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 170-174 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: 25-30 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 4:93-102 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 150:313-329 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). 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.
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 61.15 12040 IIIa PaND4 H 25 TAATAGGMGYAATAACAATAATAAT 53.1 11984 IV PaND4 H 24 TACAYATRTTYYTRTCAMCACAAA 56.02 12280 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.
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 AJ749381 AJ830259 I P. 'australis' P2 Merauke, Papua, Indonesia F. B. Yuwono CM live AJ749380 AJ830274 I P. 'australis' P4 Merauke, Papua, Indonesia F. B. Yuwono CM live AJ749408 AJ830258
II P. 'australis' JW 1 Cunnamulla, Qld, Australia John Weigel, ARP ARP live AJ749383 AJ830264 II P. 'australis' JW 2 Arnhem Hwy, ca. 100 km E Humpty John Weigel, ARP ARP live AJ749384 AJ830268 Doo, NT, Australia II P. 'australis' JW 3 Arnhem Hwy, ca. 100 km E Humpty John Weigel, ARP ARP live AJ749385 AJ830269 Doo, NT, Australia II P. 'australis' JW 4 Humpty Doo, NT, Australia John Weigel, ARP ARP live AJ749399 AJ830270 II P. 'australis' JW 5 Arnhem Hwy, ca. 100 km E Humpty John Weigel, ARP ARP live AJ749386 AJ830271 Doo, NT, Australia II P. 'australis' JW 6 Cunnamulla, Qld, Australia John Weigel, ARP ARP live - AJ830272 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 AJ749387 AJ749394 AJ830260 AJ830265
II P. 'australis' JW 11 Vicinity of Uluru, NT, Australia John Weigel, ARP ARP live AJ749395 AJ830266 II P. 'australis' JW 17 Western NSW /southwestern Qld, John Weigel, ARP Uncollected AJ749392 AJ830267 Australia II P. 'australis' ROM 15751 Road to Brewer Plain Traps, 6.6 km ROM ROM 22722 AJ749402 AJ830257 II P. 'australis' SAM R38475 II P. 'australis' SAM R31703 II P. 'australis' WAM 104125 II P. 'australis' WAM 104126 S of turnoff from airport, NT, Australia 3 km S NT-SA border, SA, Australia (26 01'S 132 58'E) 11 km SSW Maralinga, SA, Australia (30 15'20''S 131 33'00''E) Woodstock, site #22, WA, Australia (21 35'15''S 119 05'04''E) Woodstock, site #10, WA, Australia (21 40'14''S 119 02'30''E) SAM SAM R38475 AJ810712 - SAM SAM R31703 AF217824 AJ830715 WAM WAM 104125 AJ830892 AJ830276 WAM WAM 104126 AJ749407 AJ830261 II P. 'australis' WAM Spalding Park, Geraldton, WA, WAM WAM 115106 AJ749411 AJ830277
115106 Australia (28 39'00''S 114 38'00''E) II P. 'australis' WAM 22722 Ilgarari Creek, WA, Australia WAM WAM 22722 AJ749410 - II P. 'australis' WAM 123697 III P. 'australis' WAM 127363 (24 23'00''S 120 00'00E) Peron Peninsula, WA, Australia (25 58'32''S 113 34'15''E) Walcott Inlet, WA, Australia (16 27'01''S 124 45'00''E) WAM WAM 123697 AJ749404 AJ830278 WAM WAM 127363 AJ749405 AJ830275 III P. 'australis' WAM 98871 Ca. 2 km upstream from Mitchell Falls, WA, Australia (14 51'00''S 125 41'00''E) John Weigel, WAM WAM 98871 AJ749406 - III P. 'australis' SMF 83081 Kimberley Coastal Camp, Port John Weigel, ARP SMF 83081 AJ749409 - Warrender, WA, Australia III P. 'australis' JW 8 Prince Frederick Harbour, ca. 40 km John Weigel, ARP ARP live AJ749388 AJ830273 SW of Mitchell Falls, WA, Australia IV P. 'australis' PP Near Mount Isa, Qld, Australia (ca. Roy Pails Roy Pails live AJ749412 AJ830262 20 47'S 139 47'E)
IV P. 'australis' JW 9 Vicinity of Daly River township, NT, John Weigel, ARP No voucher AJ749389 AJ830263 Australia Outgr. P. butleri JW 12 Yalgoo, WA, Australia John Weigel No voucher AJ749397 AJ830279 Outgr. P. SAM 5 km E Tungkillo, Harrison Creek, SAM SAM R25056 AJ810711 AJ830256 porphyriacus R25056 SA, Australia
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 (378+697 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.
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 (%)
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.