). These pairwise comparisons also showed that, within 232
each population, there has been some gene flow between subpopulations, even though the 233
subpopulations were generally well differentiated. Linkage disequilibrium (LD) decay indicated 234
low recombination in these wild subpopulations (Figure 3B), with variability between 235
subpopulations. For the species as a whole, LD decayed to one-half at about 5 kbp, which is 236
somewhat higher than the 500bp - 3kbp observed in S. cerevisiae (Liti et al. 2009; Schacherer et 237
al. 2009; Peter et al. 2018) and lower than the 9 kbp observed in S. paradoxus (Liti et al. 2009), 238
indicating that there is less mating, outcrossing, and/or recombination in this wild species than S. 239
cerevisiae and more than in S. paradoxus. 240
0.0
0.2
0.4
0.6
0KB 30KB 60KB 90KB 120KB 150KB 180KB 210KB 240KB 270KBSNP distance
r2
SubpopPA−1
PA−2
PB−1
PB−2
PB−3
Seub
A B
0.43
0.51
0.59
0.64
0.64
0.670.69
0.70
0.72
0.73
0.78
0.78
0.80
●
●
SoAm
NoAm
PA−1
PA−2
PB−1
PB−2
PB−3
Hol
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241
Recent admixture and historical gene flow between populations: 242
We previously reported the existence of 7 strains of an admixed lineage in Wisconsin, 243
USA, and New Brunswick, Canada (Peris et al. 2014, 2016). Here, we present 14 additional 244
isolates of this same admixed lineage. These new isolates were from the same site in Wisconsin, 245
as well as two new locations in North Carolina and South Carolina (Table S1). Strikingly, all 21 246
strains shared the exact same genome-wide ancestry profile (Figure 4A), indicating that they all 247
descended from the same outcrossing event between the two main populations of S. eubayanus. 248
These admixed strains were differentiated by 571 SNPs, which also delineated these strains 249
geographically (Figure 4B). Pairwise diversity and FST comparisons across the genomes suggest 250
that the PA parent came from the PA-2 subpopulation (Figure 4C and Figure S5A) and that the 251
PB parent was from the PB-1 subpopulation (Figure 4D and Figure S5A). 252
253 Figure 4. Genomic ancestries of NoAm and SoAm admixed lineages. 254
0.0
0.5
1.0
1.5
I II III IV VVI VII VIII IX X XI XII XIII XIV XV XVIGenome Position
Pairw
ise
Dive
rgen
ce (%
)
SubpopPB−1
PB−2
PB−3
0.0
0.5
1.0
1.5
I II III IV VVI VII VIII IX X XI XII XIII XIV XV XVIGenome Position
Pairw
ise
Dive
rgen
ce (%
)
SubpopPA−1
PA−2
−5
0
5
I II III IV VVI VII VIII IX X XI XII XIII XIV XV XVIGenome Position
log 2(d
A-So
Amd B
-SoA
m)
SoAmyHDPN421
yHDPN422
yHDPN423
yHDPN424
yHKB201
yHKB210
yHKB218
yHKB242
yHKB246
yHKB251
yHKB268
yHKS210
yHKS211
yHKS212
yHKS576
yHKS689
yHKS694
yHKS810
yHKS812
yHQL1481
yHQL1621
I II III IV VVIVIIVIII IX X XI XII XIIIXIVXVXVIGenome Position
log 2(d
A-N
oAm
d B-N
oAm)
0.0
0.5
1.0
1.5
I II III IV VVI VII VIII IX X XI XII XIII XIV XV XVIGenome Position
Pairw
ise
Dive
rgen
ce (%
)
SubpopPB−1
PB−2
PB−3
0.0
0.5
1.0
1.5
I II III IV VVI VII VIII IX X XI XII XIII XIV XV XVIGenome Position
Pairw
ise
Dive
rgen
ce (%
)
SubpopPA−1
PA−2
A
C
D
E
F
G
yHDPN421yHDPN424
yHDPN423
yHDPN422
yHKS211yHKS210
yHKS212
yHKS576yHKS689
yHKS694yHKB251
yHKB201yHQL1481
yHKS810yHQL1621yHKS812
yHKB210
yHKB218
yHKB246yHKB242
yHKB268
0.1
New Brunswick,
Canada South Carolina, USA
North Carolina, USAWisconsin,
USA
NoAm
B 0.1
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(A) For all 21 NoAm admixed strains, log2 ratio of the minimum PB-NoAm pairwise nucleotide sequence 255 divergence (dB-NoAm) and the minimum PA-NoAm pairwise nucleotide sequence divergence (dA-256 NoAm) in 50-kbp windows. Colors and log2 < 0 or > 0 indicate that part of the genome is more closely 257 related to PA or PB, respectively. (B) Neighbor-Net phylogenetic network reconstructed with the 571 258 SNPs that differentiate the NoAm strains. The scale bar represents the number of substitutions per site. 259 Collection location is noted. (C) Pairwise nucleotide sequence divergence of the NoAm strain yHKS210 260 compared to strains from the PA-1 and PA-2 subpopulations of PA in 50-kbp windows. (D) Pairwise 261 nucleotide sequence divergence of the NoAm strain yHKS210 compared to strains from the PB-1, PB-2, 262 and PB-3 subpopulations of PB in 50-kbp windows. (E) log2 ratio of the minimum PB-SoAm pairwise 263 nucleotide sequence divergence (dB-SoAm) and the minimum PA-SoAm pairwise nucleotide sequence 264 divergence (dA-SoAm) in 50-kbp windows. Colors and log2 < 0 or > 0 indicate that part of the genome is 265 more closely related to PA or PB, respectively. (F) Pairwise nucleotide sequence divergence of the SoAm 266 strain compared to strains from the PA-1 and PA-2 subpopulations of PA in 50-kbp windows. (G) 267 Pairwise nucleotide sequence divergence of the SoAm strain compared to strains of the PB-1, PB-2, and 268 PB-3 subpopulations of PB in 50-kbp windows. 269 270 Here, we report a second instance of recent outcrossing between PA and PB. One other 271
strain with fairly equal contributions from the two major populations, PA (~45%) and PB 272
(~55%) (Figure 4E), was isolated from the eastern side of Nahuel Huapi National Park, an area 273
that is sympatric for all subpopulations found in South America. This strain had a complex 274
ancestry, where both PA-1 and PA-2 contributed to the PA portions of its genome (Figure 4F and 275
Figure S5B), indicating that its PA parent was already admixed between PA-1 and PA-2. As with 276
the NoAm admixed strains, the PB parent was from the PB-1 subpopulation (Figure 4G and 277
Figure S5B). Together, these two admixed lineages show that outcrossing occurs between the 278
two major populations, and that admixture and gene flow are likely ongoing within sympatric 279
regions of South America. 280
We also found examples of smaller tracts of admixture between PA and PB that were 281
detectable as 2-12% contributions. These introgressed strains included the taxonomic type strain 282
of S. eubayanus (CBS12357T), whose genome sequence was mostly inferred to be from PB-1, 283
but it had a ~4% contribution from PA-1 (Figure S6). We found several other examples of 284
admixture between PA and PB, as well as admixture between subpopulations of PA or of PB 285
(Table S3). Notably, the PB contributions were usually from PB-1, the subpopulation with the 286
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largest range, most hosts, and strongest signature of population expansion, factors that would 287
tend to make contact with other subpopulations more likely. 288
In our collection of 200 strains, we observed nuclear genome contributions from S. 289
uvarum in four strains. These four strains all shared the same introgression of ~150-kbp on 290
chromosome XIV (Figure S7A&B). When we analyzed the portion of the genome contributed by 291
S. eubayanus, we found that these strains were all embedded in the PB-1 subpopulation (Figure 292
S7C). Analysis of the 150-kbp region from S. uvarum indicated that the closest S. uvarum 293
population related to these introgressed strains was SA-B (Figure S7D), a population restricted to 294
South America that has not previously been found to contribute to any known interspecies 295
hybrids (Almeida et al. 2014). These strains thus represent an independent hybridization event 296
between South American lineages of these two sister species that is not related to any known 297
hybridization events among industrial strains (Almeida et al. 2014). These strains show that S. 298
eubayanus and S. uvarum can and do hybridize in the wild, but the limited number (n=4) of 299
introgressed strains, small introgression size (150-kbp), and shared breakpoints suggest that the 300
persistence of hybrids in the wild is rare. 301
302
Northern Patagonia is a diversity hot spot: 303
Patagonia harbors the most genetic diversity of S. eubayanus in our dataset, and four 304
subpopulations were found only there: PA-1, PA-2, PB-2, and PB-3 (Figure 1A and 4A). 305
Therefore, we examined the genetic diversity and range distributions of the isolates from South 306
America more closely. Nahuel Huapi National Park yielded isolates from all five subpopulations 307
found in South America, was the only place where PA-1 was found, and was the location where 308
the SoAm admixed strain was isolated (Figure 5A & B). All five sub-populations were found 309
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15
north of 43°S, an important boundary during the last glaciation period that affects many 310
organisms (Mathiasen and Premoli 2010; Premoli et al. 2010; Quiroga and Premoli 2010). 311
Species-wide, there was more genetic diversity north of this boundary (Figure 5B). In contrast, 312
only PB-1 and PB-3 were found south of 43°S, with both distributions reaching Tierra del Fuego. 313
The southernmost strains were primarily PB-3 (89.7%), but they included two highly admixed 314
PB-1 × PB-3 strains (Table S1 & S3). 315
316 Figure 5. South American genomic diversity versus range, diversity by area, and isolation by distance. 317 (A) Range and genomic diversity of South American sampling sites. Circle sizes correspond to nucleotide 318 diversity of all strains from that site, and pie proportions correspond to each subpopulation’s contribution 319 to 𝜋 at each site. Latitudinal range of each subpopulation is shown to the right. (B) Nucleotide diversity 320 by subpopulation by sampling site, where larger and darker circles indicate more diversity. “SA Sites” in 321 gray show the diversity of all strains found in each South American (SA) site. “World Sites” in darker 322 gray show the nucleotide diversity of all North American or non-South American strains, regardless of 323 subpopulation, compared to South American strains south or north of 43°S, aligned to mean latitude of all 324 strains included in the analysis. (C) Correlation of nucleotide diversity and the area or distance a 325 subpopulation covers. The y-axis shows the nucleotide diversity of each subpopulation, and circle sizes 326 correspond to the geographic sizes of the subpopulations on a log10 scale. Note that PA-1 (dark red) is as 327 diverse as PB-3 (dark blue) but encompasses a smaller area. (D) log10(pairwise nucleotide diversity) 328 correlated with distance between strains, which demonstrates isolation by distance. Note that y-axes are 329 all scaled the same but not the x-axes. Holarctic includes the S. eubayanus sub-genome of two lager-330 brewing strains. Figure S8A shows the individual plots for the NoAm lineage. Figure S8B shows the 331 individual plot of PB-1. 332 333 Despite the limited geographic range of some subpopulations, their genetic diversity was 334
high, and this diversity often did not scale with the geographic area over which they were found 335
(Figure 5C). The widespread distribution of some subpopulations led us to question if there was 336
isolation by distance within a subpopulation (Figure 5D). We used pairwise measures of 337
−55
−50
−45
−40
PA−1 PA−2 PB−1 PB−2 PB−3 SoAm SASites
WorldSites
Latit
ude
NucleotideDiversity (%)
0.0
0.1
0.2
0.3
0.4
0.5
−55
−50
−45
−40
−75 −70 −65 −60Longitude
