Table S1. Multiplex PCR Conditions for 16 Microsatellite Loci Optimized in This Study

Table S1. Multiplex PCR conditions for 16 microsatellite loci optimized in this study.

Locus/Multiplex / Individual
Primer
Conc. / MgCl2 / dNTP
Conc. / Annealing Temp. (°C) / Cycles
Sat12/Lsa1/Lsa8† / 0.24 μm / 3.00 mM / 0.20 mM / 56 / 35
Sat13‡ / 0.33 μm / 1.50 mM / 0.20 mM / 55 / 38
INRA100‡ / 0.64 μm / 1.00 mM / 0.20 mM / 60 / 35
INRA106‡ / 0.64 μm / 1.25 mM / 0.20 mM / 56 / 35
Sat3/Sat7‡ / 0.33 μm / 2.50 mM / 0.20 mM / 58 / 40
INRA016/
INRA021/Y† / 0.64 μm / 1.50 mM / 0.25 mM / 60 / 35
6L1F10/INRA241/
INRA259† / 0.67 μm / 1.50 mM / 0.25 mM / 60 / 35
Sol44*† / 0.20 μm / 2.50 mM / 0.20 mM / 62 / 40
Sol03*† / 0.20 μm / 3.00 mM / 0.20 mM / 52 / 40
†Cycling Conditions: / ‡Cycling Conditions:
Temperature (°C) / Time / Temperature (°C) / Time
Initial Denaturation: / 95 / 5’ / 95 / 5’
Denaturation: / 95 / 30” / 95 / 30”
Annealing: / See Table / 90” / See Table / 30”
Extension: / 72 / 30” / 72 / 60”
Final Extension: / 60 / 30’ / 72 / 7’

*12.5 μl reaction

All PCRs were in 15 μl reactions, except as noted. All reactions used 1X BSA, 1X buffer, and 0.75 U Taq polymerase.

Table S2. Genotyping error rates1 for noninvasive New England cottontail fecal pellet samples at 13 autosomal and 1 Y-chromasomal microsatellite loci.

Locus / Per Locus Error Rate / Per Allele Error Rate
Sat12 / 0.061 / 0.031
Sol03 / 0.293 / 0.150
Sol44 / 0.085 / 0.043
Lsa1 / 0.103 / 0.052
Lsa8 / 0.041 / 0.020
Sat13 / 0.174 / 0.098
INRA106 / 0.013 / 0.007
Sat3 / 0.106 / 0.063
Sat7 / 0.022 / 0.011
D6Utr4 / 0.018 / 0.010
INRA241 / 0.034 / 0.017
INRA259 / 0.028 / 0.015
INRA016 / 0.055 / 0.027
INRA326 (Y)2 / 0.082 / 0.041

1Error rates were calculated following Pompanon et al. (2005) equations 1 (ea = ma/2nt; per allele error rate) and 2 (el = ml/nt; per locus error rate), where m represents the number of allelic (or genotypic) mismatches relative to the consensus genotype, n is the number of individual single-locus genotypes and t is the number of replications.

2Error rate for the SRY - locus (INRA326) was based upon samples that produced an amplified product in more than one PCR run (2 individuals were classified as males despite only amplifying at INRA326 once in multiple runs).

Table S3. Null allele frequencies per locus by population calculated in INest (unshaded) and FreeNA (shaded). INest estimated null allele frequencies are significantly higher than those calculated in FreeNA (P < 0.001; Wilcoxon paired-sample test).

Population (N) / Sat12 / Sol03 / Sol44 / Lsa1 / Lsa8 / Sat13 / INRA106 / Sat3 / Sat7 / D6Utr4 / INRA241 / INRA259 / INRA016
MENH (58) / 0.07 / 0.11 / 0.05 / 0.15 / 0.11 / 0.18 / 0.08 / 0.05 / 0.08 / 0.07 / 0.08 / 0.08 / 0.10
0.05 / 0.11 / 0.02 / 0.14 / 0.09 / 0.18 / <0.001 / 0.02 / <0.001 / <0.001 / <0.001 / <0.001 / 0.08
NH-MV (14) / 0.09 / 0.11 / 0.20 / 0.08 / 0.09 / 0.21 / 0.14 / 0.13 / 0.15 / 0.28 / 0.15 / 0.167 / 0.19
0.02 / 0.05 / 0.16 / <0.001 / <0.001 / 0.17 / <0.001 / 0.02 / <0.001 / <0.001 / <0.001 / <0.001 / 0.12
CC (25) / 0.09 / 0.05 / 0.07 / 0.12 / 0.11 / 0.06 / 0.12 / 0.08 / 0.22 / 0.11 / 0.11 / 0.10 / 0.22
0.04 / <0.001 / <0.001 / 0.07 / 0.05 / <0.001 / <0.001 / <0.001 / 0.20 / <0.001 / <0.001 / <0.001 / 0.21
CTRI (58) / 0.04 / 0.06 / 0.11 / 0.18 / 0.09 / 0.05 / 0.08 / 0.09 / 0.12 / 0.15 / 0.11 / 0.11 / 0.15
<0.001 / 0.01 / 0.08 / 0.16 / <0.001 / <0.001 / <0.001 / 0.06 / <0.001 / 0.10 / 0.04 / 0.06 / 0.13
CTNY (38) / 0.05 / 0.09 / 0.15 / 0.05 / 0.04 / 0.05 / 0.04 / 0.05 / 0.20 / 0.07 / 0.10 / 0.08 / 0.11
0.02 / 0.07 / 0.13 / <0.01 / <0.001 / 0.02 / <0.001 / <0.001 / 0.18 / 0.03 / 0.07 / 0.01 / 0.10

