1
Appendix S1. Polymerase Chain Reaction (PCR) Protocol
2
DNA was extracted from 50 mg of desiccated needle tissue. DNA was extracted using a
3
modified version of the cetyl-trimethyl ammonium bromide (CTAB) method (Doyle 1991). All
4
reactions used 50-100 ng genomic DNA, 2 pmol of each primer, 0.5 mM each of dATP, dCTP,
5
and dTTP, 1 x PCR buffer, and 0.4 units of Taq polymerase. Thermocycling conditions followed
6
Liewlaksaneeyanawin et al. (2004) for the Lop1 and Lop5 loci and Maherali et al. (2002) for the
7
NZPR4.6 locus. For the Pico2, Pico3, Pico138, Pico104, Pico109, and Pico31 loci protocols
8
followed Lesser et al. (2012). To visualize genotypes forward primers were labeled with
9
fluorescent dyes and PCR products were detected via capillary electrophoresis on an ABI 3130
10
genetic analyzer (ABI, Foster City, CA).
11
Literature Cited
12
Doyle, J. (1991). DNA protocols for plants: a CTAB total DNA isolation. IN: Molecular
13
Techniques in Taxonomy (eds Hewitt, G.M. & Johnston, A.), pp. 283–293. Springer-
14
Verlag, Berlin.
15
Lesser, M.L., Parchman, T.L., & Buerkle, C.A. (2012). Cross-species transferability
16
of SSR loci developed from transcriptome sequencing in lodgepole pine. Molecular
17
Ecology Resources. 12: 448-455.
18
Liewlaksaneeyanawin C, Ritland CE, El-Kassaby YA, Ritland K. (2004). Single-copy, species-
19
transferable microsatellite markers developed from loblolly pine ESTs. Theoretical and
20
Applied Genetics. 109: 361–369. (doi: 10.1007/s00122-004-1635-7).
21
Maherali, H, Williams, B, Paige, K, Delucia, E. (2002). Hydraulic differentiation of Ponderosa
22
pine populations along a climate gradient is not associated with ecotypic divergence.
23
Functional Ecology. 16: 510–521. (doi: 10.1046/j.1365-2435.2002.00645.x).
24
25
Appendix S2. Input parameters for parentage simulation runs and LOD critical values at 80 and
26
50 % confidence in parentage assignment based on simulation runs. Parameters and critical
27
values are given by decade for each population.
28
Castle Garden
candidate
decade
parents
1620
102
1630
\
1640
\
1650
\
1660
\
1670
103
1680
104
1690
105
1700
106
1710
107
1720
\
1730
108
1740
112
1750
114
1760
118
1770
121
1780
125
1790
132
1800
\
1810
137
1820
138
1830
140
1840
144
1850
151
1860
154
1870
163
1880
175
1890
183
1900
189
1910
198
1920
200
1930
202
1940
206
1950
213
1960
221
1970
226
1980
230
1990
237
2000
245
prop.
