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Journal of Biogeography
SUPPORTING INFORMATION
The dissection of a Pleistocene refugium: phylogeography of the smooth newt,
Lissotriton vulgaris, in the Balkans
Maciej Pabijan, Piotr Zieliński, Katarzyna Dudek, Marta Chloupek, Konstantinos
Sotiropoulos, Marcin Liana and Wiesław Babik
Appendix S1 PCR and sequencing methods, primer sequences, GenBank accession numbers
and maximum likelihood tree for ND2 sequences in Lissotriton vulgaris.
DNA extraction, PCR and sequencing
DNA from alcohol-preserved tissues of smooth newts (Lissotriton vulgaris) was extracted
using the Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). We
amplified and sequenced two mitochondrial DNA (mtDNA) fragments. For all samples, a c.
1145 bp fragment consisting of the tRNA-Met, NADH dehydrogenase subunit 2, tRNA-Trp
and tRNA-Ala (ND2) was obtained. For a subset of samples (14 individuals), we also
obtained the 3′ portion of the gene encoding NADH dehydrogenase subunit 4, the entire
tRNA-His gene, and part of tRNA-Ser (ND4). PCRs were conducted using the ND4/Leu
primer pair (Arévalo et al., 1994) or, in the case of unsatisfactory amplification products, we
applied the alternative pair ND4-deg/Leu_MP. Twenty microlitre PCRs contained 2 µL of
10× PCR buffer with (NH4)2SO4 (Thermo Fisher Scientific, Waltham, MA, USA), 2.5 mM
MgCl2, 1µM of the forward and reverse primers, 0.2 mM of each dNTP, and 0.5 U of Taq
polymerase (Thermo Fisher Scientific, Waltham, MA, USA). The cycling scheme included
initial denaturation at 94 °C for 3 min 30 s, followed by 35–40 cycles at 94 °C for 30 s, 54–56
°C for 30 s, 72 °C for 1 min 20 s, and a final elongation step at 72 °C for 3 min. Exonuclease
I/ alkaline phosphatase (Thermo Fisher Scientific, Waltham, MA, USA) digestion was used to
purify the PCR products. Sequencing reactions were based on BigDye v3.1 Terminator Cycle
Sequencing chemistry (Applied Biosystems, Foster City, CA, USA) using the PCR primers
and additional sequencing primers. After EDTA/ethanol precipitation, the sequencing
products were run an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA,
USA).
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Table S1 Primers used for amplification and sequencing of the ND2 and ND4 genes in
Lissotriton.
Gene
Primer
Sequence (5′–3′)
Application
Reference
ND2
H5018
TCTGGGTTGCATTCAGAAGA
amplification
Arévalo et al. (1994)
ND2
L3879
TGTTGGTGGAAACCCTTCTC
amplification,
this study
sequencing
ND2
ND2 seq3
GGGCTyCCGCCAACATGTGG
sequencing
this study
ND2
ND2 seq6
GGCCCTTGGTCTTGyTATCC
sequencing
this study
ND4
ND4
CACCTATGACTACCAAAAGCTCATGTAGAAGC
amplification,
Arévalo et al. (1994)
sequencing
ND4
Leu
CATTACTTTTACTTGGATTTGCACCA
amplification,
Arévalo et al. (1994)
sequencing
ND4
ND4_deg
CACCTATGAYTACCAAAAGCYCATGTAGAAGC
amplification,
this study
sequencing
ND4
Leu_MP
CATAACTTTTACTTGGAGTTGCACCA
amplification,
this study
sequencing
ND4
ND4_seq1
AAATTATACGCACATGAGAAGG
sequencing
this study
ND4
ND4_seq2
CAAACACCATGAAGCCTTACAG
sequencing
this study
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Table S2 GenBank accession numbers, haplotypes and gene annotations of ND2 and ND4
sequences from smooth newts (Lissotriton sp.) and alpine newts (Mesotriton alpestris, used as
an outgroup) from previous studies (Babik et al., 2005; Pabijan et al., 2006) applied in
different analyses (see main text).
