Tissue samples, voucher specimens and sequence accession numbers

Transcription

1 Electronic Supplementary Material S1 Internal sequencing primers for partial ND4+tRNA His,Ser,Leu sequence Three internal oligonucleotide primers were designed to sequence overlapping portions of a PCR product (amplification primers: ND4 and Leu; Forstner et al. 1995) comprising a region of the mitochondrial genome that includes the 3'-end of the NADH dehydrogenase subunit 4 (ND4) gene, and the histidine, serine, and part of the leucine trna genes. This region spans 697 bp (ND4) and bp (trnas) in most species of Pseudechis and Bungarus (blacksnakes and kraits, respectively, Squamata: Elapidae). Cycle sequencing using these primers (Qiagen Operon, salt-free quality) and ABI BigDye chemistries v3.0 and v1.1 (Applied Biosystems, Foster City, CA, U.S.A.) was performed with the following parameters: cycles of 96 C, 1 s; 50 C, 5 s; 55 C, 4 min; and final ramping to 4 C. After ddntp and primer removal using Qiagen Dye-Ex v.2.0 spin columns according to the manufacturer's protocol, the products of the sequencing reaction were separated using an ABI PRISM Model 3100 automated sequencer (Applied Biosystems). References Forstner MRJ, Davis SK, Arévalo E (1995) Support for the hypothesis of anguimorph ancestry for the suborder Serpentes from phylogenetic analysis of mitochondrial DNA sequences. Mol Phyl Evol 4: Kumazawa Y, Ota H, Nishida M, Ozawa T (1998) The complete nucleotide sequence of a snake (Dinodon semicarinatus) mitochondrial genome with two identical control regions. Genetics 150: Electronic Supplementary Material S2 Tissue samples, voucher specimens and sequence accession numbers List of Pseudechis tissue samples, clade assignment (see Fig. 2 and article text), voucher specimens, collecting localities, collector or source, mitochondrial DNA sequences used, DDBJ/EMBL/GenBank Nucleotide Sequence Database accession numbers. Abbreviations

2 and museum acronyms: ARP, Australian Reptile Park (Gosford, NSW, Australia); CM, Christian Melaun private ; Hwy, Highway; NSW, New South Wales; NT, Northern Territory; Outgr., outgroup taxon; Qld, Queensland; ROM, Royal Ontario Museum (Toronto, Canada); SA, South Australia; SAM, South Australian Museum (Adelaide, SA, Australia); SMF, Forschungsinstitut und Naturmuseum Senckenberg (Frankfurt am Main, Germany); WAM, Western Australian Museum (Perth, WA, Australia). Electronic Supplementary Material S3 Electronic Supplementary Material S4 S4a. King brown snake (Pseudechis 'australis' complex, clade II species) from Darwin, Northern Territory, Australia ("northern" form). Photograph by John Weigel. S4b-c. King brown snakes (Pseudechis 'australis' complex, clade II species). Above: from 60 km ESE Port Hedland, northwestern Western Australia. Below: from Kununurra, northeastern Western Australia. Photographs by Brad Maryan. S4d-e. King brown snake (Pseudechis 'australis' complex, clade II species, "red" form). Above and below: from St. George, Queensland, Australia. Photographs by John Weigel. S4f. King brown snake (Pseudechis 'australis' complex, clade III species) from Bullo River Station, Northern Territory, Australia. Photograph by John Weigel. S4g. Yellow-bellied blacksnake (Pseudechis butleri), juvenile, from 23 km SW Yalgoo, Western Australia, Australia. Photograph by Brad Maryan.

3 Electronic Supplementary Material S1 Internal sequencing primers for partial ND4+tRNA His,Ser,Leu sequence Name Direction Length Sequence 5' to 3' T m Dinodon* C PaND4 H 24 GAAYTATAATRCGAGTTTGGGTGC IIIa PaND4 H 25 TAATAGGMGYAATAACAATAATAAT IV PaND4 H 24 TACAYATRTTYYTRTCAMCACAAA VI * Position in the mitochondrial genome of the 'colubrid' snake Dinodon semicarinatus (Kumazawa et al. 1998) that corresponds to the 3' nucleotide of the primer.

