The State of Squamate Genomics: Past, Present, and Future of Genome Research in the Most Speciose Terrestrial Vertebrate Order

Abstract

Squamates include more than 11,000 extant species of lizards, snakes, and amphisbaenians, and display a dazzling diversity of phenotypes across their over 200-million-year evolutionary history on Earth. Here, we introduce and define squamates (Order Squamata) and review the history and promise of genomic investigations into the patterns and processes governing squamate evolution, given recent technological advances in DNA sequencing, genome assembly, and evolutionary analysis. We survey the most recently available whole genome assemblies for squamates, including the taxonomic distribution of available squamate genomes, and assess their quality metrics and usefulness for research. We then focus on disagreements in squamate phylogenetic inference, how methods of high-throughput phylogenomics affect these inferences, and demonstrate the promise of whole genomes to settle or sustain persistent phylogenetic arguments for squamates. We review the role transposable elements play in vertebrate evolution, methods of transposable element annotation and analysis, and further demonstrate that through the understanding of the diversity, abundance, and activity of transposable elements in squamate genomes, squamates can be an ideal model for the evolution of genome size and structure in vertebrates. We discuss how squamate genomes can contribute to other areas of biological research such as venom systems, studies of phenotypic evolution, and sex determination. Because they represent more than 30% of the living species of amniote, squamates deserve a genome consortium on par with recent efforts for other amniotes (i.e., mammals and birds) that aim to sequence most of the extant families in a clade.

Keywords: genome assembly; genome sequencing; phylogenomics; squamates; transposable elements.

Figure 1

Phylogenetic relationships and evolutionary history of amniotes. Relationships and approximate divergence times for the major extant amniote lineages. Amphibians (Amphibia) are the outgroup. Amniotes arose ~310 million years ago (MYA) in the Carboniferous Period, and after the branching off of synapsids (leading to modern mammals), sauropsids began radiating in the Permian Period. The Lepidosauria emerged ~250 MYA. The only extant nonsquamate lepidosaurian is the tuatara (S. punctatus). Most major groups of Squamata (green box, represented here by Gekkota, Serpentes, Anguimorpha and the pleurodont and acrodont iguanians) diverged during the Mesozoic Era. Divergence time estimates were taken from www.timetree.org [20]. Geological timescale is approximate. Animal silhouettes from www.phylopic.org under the public domain.

Figure 2

Genome database representation for amniotes, including squamates, during the 21st century. The proportion of extant species of squamate with a complete genome assembly available on NCBI as of spring 2023 is far below that for birds and turtles, and, to a greater degree, crocodilians and mammals.

Figure 3

Comparison of genome assembly size, scaffold N50, percentage of complete and single-copy BUSCO genes, and percentage of total interspersed repeats across major Squamata clades with colors organized by clade, based on 83 publicly available genome assemblies. Assembly size and scaffold N50 are reported from NCBI metadata. Total interspersed repeat content is from de novo RepeatMasker output. Mean squamate assembly size is 1,696,615,317 bp (1.70 Gbp) with a standard deviation of 322,492,546 bp. MYA = millions of years ago.

Figure 4

Relationships between genome assembly metrics across 91 squamate genomes. We analyzed contiguity (N50), assembly size (bp), gene completeness (% BUSCOs), and repetitiveness (% total interspersed repeats). (a) Assembly contiguity predicts gene completeness. (b) Assembly contiguity predicts interspersed repeat content. (c) Genome assemblies with high predicted gene content contain a similarly greater interspersed repeat content. (d) Larger genomes do not contain a larger percentage of complete genes. (e) Larger genomes contain more interspersed repeats. (f) A positive significant relationship between estimated genome size from the Animal Genome Database and assembly size.

Figure 5

Species tree reconstruction and discordance analysis of 91 squamate species based on 6050 protein sequences extracted from complete genome assemblies. We downloaded publicly available genome assemblies for 91 squamates, and extracted orthologous protein sequences and aligned and filtered sequences according to Gable et al. (2022) [8]. Gene trees were inferred with IQ-TREE2 using model testing, and a species was constructed given the gene trees using ASTRAL-III [97]. All branches received 100% posterior support except where indicated. Gene and site concordance factors were computed in IQ-TREE2 [86].

Figure 6

(a) Abundance of the four main TE subclasses across 25 squamate families based on 91 genomes/species. Families are ordered by taxonomic relationship. Vertical bars represent median, black circles represent outliers. (b) Repeat landscapes for nine squamate genomes representing key lineages. Divergence is in terms of Kimura 2-parameter distance from family consensus.