Supplementary material A. Dna sequencing and phylogenetic methods




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Nature manuscript 2004-11-26197. Min et al. Discovery of the first Asian plethodontid salamander

Supplementary material


Supplementary material A. DNA sequencing and phylogenetic methods
DNA was extracted from fresh frozen tissues (Karsenia koreana, MVZ 246033 and MVZ 246034; Hydromantes brunus MVZ 247158) using a method modified from Brufford et al. (1992)1. A 1,503 base pair portion of the recombination activating gene-1 (Rag-1) was amplified in several overlapping fragments using standard protocols. PCR products were run out on 1.5 % agarose gels. Successful amplifications were either directly cut from the gel and extracted using Qiagen Gel Extraction kit, or cleaned with a Millipore PCR96 cleanup kit (MontágeTM). Big Dye (ABI) was used for cycle sequencing reactions sequenced on an ABI 3730 capillary sequencer. Individual sequences were edited and multiple sequence alignments were made in Sequencher TM vers 3.1 (Gene Codes Corp). The two K. koreana had identical Rag-1 sequences, so we only included one in the phylogenetic analyses. We compared our new sequences of K. koreana and H. brunus with recently published Rag-1 sequences for salamanders2,3(Appendix 1). The sequences that we chose represent all extant families of salamanders and the majority of genera of plethodontids. Modeltest vers 3.64 chose GTR + I +  as the most appropriate model of sequence evolution4. Bayesian analyses implemented via the program MRBAYES vers 35, applying a six parameter model plus invgamma, was run with four chains for 5.2 million generations (with the first 200,000 discarded as burn-in). The resulting 50% majority-rule consensus of the 50,000 post burn-in trees was computed in PAUP* to determine the topology and posterior probabilities for each node6. To determine the amount of evolutionary distance among our taxa, the sumt command in MR BAYES was used to calculate the average branch length across all post burn-in trees. Pseudobranchus (Sirenidae) was selected as the outgroup2. PAUP* was used to generate a matrix of Maximum Likelihood distances based on parameters (Base frequencies: A = 0.2797, C = 0.2477, G = 0.2446, T = 0.2280; Substitution model: [A-C] = 1.4290, [A-G] = 3.4255, [A-T] = 1.0241, [C-G] = 0.6478, [C-T] = 5.4303, [G-T] = 1.0000; I = 0.4544;  = 1.4226) chosen by Modeltest4. Maximum Likelihood bootstrapping was performed in PAUP* and run for 600 pseudoreplicates. The most likely tree was identical to the Bayesian tree.

References
1. Brufford, M. W., Hanotte, O., Brookfield, J. F. Y. & Burke. T. Single-locus and multilocus DNA fingerprint. In The South American Herpetofauna: Its Origin, Evolution, and Dispersal. Molecular Genetic Analysis in Conservation (ed Hoelzel AR), pp. 225-270. IRL Press, Oxford (1992):
2. Chippindale, P. T., Bonett, R. M., Baldwin, A. S. & Wiens, J. J. Phylogenetic evidence for a major reversal of life history evolution in plethodontid salamanders. Evol. 58, 2809-2822 (2004).
3. Wiens, J. J., Bonett, R. M. & Chippindale, P. T. Ontogeny discombobulates phylogeny: paedomorphosis and higher-level salamander relationships. Syst. Biol. In Press (2005).
4. Posada, D. & Crandall, K.A. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817-818 (1998).
5. Huelsenbeck, J.P. & Ronquist, F.R. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754-755 (2001).
6. Swofford, D.L. PAUP*: Phylogenetic analysis using parsimony (and other methods), v. 4.0. b10. (Sinauer Associates, Sunderland, MA, 2001).
Supplementary material B Appendix 1. Specimens used for molecular analyses.

Family

Genus

Species

Genbank

accession number



Amphiumidae

Amphiuma

pholeter

AY650128

Ambystomatidae

Ambystoma

opacum

AY650130

Cryptobranchidae

Cryptobranchus

alleghaniensis

AY650141

Dicamptodontidae

Dicamptodon

tenebrosus

AY650132

Hynobiidae

Hynobius

nebulosus

AY650144

Plethodontidae

Aneides

aeneus

AY691701

Plethodontidae

Aneides

lugubris

AY650118

Plethodontidae

Bolitoglossa

helmrichi

AY650124

Plethodontidae

Desmognathus

aeneus

AY691701

Plethodontidae

Desmognathus

monticola

AY691698

Plethodontidae

Desmognathus

quadramaculatus

AY650117

Plethodontidae

Desmognathus

wrighti

AY691699

Plethodontidae

Ensatina

eschscholtzii

AY691702

Plethodontidae

Eurycea

multiplicata

AY691707

Plethodontidae

Gyrinophilus

porphyriticus

AY691710

Plethodontidae

Hemidactylium

scutatum

AY691711

Plethodontidae

Hydromantes

brunus

AY887134

Plethodontidae

Karsenia

koreana

AY887135

Plethodontidae

Nyctanolis

pernix

AY691714

Plethodontidae

Phaeognathus

hubricti

AY691700

Plethodontidae

Plethodon

cinereus

AY691703

Plethodontidae

Plethodon

elongatus

AY650120

Plethodontidae

Plethodon

vandykei

AY691715

Plethodontidae

Plethodon

vehiculum

AY691716

Plethodontidae

Plethodon

welleri

AY691717

Plethodontidae

Plethodon

yonahlossee

AY691718

Plethodontidae

Pseudoeurycea

rex

AY650125

Plethodontidae

Pseudotriton

ruber

AY650123

Plethodontidae

Stereochilus

marginatus

AY691713

Proteidae

Necturus

alabamensis

AY650136

Rhyacotritonidae

Rhyacotriton

kezeri

AY650129

Salamandridae

Notophthalmus

viridescens

AY650134

Sirenidae

Pseudobranchus

sp.

AY650139


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