The oriental garden lizard Calotes versicolor (Daudin, 1802) is a medium to large-sized lizard characterized by the posterodorsal orientation of lateral scales in genus Calotes, subfamily Draconinae and family Agamidae (http://www.reptile-database.org/). Calotes is the most widely geographically distributed genus with more than 27 species being found in southern Iran, Afghanistan, Nepal, India, Sri Lanka, Brunei, Indonesia, the Malay Peninsula, Sumatra and southern China (Boulenger and Robinson, 1912; Smith, 1943; Erdelen, 1986; Zhao, 1993). Moreover, the oriental garden lizard has been introduced into Kenya, Borneo, Sulawesi, Seychelles, Mauritius, Oman and Florida (United States) (Gowande et al., 2016). The oriental garden lizard is a diurnal lizard, semi-arboreal to arboreal in habit and has been recorded mostly in scrub, deciduous forests and plantations. They usually bask in the Sun on tree trunks or branches with their heads up or down to regulate their body temperature (Diong et al., 1994). Male and female are identical in general morphology and scalation. However, females have much smaller nuchal and dorsal spines compared to the males and lack a gular sac (Pal et al., 2018). During breeding, adult males have bright body colors which can be quickly changed, and it is also called ‘chameleon tree lizard’ (Shanbhag, 2003).

Calotes versicolor is known to play a role in controlling insect populations, and also serves as a prey item of snakes and birds (Matyot, 2004; Sudasinghe and Somaweera, 2015). Studies on the mitochondrial DNA of C. versicolor conducted in Hainan island and adjacent mainland China found high genetic variation between two major lineages (Huang et al., 2013). Many researchers have mentioned the high level of morphological variation in this species across different populations, and some have considered it to be a species complex (Zug et al., 2006; Gowande et al., 2016). Recent studies have mainly focused on taxology, morphology, physiology and ecology of the C. versicolor (Gowande et al., 2016; Gowande et al., 2021; Tantrawatpan et al., 2021). However, the genomic background of C. versicolor is poorly characterized.

Rapid development of high-throughput sequencing technologies during the last decade has opened new avenues to address the genetic basis of adaptation and speciation in natural populations (Vijay et al., 2016). The use of genetic data has proven to be valuable in delimiting taxa that would not have been recognized based on morphology alone (Spinks and Shaffer, 2005; Wenner et al., 2012; Rodríguez et al., 2020). A high-quality genome is a valuable genetic resource to explore the possible genetic basis for the biological features of lizards (Alföldi et al., 2011; Lind et al., 2019; Gemmell et al., 2020). Therefore, obtaining the high-quality C. versicolor genome will be important for elucidating the genetic mechanisms underlying the species’ distinct biological characteristics and complexity.

Here, we generated the first high-quality genome of the oriental garden lizard using the oxford nanopore technologies (ONT) and DNBSEQ sequencing technology. This is the first and only high-quality genome of subfamily Draconinae. This high-quality C. versicolor genome with high contiguity and completeness become the genomic basis for the molecular studies in the subfamily Draconinae. It could be a valuable resource to conduct future research on the ecology, evolution and genetic mechanisms of biological characteristics of C. versicolor and the subfamily Draconinae.

In total, we generated ∼103.75 gigabases (Gb) of ONT long reads (×63 depth) for genome assembly. The average length of long reads was 15,716 bp, and the N50 of long reads was 27,282 bp. We also generated ∼222.61 Gb of clean whole genome sequencing (WGS) data (×138 depth) for genome assembly and ∼30.56 Gb of RNA-seq data for gene annotation (Table 1). Based on the prediction, the total number of 17-mer present in this subset was 186,766,705,020 and the peak depth was ×110 (Supplementary Figure S1). The C. versicolor genome is estimated to be 1.70 Gb in size. We yielded a draft genome assembly with 104 contigs, a total length of 1.61 Gb, and an N50 contig size of 91.60 Mb (Supplementary Table S1). Benchmarking universal Single-Copy Ortholog (BUSCO) analysis showed that 98% of 3354 BUSCO genes (vertebrata_odb10) were identified, with 97.2% single and 0.8% duplicated copy. The remaining 0.7% and 1.3% were fragmented and missing, respectively (Supplementary Table S2). This was significantly improved when compared with the published assemblies (Wilson et al., 2019) (ASM2071127v1, genome size: 0.91 Gb, contig N50: 1.62 kb and BUSCO: 6.8%, Supplementary Table S1). The C. versicolor genome (43.79% GC content) has a much more homogenous GC distribution in 500 kb windows than either the green anole lizard (Anolis carolinensis), human (Homo sapiens) or chicken (Gallus Gallus) genome (Supplementary Figure S2).

In total, we identified 650.26 Mb repetitive elements representing 40.29% of our assembled C. versicolor genome size (Supplementary Table S3). The repeat category with the highest proportion in the genome was LTRs (22.53%) followed by LINEs (6.21%), DNA elements (6.07%), and SINEs (0.88%) (Supplementary Tables S4–S5). The final 17,547 protein-coding genes was generated by combining high-quality homology-based, de novo, and RNA-seq supported genes. The average gene length, exon length, and intron length were 29,321.76 bp, 173.85 bp and 3,195.60 bp, respectively (Supplementary Figure S3), which is consistent with other animals used in annotation (Table 1, Supplementary Figure S4). BUSCO analysis showed that 95.1% and 2.1% of complete and fragmented BUSCO were identified, respectively, indicative of a high-quality gene set (Supplementary Table S2). Finally, 17,546 (99.99%) protein-coding genes were functionally annotated in at least one of the five databases that were used (Supplementary Table S6). In addition, we predicted 195 miRNA, 744 tRNA, 404 rRNA and 384 snRNA in the C. versicolor genome, respectively (Supplementary Table S7).

Comparative genomic analyses were performed between the C. versicolor and 16 other species and identified 6,121 single-copy orthologs, 2,224 multiple-copy orthologs, 8,737 other orthologs and 18 unclustered genes (Figure 1B). We further identified 4,357 single-copy genes shared by these species (Supplementary Table S8). A phylogenetic tree was constructed using these genes, with divergence times being calculated between each pair of species. It was found that the C. versicolor and Pogona vitticeps is in a clade with a divergence time of 82 (35.7–122.5) million years ago (MYA), which is much later than the divergence time between the C. versicolor and snake (Figure 1B, Supplementary Figure S5). The gene family expansion and contraction analysis showed that 545 gene families were expanded and 1,394 gene families were contracted (Figure 1B). We performed Gene Ontology (GO) enrichment analysis of 177 genes (Supplementary Table S9) in significantly expanded gene families (N = 22), which showed that they were significantly enriched in 81 GO terms (Supplementary Table S10), especially those related to sensory perception and biological regulation, including sensory perception of taste (GO:0050909, p = 1.43E-77), chemical stimulus (GO:0007606, p = 1.43E-77), and sensory perception (GO:0007600, p = 6.93E-53). We further performed Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of these significantly expanded genes. The analysis showed that they were significantly enriched in 59 KEGG pathways (Supplementary Table S11, Figure 1C), especially those related to the immune system, including NF-kappa B signaling pathway (map04064, p = 1.20E-49), B cell receptor signaling pathway (map04662, p = 6.68E-42), Natural killer cell mediated cytotoxicity (map04650, p = 2.29E-40) and T cell receptor signaling pathway (map04660, p = 1.14E-4). The biological characteristics, included Taste transduction (map04742, p = 3.06E-46), Retinol metabolism (map00830, p = 4.36E-08) and Melanogenesis (map04916, p = 1.87E-4).