Latitude
PA−1 PA−2 PB−1 PB−2 PB−3
BA D
All North AmericanAll Non-South American
CPA−1 PA−2 NoAm PB−1 PB−2 PB−3 Hol
0 10 20 0 200 400 0 500 10001500 0 5000 10000 0 100 200 0 500 1000 1500 0 5000 10000
0.0001
0.0010
0.0100
0.1000
Distance (Km)
log(
Nuc
leot
ide
Dive
rsity
(%))
● ●
● ●
●
● ●
● ●
●
● ●
● ●
0.00
0.05
0.10
0.15
0.20
Area(10 Km^2)
Distance(Km)
Nuc
leot
ide
Dive
rsity
(%)
Subpopulation●●
●●
●●
●●
●●
●●
PA−1
PA−2
PB−1
PB−2
PB−3
NoAm
PopulationGeographicSize
100000
200000
300000
400000
p-val=0.0001 p-val=0.0001 p-val=0.0001 p-val=0.0001 p-val=0.0001p-val=0.0003p-val=0.564
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diversity and geographic distance between each strain and conducted Mantel tests for each 338
subpopulation. All subpopulations showed significant isolation by distance (Table S4), except 339
PA-1, likely because it had the smallest geographic range (25 km). Even the Mantel test for the 340
least diverse lineage, NoAm, was highly significant (p-val = 0.0001, R2= 0.106), indicating that 341
each location has been evolving independently after their recent shared outcrossing and dispersal 342
event. Through these pairwise analyses, we also detected two strains from Cerro Ñielol, Chile, 343
that were unusually genetically divergent from the rest of PB-1 and could potentially be a novel 344
lineage (Figure S8). 345
346
Additional global regions are climatically suitable: 347
The sparse but global distribution of S. eubayanus raises questions about whether other 348
areas of the world could be suitable for this species. We used the maxent environmental niche 349
modeling algorithm implemented in Wallace (Kass et al. 2018) to model the global climatic 350
suitability for S. eubayanus, using GPS coordinates of all known S. eubayanus strains published 351
here and estimates of coordinates for the East Asian isolates (Bing et al. 2014). These niche 352
models were built using the WorldClim Bioclims, which are based on monthly temperature and 353
rainfall measures, reflecting both annual and seasonal trends, as well as extremes, such as the 354
hottest and coldest quarters. How climatic variables affect yeast distributions is an understudied 355
area, and building these models allowed us a novel way to explore climatic suitability. 356
Using all known locations of isolation (Figure 6), we found that the best model 357
(AIC=2122.4) accurately delineated the known distribution along the Patagonian Andes. In 358
North America, the strains from the Olympic Mountains of Washington state and the Blue Ridge 359
region of North Carolina fell within the predicted areas, and interestingly, these sites had yielded 360
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pure PB-1 and Holarctic strains. In contrast, some of the NoAm admixed strains were found in 361
regions that were on the border of suitability in this model (New Brunswick and Wisconsin). In 362
Asia, the model predicts further suitable regions along the Himalayas that are west of known 363
locations. 364
365
Figure 6. Predicted climatic suitability of S. eubayanus. 366 Minimum training presence (light green) and 10th percentile training presence (dark green) based on a 367 model that includes all known S. eubayanus isolations, as well as a scenario of dispersal and 368 diversification out of Patagonia (inset and arrows). Black arrows signify diversification events, dotted 369 lines are diversification events where the population is not found in Patagonia, and colored arrows are 370 migration events for the lineage of matching color. Roman numerals order the potential migration events. 371 S. eubayanus has not been found in the wild in Europe, but it has contributed to fermentation hybrids, 372 such as lager yeasts. This scenario proposes that the last common ancestor of PA and PB-Holarctic 373 bifurcated into PA (red) and PB-Holarctic (blue), which further radiated into PA-1 (dark red), PA-2 (light 374 red), PB-1 (blue), PB-2 (lighter blue), PB-3 (dark blue), and Holarctic (very light blue). At least four 375 migration events are needed to explain the locations where S. eubayanus has been found. I. The Holarctic 376 subpopulation was drawn from the PB-Holarctic gene pool and colonized the Holarctic ecozone. II. PB-1 377 colonized the Pacific Rim, including New Zealand and Washington state, USA. III. An independent 378 dispersal event brought PB-1 to North Carolina, USA. IV. Outcrossing between PA-2 and PB-1 gave rise 379 to a low-diversity admixed linage that has recently invaded a large swath of North America. 380 381
The uneven global distribution of S. eubayanus led us to test if models were robust to 382
being built only with the South American locations or only with the non-South American 383
locations (Figure S9). Remarkably, with just the South American isolates, the model 384
(AIC=1327.32) accurately predicted the locations of the non-South American isolates (Figure 385
Predicted Climatic Suitability
Longitude
Latitude
40°S
20°S
0°
20°N
40°N
60°N
150°W 100°W 50°W 0° 50°E 100°E 150°E
0.0
0.5
1.0
I
II
III
IV
Ancestor
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S9A). Even the model built from the limited number of isolates from outside South America 386
(AIC=558.58) still performed reasonably well, identifying the regions in Patagonia along the 387
Andes where S. eubayanus has been found (Figure S9B). Collectively, these models suggest that 388
climatic modeling can predict other suitable regions for eukaryotic microbes. These approaches 389
could be used to direct future sampling efforts or applied to other microbes to gain further insight 390
into microbial ecology. 391
Notably, all models agree that Europe is climatically a prime location for S. eubayanus 392
(Figure S9C), but no pure isolates have ever been found there, only hybrids with S. uvarum, S. 393
cerevisiae, or hybrids with even more parents (Almeida et al. 2014). These hybrids with complex 394
ancestries have been found in numerous fermentation environments, suggesting that pure S. 395
eubayanus once existed, or still exists at low abundance or in obscure locations, in Europe. Thus, 396
the lack of wild isolates from sampling efforts in Europe remains a complex puzzle. 397
398
Discussion: 399
Here, we integrated genomic, geographic, and phenotypic data for 200 strains of S. 400
eubayanus, the largest collection to date, to gain insight into its world-wide distribution, climatic 401
suitability, and population structure. All the strains belong to the two major populations 402
previously described (Peris et al. 2014, 2016), but with the extended dataset, we were able to 403
define considerable additional structure, consisting of six subpopulations and two admixed 404
lineages. These subpopulations have high genetic diversity, high FST, and long LD decay; all 405
measures indicative of large and partly isolated populations undergoing limited gene flow. 406
Despite this high genetic diversity, there was relatively little phenotypic differentiation between 407
subpopulations. This dichotomy between large genetic diversity and limited phenotypic 408
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differentiation hints at a complex demographic history where genetically differentiated 409
subpopulations are minimally phenotypically differentiated and grow well in a wide range of 410
environments. 411
Despite the strong population structure, we also observed considerable evidence of 412
admixture and gene flow. The two recently admixed lineages had nearly equal contributions 413
from the two major populations, but they were the result of independent outcrossing events. The 414
SoAm admixed strain was isolated from a hotspot of diversity and contains contributions from 415
three subpopulations. The NoAm admixed lineage has spread across at least four distant 416
locations, but all strains descended from the same outcrossing event. Since PA has only been 417
isolated in South America, it is intriguing that the NoAm admixed lineage has been successful in 418
so many locations throughout North America. The success of this lineage could be partially 419
explained by its equal or better performance in many environments in comparison to its parental 420
populations (Fig. 2), perhaps contributing to its invasion of several new locations. Several other 421
Patagonian strains also revealed more modest degrees of gene flow between PA and PB. Finally, 422
we characterized a shared nuclear introgression from S. uvarum into four Patagonian strains of S. 423
eubayanus, demonstrating that hybridization and backcrossing between these sister species has 424
occurred in the wild in South America. 425
S. eubayanus has a paradoxical biogeographical distribution; it is abundant in Patagonia, 426
but it is sparsely found elsewhere with far-flung isolates from North America, Asia, and Oceania. 427
Most subpopulations displayed isolation by distance, but genetic diversity only scaled with 428
geographic range to a limited extent. In Patagonia, some sampling sites harbor more genetic 429
diversity than all non-Patagonian locations together (Figure 5B). Although we found the most 430
genetic diversity and largest number of subpopulations north of 43°S, the pattern of genetic 431
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diversity appears to be reversed on the west side of the Andes, at least for the PB-1 432
subpopulation (Nespolo et al. 2019). This discrepancy could be due to differences in how glacial 433
refugia were distributed (Sérsic et al. 2011) and limitations on gene flow between the east and 434
west sides of the Andes. Together, the levels of S. eubayanus genetic diversity found within 435
Patagonia, as well as the restriction of four subpopulations to Patagonia, suggest that Patagonia is 436
the origin of most of the diversity of S. eubayanus, likely including the last common ancestor of 437
the PA and PB-Holarctic populations. 438
The simplest scenario to explain the current distribution and diversity of S. eubayanus is 439
a series of radiations in Patagonia, followed by a handful of out-of-Patagonia migration events 440
(Figure 6). Under this model, PA and PB would have bifurcated in Patagonia, possibly in 441
separate glacial refugia. The oldest migration event would have been the dispersal of the ancestor 442
of the Holarctic subpopulation, drawn from the PB gene pool, to the Northern Hemisphere. 443
Multiple more recent migration events could have resulted in the few PB-1 strains found in New 444
Zealand and the USA. The New Zealand and Washington state strains cluster phylogenetically 445
and could have diversified from the same migration event from Patagonia into the Pacific Rim. 446
The PB-1 strain from North Carolina (yHKB35) is genetically more similar to PB-1 strains from 447
Patagonia, suggesting it arrived in the Northern Hemisphere independently of the Pacific Rim 448
strains. Finally, the NoAm admixed strains are likely the descendants of a single, and relatively 449
recent, out-of-Patagonia dispersal. Given that PA appears to be restricted to northern Patagonia, 450
