Journal of Biogeography
SUPPORTING INFORMATION
Phylogeography of the prickly sculpin (Cottus asper) in north-western
North America reveals parallel phenotypic evolution across multiple
coastal–inland colonizations
Stefan Dennenmoser, Arne W. Nolte, Steven M. Vamosi and Sean M. Rogers
Appendix S1 List of primer sequences for mitochondrial and microsatellite
markers used for Cottus asper, and PCR protocols (see below).
Locus
Primer
Primer sequence (5’ – 3’)
D-loop, and
CotL1
CCGGAGGTTAAAATCCTCCC
partial tRNA
HN20
GTGTTATGCTTTAGTTAAGC
Cytochrome b
L14724
CGAAGCTTGATATGAAAAACCATCGTTG
H15149
AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA
FishF1
TCAACCAACCACAAAGACATTGGCAC
FishR1
TAGACTTCTGGGTGGCCAAAGAATCA
Cott170
ACATGGTGCATAATGTTGCCC
Cox1
Microsatellite
TCAGGGCCTACGCACAGAGC
Microsatellite
Cott207
AGTCCTTGTCGGGAGCCTCG
ATTGGGCGTTGCTCACCAGC
Microsatellite
Cott255
TCACTACAGCCAGGTGTCTG
GCATGTGCATGCCTCCACAG
Microsatellite
Cott54
CACGTACATGTGAAACGAACCC
AACAGAGGAAACGCCATGAC
Microsatellite
CottE30
AAGCGGACAGACGCAAAGAG
ACTCGCCCAAGAAGACCAGC
Microsatellite
LCE54
ATGTGTGAGGGCCTCAATGC
CTGTGTGGTGCGTCCATTGG
Microsatellite
Cott214
AGTAGTGCCGCTGCACCCAC
TCTCCCTCGTGGTGCAAACC
Microsatellite
Cott78
TCTGCTCCGCAGGGTCGCTG
TGACACATGGTGAACCCGTG
Microsatellite
Cott348
TGGACCACTGAGTTGCTGTG
TGGTGTGTCATACCTGCAGC
Microsatellite
CottE11
TCAAGAGGTGAGAGGCCAAGAG
CATCCTGGTGTTACCATTGCTG
Microsatellite
CottE20
CGTCTGCACAGTTGAGCCTG
AGCACGCCGTCGCTAACAGC
Microsatellite
Cott153
GGGTTCAGGTGCGTTTGGTC
ACCTTCAGCTCCGACATCAG
Microsatellite
Cott213
TTGCCATGGATTTGAGGCAG
AGCATTGCTATTATCAGGCTGC
Microsatellite
CottE13
AGTTACGCAATGCAACACGCCT
TCGAACACGAAGCTCCCGTCAC
Microsatellite
LCE81
GCTTGTGAACATTTCGGCCTC
AATGTACAAAGCTCCCACGTCC
Microsatellite
Cott50
TCCTGTGCTAATGCTGTGGA
AAACCTCACCTCGGTGAGTC
Microsatellite
Cott686
TGGGAGTGCGATCGGGTTGG
GCGGGCCTTGACACTGTTGG
Microsatellite
CottE23
TGAGCAGCTTTACAGTGTTGG
TCGTGCTCTTCTGGCATCTG
Microsatellite
EDA1
GGACAGCACACTCCTTCCAT
AGAGGCCCTTAAAAGGCAGA
PCR protocols used in this study:
For obtaining mitochondrial DNA sequences, we applied the following primer
combinations: CotL1 and HN20 (partial t-RNA, D-loop, Šlechtová et al., 2004),
L14724 (Meyer et al., 1990) and H15149 (Kocher et al., 1989, cytochrome b),
FishF1 and FishR1 (Cox1; Ward et al., 2005). Amplification of D-loop sequences
(CotL1/HN20, 583 bp) was performed in 30-L volumes containing the
following final reaction concentrations: 1× PCR-Buffer, 2mM MgCl2, 0.2 mM of
each dNTP, 10 pmol of each primer, 0.625 units of TAQ-polymerase, and ~200 ng
genomic DNA. The PCR protocol consisted of 2 minutes at 95C, followed by five
cycles of [94C -45s, 48C -45s, 72C -90s], 29 cycles of [94C -45s, 52C -45s,
72C -90s], and 5 minutes at 72C final elongation. For Cox1 sequences
(FishF1/R1, 583 bp), PCR reactions were performed in 25-L volumes
containing 1× PCR-Buffer, 2mM MgCl2, 0.2 mM of each dNTP, 10 pmol of each
primer, 0.625 units of TAQ-polymerase, and ~200 ng genomic DNA. Cycling
conditions consisted of 95C for 2 minutes, followed by 35 cycles of [94C -30s,
52C -40s, 72C -60s], and a final elongation of 72C for 10 minutes. A
cytochrome b fragment (419 bp) was amplified with the L14724/H15149 primer
pair in 30-l volumes containing 1× PCR-Buffer, 2mM MgCl2, 0.2 mM of each
dNTP, 10 pmol of each primer, 0.625 units of Taq-polymerase, and c. 200 ng
genomic DNA. Cycling conditions consisted of 95C for 2 minutes, followed by 35
cycles of [95C -50s, 50C -15s, 72C -80s], and a final elongation of 72C for 7
minutes.
