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Electronic Supplementary Material
Polymerase chain reactions (PCR)
All PCR reactions contained 1 l of template DNA (~150 ng), 3 l of reaction buffer
(Promega), 3.6 l of 25 mM MgCl2 (i.e. 3 mM MgCl2), 6 l of dNTP mixture
containing 125 mM of each dNTP, 1.2 l of each primer (5 mM dilutions), 1 unit of Taq
DNA polymerase (Promega) and water to a final volume of 30 l. PCR profiles
consisted of an initial denaturing step (94oC for 3 min), 35 cycles of denaturing (94oC
for 30 s), annealing for 45 s at a primer-specific annealing temperature (Table 1) and
extension (72oC for 45 s), and a final extension step (72oC for 10 min). PCR products
were purified using the UltraCleanTM15 DNA Purification Kit (MO BIO Laboratories,
Inc., Carlsbad, CA, USA), sequenced in both directions using Big Dye terminator
version 3.1 (Applied Biosystems) and run on a 3130xl Genetic Analyser (Applied
Biosystems) according to the manufacturer’s instructions.
Sequence alignment
The nuclear genes could readily be aligned by eye, but as the alignment of the S7 intron
was challenging, we used the program BALI-PHY 2.0.2 (Suchard & Redelings 2006) (a
program that simultaneously estimates phylogeny and alignment) to generate a
consensus-75 alignments. We specified 10 000 iterations (with a burn-in of 10%) and
applied the GTR+I+G model (Rodríguez et al. 1990). The process was repeated three
times to check for consistency of results, and poorly aligned sites were then removed
using the GBLOCKS (Talavera & Castresana) server available at
http://molevol.cmima.csic.es/castresana/Gblocks_server.html (the most stringent
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conditions were specified). The DNA sequence data-set used in all analyses included
1476 bp of RAG1, 711 bp of myh6, 447 bp of Rhodopsin, 459 bp of Tmo4c4 and 293
bp of S7 (Table 2).
Molecular dating
Molecular dating was performed using the program BEAST 1.4.8 (Drummond & Rambaut
2007). An uncorrelated lognormal relaxed clock was specified. Sequences were partitioned as
described for the phylogenetic analyses, and all nodes that were strongly supported in the
phylogenetic analyses (bootstrap values > 75% and posterior probabilites >95%) were
constrained to be monophyletic. A Yule process was used as a tree prior. A chain length of 20
million generations was specified, and parameters were saved every 200 generations. Results
were checked in TRACER (available at beast.bio.ed.ac.uk/Tracer) to ensure that effective
samples sizes for all parameters were above 200 and that posterior probabilities remained
constant after a burn-in of 10%. To check for consistency of results, the analyses were repeated
three times for each of two methods of calibrating the tree. A chronogram for one of the
methods (using three calibration points) was then produced using the programs
TREEANNOTATOR 1.4.6 (available at http://beast.bio.ed.ac.uk/TreeAnnotator) and FIGTREE 1.1
(available at http://beast.bio.ed.ac.uk/FigTree)
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Table 1. Primers used to amplify five nuclear loci in seahorses and other syngnathids.
Annealing
Locus
Primer Name
Primer Sequence
Reference
Temp. (oC)
RAG1
myh6
Rhodopsin
Tmo4c4
S7
a
RAG1-2510Fa
TGGCCATCCGGGTMAACAC
RAG1-4090Ra
CTGAGTCCTTGTGAGCTTCCATRAAYTT
RAG1-2533Fb
CTGAGCTGCAGTCAGTACCATAAGATGT
RAG1-4078Rb
TGAGCCTCCATGAACTTCTGAAGRTAYTT
myh6_F459a
CATMTTYTCCATCTCAGATAATGC
myh6_R1325a
ATTCTCACCACCATCCAGTTGAA
myh6_F507b
GGAGAATCARTCKGTGCTCATCA
myh6_R1322b
CTCACCACCATCCAGTTGAACAT
Rod-F2Ba
GTCTGCAAGCCCATCAGCAACTTCCG
Rod-5Ra
GGTGGTGATCATGCAGTGGCGGAA
Rod-F2Wb
AGCAACTTCCGCTTCGGTGAGAA
Rod-R4nb
GGAACTGCTTGTTCATGCAGATGTAGAT
Tmo4c4F
CCTCCGGCCTTCCTAAAACCTCTC
Tmo4c4R
CATCGTGCTCCTGGGTGACAAAGT
S7RPEX1F
TGGCCTCTTCCTTGGCCGTC
S7RPEX2R
AACTCGTCTGGCTTTTCGCC
55
Li & Ortí 2007
Lopez et al. 2004
55
Lopez et al. 2004
Lopez et al. 2004
50
Li et al. 2007
Li et al. 2007
60
Li et al. 2007
Li et al. 2007
62
Sevilla et al. 2007
Sevilla et al. 2007
56
Sevilla et al. 2007
Sevilla et al. 2007
55
Streelman & Karl
1997, Streelman et al.
1998
55
Chow & Hazama 1998
Primers for first round PCR; bPrimers for second round PCR using 1 in 100 dilutions of first round PCR products as template
Chow & Hazama 1998
4
Table 2. Syngnathid specimens used for the phylogeny, and their museum collection numbers and
GenBank accession numbers.
GenBank Accession Numbers
Species
Collection No.
RAG1
myh6
Rhodopsin
Tmo4c4
S7
Hippocampus reidi
RM2170
FJ905768
FJ905779
FJ905790
FJ905801
DQ288386
H. ingens
RM2530
FJ905769
FJ905780
FJ905791
FJ905802
DQ288382
H. zosterae
RM2310
FJ905770
FJ905781
-
FJ905803
DQ288379
H. breviceps
(own collection)
FJ905771
-
FJ905792
FJ905804
AY277320
H. bargibanti
(own collection)
FJ905772
FJ905782
FJ905793
-
AY277309
Idiotropiscis lumnitzeri
(own collection)
FJ905773
FJ905783
FJ905794
FJ905805
FJ905808
Hippichthys penicillus
I.41864-016
FJ905774
FJ905784
FJ905795
FJ905806
FJ905809
Vanacampus margaritifer
I.41084-042
FJ905777
FJ905787
FJ905798
-
FJ905811
Corythoichthys sp.
SAIAB 80704
-
FJ905788
FJ905799
FJ905807
-
Stigmatopora nigra
I.43656-001
FJ905776
FJ905786
FJ905797
-
FJ905810
Syngnathus temminckii
(own collection)
FJ905775
FJ905785
FJ905796
-
AY277308
Doryrhamphus excisus
SAIAB 78058
FJ905778
FJ905789
FJ905800
-
FJ905812
RM = Redpath Museum, Montreal; I = Australian Museum, Sydney; SAIAB = South African Institute of Aquatic Biodiversity,
Grahamstown, South Africa; RAG1 = recombination activating gene 1, myh6 = myosin heavy chain 6, S7 = first intron of the
S7 ribosomal protein
5
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