this region could have been where the hybridization leading to the NoAm lineage occurred. 451
While the dispersal vector that brought this admixed lineage to North America is unknown, its 452
far-flung distribution and low diversity show that it has rapidly succeeded by invading new 453
environments. 454
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21
Other more complex scenarios could conceivably explain the limited number of strains 455
found outside of Patagonia. For example, PA and PB could represent sequential colonizations of 456
Patagonia from the Northern Hemisphere. Under this model, PA would have arrived first and 457
would then have been restricted to northern Patagonia by competition with the later arrival of 458
PB. The Holarctic subpopulation could be interpreted as remnants of the PB population that did 459
not migrate to Patagonia; but the PB-1 strains from the Northern Hemisphere, especially 460
yHKB35, seem far more likely to have been drawn from a Patagonian gene pool than the other 461
way around. Furthermore, the structuring of the PA-1 and the PA-2 subpopulations and of the 462
PB-1, PB-2, and PB-3 subpopulations are particularly challenging to rectify with models that do 463
not allow for diversification within South America. Even more complex scenarios remain 464
possible, and more sampling and isolation will be required to fully elucidate the distribution of 465
this elusive species and more conclusively reject potential biogeographical models. 466
S. eubayanus has a strikingly parallel population structure and genetic diversity to its 467
sister species S. uvarum (Almeida et al. 2014; Peris et al. 2016). Both species are abundant and 468
diverse in Patagonia but can be found globally. Both have early diverging lineages, found in Asia 469
or Australasia, that border on being considered novel species. In South America, both have two 470
major populations, where one of these populations is restricted to northern Patagonia (north of 471
43°S). However, a major difference between the distribution of these species is that pure strains 472
of S. uvarum have been found in Europe. Many dimensions of biodiversity could be interacting 473
to bound the distribution and population structure of both S. eubayanus and S. uvarum. In 474
particular, we know very little about local ecology, including the biotic community and 475
availability of abiotic resources on a microbial scale, but these factors likely all influence 476
microbial success. We show here that substrate and host association vary between 477
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subpopulations. In Patagonia, S. eubayanus and S. uvarum are commonly associated with 478
Nothofagus, where N. dombeyi is the preferred host of S. uvarum (Libkind et al. 2011; Eizaguirre 479
et al. 2018). Therefore, niche partitioning of host trees could be playing role in the persistence of 480
these species in sympatry in Patagonia. However, in locations where Nothofagus is not found and 481
there are perhaps fewer hosts, competitive exclusion between the sister species S. eubayanus and 482
S. uvarum, could influence distribution. Competition for a narrower set of hosts could potentially 483
explain why only S. uvarum has been found in Europe as pure strains, while S. eubayanus has 484
not. A second factor influencing distribution and population structure could be dispersal. Yeasts 485
could migrate via many avenues, such as wind, insect, bird, or other animals (Francesca et al. 486
2012, 2014; Stefanini et al. 2012; Gillespie et al. 2012). Human mediated-dispersal has been 487
inferred for the S. cerevisiae Wine and Beer lineages and for the S. paradoxus European/SpA 488
lineage (Gallone et al. 2016; Gonçalves et al. 2016; Leducq et al. 2014; Kuehne et al. 2007). A 489
third bounding factor could be a region’s historical climate. Glacial refugia act as reservoirs of 490
isolated genetic diversity that allow expansion into new areas after glacial retreat (Stewart and 491
Lister 2001). 43°S is a significant geographic boundary due to past geological and climatic 492
variables (Mathiasen and Premoli 2010; Eizaguirre et al. 2018), and many other species and 493
genera show a distinction between their northern and southern counterparts, including 494
Nothofagus (Mathiasen and Premoli 2010; Premoli et al. 2012). S. eubayanus and S. uvarum 495
diversities are also strongly affected by the 43°S boundary (Almeida et al. 2014; Eizaguirre et al. 496
2018), and it seems likely that the microbes experienced some of the same glaciation effects as 497
their hosts. The strong correlation of S. eubayanus and S. uvarum population structures with 498
43°S further implies a longstanding and intimate association with Patagonia. 499
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23
The sparse global distribution and complex patterns of genetic diversity continue to raise 500
questions about the niche and potential range of S. eubayanus. Our climatic modeling suggests 501
that parts of Europe would be ideal for S. eubayanus. Despite extensive sampling efforts, S. 502
eubayanus has never been isolated in Europe (Sampaio 2018). However, recent environmental 503
sequencing of the fungal specific ITS1 region hinted that S. eubayanus may exist in the wild in 504
Europe (Alsammar et al. 2019). Considerable caution is warranted in interpreting this result 505
because the rDNA locus quickly fixes to one parent’s allele in interspecies hybrids, there is only 506
a single ITS1 SNP between S. uvarum and S. eubayanus, and the dataset contained very few 507
reads that mapped to S. eubayanus. Still, the prevalence of hybrids with contributions from the 508
Holarctic lineage of S. eubayanus found in Europe (Peris et al. 2016) suggests that the Holarctic 509
lineage exists in Europe, or at least existed historically, allowing it to contribute to many 510
independent hybridization events. 511
The patterns of radiation and dispersal observed here mirror the dynamics of evolution 512
found in other organisms (Czekanski-Moir and Rundell 2019), including humans (Nielsen et al. 513
2017). S. eubayanus and humans harbor diverse and structured populations in sub-Saharan 514
Africa and Patagonia, respectively. In these endemic regions, both species show signals of 515
ancient and recent admixture between these structured populations. Both species have 516
successfully colonized wide swaths of the globe, with the consequence of repeated bottlenecks in 517
genetic diversity. While anatomically modern humans underwent a single major out-of-Africa 518
migration that led to the peopling of the world (Nielsen et al. 2017), S. eubayanus has 519
experienced several migration events from different populations that have led to more punctate 520
global distribution. For both species, intraspecific admixture and interspecific hybridization 521
appear to have played adaptive roles in the success of colonizing these new locations. In humans, 522
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24
introgressions from past hybridizations with both Neanderthals and Denisovans underlie many 523
adaptive traits (Racimo et al. 2015), while the cold fermentation of lager-brewing would not be 524
possible without the cryotolerance of S. eubayanus and the aggressive fermentation of 525
domesticated ale strains of S. cerevisiae (Gibson and Liti 2015). These parallels illustrate how 526
the biogeographical and evolutionary dynamics observed in plants and animals also shape 527
microbial diversity. As yeast ecology and population genomics (Marsit et al. 2017; Yurkov 528
2017) move beyond the Baas-Becking “Everything is everywhere” hypothesis of microbial 529
ecology (Baas-Becking 1934; de Wit and Bouvier 2006), the rich dynamics of natural diversity 530
that is hidden in the soil at our feet is being uncovered. 531
532
Methods: 533
Wild strain isolations 534
All South American isolates were sampled, isolated, and identified as described 535
previously (Libkind et al. 2011; Eizaguirre et al. 2018). North American isolates new to this 536
publication were from soil or bark samples from the American states of Washington, Wisconsin, 537
North Carolina, and South Carolina (Table S1). Strain enrichment and isolation was done as 538
previously described (Sylvester et al. 2015; Peris et al. 2016, 2014), with a few exceptions in 539
temperature and carbon source of isolation (Table S1). Specifically, two strains were isolated at 540
4°C, eight strains were isolated at room temperature, and six strains were isolated on a non-541
glucose carbon source: three in galactose, two in sucrose, and one in maltose (Table S1). 542
543
Whole genome sequencing and SNP-calling 544
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25
Whole genome sequencing was completed with Illumina paired-end reads as described 545
previously (Peris et al. 2016; Shen et al. 2018). Reads were aligned to the reference genome 546
(Baker et al. 2015), SNPs were called, masked for low coverage, and retained for downstream 547
analysis as described previously (Peris et al. 2016). One strain, yHCT75, had more than 20,000 548
heterozygous SNPs called. This strain was pseudo-phased using read-backed phasing in GATK 549
(McKenna et al. 2010) and split into two phases. Short-read data is deposited in the NCBI Short 550
Read Archive under PRJNA555221. 551
552
Population genomic analyses: 553
Population structure was defined using several approaches: fastSTRUCTURE (Raj et al. 554
2014), fineSTRUCTURE (Lawson et al. 2012), SplitsTree v4 (Huson and Bryant 2006), 555
and Principal Component Analysis with the adegenet package in R (Jombart 2008). 556
fineSTRUCTURE analysis was completed using all strains and 11994 SNPs. The 557
SplitsTree network was built with this same set of strains and SNPs. fastStructure 558
analysis was completed with as subsample of 5 NoAm strains and 150165 SNPs. We tested K=1 559
through K=10 and selected K=6 using the “chooseK.py” command in fastSTRUCTURE. All 560
calculations of pairwise divergence, FST, and Tajima’s D for subpopulations were computed 561
using the R package PopGenome (Pfeifer and Wittelsbuerger 2015) in windows of 50-kbp. 562
Pairwise divergence between strains was calculated across the whole genome using PopGenome. 563
LD was calculated using PopLDdecay (Zhang et al. 2019). Geographic area and distance of 564
subpopulations was calculated using the geosphere package in R (Hijmans et al. 2019). The 565
Mantel tests were completed using ade4 package of R (Dray and Dufour 2007). The FST network 566
was built with iGraph in R (Csardi and Nepusz 2006). 567
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26
568
Niche projection with Wallace: 569
Climatic modeling of S. eubayanus was completed using the R package Wallace (Kass et 570
al. 2018). Three sets of occurrence data were tested: one that included only GPS coordinates for 571
strains from South America, one that included only non-South American isolates, and one that 572
included all known isolates (Table S1). We could use exact GPS coordinates for most strains, 573
