The first complete mitochondrial DNA of Tenuidactylus dadunensis (Squamata: Gekkonidae) and its phylogeny

TL;DR

This paper reports the first complete mitochondrial DNA of a gecko species and its evolutionary relationships.

Contribution

The first complete mitochondrial DNA sequence for Tenuidactylus dadunensis and its phylogenetic placement in Gekkonidae.

Findings

The complete mtDNA of Tenuidactylus dadunensis is 16,893 bp long with standard mitochondrial gene content.

Phylogenetic analysis shows T. dadunensis is a sister taxon to Cyrtopodion scabrum within Gekkonidae.

The mtDNA data will aid in conservation and evolutionary studies of geckonids.

Abstract

The complete mitochondrial DNA (mtDNA) of Tenuidactylus dadunensis (Squamata: Gekkonidae) was described by using next-generation sequencing. The total length of mtDNA was 16,893 bp, which contained 13 PCGs (COX1-3, ND1-6, ND4L, ATP6, ATP8, and CYTB), 22 transfer RNA(tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region (D-loop). The Bayesian inference tree showed that T. dadunensis was included in Gekkonidae and was a sister taxon to Cyrtopodion scabrum. The complete mtDNA of T. dadunensis will be an important genetic resource to the studies of conservation and research of geckonids.

Figures

Photograph of Tenuidactylus dadunensis (photo credit: Lei Shi, used with permission). Specimen for our study were collected in Turpan by Weizheng Gao, Tao Wang and Bo Ma.

Mitochondrial genome maps for Tenuidactylus dadunensis (OR537229).

The Phylogenetic tree of Tenuidactylus dadunensis (OR537229) and other available mitogenomes under these taxonomic. The numbers showed between branches indicate the posteriori probabilities from Bayesian inference (BI). The GenBank accession numbers of all species are shown in the figure and the citations are in the Table 1.

Tables

Table 1.. Mitochondrial genome sequences with GenBank accession numbers used in this study.

Species	Size (bp)	GenBank NO.	source
Tenuidactylus dadunensis	16.893	OR537229	this study
Gekko gecko	16.435	AY282753	Zhou et al. (2006)
Gekko swinhonis	16.818	JQ906550	Li et al. (2013)
Cyrtopodion scabrum	16.994	AB661665	–
Cyrtodactylus louisiadensis	17.594	AB606970	–
Cnemaspis limi	16.680	HQ896026	Yan et al. (2014)
Gehyra marginata	16.588	AB661662	–
Lepidodactylus lugubris	16.762	AB738945	Kumazawa et al. (2014)
Pachydactylus punctatus	17.475	AB612270	–
Paroedura picta	17.220	KR149293	Starostová and Musilová (2016)
Phelsuma guimbeaui	17.533	AB661664	Kumazawa et al. (2014)
Tropiocolotes tripolitanus	20.248	AB661661	Kumazawa et al. (2014)
Stenodactylus petrii	18.672	AB738952	Kumazawa et al. (2014)
Uroplatus fimbriatus	16.780	AB612276	Kumazawa et al. (2014)
Hemidactylus frenatus	16.891	GQ245970	Li et al. (2013)
Hemidactylus ulii	16.901	OL689330	Šmíd et al. (2023)
Hemidactylus mandebensis	16.830	OL689329	Šmíd et al. (2023)
Phyllodactylus unctus	16.881	HQ896027	–
Tarentola mauritanica	15.327	JQ425060	Rato et al. (2013)
Teratoscincus przewalskii	17.184	OL471044	–
Teratoscincus roborowskii	16.693	MT107158	Ma et al. (2020)
Teratoscincus microlepis	16.995	AB612275	–
Coleonyx variegatus	17.110	AB114446	Kumazawa (2007)
Eublepharis macularius	17.460	OQ420358	Pinto et al. (2023)
Goniurosaurus varius	17.778	OQ992199	Zhou et al. (2023)
Goniurosaurus luii	16.519	KM455054	–
Hemitheconyx caudicinctus	17.043	AB610502	Jonniaux et al. (2012)

