Supporting information for online publication only
Epioblasma triquetra PCR conditions
All 10 µl PCR reactions contained 1 µl (approximately 5 ng) DNA, 2l 5X PCR buffer containing 1.5 mM
MgCl2 (Qiagen), an additional 0.35 mM MgCl2, 0.5 mM each dNTPs (BioShop), 0.23 mg/ml BSA
(BioShop), 0.5 U Taq DNA polymerase (Qiagen) and the following concentrations of each forward
(fluourescently labelled) and reverse primer: Multiplex 1: 0.2 µM LabC23 (AF512385); 0.25 µM Etr140
(DQ396406); 0.12 µM Ecap9 (AY650397); Multiplex 2: 0.25 µM Ecap5 (AY650393); 0.15 µM LabD213
(AF512398); Multiplex 3: 0.1 µM LabC2 (AF512384); 0.3 µM Etr90 (DQ396403); 0.35 µM LabD206
(AF512395); Multiplex 4: 0.15 µM Etr114 (DQ396404); 0.25 µM Etr124 (DQ396405); 0.1 µM LabC24
(AF512386); 0.45 µM Etr145 (DQ396407); 0.1 µM Ecap6 (AY650394); Multiplex 5: 0.35 µM each of
Etr187 (DQ396408) and LabD111 (AF512395). Total reaction volumens were made to 10 l with PCRgrade ddH20 (BioShop). Amplification consisted of 2 min at 94oC followed by 40 cycles of 94oC (40 s), an
annealing temperature of 58oC (40 s), 72oC (60 s), with a final extension at 72oC (5 min). Due to
inconsistent amplification success loci Ecap9, LabC2, LabC24, and Etr187 were excluded from all
analyses.
Quadrula quadrula PCR conditions
PCR reactions for locus QfA103 contained 1 µl (approximately 5 ng) DNA, 2l 5X PCR buffer containing
1.5 mM MgCl2 (Qiagen), an additional 0.5 mM MgCl2, 0.2 mM each dNTPs (BioShop), 0.2 mg/ml BSA
(BioShop), 0.5 U Taq DNA polymerase (Qiagen), 0.1 µM forward primer with an M13 tail (TGT AA ACG
ACG GCC AGT) at the 5’ end, 0.4 µM reverse primer, and 0.4 µM fluorescently labelled M13 probe
(Schuelke 2000) in a total volume of 10 µl.
PCR reactions for the remaining loci were the same except
they contained 0.4 µM each of a fluorescently labeled forward primer and 0.4 µM reverse primer in a
total volume of 10 µl. Amplification consisted of 10 min at 94oC followed by 40 cycles of 94oC (45 s), 60 s
at the optimal annealing temperature, 72oC (60 s), with a final extension at 72oC (5 min). Annealing
temperatures (Genbank accession numbers) were as follows for each locus: 48oC for QfA103
(FJ785629); 51oC for QfD102 (FJ85635); 55oC for QfA112 (FJ85630) and QfA130 (FJ785631); and 59oC for
QfC114 (FJ85634) and QfC4 (FJ85632).
Lampsilis fasciola PCR conditions
All 10 µl PCR reactions contained 1 µl (approximately 5 ng) DNA, 2l 5X PCR buffer containing 1.5 mM
MgCl2 (Qiagen), an additional 0.35 mM MgCl2, 0.5 mM each dNTPs (BioShop), 0.23 mg/ml BSA
(BioShop), 0.5 U Taq DNA polymerase (Qiagen) and the following concentrations of each forward
(fluourescently labelled) and reverse primer (Genbank accession number): Multiplex 1: 0.1 µM Ecap1
(AY650389); 0.15 µM Ecap5 (AY650393); Multiplex 2: 0.1 µM each of Etr114 (DQ396404), LabC23
(AF512385), and LabC24 (AF512386); Multiplex 3: 0.1 µM LabD206 (AF512395); 0.075 µM LabD213
(AF512398); Multiplex 4: 0.1 µM LabC2 (AF512384); 0.2 µM LabD111 (AF512395). Total reaction
volumens were made to 10 l with PCR-grade ddH20 (BioShop). Amplification consisted of 2 min at 94oC
followed by 40 cycles of 94oC (40 s), an annealing temperature of 58oC (40 s), 72oC (60 s), with a final
extension at 72oC (5 min). Due to inconsistent amplification success locus LabC2 was excluded from all
analyses.
Amblema plicata PCR conditions
All 10 µl PCR reactions contained 1 µl (approximately 5 ng) DNA, 2l 5X PCR buffer containing 1.5 mM
MgCl2 (Qiagen), an additional 0.5 mM MgCl2, 0.5 mM each dNTPs (BioShop), 0.2 mg/ml BSA (BioShop),
0.25 U Taq DNA polymerase (Qiagen), 0.1 µM of each forward primer with an M13 tail (TGT AA ACG ACG
GCC AGT) at the 5’ end, 0.3 µM of each reverse primer and 0.4 µM fluroescently labelled M13 probe
(Schuelke 2000). Total reaction volumens were made to 10 l with PCR-grade ddH20 (BioShop).
Amplification consisted of 10 min at 94oC followed by 35 cycles of 94oC (45 s), 60 s at the optimal
annealing temperature, 60 s at 72oC, with a final extension at 72oC (5 min). Annealing temperatures
(Genbank accession numbers) were as follows for each locus: 58oC for Anec103 (JF719044); 51oC for
Anec114 (JF719045); 48oC for Anec126 (JF19049); 60oC for Aned103 (JF719052); 56oC for Aned104
(JF19053); 54oC for Aned108 (JF19055) and Aned140 (JF19059); and 50oC for Aned126 (JF19056).
Thames
Grand
Ausable
Sydenham
Supplemental Table 1: Summary of samples for the six mussel species, showing sampled rivers,
sites, and number of genotyped loci, and local sample sizes (numbers of sampled individuals) for
each species and location. Sites with n<10 were removed from all population-based analyses to
avoid bias, but were included for individual-based assignments. Site locations are shown in Figure 1.
E. triquetra P. fasciolaris Q. quadrula L. fasciola L. costata A. plicata
River Site
(11 loci)
(9 loci)
(6 loci)
(8 loci)
(11 loci)
(8 loci)
1
2
3
4
5
Total
6
7
8
Total
9
10
11
12
13
14
Total
15
16
17
18
19
20
Total
Grand
Total
23
4
7
3
27
64
30
0
0
30
0
0
0
0
0
0
0
0
0
0
0
0
0
0
17
14
16
9
49
105
0
25
25
50
0
0
0
0
0
0
0
0
0
0
0
0
0
0
68
38
54
23
47
230
0
0
0
0
20
19
2
21
0
0
62
21
25
67
0
0
0
113
0
0
0
0
0
0
0
0
0
0
0
0
0
0
18
38
56
0
0
0
32
50
37
119
19
4
9
10
23
65
24
9
25
58
0
0
0
0
20
19
39
4
2
0
18
28
27
79
11
9
10
7
0
37
25
10
20
55
0
0
0
0
0
0
0
0
0
0
0
0
0
0
94
155
405
175
241
92