except for the strains from East Asia, where we estimated the locations (Bing et al. 2014). 574
WorldClim bioclimatic variables were obtained at a resolution of 2.5 arcmin. The background 575
extent was set to “Minimum convex polygon” with a 0.5-degree buffer distance and 10,000 576
background points were sampled. We used block spatial partitioning. The model was built using 577
the Maxent algorithm, using the feature classes: L (linear), LQ (linear quadradic), H (Hinge), 578
LQH, and LQHP (Linear Quadradic Hinge Product) with 1-3 regularization multipliers and the 579
multiplier step value set to 1. The model was chosen based on the Akaike Information Criterion 580
(AIC) score (Table S5). The best models were then projected to the all continents, except 581
Antarctica. 582
583
Phenotyping: 584
Strains were first revived in Yeast Peptone Dextrose (YPD) and grown for 3 days at room 585
temperature. These saturated cultures were then transferred to two 96-well microtiter plates, for 586
growth rate and stress tolerance phenotyping. These plates were incubated overnight. Cells were 587
pinned from these plates into plates for growth rate measurements. For temperature growth 588
assays, cells were pinned into four fresh YPD microtiter plates and then incubated at 0°C, 4°C, 589
10°C, and 20°C. For the microtiter plates at 0°C, 4°C, and 10°C, OD was measured at least once 590
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27
a day for two weeks or until a majority of the strains had reached stationary phase. Growth on 591
different carbon sources was measured at 20°C in MM media with 2% of the respective carbon 592
source. Carbon sources tested were: glucose, galactose, raffinose, maltose, maltotriose, ethanol, 593
and glycerol. OD was read every two hours for one week or until saturation. All phenotyping 594
was completed in biological triplicate. The carbon source data was truncated to 125 hours to 595
remove artifacts due to evaporation. Growth curves were analyzed using the package grofit 596
(Kahm et al. 2010) in R to measure saturation and growth rate. We then averaged each strain 597
over the triplicates. We used an ANOVA corrected with Tukey’s HSD to test for growth rate 598
interactions between subpopulation and carbon source or subpopulation and temperature. We 599
used the R package pvclust (Suzuki and Shimodaira 2006) to cluster and build heatmaps of 600
growth rate by subpopulation. 601
Heat shock was completed by pelleting 200μl saturated culture, removing supernatant, 602
resuspending in 200μl YPD pre-heated to 37°C, and incubating for one hour at 37°C, with a 603
room temperature control. Freeze-thaw tolerance was tested by placing saturated YPD cultures in 604
a dry ice ethanol bath for two hours, with a control that was incubated on ice. After stress, the 605
strains were serially diluted 1:10 three times and pinned onto solid YPD. These dilution plates 606
were then photographed after 6 and 18 hours. CellProfiler (Lamprecht et al. 2007) was 607
used to calculate the colony sizes after 18 hours, and the 3rd (1:1000) dilutions were used for 608
downstream analyses. The heat shock measurements were normalized by the room temperature 609
controls, and the freeze thaw measurements were normalized by the ice incubation controls. 610
Statistical interactions of subpopulations and stress responses were tested as above. 611
612
Data Availability: 613
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28
All short-read genome sequencing data has been deposited in the NCBI Short Read Archive 614
under the PRJNA555221. Accessions of public data is given in Table S1. 615
616
Acknowledgements: 617
We thank Francisco A. Cubillos for coordinating publication with their study; Sean D. Schoville, 618
José Paulo Sampaio, Paula Gonçalves, and members of the Hittinger Lab, in particular 619
EmilyClare P. Baker, for helpful discussion and feedback; Amanda B. Hulfachor and Martin 620
Bontrager for preparing a subset of Illumina libraries; the University of Wisconsin 621
Biotechnology Center DNA Sequencing Facility for providing Illumina sequencing facilities and 622
services; the Agricultural Research Service (ARS) NRRL collection, Christian R. Landry, 623
Ashley Kinart, and Drew T. Doering for strains included in phenotyping; Huu-Vang Nguyen for 624
strains used for hybrid genome comparison; and Leslie Shown, Anita R. & S. Todd Hittinger, 625
EmilyClare P. Baker, and Ryan V. Moriarty for collecting samples and/or isolating strains. This 626
material is based upon work supported by the National Science Foundation under Grant Nos. 627
DEB-1253634 (to CTH) and DGE-1256259 (Graduate Research Fellowship to QKL), the USDA 628
National Institute of Food and Agriculture Hatch Project No. 1003258 to CTH, and in part by the 629
DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science Nos. DE-630
SC0018409 and DE-FC02-07ER64494 to Timothy J. Donohue). QKL was also supported by the 631
Predoctoral Training Program in Genetics, funded by the National Institutes of Health 632
(5T32GM007133). DP is a Marie Sklodowska-Curie fellow of the European Union’s Horizon 633
2020 research and innovation program (Grant Agreement No. 747775). DL was supported by 634
CONICET (PIP11220130100392CO), FONCyT (PICT 3677, PICT 2542), Universidad Nacional 635
del Comahue (B199), and NSF-CONICET grant. CTH is a Pew Scholar in the Biomedical 636
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29
Sciences and H. I. Romnes Faculty Fellow, supported by the Pew Charitable Trusts and Office of 637
the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin 638
Alumni Research Foundation (WARF), respectively. 639
640
Author Contributions: 641
QKL, DL, and CTH, conceived of the study; QLK, DP, JIE, DL, and CTH refined concept and 642
design; QKL performed all population genomic and ecological niche analyses; QKL and DAO 643
sequenced genomes, performed phenotyping and statistical analyses, and mentored KVB and 644
MJ; DL and CTH supervised the study; KVB, KS, and MJ isolated and/or identified North 645
American strains; JIE, MER, CAL, and DL isolated and/or provided South American strains; and 646
QKL and CTH wrote the manuscript with editorial input from all co-authors. 647
648
Conflict of Interest Disclosure: 649
Commercial use of Saccharomyces eubayanus strains requires a license from WARF or 650
CONICET. Strains are available for academic research under a material transfer agreement. 651
652
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817 Figure Legends: 818
Figure 1. S. eubayanus distribution and population structure. 819
S. eubayanus has a global distribution and two major populations with six subpopulations. (A) 820
Isolation locations of S. eubayanus strains included in the dataset. For visibility, circle size is not 821
scaled by the number of strains. Subpopulation abundance is shown as pie charts. The 822
Patagonian sampling sites have been collapsed to two locations for clarity. Details of sites and 823
subpopulations found in South America (B) and North America (C) with circle size scaled by the 824
number of strains. (D) Whole genome PCA of S. eubayanus strains and five hybrids with large 825
contributions from S. eubayanus. (E) PCA of just PA. (F) PCA of just PB and hybrid S. 826
eubayanus sub-genomes. Color legends in A and D apply to this and all other figures. 827
828
Figure 2. Phenotypic differences. 829
(A) Heat map of mean of maximum growth rate (change in OD/hour) (GR) on different carbon 830
sources by subpopulation. Warmer colors designate faster growth. (B) Heat map of log10 831
normalized growth at different temperatures by subpopulation. 832
833
Figure 3. Population genomic parameters. 834
(A) Network built with pairwise FST values < 0.8 between each subpopulation. FST values are 835
printed and correspond to line thickness, where lower values are thicker. Circle sizes correspond 836
to genetic diversity. (B) LD decay for each subpopulation (colors) and the species in whole 837
(black). 838
839
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34
Figure 4. Genomic ancestries of NoAm and SoAm admixed lineages. 840
(A) For all 21 NoAm admixed strains, log2 ratio of the minimum PB-NoAm pairwise nucleotide 841
sequence divergence (dB-NoAm) and the minimum PA-NoAm pairwise nucleotide sequence 842
divergence (dA-NoAm) in 50-kbp windows. Colors and log2 < 0 or > 0 indicate that part of the 843
genome is more closely related to PA or PB, respectively. (B) Neighbor-Net phylogenetic 844
network reconstructed with the 571 SNPs that differentiate the NoAm strains. The scale bar 845
represents the number of substitutions per site. Collection location is noted. (C) Pairwise 846
nucleotide sequence divergence of the NoAm strain yHKS210 compared to strains from the PA-847
1 and PA-2 subpopulations of PA in 50-kbp windows. (D) Pairwise nucleotide sequence 848
divergence of the NoAm strain yHKS210 compared to strains from the PB-1, PB-2, and PB-3 849
subpopulations of PB in 50-kbp windows. (E) log2 ratio of the minimum PB-SoAm pairwise 850
nucleotide sequence divergence (dB-SoAm) and the minimum PA-SoAm pairwise nucleotide 851
sequence divergence (dA-SoAm) in 50-kbp windows. Colors and log2 < 0 or > 0 indicate that 852
part of the genome is more closely related to PA or PB, respectively. (F) Pairwise nucleotide 853
sequence divergence of the SoAm strain compared to strains from the PA-1 and PA-2 854
subpopulations of PA in 50-kbp windows. (G) Pairwise nucleotide sequence divergence of the 855
SoAm strain compared to strains of the PB-1, PB-2, and PB-3 subpopulations of PB in 50-kbp 856
windows. 857
858
Figure 5. South American genomic diversity versus range, diversity by area, and isolation by 859
distance. 860
(A) Range and genomic diversity of South American sampling sites. Circle sizes correspond to 861
nucleotide diversity of all strains from that site, and pie proportions correspond to each 862
subpopulation’s contribution to 𝜋 at each site. Latitudinal range of each subpopulation is shown 863
to the right. (B) Nucleotide diversity by subpopulation by sampling site, where larger and darker 864
circles indicate more diversity. “SA Sites” in gray show the diversity of all strains found in each 865
South American (SA) site. “World Sites” in darker gray show the nucleotide diversity of all 866
North American or non-South American strains, regardless of subpopulation, compared to South 867
American strains south or north of 43°S, aligned to mean latitude of all strains included in the 868
analysis. (C) Correlation of nucleotide diversity and the area or distance a subpopulation covers. 869
The y-axis shows the nucleotide diversity of each subpopulation, and circle sizes correspond to 870
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35
the geographic sizes of the subpopulations on a log10 scale. Note that PA-1 (dark red) is as 871
diverse as PB-3 (dark blue) but encompasses a smaller area. (D) log10(pairwise nucleotide 872
diversity) correlated with distance between strains, which demonstrates isolation by distance. 873