sampled
0.02
\
\
\
\
0.03
0.04
0.05
0.06
0.07
\
0.07
0.11
0.12
0.15
0.17
0.20
0.24
\
0.27
0.28
0.29
0.31
0.34
0.35
0.39
0.43
0.45
0.47
0.49
0.50
0.50
0.51
0.53
0.55
0.56
0.57
0.58
0.59
prop. loci
typed
0.89
\
\
\
\
0.74
0.72
0.73
0.74
0.71
\
0.75
0.78
0.77
0.77
0.79
0.79
0.81
\
0.80
0.80
0.81
0.81
0.82
0.83
0.83
0.81
0.82
0.82
0.82
0.82
0.82
0.81
0.81
0.82
0.82
0.82
0.81
0.80
parent pair
single parent
80% critical LOD 50% critical LOD 80% critical LOD 50% critical LOD
999.00
999.00
999.00
999.00
\
\
\
\
\
\
\
\
\
\
\
\
\
\
\
\
999.00
999.00
999.00
3.38
8.50
8.25
4.50
3.56
8.25
8.13
5.00
3.57
999.00
9.50
4.81
3.50
999.00
8.21
4.44
3.70
\
\
\
\
999.00
10.00
4.86
3.43
13.75
12.00
4.78
3.22
16.50
10.13
4.74
3.16
12.64
9.63
4.69
3.10
12.45
9.46
4.53
2.90
12.23
9.50
4.55
2.87
12.41
9.24
4.53
2.73
\
\
\
\
12.73
9.43
4.60
2.79
12.62
9.25
4.54
2.73
12.41
9.18
4.47
2.60
12.70
9.24
4.51
2.61
12.57
9.10
4.50
2.53
12.28
9.02
4.48
2.50
12.59
9.03
4.50
2.43
12.68
9.14
4.59
2.53
12.64
9.12
4.61
2.48
12.92
9.13
4.65
2.45
13.00
9.24
4.70
2.51
13.02
9.22
4.67
2.50
13.03
9.19
4.70
2.49
13.04
9.24
4.74
2.57
13.06
9.24
4.74
2.55
12.99
9.09
4.78
2.46
13.06
9.19
4.79
2.48
13.05
9.12
4.80
2.49
13.15
9.32
4.88
2.61
13.36
9.42
4.96
2.75
Grass Creek
decade
29
1740
1750
1760
1770
1780
1790
1800
1810
1820
1830
1840
1850
1860
1870
1880
1890
1900
1910
1920
1930
1940
1950
1960
1970
candidate
parents
102
108
110
117
119
121
124
\
126
131
134
135
136
139
143
145
148
154
155
157
159
160
161
162
prop.
sampled
0.02
0.07
0.09
0.15
0.16
0.17
0.19
\
0.21
0.24
0.25
0.26
0.26
0.28
0.30
0.31
0.32
0.35
0.36
0.36
0.37
0.38
0.39
0.40
prop. loci
typed
0.61
0.71
0.77
0.74
0.73
0.71
0.69
\
0.70
0.71
0.70
0.70
0.70
0.70
0.69
0.69
0.70
0.71
0.71
0.72
0.73
0.72
0.73
0.73
parent pair
single parent
80% critical LOD 50% critical LOD 80% critical LOD 50% critical LOD
999.00
999.00
999.00
999.00
9.69
9.50
4.92
3.42
999.00
9.84
4.85
3.29
14.50
10.24
4.77
3.22
13.25
10.14
4.74
3.18
12.97
9.94
4.72
3.23
12.50
10.02
4.82
3.25
\
\
\
\
12.97
10.08
4.83
3.22
13.00
10.09
4.75
3.23
13.84
10.24
4.87
3.27
13.92
10.19
4.85
3.26
14.13
10.15
4.83
3.27
12.95
10.11
4.83
3.24
13.46
10.13
4.82
3.24
13.48
10.27
4.83
3.26
13.30
10.30
4.78
3.22
13.55
10.11
4.84
3.20
13.68
10.16
4.81
3.18
13.42
10.20
4.83
3.18
13.79
10.18
4.77
3.14
13.52
10.13
4.81
3.14
13.46
10.05
4.80
3.14
13.56
10.07
4.77
3.12
30
Cottonwood Creek
candidate
decade
parents
1540
101
1550
102
1560
\
1570
103
1580
105
1590
\
1600
106
1610
107
1620
109
1630
\
1640
110
1650
114
1660
121
1670
125
1680
132
1690
139
1700
144
1710
151
1720
154
1730
160
1740
163
1750
169
1760
180
1770
196
1780
225
1790
259
1800
290
1810
316
1820
346
1830
390
1840
428
1850
469
1860
515
1870
552
1880
604
1890
649
1900
699
1910
741
1920
781
1930
809
1940
824
1950
845
1960
864
1970
877
1980
887
1990
890
2000
897
prop.