Analysis
Accession
Haplotype
Gene annotation
ND2; ND2+ND4
AY951351
A1
Triturus_vulgaris_haplotype_A1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951403
A4
Triturus_vulgaris_haplotype_A4_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951342
B1
Triturus_vulgaris_haplotype_B1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951493
C1
Triturus_vulgaris_haplotype_C1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951501
D1
Triturus_vulgaris_haplotype_D1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951402
E1
Triturus_vulgaris_haplotype_E1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951390
F1
Triturus_vulgaris_haplotype_F1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951349
G1
Triturus_montandoni_haplotype_G1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951454
H1
Triturus_vulgaris_haplotype_H1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951492
I1
Triturus_montandoni_haplotype_I1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951500
J1
Triturus_vulgaris_haplotype_J1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951343
K1
Triturus_vulgaris_haplotype_K1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951471
L1
Triturus_vulgaris_haplotype_L1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951347
L24
Triturus_vulgaris_haplotype_L24_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951396
L57
Triturus_vulgaris_haplotype_L57_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951502
L. italicus
Triturus_italicus_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951506
L.boscai Tb2
Triturus_boscai_haplotype_Tb2_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951507
L.boscai Tb3
Triturus_boscai_haplotype_Tb3_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951505
L.boscai Tb1
Triturus_boscai_haplotype_Tb1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
AY951503
L.helveticus Th1
Triturus_helveticus_haplotype_Th1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
DQ282140
M. alpestris hap1
Triturus_alpestris_haplotype_hap1_NADH_dehydrogenase_subunit_2_(ND2)
ND2; ND2+ND4
DQ282138
M. alpestris hapA
Triturus_alpestris_haplotype_hapA_NADH_dehydrogenase_subunit_2_(ND2)
ND2
AY951363
E3
Triturus_vulgaris_haplotype_E3_NADH_dehydrogenase_subunit_2_(ND2)
ND2
AY951495
H4
Triturus_vulgaris_haplotype_H4_NADH_dehydrogenase_subunit_2_(ND2)
ND2
AY951393
D3
Triturus_vulgaris_haplotype_D3_NADH_dehydrogenase_subunit_2_(ND2)
ND2
AY951472
D7
Triturus_vulgaris_haplotype_D7_NADH_dehydrogenase_subunit_2_(ND2)
ND2
AY951494
C2
Triturus_vulgaris_haplotype_C2_NADH_dehydrogenase_subunit_2_(ND2)
ND2
AY951340
B4
Triturus_vulgaris_haplotype_B4_NADH_dehydrogenase_subunit_2_(ND2)
ND2+ND4
AY951447
F27
Triturus montandoni haplotype F27 NADH dehydrogenase subunit 2 (ND2)
ND2+ND4
AY951339
B2
Triturus vulgaris haplotype B2 NADH dehydrogenase subunit 2 (ND2)
ND2+ND4
AY951393
D3
Triturus vulgaris haplotype D3 NADH dehydrogenase subunit 2 (ND2)
ND2+ND4
AY951443
G14
Triturus vulgaris haplotype G14 NADH dehydrogenase subunit 2 (ND2)
ND2+ND4
AY951468
J19
Triturus vulgaris haplotype J19 NADH dehydrogenase subunit 2 (ND2)
ND2+ND4
AY951448
I5
Triturus vulgaris haplotype I5 NADH dehydrogenase subunit 2 (ND2)
4
ND2+ND4
AY951520
A1
Triturus_vulgaris_haplotype_A1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951568
A4
Triturus_vulgaris_haplotype_A4_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951510
B1
Triturus_vulgaris_haplotype_B1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951638
C1
Triturus_vulgaris_haplotype_C1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951646
D1
Triturus_vulgaris_haplotype_D1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951566
E1
Triturus_vulgaris_haplotype_E1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951557
F1
Triturus_vulgaris_haplotype_F1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951518
G1
Triturus_montandoni_haplotype_G1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951609
H1
Triturus_vulgaris_haplotype_H1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951637
I1
Triturus_montandoni_haplotype_I1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951645
J1
Triturus_vulgaris_haplotype_J1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951511
K1
Triturus_vulgaris_haplotype_K1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951621
L1
Triturus_vulgaris_haplotype_L1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951515
L24
Triturus_vulgaris_haplotype_L24_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951562
L57
Triturus_vulgaris_haplotype_L57_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951653
L. italicus
Triturus_italicus_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951651
L.boscai Tb2
Triturus_boscai_haplotype_Tb2_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951652
L.boscai Tb3
Triturus_boscai_haplotype_Tb3_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951650
L.boscai Tb1
Triturus_boscai_haplotype_Tb1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951648
L.helveticus Th1
Triturus_helveticus_haplotype_Th1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
DQ282134
M. alpestris hap1
Triturus_alpestris_haplotype_hap1_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
DQ282133
M. alpestris hapA
Triturus_alpestris_haplotype_hapA_NADH_dehydrogenase_subunit_4_(ND4)
ND2+ND4
AY951605
F27
Triturus montandoni haplotype F27 NADH dehydrogenase subunit 2 (ND4)
ND2+ND4
AY951509
B2
Triturus vulgaris haplotype B2 NADH dehydrogenase subunit 4 (ND4)
ND2+ND4
AY951559
D3
Triturus vulgaris haplotype D3 NADH dehydrogenase subunit 4 (ND4)
ND2+ND4
AY951600
G14
Triturus vulgaris haplotype G14 NADH dehydrogenase subunit 4 (ND4)
ND2+ND4
AY951617
J19
Triturus vulgaris haplotype J19 NADH dehydrogenase subunit 4 (ND4)
ND2+ND4
AY951606
I5
Triturus vulgaris haplotype I5 NADH dehydrogenase subunit 4 (ND4)
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Figure S1
A maximum likelihood tree showing the relationships among mitochondrial ND2 sequences
in Lissotriton vulgaris and outgroups. Bootstrap support > 70 is shown at nodes; * denotes
support ≥ 90–95; ** denotes support > 95. Red tip labels mark haplotypes from Babik et al.
(2005). Note that sublineages L24 and L57 represent lineages L2 and L3 (Babik et al., 2005),
respectively. Green tip labels denote collection numbers and population names for newts
sequenced in this study.
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