Note that y-axes are all scaled the same but not the x-axes. Holarctic includes the S. eubayanus 874
sub-genome of two lager-brewing strains. Figure S8A shows the individual plots for the NoAm 875
lineage. Figure S8B shows the individual plot of PB-1. 876
877
Figure 6. Predicted climatic suitability of S. eubayanus. 878
Minimum training presence (light green) and 10th percentile training presence (dark green) based 879
on a model that includes all known S. eubayanus isolations, as well as a scenario of dispersal and 880
diversification out of Patagonia (inset and arrows). Black arrows signify diversification events, 881
dotted lines are diversification events where the population is not found in Patagonia, and 882
colored arrows are migration events for the lineage of matching color. Roman numerals order the 883
potential migration events. S. eubayanus has not been found in the wild in Europe, but it has 884
contributed to fermentation hybrids, such as lager yeasts. This scenario proposes that the last 885
common ancestor of PA and PB-Holarctic bifurcated into PA (red) and PB-Holarctic (blue), 886
which further radiated into PA-1 (dark red), PA-2 (light red), PB-1 (blue), PB-2 (lighter blue), 887
PB-3 (dark blue), and Holarctic (very light blue). At least four migration events are needed to 888
explain the locations where S. eubayanus has been found. I. The Holarctic subpopulation was 889
drawn from the PB-Holarctic gene pool and colonized the Holarctic ecozone. II. PB-1 colonized 890
the Pacific Rim, including New Zealand and Washington state, USA. III. An independent 891
dispersal event brought PB-1 to North Carolina, USA. IV. Outcrossing between PA-2 and PB-1 892
gave rise to a low-diversity admixed linage that has recently invaded a large swath of North 893
America. 894
895
Supplementary Figure 1. Additional visualizations of population structure. 896
(A) SplitsTree network tree built with 11994 SNPs with subpopulations circled and labeled. (B) 897
FineStructure co-ancestry plot built with 11994 SNPs. Bluer colors correspond to more genetic 898
similarity. Boxes have been added to label the subpopulations. (C) FastSTRUCTURE plot (K=6) 899
built with 150165 SNPs and showing the same six monophyletic subpopulations found with 900
other approaches. Only five NoAm strains were included in the fastSTRUCTURE analysis. 901
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36
902
Supplementary Figure. 2. Additional phenotypic data. 903
(A) Violin plots of recovery from stress, normalized by controls. There were no significant 904
subpopulation by stress interactions. (B) Violin plots of log10 normalized mean growth rates of 905
each subpopulation at 0°C, 4°C, 10°C, and 20°C. * = p-val < 0.05 of interactions between Lager 906
and PA-2, PB-2, and PB-3 at 10°C; Lager and PA-1, PA-2, PB-1, PB-2, and PB-3 at 20°C; and 907
PB-2 and both PA-2 and NoAm at 20°C (C) Violin plots of mean growth rate on different carbon 908
sources (* = p-val < 0.05). (D) Heatmaps of significant subpopulation by temperature 909
interactions and (E) significant subpopulation by carbon source interactions. Warmer colors 910
indicate that the subpopulation-temperature or carbon source on the left hand had a faster growth 911
rate than the subpopulation-temperature or carbon source along the bottom; cooler colors 912
represent the reverse. Non-significant interactions, based on multiple test corrections, are in 913
white. More intense colors represent smaller p-values. 914
915
Supplementary Figure 3. Heterozygosity analyses. 916
(A) Summary of all SNPs vs SNPs called as heterozygous compared to the taxonomic type strain 917
for all 200 strains included in this study. The upper limit of the bar is the total SNP count. The 918
lower point corresponds to SNPs called as heterozygous. The horizontal line is 20k SNPs. (B) 919
Strain yHCT75 (CRUB 1946) is the only strain with > 20K heterozygous SNPs. (C) When the 920
heterozygous SNPs of yHCT75 were pseudo-phased (labeled), both phases clustered with PB-1. 921
922
Supplementary Figure 4. Additional population genomic statistics. 923
(A) Mean pairwise nucleotide diversity (𝜋 * 100) for each subpopulation across the genome in 924
50-kbp windows. (B) Tajima’s D across the genome in 50-kbp windows for each subpopulation. 925
(C) Mean FST in 50-kpb windows for each subpopulation compared to all subpopulations. (D) 926
Pairwise FST for each subpopulation compared to PB-1. 927
928
Supplementary Figure 5. Pairwise FST plots for NoAm and SoAm compared to all other 929
subpopulations. 930
Pairwise FST for the NoAm lineage (A) or SoAm strain (B) compared to all other subpopulations. 931
932
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37
Supplementary Figure 6. The taxonomic type strain has a mosaic genome. 933
(A) Pairwise genetic divergence of the taxonomic type strain compared to each subpopulation. 934
(B) Comparison of pairwise genetic divergence of the taxonomic type strain compared to PA-1 935
and PB-1. (C) log2 divergence plot (as in Figure 4) showing regions introgressed from PA-1 in 936
the taxonomic type strain. 937
938
Supplementary Figure 7. Four S. eubayanus strains with S. uvarum nuclear introgressions. 939
(A) Depth of coverage plots of reads from four strains mapped to both the S. uvarum (Suva) and 940
S. eubayanus (Seub) reference genomes. (B) Zoom-in of region on Chromosome XIV where 941
these four strains have the same S. uvarum (purple) introgression into a S. eubayanus 942
background. (C) A PCA plot shows that these four strains belong to the PB-1 subpopulation of S. 943
eubayanus. (D) A PCA plot shows that the introgressed region from S. uvarum came from the 944
South American SA-B subpopulation of S. uvarum. 945
946
Supplementary Figure 8. Isolation by distance plots for NoAm and PB-1. 947
(A) Isolation by distance for all NoAm strains. The y-axis has been rescaled compared to Figure 948
5 for better visualization. (B) Isolation by distance for subpopulation PB-1. Comparisons with 949
strains from Cerro Ñielol are labeled. All comparisons of South American strains with non-South 950
American strains are on the right side. 951
952
Supplementary Figure 9. Additional Wallace climatic models. 953
(A) Model built using only South American isolation locations. (B) Model built using only non-954
South American sites. (C) Comparison of models based on all known S. eubayanus collection 955
sites, only South American, or only non-South American sites. Where the models agree is in dark 956
green, where two models agree is in medium green, and where one model predicts suitability is 957
in light green. 958
959
Supplementary Table 1. Collection information for all strains whose genomes were sequenced or 960
analyzed in this study. 961
962
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38
Supplementary Table 2. Average triplicate growth rates for various temperatures and carbon 963
sources. Note that this spreadsheet has multiple sheets. 964
965
Supplementary Table 3. K=6 output of FastSTRUCTURE. 966
967
Supplementary Table 4. Mantel test results. 968
969
Supplementary Table 5. Input and output for Wallace climatic modeling. 970
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25
30
35
40
45
50
−120 −100 −80 −60Longitude
Latitude
PB−1
PB−2
PB−3Holarctic
PA−1 PA−2
NoAm
SoAm
−150
−100
−50
0
50
−100 0 100 200PC1 = 40.06%
PC2
= 9.
95%
PA−1
PA−2
PB−1
PB−2
PB−3
Holarctic
Hybrid
NoAm
SoAm
PA−1PA−2
−75
−50
−25
0
25
50
−150 −100 −50 0 50PC1 = 41.56%
PC2
= 7.
81%
PA−1
PA−2
−50
0
50
−100 0 100 200Longitude
Latitude
PA−1PA−2
PB−1PB−2
PB−3Holarctic
NoAmSoAm
Figure 1
−55
−50
−45
−40
−75 −70 −65 −60Longitude
Latitude
B
C
E
D
A
PB1
PB−2
PB−3
Holarctic−50
0
50
−50 0 50 100PC1 = 19.03%
PC2
= 13
.96%
PB−1
PB−2
PB−3
Holarctic
Hybrid
F
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Figure 2
0.0
0.2
0.4
0.6
0KB 30KB 60KB 90KB 120KB 150KB 180KB 210KB 240KB 270KBSNP distance
r2Subpop
PA−1
PA−2
PB−1
PB−2
PB−3
Seub
A B
GR
Raffinose
Maltotriose
Glycerol
EthanolGlucose
Maltose
Galactose
PA−2NoAmPA−1PB−2PB−3PB−1LagerHolarctic
0.01
0.02
0.03
0.04
0.05
0.06
Raffinose
Maltotriose
Glycerol
EthanolGlucose
Maltose
Galactose
PA−2NoAmPA−1PB−2PB−3PB−1LagerHolarctic
0.01
0.02
0.03
0.04
0.05
0.06GR
20C10C0C 4C
LagerHolarcticPA−2NoAmPB−2PB−3PB−1PA−1 0.05
0.1
0.15
20C10C0C 4C
LagerHolarcticPA−2NoAmPB−2PB−3PB−1PA−1 0.05
0.1
0.15BA
20C10C0C 4C
LagerHolarcticPA−2NoAmPB−2PB−3PB−1PA−1 0.05
0.1
0.15
20C10C0C 4C
LagerHolarcticPA−2NoAmPB−2PB−3PB−1PA−1 0.05
0.1
0.15
Figure 3
0.43
0.51
0.59
0.64
0.64
0.670.69
0.70
0.72
0.73
0.78
0.78
0.80
●
●
SoAm
NoAm
PA−1
PA−2
PB−1
PB−2
PB−3
Hol
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0.0
0.5
1.0
1.5
I II III IV VVI VII VIII IX X XI XII XIII XIV XV XVIGenome Position
Pairw
ise
Dive
rgen
ce (%
)
SubpopPB−1
PB−2
PB−3
0.0
0.5
1.0
1.5
I II III IV VVI VII VIII IX X XI XII XIII XIV XV XVIGenome Position
Pairw
ise
Dive
rgen
ce (%
)
SubpopPA−1
PA−2
−5
0
5
I II III IV VVI VII VIII IX X XI XII XIII XIV XV XVIGenome Position
log 2(d
A-So
Amd B
-SoA
m)
SoAmyHDPN421
yHDPN422
yHDPN423
yHDPN424
yHKB201
yHKB210
yHKB218
yHKB242
yHKB246
yHKB251
yHKB268
yHKS210
yHKS211
yHKS212
yHKS576
yHKS689
yHKS694
yHKS810
yHKS812
yHQL1481
yHQL1621
I II III IV VVIVIIVIII IX X XI XII XIIIXIVXVXVIGenome Position
log 2(d
A-N
oAm
d B-N
oAm)
0.0
0.5
1.0
1.5
I II III IV VVI VII VIII IX X XI XII XIII XIV XV XVIGenome Position
Pairw
ise
Dive
rgen
ce (%
)
SubpopPB−1
PB−2
PB−3
0.0
0.5
1.0
1.5
I II III IV VVI VII VIII IX X XI XII XIII XIV XV XVIGenome Position
Pairw
ise
Dive
rgen
ce (%
)
SubpopPA−1
PA−2
Figure 4 A
C
D
E
F
G
yHDPN421yHDPN424
yHDPN423
yHDPN422
yHKS211yHKS210
yHKS212
yHKS576yHKS689
yHKS694yHKB251
yHKB201yHQL1481
yHKS810yHQL1621yHKS812
yHKB210
yHKB218
yHKB246yHKB242
yHKB268
0.1
New Brunswick,
Canada South Carolina, USA
North Carolina, USAWisconsin,
USA
NoAm
B 0.1
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−55
−50
−45
−40
PA−1 PA−2 PB−1 PB−2 PB−3 SoAm SASites
WorldSites
Latit
ude
NucleotideDiversity (%)
0.0
0.1
0.2
0.3
0.4
0.5
−55
−50
−45
−40
−75 −70 −65 −60Longitude
Latitude
PA−1 PA−2 PB−1 PB−2 PB−3
Figure 5BA D
All North AmericanAll Non-South American
CPA−1 PA−2 NoAm PB−1 PB−2 PB−3 Hol
0 10 20 0 200 400 0 500 10001500 0 5000 10000 0 100 200 0 500 1000 1500 0 5000 10000
0.0001
0.0010
0.0100
0.1000
Distance (Km)
log(
Nuc
leot
ide
Dive
rsity
(%))
● ●
● ●
●
● ●
● ●
●
● ●
● ●
0.00
0.05
0.10
0.15
0.20
Area(10 Km^2)
Distance(Km)
Nuc
leot
ide
Dive
rsity
(%)
Subpopulation●●
●●
●●
●●
●●
●●
PA−1
PA−2
PB−1
PB−2
PB−3
NoAm
PopulationGeographicSize
100000
200000
300000
400000
p-val=0.0001 p-val=0.0001 p-val=0.0001 p-val=0.0001 p-val=0.0001p-val=0.0003p-val=0.564
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Figure 6
Predicted Climatic Suitability
Longitude
Latitude
40°S
20°S
0°
20°N
40°N
60°N
150°W 100°W 50°W 0° 50°E 100°E 150°E
0.0
0.5
1.0
I
II
III
IV
Ancestor
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Figure S1
PB -3
PB -2
Holarctic
PA -2
PA -1
NoAm
SoAm
PB -1
B
A
Holarctic PB -2 PA -2 PA -1 PB -3 PB -1 SoAm /NoAm
C
51.7
115
178
241
304
367
431
494
557
620
CDFM
21L.
