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Table S1. Details of specimens from used for dissection, histology, and diceCT in this study.

File containing Table S1 and S2 and Figures S1 to S6

Molecular genetic data have recently been incorporated in attempts to reconstruct the ecology of the ancestral snake, though this has been limited by a paucity of data for one of the two main extant snake taxa, the highly fossorial Scolecophidia. Here we present and analyze vision genes from the first eye-transcriptomic and genome-wide data for Scolecophidia, for Anilios bicolor, and A. bituberculatus, respectively. We also present immunohistochemistry data for retinal anatomy and visual opsin-gene expression in Anilios. Analyzed in the context of 19 lepidosaurian genomes and 12 eye transcriptomes, the new genome-wide and transcriptomic data provide evidence for a much more reduced visual system in Anilios than in non-scolecophidian (=alethinophidian) snakes and in lizards. In Anilios, there is no evidence of the presence of 7 of the 12 genes associated with alethinophidian photopic (cone) phototransduction. This indicates extensive gene loss and many of these candidate gene losses occur also in highly fossorial mammals with reduced vision. Although recent phylogenetic studies have found evidence for scolecophidian paraphyly, the loss in Anilios of visual genes that are present in alethinophidians implies that the ancestral snake had a better-developed visual system than is known for any extant scolecophidian.

SI Figure 1 - Maximum likelihood phylogeny of curated and Repbase Harbinger sequences. Tree constructed using FastTree [35] from a MAFFT [33] aligned nucleotide MSA of the Harbinger transposase sequences trimmed using Gblocks [34]. Grey segment encompasses all curated Harbinger-Snek-like sequences. SI Figure 2 - Maximum likelihood phylogeny constructed from the aligned nucleotide sequences of the Harbinger transposases present in curated elements constructed using IQ-TREE 2 [36]. Clade phylogeny constructed with TimeTree [41].