Table S4. FIS and P values of the 5 geographic populations of New England cottontails at 15 autosomal loci, as computed in Genepop. P-values are shaded below FIS values. Significance at the 0.05 level after false discovery rate correction is indicated with an asterisk.

Population (N) / Sat12 / Sol03 / Sol44 / Lsa1 / Lsa8 / Sat13 / INRA 106 / Sat3 / Sat7 / D6Utr4 / INRA 241 / INRA 259 / INRA 016 / Overall Probability
MENH (58) / 0.153 / 0.221 / 0.050 / 0.388 / 1.000 / 0.453 / — / -0.018 / — / -0.009 / — / — / 0.319
0.223 / 0.002* / 0.074 / <0.001* / 0.009 / 0.000* / — / 0.470 / — / 1.000 / — / — / 0.040 / <0.0001*
NH-MV (14) / 0.060 / 0.050 / 0.498 / -0.247 / -0.238 / 0.472 / -0.040 / 0.178 / — / — / — / — / 0.464
0.138 / 0.132 / 0.020 / 1.000 / 1.000 / 0.051 / 1.000 / 0.432 / — / — / — / — / 0.220 / 0.088
CC (25) / 0.166 / -0.037 / 0.124 / 0.267 / 0.189 / -0.065 / — / 0.080 / 0.613 / -0.021 / -0.021 / -0.029 / 0.579
0.553 / 0.610 / 0.153 / 0.101 / 0.387 / 1.000 / — / 0.423 / 0.004* / 1.000 / 1.000 / 1.000 / 0.001* / 0.028
CTRI (58) / -0.001 / 0.095 / 0.198 / 0.436 / -0.061 / 0.008 / 0.018 / 0.137 / — / 0.350 / 0.171 / 0.208 / 0.327
0.883 / 0.019 / 0.005 / <0.001* / 1.000 / 0.423 / 0.478 / 0.057 / — / 0.028 / 0.281 / 0.187 / 0.004* / <0.0001*
CTNY (38) / 0.096 / 0.236 / 0.390 / -0.009 / -0.198 / -0.039 / -0.112 / 0.073 / 0.698 / 0.045 / 0.188 / 0.048 / 0.244
0.198 / 0.009 / <0.001* / 0.852 / 0.539 / 0.309 / 0.341 / 0.457 / <0.001* / 0.183 / 0.156 / 1.000 / 0.040 / <0.0001*
Overall Probability / 0.314 / <0.001* / <0.0001* / <0.0001* / 0.254 / <0.0001* / 0.166 / 0.108 / <0.0001* / 0.041 / 0.045 / 0.147 / <0.0001*

Table S5. Allele frequencies per locus of each geographic population of New England cottontails. Private alleles are highlighted in bold.