1_Se
ub_H
olar
ctic
Y84
6_yH
QL5
60_F
RUIT
_Sce
rXSk
udXS
eub
CitK
P_yH
QL6
94_F
RUIT
_lag
erSa
azY
1264
6_yH
QL5
72_F
RUIT
_Suv
aXSe
ubY
1527
_yHQ
L580
_FRU
IT_S
uvaX
Seub
Y12
693_
FM47
1_BE
ER_l
ager
Saaz
WLP
862_
yHQ
L618
_BEE
R_la
ger
WY2
042_
yHQ
L627
_BEE
R_la
ger
yHRV
M10
7_Se
ub_H
olar
ctic
yHRV
M10
8_Se
ub_H
olar
ctic
yHKS
509_
Seub
_PB1
yHRV
M83
_Seu
b_PB
1P1
C1_S
eub_
PB1
yHAB
575_
NR7
_Seu
b_PB
1yH
AB57
6_N
R9_S
eub_
PB1
yHAB
121_
CRUB
2058
_Seu
b_PB
1yH
AB93
_CRU
B203
0_Se
ub_P
B1yH
CT53
_CRU
B193
8_Se
ub_P
B1yH
AB71
_CRU
B200
9_Se
ub_P
B1yH
CT94
_CRU
B196
4_Se
ub_P
B1yH
AB50
5_TC
64_S
eub_
PB1
yHAB
61_C
RUB2
001_
Seub
_PB1
yHAB
75_C
RUB1
582_
Seub
_PB1
yHCT
62_C
RUB1
593_
Seub
_PB1
yHCT
92_C
RUB1
962_
Seub
_PB1
yHAB
65_C
RUB1
575_
Seub
_PB1
yHCT
61_C
RUB1
576_
Seub
_PB1
yHAB
54_C
RUB1
995_
Seub
_PB1
yHCT
95_C
RUB1
965_
Seub
_PB1
yHAB
55_C
RUB1
996_
Seub
_PB1
yHAB
57_C
RUB1
998_
Seub
_PB1
yHAB
68_C
RUB1
579_
Seub
_PB1
yHCT
108_
CRUB
1581
_Seu
b_PB
1yH
CT93
_CRU
B196
3_Se
ub_P
B1yH
AB77
_CRU
B201
4_Se
ub_P
B1yH
AB59
_CRU
B200
0_Se
ub_P
B1yH
AB62
_CRU
B200
2_Se
ub_P
B1yH
CT12
0_CR
UB19
86_S
eub_
PB1
yHCT
87_C
RUB1
957_
Seub
_PB1
yHCT
98_C
RUB1
968_
Seub
_PB1
yHKB
35_S
eub_
PB1
yHCT
63_C
RUB1
761_
Seub
_PB1
yHAB
97_C
RUB2
034_
Seub
_PB1
yHCT
69_C
RUB1
940_
Seub
_PB1
yHAB
100_
CRUB
2037
_Seu
b_PB
1yH
AB99
_CRU
B203
6_Se
ub_P
B1yH
AB57
0_CB
72
yHAB
571_
CB8
5_Se
ub_P
B1yH
AB57
3_CB
95_
Seub
_PB1
yHAB
572_
CB9
2_Se
ub_P
B1yH
CT16
0_CR
UB15
68_S
eub_
PB1
yHCT
129_
1568
.2_S
eub_
PB1
yHCT
76_C
RUB1
568T
_Seu
b_PB
1yH
CT48
_CRU
B193
1_Se
ub_P
B1yH
AB10
7_CR
UB20
44_S
eub_
PB1
yHAB
89_C
RUB2
026_
Seub
_PB1
yHAB
109_
CRUB
2046
_Seu
b_PB
1yH
AB48
9_TC
48_S
eub_
PB1
yHAB
108_
CRUB
2045
_Seu
b_PB
1yH
AB87
_CRU
B202
4_Su
vaXS
eub
yHAB
88_C
RUB2
025_
Suva
XSeu
byH
AB96
_CRU
B203
3_Se
ub_P
B1yH
CT50
_CRU
B193
3_Se
ub_P
B1yH
CT64
_CRU
B176
2_Se
ub_P
B1yH
CT75
_CRU
B194
6_Se
ub_P
B1yH
CT51
_Suv
aXSe
ubyH
CT10
6_CR
UB19
76_S
uvaX
Seub
yHAB
90_C
RUB2
027_
Seub
_PB1
yHAB
91_C
RUB2
028_
Seub
_PB1
yHCT
49_C
RUB1
932_
Seub
_PB1
yHCT
84_C
RUB1
954_
Seub
_PB1
yHAB
105_
CRUB
2042
_Seu
b_PB
3yH
CT10
2_CR
UB19
72_S
eub_
PB3
yHAB
116_
CRUB
2053
_Seu
b_PB
1yH
CT57
_CRU
B195
0_Se
ub_P
B1yH
AB11
7_CR
UB20
54_S
eub_
PB1
yHAB
106_
CRUB
2043
_Seu
b_PB
1yH
CT11
6_CR
UB19
82_S
eub_
PB1
yHCT
47_C
RUB1
930_
Seub
_PB1
yHAB
566_
B33
5_Se
ub_P
B3yH
AB56
4_B3
13_
Seub
_PB3
yHCT
119_
CRUB
1985
_Seu
b_PB
2yH
QL7
26_N
PCC1
445_
Seub
_PB2
yHQ
L738
_NPC
C129
7_Se
ub_P
B2yH
QL7
32_N
PCC1
294_
Seub
_PB2
yHQ
L736
_NPC
C144
0_Se
ub_P
B2yH
CT10
7_CR
UB19
77_S
eub_
PB2
yHAB
110_
CRUB
2047
_Seu
b_PB
2yH
AB11
1_CR
UB20
48_S
eub_
PB2
yHCT
70_C
RUB1
941_
Seub
_PB2
yHCT
71_C
RUB1
942_
Seub
_PB2
yHQ
L744
_NPC
C131
1_Se
ub_P
B2yH
QL7
58_N
PCC1
292_
Seub
_PB2
yHQ
L743
_NPC
C144
4_Se
ub_P
B2yH
QL7
47_N
PCC1
301_
Seub
_PB2
yHQ
L733
_NPC
C144
2_Se
ub_P
B2yH
AB12
4_CR
UB20
61_S
eub_
PB2
yHAB
126_
CRUB
2062
_Seu
b_PB
2yH
AB56
_CRU
B199
7_Se
ub_P
B2yH
CT11
7_CR
UB19
83_S
eub_
PB2
yHCT
97_C
RUB1
967_
Seub
_PB2
yHCT
91_C
RUB1
961_
Seub
_PB2
yHCT
88_C
RUB1
958_
Seub
_PB2
yHAB
66_C
RUB2
005_
Seub
_PB2
yHAB
74_C
RUB2
012_
Seub
_PB2
yHAB
67_C
RUB2
006_
Seub
_PB2
yHCT
89_C
RUB1
959_
Seub
_PB2
yHCT
100_
CRUB
1970
_Seu
b_PB
2yH
AB12
5_CR
UB18
92_S
eub_
PB2
yHAB
565_
B32
1_Se
ub_P
B3yH
CT10
5_CR
UB19
75_S
eub_
PB3
yHCT
115_
CRUB
1981
_Seu
b_PB
3yH
AB69
_CRU
B200
7_Se
ub_P
B3yH
AB78
_CRU
B201
5_Se
ub_P
B3yH
AB80
_CRU
B201
7_Se
ub_P
B3yH
AB12
0_CR
UB20
57_S
eub_
PB3
yHAB
113_
CRUB
2050
_Seu
b_PB
3yH
AB11
5_CR
UB20
52_S
eub_
PB3
yHAB
86_C
RUB2
023_
Seub
_PB3
yHCT
113_
S14
1_Se
ub_P
B3yH
AB11
2_CR
UB20
49_S
eub_
PB3
yHAB
118_
CRUB
2055
_Seu
b_PB
3yH
CT66
_CRU
B193
5_Se
ub_P
B3yH
CT59
_CRU
B195
2_Se
ub_P
B3yH
CT58
_CRU
B195
1_Se
ub_P
B3yH
AB84
_CRU
B202
1_Se
ub_P
B3yH
AB85
_CRU
B202
2_Se
ub_P
B3yH
CT56
_CRU
B194
9_Se
ub_P
B3yH
AB49
1_TC
50_S
eub_
PB3
yHCT
52_C
RUB1
936_
Seub
_PB3
yHAB
114_
CRUB
2051
_Seu
b_PB
3yH
AB82
_CRU
B201
9_Se
ub_P
B3yH
AB81
_CRU
B201
8_Se
ub_P
B3yH
AB12
2_CR
UB20
59_S
eub_
PB3
yHAB
83_C
RUB2
020_
Seub
_PB3
yHCT
60_C
RUB1
953_
Seub
_PB3
yHCT
112_
S13
1_Se
ub_P
B3yH
AB92
_CRU
B202
9_Se
ub_P
B3yH
CT54
_CRU
B194
7_Se
ub_P
B3yH
CT55
_CRU
B194
8_Se
ub_P
B3yH
QL7
30_N
PCC1
286_
Seub
_PA2
yHAB
94_C
RUB2
031_
Seub
_PA2
yHQ
L755
_NPC
C129
6_Se
ub_P
A2yH
AB70
_CRU
B200
8_Se
ub_P
A2yH
AB57
7_B3
91_
Seub
_PA2
yHAB
102_
CRUB
2039
_Seu
b_PA
2yH
AB10
1_CR
UB20
38_S
eub_
PA2
yHAB
103_
CRUB
2040
_Seu
b_PA
2yH
AB10
4_CR
UB20
41_S
eub_
PA2
yHCT
68_C
RUB1
939_
Seub
_PA2
yHAB
567_
B35
5_Se
ub_P
A2yH
AB56
8_B3
56_
Seub
_PA2
yHCT
46_C
RUB1
929_
Seub
_PA2
yHCT
99_C
RUB1
969_
Seub
_PA2
yHCT
90_C
RUB1
960_
Seub
_PA2
yHQ
L710
_NPC
C144
1_Se
ub_P
A2yH
QL7
09_N
PCC1
291_
Seub
_PA2
yHQ
L717
_NPC
C144
3_Se
ub_P
A2yH
AB63
_CRU
B200
3_Se
ub_P
A2yH
AB95
_CRU
B203
2_Se
ub_P
A2yH
AB79
_CRU
B201
6_Se
ub_P
A2yH
CT11
4_CR
UB19
80_S
eub_
PA2
yHAB
119_
CRUB
2056
_Seu
b_PA
2yH
CT72
_CRU
B194
3_Se
ub_P
A2yH
QL7
08_N
PCC1
285_
Seub
_PA2
yHQ
L705
_NPC
C143
9_Se
ub_P
A2yH
QL7
15_N
PCC1
284_
Seub
_PA2
yHQ
L740
_NPC
C128
2_Se
ub_P
A2yH
QL7
27_N
PCC1
287_
Seub
_PA2
yHQ
L714
_NPC
C142
9_Se
ub_P
A2yH
QL7
51_N
PCC1
430_
Seub
_PA2
yHQ
L754
_NPC
C143
5_Se
ub_P
A2yH
QL7
29_N
PCC1
431_
Seub
_PA2
yHQ
L756
_NPC
C143
2_Se
ub_P
A2yH
QL7
75_N
PCC1
437_
Seub
_PA2
yHAB
73_C
RUB2
011_
Seub
_PA1
yHCT
96_C
RUB1
966_
Seub
_PA1
yHAB
58_C
RUB1
999_
Seub
_PA1
yHAB
569_
B36
6_Se
ub_P
A1yH
AB64
_CRU
B200
4_Se
ub_P
A1yH
CT10
1_Ex
CR10
7b_S
eub_
PA1
yHCT
103_
CRUB
1973
_Seu
b_PA
1yH
AB57
4_Q
C15_
Seub
_PA1
yHAB
76_C
RUB2
013_
Seub
_PA1
yHCT
104_
CRUB
1974
_Seu
b_PA
1yH
AB57
8_C3
94_
Seub
_SoA
myH
DPN
424_
LL20
1322
1_Se
ub_A
dmix
_NoA
myH
KS68
9_Se
ub_A
dmix
_NoA
myH
KS21
0_Se
ub_A
dmix
_NoA
myH
KS21
2_Se
ub_A
dmix
_NoA
myH
DPN
422_
LL20
1321
9_Se
ub_A
dmix
_NoA
myH
KB24
6_Se
ub_A
dmix
_NoA
myH
KS57
6_Se
ub_A
dmix
_NoA
myH
KS69
4_Se
ub_A
dmix
_NoA
myH
KB21
0_Se
ub_A
dmix
_NoA
myH
KB20
1_Se
ub_A
dmix
_NoA
myH
KS21
1_Se
ub_A
dmix
_NoA
myH
KB25
1_Se
ub_A
dmix
_NoA
myH
KB21
8_Se
ub_A
dmix
_NoA
myH
DPN
421_
LL20
1321
8_Se
ub_A
dmix
_NoA
myH
DPN
423_
LL20
1322
0_Se
ub_A
dmix
_NoA
myH
KB26
8_Se
ub_A
dmix
_NoA
myH
KB24
2_Se
ub_A
dmix
_NoA
myH
KS81
0_Se
ub_A
dmix
_NoA
myH
KS81
2_Se
ub_A
dmix
_NoA
myH
QL1
481_
Seub
_Adm
ix_N
oAm
yHQ
L162
1_Se
ub_A