Emydocephalus orarius sp. nov. Fig. 5, 7 A–B Holotype. WAM R165708. Adult male collected in Shark Bay (25°15`38”S, 113°08`19”E), WA on 10/02/2006 by G. Parry. Paratypes. WAM R73661 (female), Pilbara coast (20°00`00”S, 116°40`00”E); WAM R47852 (male), Barrow Island (20°48`00”S, 115°24`00”E); WAM R73651 (male), Pilbara coast (19°26`00”S, 118°49`00”E); WAM R73662 (male), Legendre Island (19°30`00”S, 116°42`00”E); WAM R174521 (female), Dampier (20°39`00”S, 116°41`00”E). Generic Assignment. Emydocephalus orarius sp. nov. can be assigned to the genus Emydocephalus by possessing three supralabial scales, the second of which is much larger than the others and occupies most of the border of the mouth. Diagnosis. A large (maximum recorded 116 cm TL) Emydocephalus species that usually differs from its congeners by the possession of two prefrontal scales, first supralabial in contact with preocular scale, enlarged tubercules on ventral scales of adult males, body pattern consisting of 19–21 bands. Genetically diagnosed from all other sequenced Emydocephalus by the following ND4 sites: 84(C), 111(A), 177(C), 195(C), 234(C), 261(C), 273(T), 288(C), 294(C), 342(T), 357(C), 396(T), 414(C), 486(G), 510(G), 569(C), 576(C), 609(C), 651(A), 667(G), 669(T). Comparisons. Emydocephalus orarius sp. nov. can usually be distinguished from E. ijimae in having two prefrontal scales (versus usually three or four pre-frontal scales), an elongated first supralabial scale which contacts the pre-ocular and often in colour pattern with a lower and non-overlapping number of bands when they are present (19–21 versus 24–32 in E. ijimae). Male E. orarius sp. nov. also have higher mean numbers of ventral scales (144.8 versus 141.7 in E. ijimae) and subcaudal scales (32.3 versus 27.8 in E. ijimae) and lower mean scale rows around the midbody (17 versus 18.1 in E. ijimae) and neck (15 versus 16.1 in E. ijimae) (Table 1). Mature male E. orarius sp. nov. also possess tubercules on the body whereas in E. ijimae these are absent. The two female E. orarius sp. nov. available for study also have a higher number of ventral scales (at least 144 versus less than 142 in E. ijimae). Emydocephalus orarius sp. nov. can usually be distinguished E. annulatus populations in the Australian region (both Timor Sea and Coral Sea) in having a higher number of ventral scales (mean 144.7 versus 136.9 in E. annulatus (Tables 1 and 2); usually possessing a distinctively elongated first supralabial scale that contacts the pre-ocular scale (10 out of 11 individuals examined) versus a much smaller first supralabial that does not contact the pre-ocular (4 of 32 E. annulatus examined did have an enlarged first supralabial contacting the pre-ocular) (Fig. 6). Description of holotype. Adult male, snout to vent length (SVL) 60.4 cm. Tail length 12.6 cm. In an excellent state of preservation. Body moderately slender, increasingly laterally compressed posteriorly. Head approximately the same width as the neck, short and rounded both from above and in profile. Rostral scale is small, five-sided, with a slight projection pointing anterio-ventrally. Scale sensilla present all over head and particularly numerous on snout. Nasal scales large and symmetrical, with valvular nostrils on the posterior edge of the nasal scales orientated dorsally. Two prefrontals each penetrating shallowly between frontal and supraocular. Frontal rounded, penetrating shallowly between prefrontals. Two parietals, each large and symmetrical. One preocular and two postoculars on each side. Three supralabials on each side. First supralabial contacts nasal and preocular excluding the second supralabial from touching the nasal, second supralabial is larger than other supralabials and contacts the preocular, eye, postocular and lower anterior temporal scale. Three anterior temporals on right side due to fragmentation of the upper, two anterior temporals on the left side. Mental scale small and triangular. Four infralabials on each side. First two infralabials barely contacting the margin of the mouth, while the third infralabial is enlarged and occupies most of the border of the mouth. Two pairs of sublinguals, well developed and broadly in contact with each other, anterior pair of sublinguals in contact with first, second and third infralabials, posterior pair of sublinguals only in contact with the third supralabial. Body scales large and imbricate, 15 rows near the neck, 17 at midbody and 15 anterior to the vent. Posterior dorsal scales are enlarged from just anterior to the vent until the tip of the tail. Ventrals broad, three to four times the width of adjacent body scales with a slight median keel, numbering 145 excluding the anal plate. Paddle-shaped tail is long (21% of total length) with a highly enlarged terminal scale, with nine scale rows around the mid-tail, 36 subcaudal scales.. Posterior tip of heart and anterior tip of liver are at ventral scale number 44, 30.3 % of the total ventral count. Colouration in preservative. Head dark brown, with cream markings along the posterior edge of the prefrontal scales and around the postocular and temporal scales. A single thin cream ring encircles the posterior edge of the head. Ground colour is dark brown with 21 white bands. White bands are usually separated by at least one dark scale on the dorsal surface and are usually offset from each other. Dark centres to white scales on