Full text

Introduction

Tenuidactylus dadunensis (Shi and Zhao 2011) (ICUN: DD) (Figure 1) is a species of Tenuidactylus in the family Gekkoidea which is endemic to Xinjiang, China. Shi and Zhao (2011) were the first to discover this species in Turpan Basin, western China and named it Cyrtopodion dadunense. Based on morphological analysis, they concluded that this species was significantly different from two other species of the same genus (Cyrtopodion elongatum and Cyrtopodion russowi) distributed in Xinjiang. The phylogeny and classification of the Palearctic naked-toed geckos (Cyrtodactylus seso lato) was subsequently revised by Bauer et al. (2013), which supported that three subgenus of Cyrtopodion, including Cyrtopodion, Tenuidactylus and Mediodactylus should be arised to valid genus. Meanwhile, Bauer et al. (2013) tentatively place Cyrtopodion dadunense in this genus, as Tenuidactylus daduensis. Kai et al. (2020) followed Bauer et al. (2013) in their update of the Chinese amphibian and reptile list. Since then, no further studies on T. daduensis have been reported. Tenuidactylus is Palearctic thin-toed gecko, which has a total of eight species, including Tenuidactylus bogdanovi, T. caspius, T. dadunensis, T. elongatus, T. fedtschenkoi, T. longipes, T. turcmenicus, T. voraginosus (Uetz and Stylianou 2018), no more molecular infomation reported untill now. In this study, we used Illumina NovaSeq 6000 sequencing technology for paired-end sequencing of the sample DNA to obtain the complete mitochondrial genome of T. daduensis. This is the first complete mitochondrial sequence of T. daduensis and the first complete sequence of genus Tenuidactylus, which will provide data support for the phylogenetic studies of Gekkonidae in the future.

Photograph of Tenuidactylus dadunensis (photo credit: Lei Shi, used with permission). Specimen for our study were collected in Turpan by Weizheng Gao, Tao Wang and Bo Ma.

Materials and methods

Samples of T. dadunensis were collected on 28 June 2023 in Turpan City (42.85 N, 88.87E), Xinjiang UygurAutonomous Region, China. The specimen (NO: THAT5702-9) was identified by Lei Shi. We based our identification on the description of T. dadunensis by Shi and Zhao (2011): nostril bordered by rostral, first supralabial, one supranasal, and two subequal postnasals; back tubercles arranged in regular longitudinal rows; 23–26 ventral scales across midabdomen; 97–108 scales along the ventrum of body from postmental to cloaca; 18–22 subdigital lamellae beneath fourth toe; 8–10 precloacal pores in males; caudal tubercles broadly in contact laterally with each other; a single row of transversally enlarged subcaudals; dorsal bands in waving shape, much thinner than interspaces. We cut a portion of tissue from the tail, and the specimen was preserved in the Animal Herbarium of the College of Life Sciences, Xinjiang Agricultural University, Xinjiang, China (Lei Shi, leis@xjau.edu.cn).

Total genomic DNA was extracted from the adductor muscle tissue using a DNeasy tissue kit (Qiagen, Beijing, China) following the manufacturer’s protocols. After DNA isolation, 1 μg of purified DNA was fragmented to ∼500 bp using the Covaris M220 system, used to construct short-insert libraries according to the manufacturer’s instructions (TruSeq™Nano DNA Sample Prep Kit, Illumina), and then sequenced on an Illumina NovaSeq 6000 platform (Modi et al. 2021) (BIOZERON Co., Ltd, Shanghai, China). The mitochondrial genome was reconstructed using a combination of denovo and reference-guided assemblies, and the following three steps were used to assemble the mitogenome. First, the filtered reads were assembled into contigs using GetOrganelle v1.7.5 (Jin et al. 2020), potential mitochondrial contigs were extracted by aligning against the NCBI mitogenome database. Second, the potential mitochondrial contigs were aligned to the reference mitogenomes using BLAST v 2.8.1+, and aligned contigs (>80% query coverage) were ordered and connected manually according to the reference mitogenomes. Finally, MUMmer 3.23 (Kurtz et al. 2004) was used to check whether these contigs were circular. Based on the above assembly steps, we obtained a circle of T. dadunensis mitogenome. We used GetOrganelle v1.7.5 software (https://github.com/Kinggerm/GetOrganelle) (Jin et al. 2020) for mitochondrial genome assembly. The software cycles through the target reads using the seed database and then calls SPAdes to assemble the genome. We performed reads mapping with Bowtie2 (Langdon 2015) and calculated the reads coverage of the mitochondrial region by SAMtools depth (Li et al. 2009). We compared the sequencing reads to the assembly results to get a depth of coverage map of the whole genome sequence (Supplementary Figure S1). The mitochondrion genes were annotated using the online MITOS tool (Bernt et al. 2013), using default parameters to predict protein coding genes, transfer RNA (tRNA) genes, and ribosome RNA (rRNA) genes.

