GoKawiilMicro-CT scanning and digital reconstruction of T. mirim
The holotype MPM 420 of T. mirim was scanned in the Laboratório de Caracterização Tecnológica of the Escola Politécnica at the University of São Paulo using a Zeiss Xradia Versa XRM-510 Scanner. The specimen was scanned in three parts: one for the skull and anteriormost cervicals, the second one focusing on posterior cervical vertebrae, and the third one on dorsal vertebrae. Parameters were as follows for all scans: 110 kV, 10 W, 25-s exposure (skull and cervicals) or 8 s (dorsals), and a 0.4× objective with HE6 filter resulting in 1,301 images. Voxel sizes were as follows: skull = 17.19 µm, cervicals = 16.2 µm and dorsals = 34 µm. Reconstruction was made with the software Xradia. Reconstruction and the parameters included a beam hardening of 0.08 and a smooth filter with a 0.5 kernel size. Segmentations of osteological structures, cranial endocasts and inner ear were performed using VGSTUDIO MAX v3.5., and 3D cinematic renderings were produced using the SmARTR pipeline36.
Morphological and molecular phylogenetic data
Here we used the last iteration41 of the total evidence dataset originally published by Simões et al.5, to assess the placement of Tametara not only among snakes but also its impact in the placement and divergence times of snakes relative to other groups of squamates. Compared with its most recent iterations, the morphological data now include 63 additional squamates (both fossil and extant), mostly representing snakes. This yields a total of 166 species: 95 extant squamates, 50 fossil squamates, 12 sphenodontians (including the single extant Sphenodon punctatus) and 9 stem lepidosaurs and non-lepidosaurian reptiles; sampled taxa and accession numbers for morphological data are provided in Supplementary Data 2. Furthermore, we expanded the sampling of characters by adding 69 characters, totalling 394 characters scored for all 166 taxa. This followed character construction guidelines, such as utilization of contingent coding schemes and homology assessment to avoid logical and biological biases in character construction, or artificial splitting of continuous traits50,51. Here we provide 180 detailed character illustrations for easy reproducibility of character scoring for this dataset. For additional details, we refer the reader to ref. 41.
Molecular data for total evidence dating (TED) analyses and relaxed-clock inference must necessarily consist of a small number of molecular loci due to the exceptionally demanding computing requirements of such inferences. Here we focused on molecular loci that yield a species tree consistent with much larger phylogenomic datasets—for example, refs. 52,53—but matching our morphological taxon sample and also small enough for TED. As a result, we sampled 11 genetic markers (eight nDNA, two mDNA and one rRNA) for all 95 extant taxa obtained from GenBank; sampled taxa and accession numbers for molecular data are provided in Supplementary Data 2. The sampling was performed to match the morphological data to the species level as much as possible, which was achieved in most cases. In a few instances where species did not match morphological data, a congeneric taxon was used.
Sequences were aligned in MAFFT (v7.490)54 online server using the global alignment strategy with iterative refinement and consistency scores. For the protein-coding genes, MAFFT alignments were further verified by translating nucleotide sequences to amino acids and visually inspecting and trimming regions of poor alignment with Geneious Prime (v2023.2.1). The final multiple sequence alignment was concatenated for phylogenetic inference, but both concatenated and individual locus alignments are provided as Supplementary Data 2. Molecular sequences from all extant taxa were analysed for the best partitioning scheme and model of evolution using the PartitionFinder algorithm as implemented in IQ-TREE55 under Akaike information criterion.
Maximum parsimony phylogenetic analyses
Despite the currently recognized lower accuracy of maximum parsimony in analysing morphological data (for example, ref. 56), especially in the light of inapplicable characters51—widely frequent among squamates—we provide maximum parsimony results for comparisons with previous and historical studies in squamate systematics.
All maximum parsimony analyses were conducted in T.N.T (v1.1)57, which allows a better sampling of all possible local optima of most parsimonious trees for datasets with a large taxon sample. Searches were conducted using a combination of multiple New Technology Search algorithms, namely, successive rounds of Ratchet (1,000 iterations), Sectorial Search (1,000 rounds) and Tree Fusing (1,000 rounds) upon 1,000 initial trees obtained with random addition sequences.
Bayesian phylogenetic inference
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