the dorsal three or four scales. Dark brown bands are immaculate. Five offset white bands on the tail. Ventral surface entirely dark brown. In life. Colour pattern similar but dark brown regions were black resulting in higher contrast pattern. Variation in Emydocephalus orarius sp. nov. The following description refers to all seven Emydocephalus orarius sp. nov. specimens examined (5 males, 2 females) including the holotype and all paratypes. External morphological characters. One preocular and two postoculars in all seven specimens. Three supralabials in 7/7. Only the first supralabial in contact with nasal (6/7), first and second supralabials in contact with the nasal (1/7). First and second supralabials in contact with the preocular (6/7), only the second supralabial in contact with nasal in one specimen. Only the second supralabial in contact with the eye (7/7). Two anterior temporals on each side except in the holotype, which has three on the right side. Infralabials 3/3 (left/right) in five specimens, 3/ 2 in one specimen and 4/ 4 in the holotype. Two pairs of large well-developed sublinguals in all specimens. Anterior pair of sublinguals in contact with only the first infralabial in three specimens, the first and second in one specimen and the first three infralabials in one specimen. Posterior pair of sublinguals in contact with first and second infralabials in three specimens, the first, second and third infralabials in contact in one specimen and only the second infralabial in contact in one specimen. Fifteen scale rows on the neck (7/7), 17 scale rows near midbody (7/7) and 15 scale rows ten ventral anterior to the vent (7/7). Nine scale rows around the tail (6/7), eight scale rows around the tail in one specimen. Ventrals 144–146, enlarged and three to four the size of adjacent body scales in all specimens. Subcaudals 31–36 in male specimens, unknown in females as neither specimen has a complete tail. Mean SVL in males 69.1 cm (49.5–85.0 cm, N=5), mean tail length 12.2 cm (9–13.8 cm, N=4). SVL of the two females are 85.5 cm and 87 cm. Numerous specimens released following capture in research trawls from the Pilbara coast were in excess of 110 cm total length with a maximum of 116 cm (DPIRD, unpublished data), combined data is presented in results. Internal morphological characters. In males posterior tip of heart between ventral scale numbers 42–44, %VSheart 29.2%–30.3%, anterior tip of liver at ventral scale 44, heart-liver gap 0–2. In the only female measured posterior tip of heart at ventral scale 47, %VS-heart 32.4 %, liver at anterior tip of liver at ventral scale 48 in female, %VS-liver 33.1 % in female, heart-liver gap of 1. Colouration. Colour pattern highly variable but appears to consist of two broad geographic variants. (1) a band- ed form from Shark Bay and (2) a form with indistinct bands and often heavy spotting from the Pilbara coast. Animals from Shark Bay usually have 19–22 highly contrasting black and white bands on the body, although sometimes these do not meet on the dorsal surface, instead forming off-centre bars. Relative width of the bands is variable, with black bands broader in some specimens and white bands broader in others. Black spotting is sometimes present in the white bands on only the dorsal surface, white spotting in the black bands is usually absent. The head ranges from almost entirely white to cream to black with a white bar across the pre-frontal scales. Animals from the Pilbara coast usually have indistinct white bands on a black or dark brown body. Scales in the white bands usually have a black centre, which in some highly contrasting animals forms a densely speckled pattern. Some animals are largely black with only a trace of white bars on the side, but no completely melanistic specimens were recorded. The Pilbara coast animals usually have a black head with white or cream mottling, but it is highly variable among individuals. Distribution. The new species is endemic to coastal Western Australia, with specimens from Shark Bay, Exmouth Gulf, Pilbara coast and Broome but not from the intervenening Ningaloo Reef.. Etymology. The species epithet ‘orarius’ (Latin, ‘coastal’) refers to the coastal Western Australian distribution of the new species. Other species of Emydocephalus are found on coral reefs, typically on clear oceanic reefs some distance away from coastlines of major landmasses. Natural History. No diet records are reported for Emydocephalus orarius sp. nov. but it likely feeds exclusively on fish eggs like other members of the genus. Habitat preferences are poorly known but it appears to inhabit soft-bottomed sea floors as the majority of specimens are known from trawls over these substrates. Animals from the Pilbara coast have mostly been caught in waters 40–60 m deep, while those from Shark Bay have been caught in water E. orarius sp. nov. is diurnal like its congeners.

Supplementary Figures S1-S6.

Primary and secondary antibody controls for PGP9.5 in A) taipan (Oxyuranus scutellatus) brain tissue and B) sea snake (Hydrophis stokesii) cephalic skin tissue. Negative controls were performed by omitting primary antibody incubation step. Note the generalised staining of the epidermis in snake skin, likely resulting from cross-reactivity of secondary and / or tertiary antibodies during immunohistochemistry procedure; nerve bundles (n) do not show same cross-reactivity.