sampled
0.02
0.02
\
0.03
0.05
\
0.06
0.07
0.09
\
0.09
0.12
0.17
0.20
0.24
0.28
0.31
0.34
0.35
0.38
0.39
0.41
0.44
0.49
0.56
0.61
0.66
0.68
0.71
0.74
0.77
0.79
0.81
0.82
0.83
0.85
0.86
0.87
0.87
0.88
0.88
0.88
0.88
0.89
0.89
0.89
0.89
prop. loci
typed
0.78
0.85
\
0.89
0.87
\
0.89
0.90
0.91
\
0.86
0.87
0.86
0.87
0.85
0.85
0.84
0.84
0.84
0.84
0.83
0.83
0.84
0.82
0.82
0.82
0.81
0.80
0.80
0.80
0.81
0.82
0.82
0.83
0.83
0.83
0.83
0.83
0.83
0.83
0.84
0.84
0.84
0.84
0.84
0.84
0.84
parent pair
single parent
80% critical LOD 50% critical LOD 80% critical LOD 50% critical LOD
999.00
999.00
3.50
3.50
999.00
999.00
999.00
3.65
\
\
\
\
999.00
999.00
999.00
3.94
999.00
999.00
4.81
3.29
\
\
\
\
999.00
999.00
5.23
3.36
999.00
9.94
4.70
3.27
999.00
9.75
4.82
3.23
\
\
\
\
13.25
9.44
4.68
3.06
13.59
9.88
4.45
2.84
14.00
9.55
4.47
2.81
12.83
9.52
4.33
2.61
12.59
9.36
4.23
2.49
13.07
9.42
4.26
2.51
12.91
9.33
4.18
2.50
13.16
9.41
4.18
2.47
12.94
9.29
4.20
2.44
13.18
9.21
4.19
2.42
13.06
9.32
4.20
2.43
12.87
8.99
4.19
2.35
13.02
8.73
4.09
2.18
12.55
8.62
4.19
2.17
12.50
8.35
4.17
1.99
12.40
8.05
4.18
1.69
12.42
8.10
4.20
1.73
12.66
8.11
4.26
1.64
12.42
8.06
4.28
1.68
12.38
7.93
4.31
0.97
12.52
7.85
4.32
0.00
12.47
7.74
4.33
0.00
12.33
7.78
4.36
0.00
12.38
7.54
4.37
0.00
12.51
7.68
4.42
0.00
12.68
7.77
4.48
0.00
12.89
7.87
4.59
0.00
12.77
7.93
4.51
0.00
12.67
7.82
4.51
0.00
12.81
8.05
4.59
0.00
13.00
8.10
4.59
0.00
12.82
8.05
4.59
0.00
12.81
8.12
4.58
0.00
12.82
8.08
4.58
0.00
12.83
8.01
4.57
0.00
12.97
8.05
4.61
0.00
14.39
9.63
5.11
2.87
31
32
33
34
35
36
37
38
39
40
Anchor Dam
candidate
decade
parents
1700
102
1710
\
1720
\
1730
\
1740
103
1750
\
1760
104
1770
105
1780
106
1790
107
1800
110
1810
114
1820
116
1830
119
1840
125
1850
129
1860
133
1870
138
1880
144
1890
147
1900
157
1910
160
1920
165
1930
177
1940
196
1950
216
1960
222
prop.
