Table S1. Oligonucleotides Used for the AFLP Analysis of Oeneis Melissa Semidea

Laboratory Protocol Used to Generate Amplified Fragment Length Polymorphisms (AFLPs) for Oeneis melissa semidea

Genomic DNA (50 – 100 ng) was digested with MseI and EcoRI restriction enzymes and ligated to MseI and EcoRI adaptors (Table S1) simultaneously in a total reaction volume of 11 μl (see Tables S2 and S3 for detailed restriction-ligation protocol). Restriction-ligation (R-L) fragments were then diluted with 89 μl of TE0.1 buffer and stored at -20°C. Diluted R-L fragments were amplified in a pre-selective PCR using the primers Eco-A and Mse-C (Table S1) in a total reaction volume of 10 μl (8 μl master mix + 2 μl diluted R-L template; see Table S4 for detailed pre-selective PCR protocol). The pre-selective primers are complementary to the adaptors with one additional nucleotide (A for EcoRI and C for MseI) at the 3’ end. Only restriction fragments with bases complementary to these additional nucleotides (i.e., T and G) next to the restriction sites will be amplified, reducing the number of fragments to approximately 1/16 of the original amount (Meudt and Clarke 2007). Pre-selective products were diluted with 12.5 μl of TE0.1 buffer and stored at -20°C. Diluted pre-selective products were then amplified by PCR using selective primers (Eco-AXX and Mse-CXX; Table S1) in a total reaction volume of 10 μl (8.5 μl master mix + 1.5 μl diluted pre-selective PCR product; see Table S5 for detailed selective amplification protocol). The selective primers contain two additional nucleotides at the 3’ end, which further reduces the number of fragments to approximately 1/256 of the initial amount. Six selective primer pairs (EcoRI-AAC + MseI-CAC, EcoRI-AAC + MseI-CTC, EcoRI-AAG + MseI-CAG, EcoRI-ACA + MseI-CAA, EcoRI-ACC + MseI-CTA, EcoRI-ACG + MseI-CAA) were used for the selective amplification of Oeneis melissa semidea (Table S1). Prior to fragment analysis, selective PCR products were diluted 25x with water. Negative controls were included in each step of the protocol to ensure that no contamination had occurred.

Table S1. Oligonucleotides used for the AFLP analysis of Oeneis melissa semidea.

Protocol Step / Oligonucelotide / Sequence (5'-3') / Size (bp)
Restriction-Ligation / *EcoRI Adaptor 1 / CTCGTAGACTGCGTACC / 17
EcoRI Adaptor 2 / AATTGGTACGCAGTCTAC / 18
MseI Adaptor 1 / GACGATGAGTCCTGAG / 16
MseI Adaptor 2 / TACTCAGGACTCAT / 14
Pre-Selective PCR / Eco+A / GACTGCGTACCAATTCA / 17
Mse+C / GATGAGTCCTGAGTAAC / 17
Selective PCR / †Eco+AXX / GACTGCGTACCAATTCAAC / 19
GACTGCGTACCAATTCAAG / 19
GACTGCGTACCAATTCACA / 19
GACTGCGTACCAATTCACC / 19
GACTGCGTACCAATTCACG / 19
Mse+CXX / GATGAGTCCTGAGTAACAA / 19
GATGAGTCCTGAGTAACAC / 19
GATGAGTCCTGAGTAACAG / 19
GATGAGTCCTGAGTAACTC / 19
GATGAGTCCTGAGTAACTA / 19

*EcoRI and MseI adaptors must be annealed separately before use in restriction-ligation reactions (Table S2).

† All Eco-AXX primers were fluorescently-labelled with FAM dye.

Table S2. Protocol for annealing EcoRI and MseI adaptors for use in restriction-ligation reactions.

Annealing Reaction / Reaction Component / Concentration / Volume (μl)
EcoRI / EcoRI Adaptor 1 / 1000 μM / 1
EcoRI Adaptor 2 / 1000 μM / 1
water / 108
*T10E1 / 90
Total / 200
MseI / MseI Adaptor 1 / 1000 μM / 10
MseI Adaptor 2 / 1000 μM / 10
water / 90
*T10E1 / 90
Total / 200

*T10E1 = 10 mM Tris, 1 mM EDTA

Each master mix was prepared separately, placed in a thermalcycler for 8 min at 94°C followed by 10 min at 22°C, and centrifuged at 1400 g for 10 s.

Table S3. Protocol for restriction-ligation (R-L) reactions.

Reaction Component / Concentration / Volume (μl)
water / - / 0.55
T4 DNA ligase buffer with ATP / 10x / 1.1
NaCl / 0.5 M / 1.1
BSA / 1 mg/ml / 0.55
MseI Adaptor* / 100 U / 1
EcoRI Adaptor* / 500 U / 1
MseI (1 U) / 1 U / 0.1
EcoRI (5 U) / 5 U / 0.05
T4 DNA ligase / 100 Weiss U / 0.05
genomic DNA (μl) / 10 - 50 ng / 5.5
Total / 11

*MseI and EcoRI adaptors must be annealed as described in Table S2 before

use in R-L reaction.

R-L reactions were held at 24°C for 16 h in a thermalcycler, diluted with 89 μl TE buffer, and stored at -20°C.

Table S4.Protocol for pre-selective PCR amplification.

Reaction Component / Concentration / Volume (μl)
water / - / 1.5
betaine / 3 M / 3.5
PCR buffer / 10x / 1
MgCl2 / 25 mM / 0.65
dNTPs / 10 mM / 0.25
Mse-C primer / 10 μM / 0.5
Eco-A primer / 10 μM / 0.5
*Platinum® Taq DNA polymerase / 5 U/μl / 0.1
diluted R-L product / - / 2
Total / 10

*produced by Life TechnologiesTM

Preselective PCR thermalcycling conditions: one cycle of 72°C for 2 min; 25 cycles of 94°C for 20s, 56°C for 30 s, and 72°C for 2 min; and a final hold of 60°C for 30 min.

Table S5. Protocol for selective PCR amplification.

Reaction Component / Concentration / Volume (μl)
water / - / 4.4
PCR buffer / 10x / 1
MgCl2 / 25 mM / 1.75
dNTPs / 10 mM / 0.25
*Platinum® Taq DNA polymerase / 5 U/μl / 0.1
Eco+AXX primer / 10 μM / 0.5
Mse+CXX primer / 10 μM / 0.5
diluted pre-selective PCR product / - / 1.5
Total / 10

*produced by Life TechnologiesTM

Selective PCR thermalcycling conditions: one cycle of 94°C for 2 min; 94°C for 20 s, 66°C for 30s decreasing by 1°C/cycle, and 72°C for 2 min for 10 cycles; 94°C for 20 s, 56°C for 20 s, and 72°C for 2 min for 22 cycles; and a final hold of 60°C for 30 min.