Microsatellites were multiplexed with up to six fluorescently-labelled primers
using QIAGEN Multiplex Kits (Qiagen, Inc.), and amplified in 10 L-reactions (5
L Qiagen-Multiplex-Kit, 0.3-0.6 M primer, 40 ng DNA-template). PCR-protocols
consisted of an initial denaturation step of 95C for 15 minutes, followed by 38
cycles of [94C -30s, 60C -90s, 72C -60s], and a final elongation of 72C for 30
minutes.
REFERENCES
Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Pääbo, S., Villablanca, F.X. &
Wilson, A.C. (1998) Dynamics of mitochondrial DNA evolution in animals:
amplification and sequencing with conserved primers. Proceedings of the
National Academy of Sciences USA, 86, 6196-6200.
Meyer, A., Kocher, T.D., Basasibwaki, P. & Wilson, A.C. (1990) Monophyletic
origin of Lake Victoria cichlid fishes suggested by mitochondrial DNA
sequences. Nature, 347, 550-553.
Šlechtová, V., Bohlen, J., Freyhof, J., Persat, H. & Delmastro, G.B. (2004) The Alps
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Ward, R.D., Zemlak, T.S., Innes, B.H., Last, P.R. & Hebert, P.D.N. (2005) DNA
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Appendix S2 Prickling and body shape differences between inland and coastal
sculpins.
Figure S2 A) Landmark configuration used for Cottus asper (1 = tip of nasal, 2 =
interorbital pore, 3 = end of head, 4 = first dorsal fin origin, 5 = second dorsal fin
origin, 6 = end of second dorsal fin, 7 = upper caudal fin origin, 8 = midpoint
caudal, 9 = lower caudal fin origin, 10 = anal fin end, 11 = anal fin origin, 12 =
lower pectoral fin origin, 13 = upper pectoral fin origin, 14 = upper origin of gill
opening, 15 = opercular spine, 16 = 3rd pore of the preopercular-mandibular
canal, 17 = posterior end of maxilla, 18 = posterior end of eye, 19 = midpoint of
eye, 20 = distal end of eye, 21 = nasal nostril, 22 = midpoint between 7 and 9, 23
= mid of caudal peduncle). B) Wireframe graph showing shape differences
between coastal (black line) and inland (grey line) groups (scale factor × 6). C)
Frequencies (circle sizes represent percentages) of prickling categories (high,
medium, low) in coastal and inland populations. D) PCA plot visualizing shape
variation among coastal (black) and inland (grey) individuals along the first two
principal components (with 95% confidence ellipses).
Appendix S3 Fifty per cent majority-rule consensus tree of mtDNA sequences.
Figure S3 Fifty per cent majority-rule consensus tree of mtDNA sequences of
Cottus asper. Numbers above branches indicate Bayesian inference posterior
probabilities, and numbers below branches maximum likelihood bootstrap
proportions. Genetic lineages inferred from tree clusters are indicated by vertical
bars (black = A, grey = B, white = C).