dmix
_NoA
m
CDFM21L.1_Seub_HolarcticY 846_yHQL560_FRUIT_ScerXSkudXSeubCitKP_yHQL694_FRUIT_lager SaazY 12646_yHQL572_FRUIT_SuvaXSeubY 1527_yHQL580_FRUIT_SuvaXSeubY 12693_FM471_BEER_lager SaazWLP862_yHQL618_BEER_lagerWY2042_yHQL627_BEER_lageryHRVM107_Seub_HolarcticyHRVM108_Seub_HolarcticyHKS509_Seub_PB1yHRVM83_Seub_PB1P1C1_Seub_PB1yHAB575_NR7_Seub_PB1yHAB576_NR9_Seub_PB1yHAB121_CRUB2058_Seub_PB1yHAB93_CRUB2030_Seub_PB1yHCT53_CRUB1938_Seub_PB1yHAB71_CRUB2009_Seub_PB1yHCT94_CRUB1964_Seub_PB1yHAB505_TC64_Seub_PB1yHAB61_CRUB2001_Seub_PB1yHAB75_CRUB1582_Seub_PB1yHCT62_CRUB1593_Seub_PB1yHCT92_CRUB1962_Seub_PB1yHAB65_CRUB1575_Seub_PB1yHCT61_CRUB1576_Seub_PB1yHAB54_CRUB1995_Seub_PB1yHCT95_CRUB1965_Seub_PB1yHAB55_CRUB1996_Seub_PB1yHAB57_CRUB1998_Seub_PB1yHAB68_CRUB1579_Seub_PB1yHCT108_CRUB1581_Seub_PB1yHCT93_CRUB1963_Seub_PB1yHAB77_CRUB2014_Seub_PB1yHAB59_CRUB2000_Seub_PB1yHAB62_CRUB2002_Seub_PB1yHCT120_CRUB1986_Seub_PB1yHCT87_CRUB1957_Seub_PB1yHCT98_CRUB1968_Seub_PB1yHKB35_Seub_PB1yHCT63_CRUB1761_Seub_PB1yHAB97_CRUB2034_Seub_PB1yHCT69_CRUB1940_Seub_PB1yHAB100_CRUB2037_Seub_PB1yHAB99_CRUB2036_Seub_PB1yHAB570_CB7 2yHAB571_CB8 5_Seub_PB1yHAB573_CB9 5_Seub_PB1yHAB572_CB9 2_Seub_PB1yHCT160_CRUB1568_Seub_PB1yHCT129_1568.2_Seub_PB1yHCT76_CRUB1568T_Seub_PB1yHCT48_CRUB1931_Seub_PB1yHAB107_CRUB2044_Seub_PB1yHAB89_CRUB2026_Seub_PB1yHAB109_CRUB2046_Seub_PB1yHAB489_TC48_Seub_PB1yHAB108_CRUB2045_Seub_PB1yHAB87_CRUB2024_SuvaXSeubyHAB88_CRUB2025_SuvaXSeubyHAB96_CRUB2033_Seub_PB1yHCT50_CRUB1933_Seub_PB1yHCT64_CRUB1762_Seub_PB1yHCT75_CRUB1946_Seub_PB1yHCT51_SuvaXSeubyHCT106_CRUB1976_SuvaXSeubyHAB90_CRUB2027_Seub_PB1yHAB91_CRUB2028_Seub_PB1yHCT49_CRUB1932_Seub_PB1yHCT84_CRUB1954_Seub_PB1yHAB105_CRUB2042_Seub_PB3yHCT102_CRUB1972_Seub_PB3yHAB116_CRUB2053_Seub_PB1yHCT57_CRUB1950_Seub_PB1yHAB117_CRUB2054_Seub_PB1yHAB106_CRUB2043_Seub_PB1yHCT116_CRUB1982_Seub_PB1yHCT47_CRUB1930_Seub_PB1yHAB566_B33 5_Seub_PB3yHAB564_B31 3_Seub_PB3yHCT119_CRUB1985_Seub_PB2yHQL726_NPCC1445_Seub_PB2yHQL738_NPCC1297_Seub_PB2yHQL732_NPCC1294_Seub_PB2yHQL736_NPCC1440_Seub_PB2yHCT107_CRUB1977_Seub_PB2yHAB110_CRUB2047_Seub_PB2yHAB111_CRUB2048_Seub_PB2yHCT70_CRUB1941_Seub_PB2yHCT71_CRUB1942_Seub_PB2yHQL744_NPCC1311_Seub_PB2yHQL758_NPCC1292_Seub_PB2yHQL743_NPCC1444_Seub_PB2yHQL747_NPCC1301_Seub_PB2yHQL733_NPCC1442_Seub_PB2yHAB124_CRUB2061_Seub_PB2yHAB126_CRUB2062_Seub_PB2yHAB56_CRUB1997_Seub_PB2yHCT117_CRUB1983_Seub_PB2yHCT97_CRUB1967_Seub_PB2yHCT91_CRUB1961_Seub_PB2yHCT88_CRUB1958_Seub_PB2yHAB66_CRUB2005_Seub_PB2yHAB74_CRUB2012_Seub_PB2yHAB67_CRUB2006_Seub_PB2yHCT89_CRUB1959_Seub_PB2yHCT100_CRUB1970_Seub_PB2yHAB125_CRUB1892_Seub_PB2yHAB565_B32 1_Seub_PB3yHCT105_CRUB1975_Seub_PB3yHCT115_CRUB1981_Seub_PB3yHAB69_CRUB2007_Seub_PB3yHAB78_CRUB2015_Seub_PB3yHAB80_CRUB2017_Seub_PB3yHAB120_CRUB2057_Seub_PB3yHAB113_CRUB2050_Seub_PB3yHAB115_CRUB2052_Seub_PB3yHAB86_CRUB2023_Seub_PB3yHCT113_S14 1_Seub_PB3yHAB112_CRUB2049_Seub_PB3yHAB118_CRUB2055_Seub_PB3yHCT66_CRUB1935_Seub_PB3yHCT59_CRUB1952_Seub_PB3yHCT58_CRUB1951_Seub_PB3yHAB84_CRUB2021_Seub_PB3yHAB85_CRUB2022_Seub_PB3yHCT56_CRUB1949_Seub_PB3yHAB491_TC50_Seub_PB3yHCT52_CRUB1936_Seub_PB3yHAB114_CRUB2051_Seub_PB3yHAB82_CRUB2019_Seub_PB3yHAB81_CRUB2018_Seub_PB3yHAB122_CRUB2059_Seub_PB3yHAB83_CRUB2020_Seub_PB3yHCT60_CRUB1953_Seub_PB3yHCT112_S13 1_Seub_PB3yHAB92_CRUB2029_Seub_PB3yHCT54_CRUB1947_Seub_PB3yHCT55_CRUB1948_Seub_PB3yHQL730_NPCC1286_Seub_PA2yHAB94_CRUB2031_Seub_PA2yHQL755_NPCC1296_Seub_PA2yHAB70_CRUB2008_Seub_PA2yHAB577_B39 1_Seub_PA2yHAB102_CRUB2039_Seub_PA2yHAB101_CRUB2038_Seub_PA2yHAB103_CRUB2040_Seub_PA2yHAB104_CRUB2041_Seub_PA2yHCT68_CRUB1939_Seub_PA2yHAB567_B35 5_Seub_PA2yHAB568_B35 6_Seub_PA2yHCT46_CRUB1929_Seub_PA2yHCT99_CRUB1969_Seub_PA2yHCT90_CRUB1960_Seub_PA2yHQL710_NPCC1441_Seub_PA2yHQL709_NPCC1291_Seub_PA2yHQL717_NPCC1443_Seub_PA2yHAB63_CRUB2003_Seub_PA2yHAB95_CRUB2032_Seub_PA2yHAB79_CRUB2016_Seub_PA2yHCT114_CRUB1980_Seub_PA2yHAB119_CRUB2056_Seub_PA2yHCT72_CRUB1943_Seub_PA2yHQL708_NPCC1285_Seub_PA2yHQL705_NPCC1439_Seub_PA2yHQL715_NPCC1284_Seub_PA2yHQL740_NPCC1282_Seub_PA2yHQL727_NPCC1287_Seub_PA2yHQL714_NPCC1429_Seub_PA2yHQL751_NPCC1430_Seub_PA2yHQL754_NPCC1435_Seub_PA2yHQL729_NPCC1431_Seub_PA2yHQL756_NPCC1432_Seub_PA2yHQL775_NPCC1437_Seub_PA2yHAB73_CRUB2011_Seub_PA1yHCT96_CRUB1966_Seub_PA1yHAB58_CRUB1999_Seub_PA1yHAB569_B36 6_Seub_PA1yHAB64_CRUB2004_Seub_PA1yHCT101_ExCR10 7b_Seub_PA1yHCT103_CRUB1973_Seub_PA1yHAB574_QC15_Seub_PA1yHAB76_CRUB2013_Seub_PA1yHCT104_CRUB1974_Seub_PA1yHAB578_C39 4_Seub_SoAmyHDPN424_LL2013 221_Seub_Admix_NoAmyHKS689_Seub_Admix_NoAmyHKS210_Seub_Admix_NoAmyHKS212_Seub_Admix_NoAmyHDPN422_LL2013 219_Seub_Admix_NoAmyHKB246_Seub_Admix_NoAmyHKS576_Seub_Admix_NoAmyHKS694_Seub_Admix_NoAmyHKB210_Seub_Admix_NoAmyHKB201_Seub_Admix_NoAmyHKS211_Seub_Admix_NoAmyHKB251_Seub_Admix_NoAmyHKB218_Seub_Admix_NoAmyHDPN421_LL2013 218_Seub_Admix_NoAmyHDPN423_LL2013 220_Seub_Admix_NoAmyHKB268_Seub_Admix_NoAmyHKB242_Seub_Admix_NoAmyHKS810_Seub_Admix_NoAmyHKS812_Seub_Admix_NoAmyHQL1481_Seub_Admix_NoAmyHQL1621_Seub_Admix_NoAm
PAAd
mix
AdmixPB -1 PB -2 PB -3 PA -1PA -2
PB
Holarctic & Hybrids
.CC-BY-NC 4.0 International licenseis made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It. https://doi.org/10.1101/709535doi: bioRxiv preprint
● ●
●● ● ● ● ●
●● ●
●●●●
●●
●
●●
FT HS
−15
−10
−5
0
5
subPop
Rec
over
y Po
st S
tress
subPop●●
●●
●●
●●
●●
●●
●●
●●
PA−1
PA−2
NoAm
PB−1
PB−2
PB−3
Holarctic
Lager
Figure S2A
●●
●
●
● ●●●
●
●
●
●
●●● ●
●●
●●
●
●
●●● ●●●
●
●
●●●●● ●●●
●●
0C 4C 10C 20C
1e−04
1e−03
1e−02
1e−01
subPop
log(
Gro
wth
Rat
e)
subPop●●
●●
●●
●●
●●
●●
●●
●●
PA−1
PA−2
NoAm
PB−1
PB−2
PB−3
Holarctic
Lager
● ●
●
●
● ●
● ●
●●
●
●
●● ●
●●
●
●●
●
●
●● ●
● ●
●
●●●
●●
●
●
●
●●
Galactose Glucose Maltose Raffinose
0.00
0.02
0.04
0.06
0.08
Subpop
Gro
wth
Rat
e
Subpop●●
●●
●●
●●
●●
●●
●●
●●
PA−1
PA−2
NoAm
PB−1
PB−2
PB−3
Holarctic
Lager
●
●
●●
●●
●
●
●●
●●
● ● ●●
●
●
● ●● ●
●●
●●
●
Ethanol Glycerol Maltotriose
0.000
0.005
0.010
0.015
0.020
Subpop
Gro
wth
Rat
e
Subpop●●
●●
●●
●●
●●
●●
●●
●●
PA−1
PA−2
NoAm
PB−1
PB−2
PB−3
Holarctic
Lager
*
*
*C
B
Holarctic:10C
Holarctic:20C
Holarctic:4C
Lager:0C
Lager:10C
Lager:20C
Lager:4C
NoAm:0C
NoAm:10C
NoAm:20C
NoAm:4C
PA−1:0C
PA−1:10C
PA−1:20C
PA−1:4C
PA−2:0C
PA−2:10C
PA−2:20C
PA−2:4C
PB−1:0C
PB−1:10C