sampled
0.02
\
\
\
0.03
\
0.04
0.05
0.06
0.07
0.09
0.12
0.14
0.16
0.20
0.22
0.25
0.28
0.31
0.32
0.36
0.38
0.39
0.44
0.49
0.54
0.55
prop. loci
typed
0.94
\
\
\
0.93
\
0.94
0.93
0.94
0.95
0.96
0.97
0.97
0.95
0.94
0.92
0.92
0.90
0.91
0.91
0.88
0.88
0.88
0.87
0.88
0.88
0.89
parent pair
80% critical LOD 50% critical LOD
999.00
999.00
\
\
\
\
\
\
8.00
8.00
\
\
9.50
9.00
999.00
10.00
999.00
10.25
11.88
9.22
12.50
9.57
13.10
9.84
12.82
9.58
12.59
9.31
12.46
9.41
12.88
9.36
12.78
9.62
13.26
9.72
13.44
9.62
13.21
9.49
13.38
9.72
13.32
9.57
13.71
9.80
13.65
9.87
13.66
9.69
13.73
9.72
13.93
9.88
single parent
80% critical LOD 50% critical LOD
3.50
3.50
\
\
\
\
\
\
4.66
3.48
\
\
4.94
3.48
5.03
3.33
4.77
3.21
4.70
2.96
4.41
2.60
4.34
2.53
4.34
2.53
4.30
2.46
4.32
2.52
4.35
2.57
4.40
2.61
4.44
2.66
4.47
2.66
4.47
2.60
4.56
2.77
4.57
2.75
4.59
2.80
4.70
2.87
4.75
2.86
4.86
2.93
4.87
2.96
41
Appendix S3. Characteristics of nine microsatellite loci used in the study. Expected
42
heterozygosity is the estimated fraction of individuals that would be heterozygous under Hardy-
43
Weinburg equilibrium.
44
locus # of alleles
lop1
16
lop5
13
NZPR4.6
21
Pico2
15
Pico3
11
Pico138
22
Pico104
17
Pico109
25
Pico31
9
45
Appendix S4. Mean number of alleles and mean polymorphic information content (PIC) across
46
all nine microsatellite loci at each study site. Combined exclusion probabilities for first parent,
47
second parent, and parent pairs at each study site
min. allele size
146
165
195
183
160
126
121
122
162
max. allele size
180
210
238
211
188
170
169
170
187
het obs.
0.71
0.73
0.44
0.65
0.07
0.43
0.49
0.46
0.07
Combined nonexclusion prob.
(first parent)
0.005
0.010
0.027
0.047
het exp.
0.81
0.79
0.66
0.78
0.30
0.79
0.89
0.79
0.28
Combined nonexclusion prob.
(second parent)
<0.001
<0.001
0.002
0.004
Combined nonexclusion prob.
( parent pair)
<0.001
<0.001
<0.001
<0.001
site
mean number of
alleles per locus
mean PIC
48
Castle Garden
Grass creek
Cottonwood Creek
Anchor Dam
12.44
9.67
14.78
10.00
0.71
0.70
0.60
0.61
49
Appendix S5. Polymorphic information content (PIC) for nine microsatellite loci at each of the
50
four study populations.
loci
51
lop1
lop5
NZPR4.6
Pico2
Pico3
Pico138
Pico104
Pico109
Pico31
PIC
Castle Garden Grass Creek Cottonwood Creek Anchor Dam
0.79
0.70
0.74
0.62
0.70
0.73
0.71
0.61
0.76
0.65
0.48
0.59
0.78
0.84
0.73
0.73
0.41
0.55
0.20
0.39
0.84
0.81
0.76
0.67
0.90
0.81
0.86
0.83
0.86
0.83
0.74
0.68
0.37
0.40
0.20
0.39
52
Appendix S6. Estimated null allele frequencies for each of the four study populations. a indicates
53
loci where null alleles are present based on van Oosterhout et al. (2004).
54
55
56
57
58
59
60
61
62
63
64
65
66
locus
Castle
Garden
null allele frequency
Grass
Cottonwood
Creek
Creek
lop1
lop5
0.0035
-0.0160
0.1351a
-0.0078
0.0297a
0.0056
NZPR4.6
0.1745a
0.1655a
0.0856a
0.0317
a
a
Anchor
Dam
0.159a
0.047
Pico2
0.0751
0.1800
0.0461a
0.0791a
Pico3
0.2723a
0.3670a
0.2493a
0.2903a
a
a
a
Pico138
0.2214
0.3086
0.1955
0.3083a
Pico104
0.1867a
0.2060a
0.2113a
0.2701a
Pico109
0.2518a
0.1784a
0.1782a
0.1736a
a
a
a
0.3022a
Pico31
0.2449
0.3278
0.2439
67
Appendix S7. Model parameters for age to first reproduction against time since population initiation fit to a weibull distributiona for
68
the four study sites with 80 and 50% parent assignments.