Locus / Allele/n / MENH / NH-MV / CC / CTRI / CTNY
Sat12 / N / 58 / 14 / 23 / 57 / 38
117 / 0.009 / 0.000 / 0.000 / 0.237 / 0.224
121 / 0.543 / 0.536 / 0.065 / 0.368 / 0.316
125 / 0.138 / 0.143 / 0.457 / 0.158 / 0.145
129 / 0.147 / 0.107 / 0.478 / 0.184 / 0.224
133 / 0.138 / 0.107 / 0.000 / 0.044 / 0.053
137 / 0.026 / 0.107 / 0.000 / 0.009 / 0.039
Sol03 / N / 55 / 13 / 25 / 57 / 37
257 / 0.236 / 0.000 / 0.040 / 0.000 / 0.000
261 / 0.045 / 0.000 / 0.000 / 0.035 / 0.000
263 / 0.000 / 0.077 / 0.000 / 0.026 / 0.014
265 / 0.327 / 0.577 / 0.700 / 0.263 / 0.324
267 / 0.227 / 0.115 / 0.020 / 0.342 / 0.378
269 / 0.145 / 0.038 / 0.020 / 0.123 / 0.108
271 / 0.000 / 0.000 / 0.000 / 0.000 / 0.027
273 / 0.000 / 0.154 / 0.000 / 0.000 / 0.041
275 / 0.000 / 0.000 / 0.060 / 0.026 / 0.081
277 / 0.018 / 0.038 / 0.020 / 0.167 / 0.027
279 / 0.000 / 0.000 / 0.100 / 0.009 / 0.000
281 / 0.000 / 0.000 / 0.000 / 0.009 / 0.000
285 / 0.000 / 0.000 / 0.040 / 0.000 / 0.000
Sol44 / N / 58 / 14 / 25 / 57 / 38
204 / 0.000 / 0.000 / 0.000 / 0.000 / 0.066
206 / 0.009 / 0.000 / 0.000 / 0.026 / 0.000
208 / 0.000 / 0.000 / 0.020 / 0.009 / 0.053
210 / 0.655 / 0.607 / 0.320 / 0.237 / 0.053
212 / 0.190 / 0.286 / 0.560 / 0.360 / 0.513
214 / 0.026 / 0.107 / 0.040 / 0.254 / 0.303
216 / 0.009 / 0.000 / 0.000 / 0.114 / 0.013
218 / 0.112 / 0.000 / 0.020 / 0.000 / 0.000
220 / 0.000 / 0.000 / 0.040 / 0.000 / 0.000
Lsa1 / N / 57 / 14 / 25 / 57 / 37
164 / 0.000 / 0.000 / 0.040 / 0.000 / 0.000
166 / 0.158 / 0.214 / 0.060 / 0.079 / 0.122
168 / 0.711 / 0.750 / 0.820 / 0.553 / 0.554
170 / 0.132 / 0.036 / 0.040 / 0.368 / 0.311
172 / 0.000 / 0.000 / 0.000 / 0.000 / 0.014
190 / 0.000 / 0.000 / 0.040 / 0.000 / 0.000
Lsa8 / N / 58 / 14 / 25 / 57 / 38
175 / 0.017 / 0.000 / 0.000 / 0.000 / 0.000
179 / 0.983 / 0.786 / 0.860 / 0.921 / 0.724
181 / 0.000 / 0.000 / 0.000 / 0.018 / 0.026
185 / 0.000 / 0.214 / 0.140 / 0.061 / 0.250

Table S5, continued. Allele frequencies per locus of each geographic population of New England cottontails. Private alleles highlighted in bold.