PB−1:20C
PB−1:4C
PB−2:0C
PB−2:10C
PB−2:20C
PB−2:4C
PB−3:0C
PB−3:10C
PB−3:20C
PB−3:4C
Holarctic:0C
Holarctic:10C
Holarctic:20C
Holarctic:4C
Lager:0C
Lager:10C
Lager:20C
Lager:4C
NoAm:0C
NoAm:10C
NoAm:20C
NoAm:4C
PA−1:0C
PA−1:10C
PA−1:20C
PA−1:4C
PA−2:0C
PA−2:10C
PA−2:20C
PA−2:4C
PB−1:0C
PB−1:10C
PB−1:20C
PB−1:4C
PB−2:0C
PB−2:10C
PB−2:20C
PB−2:4C
PB−3:0C
PB−3:10C
PB−3:20C
Subpop|Temperature
Subpop|Tem
perature
−0.1
0.0
0.1
diff
Holarctic:GalactoseHolarctic:GlucoseHolarctic:GlycerolHolarctic:Maltose
Holarctic:MaltotrioseHolarctic:Raffinose
Lager:EthanolLager:GalactoseLager:GlucoseLager:GlycerolLager:Maltose
Lager:MaltotrioseLager:RaffinoseNoAm:Ethanol
NoAm:GalactoseNoAm:GlucoseNoAm:GlycerolNoAm:Maltose
NoAm:MaltotrioseNoAm:RaffinosePA−1:Ethanol
PA−1:GalactosePA−1:GlucosePA−1:GlycerolPA−1:Maltose
PA−1:MaltotriosePA−1:RaffinosePA−2:Ethanol
PA−2:GalactosePA−2:GlucosePA−2:GlycerolPA−2:Maltose
PA−2:MaltotriosePA−2:RaffinosePB−1:Ethanol
PB−1:GalactosePB−1:GlucosePB−1:GlycerolPB−1:Maltose
PB−1:MaltotriosePB−1:RaffinosePB−2:Ethanol
PB−2:GalactosePB−2:GlucosePB−2:GlycerolPB−2:Maltose
PB−2:MaltotriosePB−2:RaffinosePB−3:Ethanol
PB−3:GalactosePB−3:GlucosePB−3:GlycerolPB−3:Maltose
PB−3:MaltotriosePB−3:Raffinose
Holarctic:Ethanol
Holarctic:Galactose
Holarctic:Glucose
Holarctic:Glycerol
Holarctic:Maltose
Holarctic:Maltotriose
Holarctic:Raffinose
Lager:E
thanol
Lager:G
alactose
Lager:G
lucose
Lager:G
lycerol
Lager:M
altose
Lager:M
altotriose
Lager:R
affinose
NoAm:Ethanol
NoAm:Galactose
NoAm:Glucose
NoAm:Glycerol
NoAm:Maltose
NoAm:Maltotriose
NoAm:Raffinose
PA−1:Ethanol
PA−1:Galactose
PA−1:Glucose
PA−1:Glycerol
PA−1:Maltose
PA−1:Maltotriose
PA−1:Raffinose
PA−2:Ethanol
PA−2:Galactose
PA−2:Glucose
PA−2:Glycerol
PA−2:Maltose
PA−2:Maltotriose
PA−2:Raffinose
PB−1:Ethanol
PB−1:Galactose
PB−1:Glucose
PB−1:Glycerol
PB−1:Maltose
PB−1:Maltotriose
PB−1:Raffinose
PB−2:Ethanol
PB−2:Galactose
PB−2:Glucose
PB−2:Glycerol
PB−2:Maltose
PB−2:Maltotriose
PB−2:Raffinose
PB−3:Ethanol
PB−3:Galactose
PB−3:Glucose
PB−3:Glycerol
PB−3:Maltose
PB−3:Maltotriose
Subpop|Carbon
Subpop|Carbon
−0.03
0.00
0.03
diff
E
D
*
*
* **
***
***
**
●
●
●●
●●
●
●
●●
●●
● ● ●●
●
●
● ●● ●
●●
●●
●
Ethanol Glycerol Maltotriose
0.000
0.005
0.010
0.015
0.020
Subpop
Gro
wth
Rat
e
Subpop●●
●●
●●
●●
●●
●●
●●
●●
PA−1
PA−2
NoAm
PB−1
PB−2
PB−3
Holarctic
Lager
●
●
●●
●●
●
●
●●
●●
● ● ●●
●
●
● ●● ●
●●
●●
●
Ethanol Glycerol Maltotriose
0.000
0.005
0.010
0.015
0.020
Subpop
Gro
wth
Rat
e
Subpop●●
●●
●●
●●
●●
●●
●●
●●
PA−1
PA−2
NoAm
PB−1
PB−2
PB−3
Holarctic
Lager
.CC-BY-NC 4.0 International licenseis made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It. https://doi.org/10.1101/709535doi: bioRxiv preprint
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0
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yHCT75yHCT75
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popPB−1
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Figure S3
BA
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.CC-BY-NC 4.0 International licenseis made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It. https://doi.org/10.1101/709535doi: bioRxiv preprint
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PA−1PA−2
PB−1PB−2
PB−3H
olN
oAm
I II III IV IXV VI VII VIII X XI XII XIII XIV XV XVI
0.000.250.500.751.00
0.000.250.500.751.00
0.000.250.500.751.00
0.000.250.500.751.00
0.000.250.500.751.00
0.000.250.500.751.00
0.000.250.500.751.00
Genome Position
F ST
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●
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●●●●● 0.99
PA−1PA−2
PB−1PB−2
PB−3H
olSoAm
I II III IV IXV VI VII VIII X XI XII XIII XIV XV XVI
0.000.250.500.751.00
0.000.250.500.751.00
0.000.250.500.751.00
0.000.250.500.751.00
0.000.250.500.751.00
0.000.250.500.751.00
0.000.250.500.751.00
Genome Position
F ST
.CC-BY-NC 4.0 International licenseis made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It. https://doi.org/10.1101/709535doi: bioRxiv preprint
Figure S6
PA−1PA−2
PB−1PB−2
PB−3H
ol
I II III IV IXV VI VII VIII X XI XII XIII XIV XV XVI
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
Genome Position
Avg
Pairw
ise
Dive
rgen
ce (%
)
0.0
0.5
1.0
1.5
I II III IV IXV VI VII VIII X XI XII XIII XIV XV XVIGenome Position
Avg
Pairw
ise
Dive
rgen
ce (%
)
−5
0
5
I II III IV IXV VI VII VIII X XI XII XIII XIV XV XVIGenome Position
log2
(Pai
rwis
e D
iverg
ence
BA
C
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yHCT51
yHCT106
yHAB88
yHAB87
0 200000 400000 600000
0 200000 400000 600000
0 200000 400000 600000
0 200000 400000 6000000
100
200
300
0
10
20
30
40
50
0
20
40
60
80
0
5
10
15
ChrXIV
Mea
n D
epth Species
Seub
Suva SA−A
SA−B
HOL
Australasia
yHAB87
yHAB88yHCT106
yHCT51
−10
0
10
20
30
−60 −40 −20 0PC1 = 52.77%
PC2
= 21
.81%
popAustralasia
HOL
Hybrid
SA−A
SA−B
SuvaSAintro PCA
PB−1
PB−2
PB−3
Holarctic
yHAB87
yHAB88yHCT51
yHCT106
−50
0
50
100
−50 0 50 100PC1 = 20.66%
PC2
= 14
.9%
popPB−1
PB−2
PB−3
Holarctic
SeubPopBintroPhase PCAFigure S7
0
5
10
15
Suva SeubGenome Position
Aver
age
Dep
th
SpeciesSeub
Suva
yHCT51
0
20
40
60
Suva SeubGenome Position
Aver
age
Dep
th
SpeciesSeub
Suva
yHCT106
0
100
200
300
Suva SeubGenome Position
Aver
age
Dep
th
SpeciesSeub
Suva
yHAB87
0
20
40
Suva SeubGenome Position
Aver
age
Dep
th
SpeciesSeub
Suva
yHAB88
B
AC
D
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0.001
0.002
0.003
0 500 1000 1500Distance (KM)
Nuc
leot
ide
Dive
rsity
(%)
0.0
0.1
0.2
0.3
0.4
0.5
0 5000 10000Distance (KM)
Nuc
leot
ide
Dive
rsity
(%)
Comparisons with Cerro Ñielol strains
Comparisons of SA strains with NZ, WA, and NC strains
Figure S8
BA
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Figure S9
Predicted Climatic Suitability without South American Sites
Longitude
Latitude
40°S
20°S
0°
20°N
40°N
60°N
150°W 100°W 50°W 0° 50°E 100°E 150°E
0.0
0.2
0.4
0.6
0.8
1.0
Predicted Climatic Suitability with Only South American Sites
Longitude
Latitude
40°S
20°S
0°
20°N
40°N
60°N
150°W 100°W 50°W 0° 50°E 100°E 150°E
0.0
0.2
0.4
0.6
0.8
1.0
B
Predicted Climatic Suitability Comparision of Three Different Strain Sets
Longitude
Latitude
40°S
20°S
0°
20°N
40°N
60°N
150°W 100°W 50°W 0° 50°E 100°E 150°E
0.0
0.5
1.0
1.5
2.0
2.5
3.0
C
A
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