80% parent assignments
site
parameter
Castle Garden
intercept (α )
time of establishment from pop. Initiation (t)
Grass Creek
Cottonwood Creek
Anchor Dam
73
74
75
76
77
a
b
* P>0.001
SE
z
5.42
0.56
9.72
-0.01
0.00
-2.39
log(scale) (k)
-0.83
0.00
\
intercept (α)
4.47
0.06
75.25
time of establishment from pop. Initiation (t)
0.00
0.00
\
50% parent assignments
Pb
2.46e -22 *
1.70e
-2
*
\
0.00
*
\
z
Pb
coefficent
SE
5.79
0.25
23.44 1.65e -121 *
-0.01
0.00
-5.47 4.43e -8
*
-0.74
0.13
-5.76 8.62e
-9
*
6.62
0.34
19.65 6.08e -86 *
-0.01
0.00
-5.98 2.27e -9
*
log(scale) (k)
intercept (α)
-2.53
5.78
0.59
0.12
-4.29
47.60
1.76e
0.00
*
*
-1.53
5.29
0.22
0.10
-6.86 6.86e -12 *
50.80 0.00
*
time of establishment from pop. Initiation (t)
-0.54
0.00
-12.20
4.09e -34 *
0.00
0.00
-11.30 1.63e -29 *
log(scale) (k)
-0.78
0.05
-15.30
3.71e -53 *
-0.63
0.04
-15.70 7.44e -56 *
-5
intercept (α)
6.23
0.47
13.24
5.12e
*
6.04
0.19
32.31 5.18e -229 *
time of establishment from pop. Initiation (t)
-0.01
0.00
-4.07
4.62e -5 *
-0.01
0.00
-8.67 4.32e -18 *
-1.14
0.12
-9.20 3.64e -20 *
log(scale) (k)
69
70
71
72
coefficent
-1.18
0.23
-5.09
3.57e
-40
-7
*
78
79
80
Appendix S8. Slope and breakpoint coefficients for regression analysis of per capita growth rate against population size for the study
populations. Coefficients are given for the total population, for individuals with compatible local parents and individuals with parents
assigned at 50 and 80% confidence. (*** P>0.001, **P>0.01, *P>0.05)
Parameter
Slope1
Slope 2
Breakpoint (pop.size)
Parameter
81
Slope1
Slope 2
Breakpoint (pop.size)
Total Pop.
-0.0009**
\
\
Total Pop.
-0.0002***
\
\
Castle Garden
Compat.
50% Assign.
Estab.
Estab.
0.0002*
0.0005**
\
-0.0005**
\
60
Cottonwood Creek
Compat.
50% Assign.
Estab.
Estab.
0.0013***
0.0007***
-0.0016***
-0.0009***
124
171
80% Assign.
Estab.
0.0001***
-0.0003***
75
80% Assign.
Estab.
0.0001***
-0.0003***
401
Total Pop.
-0.0063***
\
\
Total Pop.
-0.0011**
\
\
Grass Creek
Compat.
50% Assign.
Estab.
Estab.
0.0011**
0.0006**
-0.0067**
-0.0039**
52
54
80% Assign.
Estab.
0.0001
\
\
Anchor dam
Compat.
50% Assign.
Estab.
Estab.
0.0012***
0.0008***
-0.045***
-0.0135***
118
115
80% Assign.
Estab.
0.0004***
-0.0074***
117
Appendix S9. Number and timing of extra-local parentage assignments for the four study
populations. All assignments are based on 95% confidence LOD critical values.