DNA sequences were aligned using ClustalW (Thompson et al. 1994) implemented in MEGA7.0 (Kumar et al. 2016). In order to clarify the phylogenetic position of the entire mitochondrial sequence of T. dadunensis, we obtained 27 complete mitochondrial genome sequences from the GenBank database from Gekkonidae, Phyllodactylidae, Sphaerodactylidae and Eublepharidae, respectively (Table 1) (Ma et al. 2020). We selected all available mitogenomes under these taxonomic designation to determine a clear phylogenetic position for T. dadunensis with a relatively complete phylogenetic tree. We used Partitionfinder-2.1.1 (Lanfear et al. 2017) to estimate the best model for the molecular dataset using the Akaike information criterion. We used Bayesian Inference (BI) to construct phylogenetic relationships for T. dadunensis, with the species of Eublepharidae as the outgroup. We then used MrBayes 3.2.2 (Ronquist et al. 2012) to perform Bayesian inference (BI). We used the Markov chain Monte Carlo (MCMC) theory approach in BI, running 8,000,000 generations, drawing one sample every 1000, running four chains per analysis, and removing the top 25% of the data with the other parameters as the default. The final results were checked using Tracer 1.7.2 (Rambaut et al. 2018) and had effective sample size (ESS) values > 200 for all parameter values. Finally, a phylogenetic tree was plotted using ITOLV6 (Letunic and Bork 2021). We considered an a posteriori probability (BPP) value of > 0.95 as strong support for monophyly.

Results

The length of T. dadunensis complete mtDNA (GenBank accession: OR537229) was 16,893 bp, which contained 13 PCGs (COX1-3, ND1-6, ND4L, ATP6, ATP8, and CYTB), 22 tRNA genes, 2 rRNA genes, and a control region (D-loop) (Figure 2). Depth of coverage maps for whole genome sequences are shown in Supplementary Figure S1. The gene order was similar to the known mitogenomes of Gekkonidae (Zhou et al. 2006; Yan et al. 2009; Ma et al. 2020). The nucleotide composition of T. dadunensis was 32.68% A, 24.02% T, 28.78% C and 14.52% G. Based on the results of the Bayesian phylogenetic tree (Figure 3), we can know the location of our mitotic genome samples (T. dadunensis) in the the genomic dataset of Gekkonidae. The results of the Bayesian phylogenetic tree (Figure 3) show that T. dadunensis was clustered into the clade of genomic dataset of Gekkonidae, and was a close sister clade to C. scabrum, with strongly support (BI = 0.99). More mitogenomic data may be needed at a later stage to analyze and clarify the specific position of T. dadunensis within the Gekkonidae.

Mitochondrial genome maps for Tenuidactylus dadunensis (OR537229).

The Phylogenetic tree of Tenuidactylus dadunensis (OR537229) and other available mitogenomes under these taxonomic. The numbers showed between branches indicate the posteriori probabilities from Bayesian inference (BI). The GenBank accession numbers of all species are shown in the figure and the citations are in the Table 1.

Discussion and conclusion

In this study, mitochondrial genomes were sequenced and assembled for T. dadunensis. The structure of the whole mitochondrial sequence of T. dadunensis is similar to that of other species of the same family. The mitogenome of T. dadunensis was 16,893 bp in size, including 13 PCGs, 22 transfer RNA(tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region (D-loop). The complete mtDNA of T. dadunensis reported in this study will play an important role in understanding the evolution and systematic biology of the family Gekkonidae. The phylogenetic results show that T. dadunensis is closely related to C. scabrum, and also show that the Gekkonidae are monophyletic. Furthermore, it will be an important genetic resource to the studies of conservation and restoration of T. dadunensis.

Supplementary Material

Supplemental Material

Supplemental Material