Locus / Allele/n / MENH / NH-MV / CC / CTRI / CTNY
Sat13 / N / 55 / 14 / 18 / 57 / 35
124 / 0.000 / 0.000 / 0.000 / 0.009 / 0.029
126 / 0.027 / 0.000 / 0.000 / 0.009 / 0.000
128 / 0.018 / 0.036 / 0.083 / 0.193 / 0.071
130 / 0.409 / 0.500 / 0.500 / 0.333 / 0.186
132 / 0.100 / 0.250 / 0.361 / 0.430 / 0.471
134 / 0.445 / 0.214 / 0.056 / 0.009 / 0.243
136 / 0.000 / 0.000 / 0.000 / 0.018 / 0.000
INRA106 / N / 58 / 14 / 25 / 57 / 38
154 / 1.000 / 0.929 / 1.000 / 0.895 / 0.592
156 / 0.000 / 0.071 / 0.000 / 0.044 / 0.000
158 / 0.000 / 0.000 / 0.000 / 0.009 / 0.053
160 / 0.000 / 0.000 / 0.000 / 0.053 / 0.329
162 / 0.000 / 0.000 / 0.000 / 0.000 / 0.026
Sat3 / N / 58 / 13 / 25 / 57 / 38
146 / 0.000 / 0.000 / 0.000 / 0.026 / 0.039
148 / 0.009 / 0.000 / 0.000 / 0.018 / 0.303
150 / 0.741 / 0.692 / 0.860 / 0.623 / 0.434
152 / 0.000 / 0.000 / 0.000 / 0.026 / 0.039
154 / 0.164 / 0.038 / 0.020 / 0.158 / 0.118
156 / 0.043 / 0.000 / 0.020 / 0.000 / 0.000
158 / 0.000 / 0.000 / 0.000 / 0.000 / 0.053
160 / 0.000 / 0.000 / 0.000 / 0.000 / 0.013
162 / 0.000 / 0.000 / 0.060 / 0.000 / 0.000
164 / 0.043 / 0.269 / 0.020 / 0.149 / 0.000
174 / 0.000 / 0.000 / 0.020 / 0.000 / 0.000
Sat7 / N / 58 / 13 / 25 / 57 / 37
188 / 0.000 / 0.000 / 0.000 / 0.000 / 0.108
192 / 1.000 / 0.962 / 0.500 / 0.991 / 0.851
194 / 0.000 / 0.038 / 0.500 / 0.009 / 0.041
D6Utr4 / N / 58 / 13 / 25 / 49 / 35
155 / 0.000 / 0.038 / 0.000 / 0.204 / 0.371
161 / 0.983 / 0.962 / 0.960 / 0.765 / 0.543
163 / 0.017 / 0.000 / 0.000 / 0.031 / 0.086
169 / 0.000 / 0.000 / 0.040 / 0.000 / 0.000
INRA241 / N / 58 / 14 / 25 / 57 / 38
147 / 0.000 / 0.000 / 0.000 / 0.070 / 0.105
149 / 1.000 / 0.963 / 0.960 / 0.921 / 0.816
151 / 0.000 / 0.036 / 0.000 / 0.000 / 0.013
153 / 0.000 / 0.000 / 0.000 / 0.009 / 0.066
155 / 0.000 / 0.000 / 0.040 / 0.000 / 0.000
INRA259 / N / 58 / 14 / 25 / 57 / 36
159 / 1.000 / 1.000 / 0.940 / 0.877 / 0.847
161 / 0.000 / 0.000 / 0.040 / 0.018 / 0.000
163 / 0.000 / 0.000 / 0.000 / 0.105 / 0.153
193 / 0.000 / 0.000 / 0.020 / 0.000 / 0.000

Table S5, continued. Allele frequencies per locus of each geographic population of New England cottontails. Private alleles highlighted in bold.

Locus / Allele/n / MENH / NH-MV / CC / CTRI / CTNY
INRA016 / N / 58 / 14 / 24 / 57 / 35
181 / 0.000 / 0.000 / 0.021 / 0.009 / 0.057
183 / 0.129 / 0.107 / 0.479 / 0.596 / 0.143
185 / 0.871 / 0.857 / 0.438 / 0.254 / 0.514
187 / 0.000 / 0.036 / 0.063 / 0.105 / 0.057
189 / 0.000 / 0.000 / 0.000 / 0.035 / 0.200
193 / 0.000 / 0.000 / 0.000 / 0.000 / 0.029

Table S6. Possible migrants and individuals of admixed ancestry as identified by STRUCTURE migrant detection and GENECLASS assignment tests for the New England cottontail genetic clusters identified in STRUCTURE.†

