The complete mitochondrial genome and phylogenetic analysis of Hemidactylus bowringii (Squamata: Gekkonidae)

Abstract

Hemidactylus bowringii Gray, 1845, a species of the family Gekkonidae, has garnered attention due to its ecological significance and adaptability to diverse environments. The mitochondrial DNA of H. bowringii was packaged as a compact 17,091 bp circular molecule with an A + T content of 53.8%. It contains 37 typical mitochondrial genes, including 13 protein-coding genes, 2 rRNAs, 22 tRNAs, and 1 non-coding region. We reconstructed a phylogenetic tree based on the mitogenome sequences of 17 species and two outgroups (Sphenomorphus indicus and Calotes versicolor). This newly characterized mitogenome provides a foundational resource for conservation genomics and phylogenetic studies of gekkonid lizards.

1. Introduction

Hemidactylus bowringii Gray, 1845, a gekkonid lizard (suborder Lacertilia, order Squamata) has been extensively studied using mitochondrial sequences (Carranza & Arnold, 2006). Hemidactylus bowringii exhibits male-biased sexual size dimorphism, with females exhibiting an extended oviposition period (late May to July). Dried H. bowringii carcasses are used in traditional Chinese medicine, driving commercial exploitation and captive breeding (Lan et al. 2011). Southern Chinese populations demonstrate notable thermal tolerance; for example, neonates incubated at 30 °C have larger body sizes and better locomotor performance than those incubated at 24 °C (Xu et al. 2007; Xu & Ji, 2007). Furthermore, unlike many other congeners, H. bowringii maintains locomotion capacity after caudal autotomy (Ding et al. 2012).

However, the potential genetic links between mitochondrial DNA and these traits remain unclear. Only one H. bowringii mitochondrial genome (KM508815) has previously been reported, but it remains unpublished and lacks raw data, metadata, and annotations. Here, we report the complete mitochondrial genome of geographically isolated H. bowringii. By integrating recent data, we reconstructed phylogenies across 17 gekkotan species (infraorder Gekkota), using Sphenomorphus indicus and Calotes versicolor as outgroups. This resource clarifies the phylogenetic placement within Gekkonidae and establishes a foundation for investigating mitochondrial functional adaptations.

2. Materials and methods

2.1. Sample collection and preservation

A single adult male specimen of H. bowringii (snout-vent length [SVL] = 51.94 mm, body mass [BM] = 2.90 g) was collected on July 6, 2024, from Neilingding Island, Shenzhen, Guangdong Province, China (113°48′59″E, 22°24′3″N; Figure 1). The specimen exhibited granular scales on the dorsum and lacked tuberculated spines on the tail. It possessed two pairs of chin shields, with the inner pair being significantly larger than the outer pair, confirming its identification as H. bowringii. Muscle tissue sub-samples were excised from the hind leg of this individual and preserved at −20 °C in RNA fixative. The collected specimens were transferred to the College of Wildlife and Nature Conservation at the Northeast Forestry University (https://wildlife.nefu.edu.cn, voucher number YX1-20240706, Contact person: Zhensheng Liu, Email: zhenshengliu@163.com).

Figure 1.

Dorsal (a), ventral head (B), and plantar hind foot (C) views of H. bowringii. Photograph by hongbo jiao.

2.2. DNA extraction, sequencing, and phylogenetic analysis

Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Following library preparation targeting 300 bp insert fragments, high-throughput sequencing was performed on an Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads. Initial data processing involved quality control using Trimmomatic software v0.36 (Bolger et al. 2014) using standard parameters, with subsequent quality verification via FastQC v0.11.8 (Brown et al. 2017). Considering the possible contamination from heterologous sequences, the metaSPAdes assembler (Bankevich et al. 2012) was implemented for contig reconstruction, given its demonstrated efficacy in managing multi-organism genomic data. A mitochondrial genome assembly with near-complete coverage was generated using MitoMaker (Prosdocimi & Schomaker-Bastos, 2018; Meng et al. 2019) for automated annotation. The final sequence was deposited in GenBank (Accession: PQ462284).

For phylogenetic analysis, 20 mitochondrial genomes of H. bowringii and two outgroup species (Sphenomorphus indicus and Calotes versicolor) were retrieved. GenBank BLAST matches that exhibited superior query coverage and sequence similarity rankings were prioritized. Individual alignments of the 13 protein-coding genes were performed using ClustalW under standard configurations before generating a concatenated supermatrix. Model selection analysis using MEGA 11 (Tamura et al. 2021) identified the GTR+G + I model as optimal for phylogenetic reconstruction. A maximum likelihood phylogenetic tree was constructed with 1000 bootstrap replicates using MEGA 11.

