1 Supplementary Information 2 Methods 3 Phylogenetic analysis for Australo-Papuan Hylidae 4 DNA was extracted from tissues with a standard phenol chloroform method. Details of the 5 specimens sequenced are available from SCD [donnellan.steve@saugov.sa.gov.au]. The 6 South American phyllomedusines A. saltator, A. litodryas, Ph. palliata Ph. tomopterna and 7 Pachymedusa were used as outgroups. An approximately 800 bp portion of the 12S rRNA 8 gene was amplified in two overlapping segments with one segment amplified with H1478 and 9 L1091, (Kocher et al. 1989) and the other amplified with either L669 (Donnellan et al. 1999) 10 or L675 (5'-TTG GTC CTR RCC TTG AAA TC-3') with H1160 (Donnellan et al. 1999). A 11 portion of the 16S rRNA gene was amplified and sequenced with the primers 16sar and 16sbr 12 (Cunningham et al. 1992). DNA was amplified by PCR (one cycle 94oC 3 min, 55oC 45 s, 13 72oC 1min; 29 cycles 94oC 45 s, 55oC 45 s, 72oC 1 min). PCR products were purified for 14 sequencing using a Bresa-Clean DNA Purification Kit (Bresatec). Each sample had both 15 strands cycle-sequenced directly from the PCR product using the original PCR primers using 16 the PRISM Ready Reaction DyeDeoxy Terminator Cycle sequencing kit (Applied 17 Biosystems). Sequence product was electrophoresed and viewed on an Applied Biosystems 18 Model 373A Sequencing System. 19 GenBank accession numbers for all Hylidae sequences included in the present study are: 20 FJ965851 - FJ965949; FJ945355 - FJ945451. Sequence alignments were made by eye using 21 the conserved motif (Hickson et al. 1996) and secondary structure (Kjer 1995; Kjer 1997) 22 approaches to align stems and loops according to the latest secondary structure models for 23 RNA secondary structure. Regions of doubtful homology were discarded. 24 We conducted Bayesian inference maximum likelihood (BI) analyses using MrBayes version 25 3.0 (Ronquist & Huelsenbeck 2003). Modeltest version 3.0 (Posada and Crandall 1998) was 26 used to assess the most suitable model of nucleotide substitution for the data by the AIC. The 27 model parameters were not specified a priori and were treated as unknown variables with 28 uniform priors. Bayesian analyses were run for 5,000,000 generations, saving trees from 29 every 100 generations. Four simultaneous Markov Chain Monte Carlo (MCMC) chains were 30 run with the temperature of the heated chains set at the default of 0.2. The likelihoods of trees 31 were inspected to determine whether the Markov chains had reached stationarity, ie relatively 32 stable likelihood scores over time. Sample points generated before stationarity was reached 33 were discarded as “burn-in” samples and were not considered in calculation of a posteriori 34 node probabilities or parameter estimates. To ensure that Bayesian analyses were not trapped 35 in local optima, analyses were performed four times with each analysis starting from a 36 random tree. Apparent stationarity levels were compared for convergence which was 37 considered to have occurred when likelihood values from independent Bayesian analyses had 38 similar mean values. In addition, the posterior probabilities of nodes from independent 39 analyses were compared for convergence. After verifying convergence and discarding burn- 40 in samples, the remaining samples were pooled for summary analysis. The percentage of 41 samples recovering a particular clade, determined from 50% majority rule consensus trees, 42 represents the posterior probability of the clade. Because the posterior probabilities represent 43 true P values, clades with P values >95% were considered significantly supported. 44 45 Phylogenetic analysis for Daviesia 46 Total genomic DNA was extracted from fresh, NaCl-CTAB-stored, or dried specimens using 47 CTAB/chloroform extraction. The ITS region (nuclear ribosomal Internal Transcribed 48 Spacer) was amplified using primers P1L and P2R (Crisp et al. 1999) under standard PCR 49 conditions with an annealing temperature of 55˚C. PCR fragments were purified using 50 ammonium acetate precipitation and sequenced using the same primers on an ABI Prism 3100 51 genetic analyser using Big-Dye chemistry. The resultant fully overlapping contigs in both the 52 forward and reverse directions were edited using Sequencher 4.5 (GeneCodes Corporation) 53 and manually aligned using Se-Al (Rambaut 1996). 54 DNA sequence data were analysed under a Bayesian framework using MrBayes version 3.1.2 55 (Ronquist & Huelsenbeck 2003) allowing a GTR+I+G model. The Bayesian phylogeny 56 estimation used default priors and was run for two million generations (determined to be 57 sufficient for convergence by comparing log likelihoods and other statistics recommended by 58 Ronquist & Huelsenbeck [2003]), with trees sampled every 1000 generations. Trees sampled 59 during the burn-in period (500 samples) were discarded prior to constructing the posterior 2 60 distribution consensus tree (with mean posterior probability branch lengths), which was used 61 for subsequent analyses. 62 References 63 64 65 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. 66 67 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 74 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. 75 76 77 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. 78 79 Kjer K (1997) Conserved primary and secondary structural motifs of amphibian 12S rRNA, domain III. Journal of Herpetology 31, 599-604. 80 81 82 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. 83 84 Posada D, Crandell KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14, 817-818. 85 86 Rambaut A (1996) Se-Al: Sequence Alignment Editor. Available at http://evolve.zoo.ox.ac.uk/, University of Oxford, Department of Zoology. 87 88 89 Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572-1574. 3 90 4 91 Supplementary Fig. 1. Bayesian inference majority rule consensus tree of phylogenetic 92 relationships among Australo-papuan hylid mtDNA nucleotide sequences. Light grey 93 branches had a posterior probability > 0.95. The scale bar indicates percent of substitutions 94 per site. The tree was rooted with a selection of South American phyllomedusine taxa, based 95 on a sister relationship with the Australo-papuan hylids (Frost et al. 2006). The species 96 included in our analysis that were formerly placed in Nyctimystes are now placed in Litoria 97 following the nomenclatural revision of Frost et al. (2006). This includes the northern 98 Australian species "dayi" and a further 12 species from New Guinea, which form a single 99 clade that is the sister to L. brevipalmata. 100 For the purpose of the phylogenetic endemism analysis, four Litoria species which were not 101 available for the phylogeny were included in the phylogeny based on a conservative expert 102 estimate (SD) of their phylogenetic position. Those species were Litoria castanea, L. 103 cooloolensis, L. lorica and L. staccato. 104 5