Sample / Geographic Origin / STRUCTURE Mean Q
No prior population information; K = 5 / GENECLASS cluster assignment (probability) / STRUCTURE resident probability (MIGPRIOR=0.05) / STRUCTURE migrant probabilities (immigrant/ immigrant parent/ immigrant grandparent) / Migrant/ Admixed classification
ME/NH / CC / CT/RI / Bluff Point / CT/NY
35 / MENH / 0.040 / 0.009 / 0.804 / 0.148 / 0.000 / NH-MV (89.670%)§ / 0.433* / NH-MV 0.119/0.032/0.026
Bluff Pt. 0.001/0.055/0.203 / Possible Migrant
161 / MENH / 0.464 / 0.053 / 0.480 / 0.003 / 0.000 / NH-MV (99.277%)§ / 0.756 / NH-MV 0.195/0.011/0.018 / Possible Migrant
MM5 / CC / 0.000 / 0.000 / 0.995 / 0.004 / 0.000 / NH-MV (50.221%)§
CT/RI (49.650%) / 0.008*** / NH-MV 0.328/0.046/0.019
CT/RI 0.328/0.064/0.024 / Possible Migrant
MM6 / CC / 0.004 / 0.002 / 0.988 / 0.001 / 0.005 / NH-MV (91.996%)§ / 0.402* / NH-MV 0.239/0.052/0.034 / Possible Migrant
232 / CTRI‡ / 0.000 / 0.000 / 0.000 / 1.000 / 0.000 / Bluff Pt. (99.992%)§ / 0.044*** / Bluff Pt. 0.917/0.031/0.008 / Possible Migrant
100 / CTNY / 0.097 / 0.000 / 0.739 / 0.164 / 0.000 / CT/RI (55.083%)§
NH-MV (40.824%) / 0.021*** / CT/RI 0.698/0.004/0.002
NH-MV 0.218/0.002/0.004 / Possible Migrant
175 / CTNY / 0.000 / 0.001 / 0.999 / 0.000 / 0.000 / CT/RI (88.750%)§ / 0.106** / CT/RI 0.463/0.017/0.027 / Possible Migrant
33 / MENH / 0.002 / 0.000 / 0.007 / 0.991 / 0.000 / CT/RI (92.942%)§ / 0.755 / Negligible / Admixed Ancestry
4 / MENH / 0.064 / 0.000 / 0.841 / 0.094 / 0.000 / CT/RI (72.029%) / 0.920 / Negligible / Admixed Ancestry
138 / MENH / 0.454 / 0.000 / 0.347 / 0.199 / 0.000 / NH-MV (75.985%) / 0.885 / Negligible / Admixed Ancestry
155 / NH-MV / 0.001 / 0.152 / 0.637 / 0.210 / 0.001 / CC (81.101%) / 0.784 / Negligible / Admixed Ancestry
NH3 / NH-MV / 0.000 / 0.001 / 0.999 / 0.000 / 0.000 / CT/RI (88.051%) / 0.613 / Negligible / Admixed Ancestry
MM2 / CC / 0.814 / 0.003 / 0.154 / 0.029 / 0.000 / MENH (86.247%) / 0.030*** / ME/NH 0.025/0.609/0.195 / Admixed Ancestry
213 / CC / 0.072 / 0.585 / 0.337 / 0.000 / 0.006 / CC (56.540%)
NH-MV (41.497%) / 0.345* / MENH 0.000/0.161/0.197
NH-MV 0.002/0.123/0.120 / Admixed Ancestry
1195 / CTRI‡ / 0.023 / 0.000 / 0.895 / 0.082 / 0.000 / CT/RI (57.333%)
CTNY (19.752%)
MENH (19.166%) / 0.257** / ME/NH 0.000/0.404/0.221 / Admixed Ancestry
85 / CTRI‡ / 0.000 / 0.000 / 0.504 / 0.046 / 0.450 / CT/NY (78.707%) / 0.530 / CT/NY 0.024/0.161/0.152 / Admixed Ancestry
248 / CTRI‡ / 0.000 / 0.000 / 0.113 / 0.885 / 0.002 / Bluff Pt. (97.993%) / 0.702 / Bluff Pt. 0.060/0.144/0.067 / Admixed Ancestry
219 / CTRI‡ / 0.992 / 0.000 / 0.007 / 0.001 / 0.000 / MENH (92.022%)‡ / 0.738 / MENH 0.128/0.030/0.021 / Admixed Ancestry
226 / CTRI‡ / 0.688 / 0.003 / 0.307 / 0.002 / 0.000 / NH-MV (81.436%) / 0.710 / Negligible / Admixed Ancestry
70 / CTRI‡ / 0.000 / 0.000 / 0.052 / 0.001 / 0.947 / CT/NY (75.806%) / 0.858 / Negligible / Admixed Ancestry
227 / CTRI‡ / 0.005 / 0.000 / 0.031 / 0.103 / 0.862 / CT/NY (62.275%) / 0.785 / Negligible / Admixed Ancestry
CT23.1 / CTNY / 0.013 / 0.000 / 0.978 / 0.002 / 0.007 / CT/NY (46.577%)
CT/RI (33.552%) / 0.137** / ME/NH 0.000/0.303/0.114 / Admixed Ancestry
172 / CTNY / 0.000 / 0.000 / 0.977 / 0.002 / 0.021 / CT/RI (75.503%) / 0.876 / Negligible / Admixed Ancestry
JJSM / CTNY / 0.016 / 0.001 / 0.785 / 0.174 / 0.024 / NH-MV (49.271%)
CT/RI (33.044%) / 0.889 / Negligible / Admixed Ancestry

Table S6, continued. Possible migrants and individuals of admixed ancestry as identified by STRUCTURE migrant detection and GENECLASS assignment tests for the New England cottontail genetic clusters identified in STRUCTURE.†

†MENH and NH-MV geographic populations were not combined for this analysis (see text). ‡No putative migrants originated from Bluff Point.

§Individuals detected by GENECLASS as first-generation migrants using the resampling method (P<0.01). Asterisks indicate relative probability of being a migrant or admixed, as determined by STRUCTURE (* = resident probability < 50%; ** = resident probability < 30%; *** = resident probability < 10%).

Values in bold highlight strongest assignment probabilities.