3. Results

The mitochondrial genome of H. bowringii was sequenced to an average depth of 506× (Figure S1). This 17,091 bp circular molecule contained 13 PCGs, two rRNAs (12S and 16S rRNA), 22 tRNAs, and a non-coding control region (Figure 2). Base composition analysis results revealed nearly equal frequencies of A (30.6%) and C (30.6%), whereas T (23.2%) and G (15.6%) collectively accounted for less than 40% of the total nucleotides. Gene annotation revealed a strand-specific distribution: ND6 and 8 tRNA genes were located on the light strand, whereas the remaining genes (12 PCGs, 14 tRNAs, and 2 rRNAs) were located on the heavy strand. The 13 PCGs collectively span 11,262 bp, displaying distinct base compositions of 28.8% A, 30.8% C, 25.1% T, and 15.4% G. Initiation codon usage showed variability; ATG served as the start codon for most PCGs, whereas ATA initiated ND2, ND3, and ND5, and GTG initiated ND1, ND4L, and ND6. Termination codes included TAA (ND1, ATP8, ATP6, ND4L, and Cytb), TAG (ND4 and ND6), AGA (COX1), and ND5 (AGG). Additionally, incomplete stop codons (T or TA) were identified in ND2, COX2, COX3, and ND3.

Figure 2.

Circular map of the H. bowringii mitochondrial genome, illustrating the positions of 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and the control region. Genes on the heavy (outer ring) and light (inner ring) strands are color-coded by their functional category (see legend).

A phylogenetic tree of 19 species (with H. bowringii represented by two individuals) was constructed based on the complete mitogenome using maximum-likelihood (ML), which produced a well-supported and well-resolved tree (Figure 3). Comparison of our newly sequenced H. bowringii mitogenome (PQ462284) with a previously deposited sequence (KM508815) revealed an uncorrected p-distance of approximately 0.002. Both assemblies shared identical incomplete stop codons in four genes (ND2, COX2, COX3, and ND3). Notably, KM508815 possessed an additional incomplete stop codon at the ND4 gene, which was absent in PQ462284. Furthermore, the total lengths of both H. bowringii mitogenomes exceeded those reported for other congeneric Hemidactylus species.

Figure 3.

Phylogenetic analysis of 19 species (including two H. bowringii individuals) based on maximum-likelihood (ML) in MEGA11 with 1000 bootstrap replicates. Bootstrap values are indicated above the respective branches. GenBank accession numbers of sequences are indicated after the species label. The focal individual is highlighted in red. The sequences following were used: Hemidactylus farasani OL689328, hemidactylus mandebensis OL689329, hemidactylus almakhwah OL689327, hemidactylus ulii OL689330 (Šmíd et al. 2023), hemidactylus frenatus GQ245970 (Yan et al. 2009), cyrtodactylus chanhomeae AP018117, cyrtodactylus thirakhupti AP018115, cyrtodactylus peguensis AP018114 (Areesirisuk et al. 2018), cnemaspis limi HQ896026 (Yan et al. 2014), tropiocolotes tripolitanus AB661661 (Kumazawa et al. 2014), teratoscincus roborowskii KP115216 (Li et al. 2016), teratoscincus przewalskii OL471044 (Li & Guo, 2023), sphenomorphus indicus MK45048 (Tang et al. 2019), calotes versicolor AB183287 (Amer & Kumazawa, 2007), hemidactylus bowringii KM508815, cyrtodactylus louisiadensis AB606970, cyrtopodion scabrum AB661665, homonota fasciata AB738953, teratoscincus microlepis AB612275 (unpublished).

4. Discussion and conclusions

We characterized the complete mitogenome of H. bowringii (17,091 bp; GC content: 46.2%), which exhibits a conserved architecture typical of gekkotan mitochondrial genomes. The mitochondrial gene arrangement observed in H. bowringii is identical to that found in most Gekkota species (Areesirisuk et al. 2018). Moreover, the overall architecture of the mitogenome is similar to that of other Gekkotan lineages (Yan et al. 2009; Kumazawa et al. 2014; Areesirisuk et al. 2018; Li & Guo, 2023; Šmíd et al. 2023). The divergent incomplete stop codon pattern in ND4 between PQ462284 (island population) and KM508815 (likely of mainland origin) may be associated with the prolonged geographic isolation (>7000 years) of the insular population sampled in this study, although broader population-level genomic analyses are required for confirmation.

Maximum likelihood analysis of the complete mitochondrial genomes resolved the phylogenetic relationships among 17 gekkotan species, with two lacertilian outgroups. The results of the phylogenetic analyses were consistent with those of previous studies that analyzed DNA (Carranza & Arnold, 2006; Pyron et al. 2013). However, the phylogenetic tree shows that the Hemidactylus species that we selected for tree construction do not have a particularly close phylogenetic relationship with H. bowringii, which is limited by the lack of mitochondrial genomes in Hemidactylus species. The reconstruction of the squamate phylogeny remains challenging owing to sparse mitogenomic sampling despite the extensive morphological diversity within this reptilian order. This study provides the complete mitogenome of H. bowringii, which will facilitate species identification, conservation of genomics, and adaptive evolutionary studies in Gekkonidae.

Funding Statement

This research was funded by the Neilingding Island Amphibian and Reptile Diversity Monitoring Project of the Guangdong Neilingding-Futian National Nature Reserve (0722-2024FE1108SZF-2).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

The study was approved by the institutional review board of Northeast Forestry University, Heilongjiang, China. Collection of muscle tissue was performed following the guidelines provided by Northeast Forestry University under reference number YX1-20240706. The field research complies with Guangdong Province regulations.

Data availability statement

The data supporting the findings of this investigation may be found at https://www.ncbi.nlm.nih.gov/ under the reference number PQ462284. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA1235681, SRR32683908, and SAMN47339778, respectively.