LSJ3 TH1 CBS130569 BC7 BC4 AB1 AB2 LSJ1 TH2 SO1 SO6 LEC1 LEC2 ONT6 ONT3 KEN1 KEN3 PEN1 PEN2 MIN2 MIN1 MIS2 MIS1 ND2 WIS2 ALB2 ALB1 SAS5 SAS3 BC12 CBS130562 ND1 WIS1 QC9 AGASSIZ CBS100042 QC8 QC11 ARCADIA DAVID5 pn02.02 pn01.03 20.0 98/1 100/1 From USA From Canada Sphaerulina populicola Sphaerulina musiva Sphaerulina populi Fig. S1 ITSrDNA maximum parsimony phylogram of Sphaerulina species. Statistical support for the topology is presented when above 70% for bootstrap and posterior probabilities above 95%. Bootstrap values are in front of the forward slash whilst posterior probabilities are after the forward slash. Isolate names in bold were sequenced as part of this study. All other isolate sequences were obtained from GenBank. The aligned ITS dataset contained 42 taxa, 395 characters of which 58 were informative. Parsimony analysis produced 10 trees of 63 steps (CI=0.952, R1=0.975, g1=-3.537537, minimum length= 60, maximum length=179) (tree 1 of 10 is presented here).

CBS 130562 SO4 LSJ2 BC7 LSJ3 BC9 BC8 BC6 BC10 BC5 BC1 BC2 BC3 BC14 BC4 BC15 BC11 BC12 BC13 CBS 130569 LSJ1 QC8 QC11 QC9 CBS 391.59 CBS 100042 AGASSIZ ARCADIA DAVID5 a. Sphaerulina musiva 100 72 Sphaerulina populicola 92 100 Fig. S2 Concatenated five-gene region maximum parsimony (a) and Bayesian (b) phylogram of Sphaerulina species. Statistical support for the topology is presented when bootstrap values are above 70% and posterior probabilities are above 95%. Isolate names in bold were sequenced as part of this study. All other isolate sequences were obtained from GenBank. For the maximum parsimony tree, the combined dataset of five gene regions contained 1764 characters of which 82 were informative and produced 400 equal MP trees of 141 steps (CI=0.74, RI=0.91 g1=-1.0635, minimum length=105, maximum length=522) (tree 1 of 400 is presented). Sphaerulina populi b. 0.02 BC5 BC1 BC2 BC3 BC14 CBS 130562 CBS 130569 LSJ1 BC11 BC4 BC15 BC12 BC13 SO4 LSJ2 BC7 LSJ3 BC9 BC6 BC8 BC10 AGASSIZ CBS100042 QC9 ARCADIA DAVID5 QC8 QC11 CBS391.59 Sphaerulina musiva Sphaerulina populicola Sphaerulina populi 1 0.99 0.98 0.97 From eastern Canada From western Canada

Fig. S3 Statistical parsimony network of the 73 Sphaerulina musiva isolates using the sequence dataset (concatenated sequence of eight genes used in the population genetic analysis). Each hatch mark indicates a single mutation.

Fig. S4 STRUCTURE analysis (with no priors) of 301 unlinked SNPs found amongst clone-corrected populations of 65 Sphaerulina musiva isolates. (a) Log likelihood values of DK values against the ranging K values for the STRUCTURE analysis. (b) Standard deviation of the mean likelihoods against the ranging K values for the STRUCTURE analysis. (c) STRUCTURE barplots from K2- K6; supported K are highlighted in orange. Abbreviations: LSJ= Leclercville, LEC= Lac Saint-Jean, SO= Saint-Ours, TH= Thetford-Mines, AB= Abitibi-Témiscamingue, KEN= Kentucky, PEN= Pennsylvania and MISS= Missouri, ND= North Dakota, WIS= Wisconsin and MIN= Minnesota.