4 Electronic Supplementary Material S2 Tissue samples, voucher specimens and sequence accession numbers List of Pseudechis tissue samples, clade assignment (see Fig. 2 and article text), voucher specimens, collecting localities, collector or source, mitochondrial DNA sequences used, DDBJ/EMBL/GenBank Nucleotide Sequence Database accession numbers. Abbreviations and museum acronyms: ARP, Australian Reptile Park (Gosford, NSW, Australia); CM, Christian Melaun private ; Hwy, Highway; NSW, New South Wales; NT, Northern Territory; Outgr., outgroup taxon; Qld, Queensland; ROM, Royal Ontario Museum (Toronto, Canada); SA, South Australia; SAM, South Australian Museum (Adelaide, SA, Australia); SMF, Forschungsinstitut und Naturmuseum Senckenberg (Frankfurt am Main, Germany); WAM, Western Australian Museum (Perth, WA, Australia). Clade Species Sample Locality Collector/Source Voucher Cyt b ND4/tRNA Code I P. 'australis' P1 Merauke, Papua, Indonesia F. B. Yuwono CM live AJ AJ I P. 'australis' P2 Merauke, Papua, Indonesia F. B. Yuwono CM live AJ AJ I P. 'australis' P4 Merauke, Papua, Indonesia F. B. Yuwono CM live AJ AJ830258

5 II P. 'australis' JW 1 Cunnamulla, Qld, Australia John Weigel, ARP ARP live AJ AJ II P. 'australis' JW 2 Arnhem Hwy, ca. 100 km E Humpty John Weigel, ARP ARP live AJ AJ Doo, NT, Australia II P. 'australis' JW 3 Arnhem Hwy, ca. 100 km E Humpty John Weigel, ARP ARP live AJ AJ Doo, NT, Australia II P. 'australis' JW 4 Humpty Doo, NT, Australia John Weigel, ARP ARP live AJ AJ II P. 'australis' JW 5 Arnhem Hwy, ca. 100 km E Humpty John Weigel, ARP ARP live AJ AJ Doo, NT, Australia II P. 'australis' JW 6 Cunnamulla, Qld, Australia John Weigel, ARP ARP live - AJ II P. 'australis' JW 7 S Queensland, Australia John Weigel, ARP ARP live II P. 'australis' JW 10 Vicinity of Uluru, NT, Australia John Weigel, ARP ARP live AJ AJ AJ AJ830265

6 II P. 'australis' JW 11 Vicinity of Uluru, NT, Australia John Weigel, ARP ARP live AJ AJ II P. 'australis' JW 17 Western NSW /southwestern Qld, John Weigel, ARP Uncollected AJ AJ Australia II P. 'australis' ROM Road to Brewer Plain Traps, 6.6 km ROM ROM AJ AJ II P. 'australis' SAM R38475 II P. 'australis' SAM R31703 II P. 'australis' WAM II P. 'australis' WAM S of turnoff from airport, NT, Australia 3 km S NT-SA border, SA, Australia (26 01'S 'E) 11 km SSW Maralinga, SA, Australia (30 15'20''S '00''E) Woodstock, site #22, WA, Australia (21 35'15''S '04''E) Woodstock, site #10, WA, Australia (21 40'14''S '30''E) SAM SAM R38475 AJ SAM SAM R31703 AF AJ WAM WAM AJ AJ WAM WAM AJ AJ II P. 'australis' WAM Spalding Park, Geraldton, WA, WAM WAM AJ AJ830277

