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Supplementary Information
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Methods
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Phylogenetic analysis for Australo-Papuan Hylidae
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DNA was extracted from tissues with a standard phenol chloroform method. Details of the
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specimens sequenced are available from SCD [donnellan.steve@saugov.sa.gov.au]. The
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South American phyllomedusines A. saltator, A. litodryas, Ph. palliata Ph. tomopterna and
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Pachymedusa were used as outgroups. An approximately 800 bp portion of the 12S rRNA
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gene was amplified in two overlapping segments with one segment amplified with H1478 and
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L1091, (Kocher et al. 1989) and the other amplified with either L669 (Donnellan et al. 1999)
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or L675 (5'-TTG GTC CTR RCC TTG AAA TC-3') with H1160 (Donnellan et al. 1999). A
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portion of the 16S rRNA gene was amplified and sequenced with the primers 16sar and 16sbr
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(Cunningham et al. 1992). DNA was amplified by PCR (one cycle 94oC 3 min, 55oC 45 s,
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72oC 1min; 29 cycles 94oC 45 s, 55oC 45 s, 72oC 1 min). PCR products were purified for
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sequencing using a Bresa-Clean DNA Purification Kit (Bresatec). Each sample had both
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strands cycle-sequenced directly from the PCR product using the original PCR primers using
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the PRISM Ready Reaction DyeDeoxy Terminator Cycle sequencing kit (Applied
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Biosystems). Sequence product was electrophoresed and viewed on an Applied Biosystems
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Model 373A Sequencing System.
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GenBank accession numbers for all Hylidae sequences included in the present study are:
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FJ965851 - FJ965949; FJ945355 - FJ945451. Sequence alignments were made by eye using
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the conserved motif (Hickson et al. 1996) and secondary structure (Kjer 1995; Kjer 1997)
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approaches to align stems and loops according to the latest secondary structure models for
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RNA secondary structure. Regions of doubtful homology were discarded.
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We conducted Bayesian inference maximum likelihood (BI) analyses using MrBayes version
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3.0 (Ronquist & Huelsenbeck 2003). Modeltest version 3.0 (Posada and Crandall 1998) was
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used to assess the most suitable model of nucleotide substitution for the data by the AIC. The
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model parameters were not specified a priori and were treated as unknown variables with
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uniform priors. Bayesian analyses were run for 5,000,000 generations, saving trees from
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every 100 generations. Four simultaneous Markov Chain Monte Carlo (MCMC) chains were
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run with the temperature of the heated chains set at the default of 0.2. The likelihoods of trees
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were inspected to determine whether the Markov chains had reached stationarity, ie relatively
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stable likelihood scores over time. Sample points generated before stationarity was reached
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were discarded as “burn-in” samples and were not considered in calculation of a posteriori
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node probabilities or parameter estimates. To ensure that Bayesian analyses were not trapped
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in local optima, analyses were performed four times with each analysis starting from a
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random tree. Apparent stationarity levels were compared for convergence which was
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considered to have occurred when likelihood values from independent Bayesian analyses had
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similar mean values. In addition, the posterior probabilities of nodes from independent
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analyses were compared for convergence. After verifying convergence and discarding burn-
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in samples, the remaining samples were pooled for summary analysis. The percentage of
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samples recovering a particular clade, determined from 50% majority rule consensus trees,
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represents the posterior probability of the clade. Because the posterior probabilities represent
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true P values, clades with P values >95% were considered significantly supported.
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Phylogenetic analysis for Daviesia
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Total genomic DNA was extracted from fresh, NaCl-CTAB-stored, or dried specimens using
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CTAB/chloroform extraction. The ITS region (nuclear ribosomal Internal Transcribed
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Spacer) was amplified using primers P1L and P2R (Crisp et al. 1999) under standard PCR
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conditions with an annealing temperature of 55˚C. PCR fragments were purified using
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ammonium acetate precipitation and sequenced using the same primers on an ABI Prism 3100
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genetic analyser using Big-Dye chemistry. The resultant fully overlapping contigs in both the
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forward and reverse directions were edited using Sequencher 4.5 (GeneCodes Corporation)
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and manually aligned using Se-Al (Rambaut 1996).