Fig. S5 Scenarios tested. 1-4) Round One- Determining the source population: 1) independent introduction scenario, 2) three-way split at t7, 3) three-way split at T6, 4) serial introduction scenario; 5-14) Round Two- Formation of MWUS: 5-8) Single-source population contributing to the formation of the MWUS group- Class I (without a bottleneck) and Class II (with a bottleneck), 9-14) Two-source populations contributing to the formation of the MWUS group- Class III (without a bottleneck) and Class IV (with a bottleneck); 15-29) Round Three- Formation of the BC population: 15-20) Single source population contributing to the formation of the BC population- Class I (without a bottleneck) and Class II (with a bottleneck), 21-29) Two source populations contributing to the formation of the BC population- Class III (without a bottleneck) and Class IV (with a bottleneck); 30-34) Round Four- Formation of the Alberta population: Class I (without a bottleneck) and Class II (with a bottleneck). *Tested with and without a bottleneck after the formation of each population. # Indicates the admixed population is formed with an admixture rate relative to the (ra) population (and 1-rate to the other contributing population) this was tested in both directions. Abbreviations: ?= unsampled ghost population; EC= eastern Canada [QUE= Québec (Lac Saint-Jean, Leclercville, Saint-Ours and Thetford-Mines) and ONT= Ontario]; EUS= eastern US (KEN= Kentucky, PEN= Pennsylvania and MISS= Missouri); MWUS=mid-west US (ND= North Dakota, WIS= Wisconsin and MIN= Minnesota); and WC= western Canada (SAS= Saskatchewan, ALB= Alberta and BC= British Columbia).

Round 1: ancestral populations 1. 2. 3. 4. t9 19000-100000 t8 10000-26500 t7 6-10000 t6 6-10000 PI PJ PK PI PJ PK PI PJ PK PI PJ PK pd Class I* Class II* Class III* Class IV* Round 2: formation of MWUS 7. SAS WUS EUS EC 8. 6. 5. t9 19000-100000 WUS EUS SAS EC SAS WUS EUS EC WUS EUS SAS EC t8 10000-26500 t7 6-10000 t6 6-10000 t5 6-10000 t4 6-10000 pd Class I and Class II*: single source t9 19000-100000 t8 10000-26500 t7 6-10000 t6 6-10000 pd t4 6-10000 t5 6-10000 10. 11. 9. WUS EUS SAS EC 1- ra ra WUS EUS SAS 1- ra ra EC WUS EUS SAS 1- ra ra EC t9 19000-100000 t8 10000-26500 t7 6-10000 t6 6-10000 pd t4 6-10000 t5 6-10000 12. WUS EUS SAS EC 1- ra ra 13. EC WUS EUS SAS 1- ra ra 14. EC WUS EUS SAS 1- ra ra Class III and Class IV*#: multiple sources Fig. S5 Scenarios tested. 1-4) Round One- Determining the source population: 1) independent introduction scenario, 2) three-way split at t7, 3) three-way split at T6, 4) serial introduction scenario; 5-14) Round Two- Formation of MWUS: 5-8) Single-source population contributing to the formation of the MWUS group- Class I (without a bottleneck) and Class II (with a bottleneck), 9-14) Two-source populations contributing to the formation of the MWUS group- Class III (without a bottleneck) and Class IV (with a bottleneck). Continued next page. *Tested with and without a bottleneck after the formation of each population. # Indicates the admixed population is formed with an admixture rate relative to the (ra) population (and 1-rate to the other contributing population) this was tested in both directions. Abbreviations: ?= unsampled ghost population; EC= eastern Canada [QUE= Québec (Lac Saint-Jean, Leclercville, Saint-Ours and Thetford-Mines) and ONT= Ontario]; EUS= eastern US (KEN= Kentucky, PEN= Pennsylvania and MISS= Missouri); MWUS=mid-west US (ND= North Dakota, WIS= Wisconsin and MIN= Minnesota); and WC= western Canada (SAS= Saskatchewan, ALB= Alberta and BC= British Columbia)