7 Australia (28 39'00''S '00''E) II P. 'australis' WAM Ilgarari Creek, WA, Australia WAM WAM AJ II P. 'australis' WAM III P. 'australis' WAM (24 23'00''S '00E) Peron Peninsula, WA, Australia (25 58'32''S '15''E) Walcott Inlet, WA, Australia (16 27'01''S '00''E) WAM WAM AJ AJ WAM WAM AJ AJ III P. 'australis' WAM Ca. 2 km upstream from Mitchell Falls, WA, Australia (14 51'00''S '00''E) John Weigel, WAM WAM AJ III P. 'australis' SMF Kimberley Coastal Camp, Port John Weigel, ARP SMF AJ Warrender, WA, Australia III P. 'australis' JW 8 Prince Frederick Harbour, ca. 40 km John Weigel, ARP ARP live AJ AJ SW of Mitchell Falls, WA, Australia IV P. 'australis' PP Near Mount Isa, Qld, Australia (ca. Roy Pails Roy Pails live AJ AJ 'S 'E)

8 IV P. 'australis' JW 9 Vicinity of Daly River township, NT, John Weigel, ARP No voucher AJ AJ Australia Outgr. P. butleri JW 12 Yalgoo, WA, Australia John Weigel No voucher AJ AJ Outgr. P. SAM 5 km E Tungkillo, Harrison Creek, SAM SAM R25056 AJ AJ porphyriacus R25056 SA, Australia

9 Electronic Supplementary Material S3 Proportional Pairwise Divergences (%) cytb + ND4 all positions ti + tv Kimura 2-Parameter Pairwise Divergences (%) S3a. Plot of uncorrected pairwise sequence divergence (p-distance) vs. Kimura 2-parameter corrected distances for transitions and transversions at all three codon positions of 1075 bp of combined cytb and ND4 ( bp, respectively). The plot represents all possible pairwise comparisons of Pseudechis australis complex members, and the outgroup taxon P. butleri. Cytb Proportional Pairwise Divergences (%) cytb vs. ND4 ND4 Proportional Pairwise Divergences (%) S3b. Plot of uncorrected pairwise sequence divergence (p-distance) of 378 bp from the 5 -end of cytb vs. 697 bp from the 3 -end of ND4. The plot represents all possible pairwise comparisons of Pseudechis australis complex members, and the outgroup taxa P. butleri and P. porphyriacus. The regression slope (1.08, bold line) suggests that, in blacksnakes, the two gene segments evolve at approximately the same rate.

10 Electronic Supplementary Material S3. S3c. Plots of uncorrected pairwise sequence divergence (p-distance) vs. Kimura 2-parameter corrected distances for transitions (ti) and transversions (tv) at first, second, and third codon positions. Each plot represents all possible pairwise comparisons of Pseudechis australis complex members, and the outgroup taxon P. butleri. (Top) Cytb (378 bp), lacking transversions at second codon positions. (Bottom) ND4 (697 bp). Deviation from the X = Y line in the plots is a measure of the degree of saturation for the indicated class of substitution. Proportional Pairwise Divergences (%) cytb 1 st position ti cytb 1 st position tv ND4 1 st position ti ND4 1 st position tv cytb 2 nd position ti ND4 2 nd position ti ND4 2 nd position tv cytb 3 rd position ti cytb 3 rd position tv ND4 3 rd position ti ND4 3 rd position tv Kimura 2-Parameter Pairwise Divergences (%)

11 Electronic Supplementary Material S4 S4a. King brown snake (Pseudechis 'australis' complex, clade II species) from Darwin, Northern Territory, Australia ("northern" form). Photograph by John Weigel.

12 S4b-c. King brown snakes (Pseudechis 'australis' complex, clade II species). Above: from 60 km ESE Port Hedland, northwestern Western Australia. Below: from Kununurra, northeastern Western Australia. Photographs by Brad Maryan.

13 S4d-e. King brown snake (Pseudechis 'australis' complex, clade II species, "red" form). Above and below: from St. George, Queensland, Australia. Photographs by John Weigel.

14 S4f. King brown snake (Pseudechis 'australis' complex, clade III species) from Bullo River Station, Northern Territory, Australia. Photograph by John Weigel. S4g. Yellow-bellied blacksnake (Pseudechis butleri), juvenile, from 23 km SW Yalgoo, Western Australia, Australia. Photograph by Brad Maryan.