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DNA sequence data were analysed under a Bayesian framework using MrBayes version 3.1.2
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(Ronquist & Huelsenbeck 2003) allowing a GTR+I+G model. The Bayesian phylogeny
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estimation used default priors and was run for two million generations (determined to be
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sufficient for convergence by comparing log likelihoods and other statistics recommended by
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Ronquist & Huelsenbeck [2003]), with trees sampled every 1000 generations. Trees sampled
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during the burn-in period (500 samples) were discarded prior to constructing the posterior
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distribution consensus tree (with mean posterior probability branch lengths), which was used
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for subsequent analyses.
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References
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Crisp MD, Gilmore SR & Weston PH (1999) Phylogenetic relationships of two anomalous
species of Pultenaea (Fabaceae: Mirbelieae), and description of a new genus. Taxon
48, 701-704.
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Cunningham CW, Blackstone NW, Buss LW (1992) Evolution of king crabs from hermit crab
ancestors. Nature 355, 539-542.
68
69
Donnellan SC, Hutchinson MN, Saint KM (1999) Molecular evidence for the phylogeny of
Australian gekkonoid lizards. Biological Journal of the Linnean Society 67, 97-118.
70
71
Frost DR, Grant T, Faivovich J, et al. (2006) The amphibian tree of life. Bulletin of the
American Museum of Natural History, 1-370.
72
73
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Hickson RE, Simon C, Cooper AJ, Spicer G, Sullivan J, Penny D (1996) Conserved motifs,
alignment and secondary structure for the third domain of animal 12S rRNA.
Molecular Biology and Evolution 13, 150-169.
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76
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Kjer K (1995) Use of rRNA secondary structure in phylogenetic studies to identify
homologous positions: an example of alignment and data presentation from the frogs.
Molecular Phylogenetics and Evolution 4, 314-330.
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Kjer K (1997) Conserved primary and secondary structural motifs of amphibian 12S rRNA,
domain III. Journal of Herpetology 31, 599-604.
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81
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Kocher TD, Thomas WK, Meyer A, et al. (1989) Dynamics of mitochondrial DNA evolution
in animals: amplification and sequencing with conserved primers. Proceedings of the
National Academy of Sciences, USA 86, 6196-6200.
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Posada D, Crandell KA (1998) Modeltest: testing the model of DNA substitution.
Bioinformatics 14, 817-818.
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Rambaut A (1996) Se-Al: Sequence Alignment Editor. Available at
http://evolve.zoo.ox.ac.uk/, University of Oxford, Department of Zoology.
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Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed
models. Bioinformatics 19, 1572-1574.
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Supplementary Fig. 1. Bayesian inference majority rule consensus tree of phylogenetic
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relationships among Australo-papuan hylid mtDNA nucleotide sequences. Light grey
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branches had a posterior probability > 0.95. The scale bar indicates percent of substitutions
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per site. The tree was rooted with a selection of South American phyllomedusine taxa, based
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on a sister relationship with the Australo-papuan hylids (Frost et al. 2006). The species
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included in our analysis that were formerly placed in Nyctimystes are now placed in Litoria
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following the nomenclatural revision of Frost et al. (2006). This includes the northern
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Australian species "dayi" and a further 12 species from New Guinea, which form a single
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clade that is the sister to L. brevipalmata.
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For the purpose of the phylogenetic endemism analysis, four Litoria species which were not
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available for the phylogeny were included in the phylogeny based on a conservative expert
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estimate (SD) of their phylogenetic position. Those species were Litoria castanea, L.
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cooloolensis, L. lorica and L. staccato.
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