Journal of Biogeography
SUPPORTING INFORMATION
Evolution of codfishes (Teleostei: Gadinae) in geographical and ecological space:
evidence that physiological limits drove diversification of subarctic fishes
Hannah L. Owens
APPENDIX S1 Detailed phylogenetic methods.
Gene sequencing
PCR reactions for 12S, CYTB and ZIC1 consisted of 1µL extract, 1.5 µL TAQ buffer, 0.125 µL
TAQ, 0.75 µL dNTP and MgCl2, 0.25µL forward and reverse primers, and dH2O, to a final
volume of 13 µL. For RAG1, reactions consisted of 5 µL extract, 1.25 µL forward and reverse
primers, one PuReTaq Ready-To-Go™ PCR Bead (GE Healthcare Bio-Sciences, Pittsburgh, PA,
USA), and 17.5 µL dH2O, for a final volume of 25 µL. RAG1 was amplified using a custom PCR
profile, beginning with an initial cycle of 180 s at 94 °C, followed by 10 cycles of 45 s at 94 °C,
45 s at 52 °C, and 75 s at 72 °C, followed by 30 cycles of 45 s at 94 °C, 45 s at 47 °C, and 75 s at
72 °C, with a final extension of 7 min at 72 °C. PCR bands were visualized on 1% low meltingpoint agar gels, and PCR products were purified with QIAquick PCR purification kits (Qiagen,
Valencia, CA, USA). DNA sequencing was done using an ABI BigDye Terminator ver. 3.1
cycle sequencing kit (Perkin Elmer, Waltham, MA, USA) for dye-terminator chemistry
following manufacturer’s instructions. Cycle-sequencing reactions were precipitated in 75%
solution of ethanol following ABI protocol. Dried cycle-sequencing reactions were resuspended
and electrophoresed on an AB 3730 Genetic Analyzer (Applied Biosystems, Grand Island, NY,
USA). Both strands were sequenced to verify accuracy of sequences.
Phylogenetic inference
The Akaike information criterion (AIC) as implemented in JMODELTEST 2.1 (Posada & Crandall,
1998) was used to determine appropriate data partitioning and model selection for phylogenetic
inference. Nine data partitions were tested, and those with the simplest model of evolution were
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used to conduct several preliminary BEAST analyses (Appendix S1: Table S2). Analyses failed to
converge after 5 million generations due to CG and GT transversions in 12S and AT
transversions in RAG1 position 3 as indicated by TRACER 1.5 (http://beast.bio.ed.ac.uk/tracer).
As such, simpler models were used in final BEAST analysis. The standard deviation of the RAG1
uncorrelated lognormal clock model in preliminary BEAST analysis included 0, so a strict clock
model was applied in the final analyses of that gene.
For final BEAST analyses, two independent runs of 106 generations were conducted, and
the resulting tree and log files for each run were combined using LOGCOMBINER 1.6.1
(http://beast.bio.ed.ac.uk/logcombiner). Convergence of model parameter values was assessed by
examination of convergence and likelihood stationarity in TRACER 1.5 from combined posterior
samples to ensure adequate mixing of the Markov chain Monte Carlo (MCMC). A cutoff of 20%
was used for the burn-in. The posterior probability density of the combined tree and log files was
summarized as a maximum clade credibility tree using TREEANNOTATOR 1.6.1
(http://beast.bio.ed.ac.uk/TreeAnnotator).
REFERENCE
Posada, D. & Crandall, K.A. (1998) MODELTEST: testing the model of DNA substitution.
Bioinformatics, 14, 817-818.
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