Round 3: formation of BC 15. EC EUS SAS BC 16. 17. t9 19000-100000 t8 10000-26500 t7 6-10000 t6 6-10000 pd t4 6-10000 t5 6-10000 t3 6-10000 t2 6-100 EC EUS SAS BC ra ra-1 ra ra-1 ra ra-1 WUS BC EC EUS WUS SAS WUS 20. t9 19000-100000 t8 10000-26500 t7 6-10000 t6 6-10000 pd t4 6-10000 t5 6-10000 t3 6-10000 t2 6-100 18. EC EUS SAS BC 19. EC SAS EUS ra ra-1 WUS BC ra ra-1 WUS ra ra-1 WUS EC EUS BC SAS Class I and Class II*: single source 22. 23. 21. t9 19000-100000 t8 10000-26500 t7 6-10000 t6 6-10000 pd t4 6-10000 t5 6-10000 t3 6-10000 t2 6-100 EC SAS EUS ra ra-1 WUS BC EC SAS EUS ra ra-1 WUS BC EC SAS EUS ra ra-1 WUS BC t9 19000-100000 t8 10000-26500 t7 6-10000 t6 6-10000 pd t4 6-10000 t5 6-10000 t3 6-10000 t2 6-100 24. EC SAS EUS ra ra-1 WUS BC 25. 26. EUS SAS BC ra ra-1 EC SAS EUS ra ra-1 WUS BC ra ra-1 EC WUS Class III and Class IV*#: multiple sources Fig. S5 cont 15-29) Round Three- Formation of the BC population: 15-20) Single source population contributing to the formation of the BC population- Class I (without a bottleneck) and Class II (with a bottleneck), 21-29) Two source populations contributing to the formation of the BC population- Class III (without a bottleneck) and Class IV (with a bottleneck). Continued next page. *Tested with and without a bottleneck after the formation of each population. # Indicates the admixed population is formed with an admixture rate relative to the (ra) population (and 1-rate to the other contributing population) this was tested in both directions. Abbreviations: ?= unsampled ghost population; EC= eastern Canada [QUE= Québec (Lac Saint-Jean, Leclercville, Saint-Ours and Thetford-Mines) and ONT= Ontario]; EUS= eastern US (KEN= Kentucky, PEN= Pennsylvania and MISS= Missouri); MWUS=mid-west US (ND= North Dakota, WIS= Wisconsin and MIN= Minnesota); and WC= western Canada (SAS= Saskatchewan, ALB= Alberta and BC= British Columbia)

Round 3: formation of BC cont. 27. 28. 29. t9 19000-100000 t8 10000-26500 t7 6-10000 t6 6-10000 pd t4 6-10000 t5 6-10000 t3 6-10000 t2 6-100 EC EUS SAS BC ra ra-1 WUS EUS SAS ra ra-1 BC EC EUS SAS BC ra ra-1 WUS ra ra-1 WUS ra ra-1 EC Class III and Class IV*#: multiple sources cont Round 4: formation of Alberta t9 19000-100000 t8 10000-26500 t7 6-10000 t6 6-10000 pd t4 6-10000 t5 6-10000 t3 6-10000 t2 6-100 EC EUS SAS BC ra ra-1 ALB WUS 30. EC EUS SAS BC ra ra-1 ALB WUS 31. EC EUS SAS BC ra ra-1 ALB WUS 32. t1 6-100 33. 34. EC EUS SAS BC ra ra-1 ALB WUS t9 19000-100000 t8 10000-26500 t7 6-10000 t6 6-10000 pd t4 6-10000 t5 6-10000 t3 6-10000 t2 6-100 EC EUS SAS BC ra ra-1 ALB WUS t1 6-100 Class I and Class II*: single source Fig. S5 cont 21-29) Two source populations contributing to the formation of the BC population- Class III (without a bottleneck) and Class IV (with a bottleneck); 30-34) Round Four- Formation of the Alberta population: Class I (without a bottleneck) and Class II (with a bottleneck). *Tested with and without a bottleneck after the formation of each population. # Indicates the admixed population is formed with an admixture rate relative to the (ra) population (and 1-rate to the other contributing population) this was tested in both directions. Abbreviations: ?= unsampled ghost population; EC= eastern Canada [QUE= Québec (Lac Saint-Jean, Leclercville, Saint-Ours and Thetford-Mines) and ONT= Ontario]; EUS= eastern US (KEN= Kentucky, PEN= Pennsylvania and MISS= Missouri); MWUS=mid-west US (ND= North Dakota, WIS= Wisconsin and MIN= Minnesota); and WC= western Canada (SAS= Saskatchewan, ALB= Alberta and BC= British Columbia)