Mitochondrial genome of Diploderma micangshanense and its implications for phylogeny of the genus Diploderma

Abstract The lizard Diploderma micangshanense, which belongs to the family Agamidae is endemic to China. Here, we determined the complete mitogenome of D. micangshanense using an Illumina Hiseq X Ten sequencer. This mitogenome’s structure is a typical circular molecule of 16,467 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region. The overall base composition of D. micangshanensis is 34.1% A, 23.64% T, 13.62% C, and 28.64% G with a slight AT bias of 57.74%. Most mitochondrial genes except ND6 and seven tRNAs were encoded on the heavy strand. Notably, the trnP gene was encoded on the heavy strand instead of its typical light strand position, providing an example of gene inversion in vertebrate mitogenomes. Phylogenetic analysis indicated that D. micangshanensis had a close relationship with D. zhaoermii.


Introduction
Animals in the genus Japalura sensu lato are important components of species diversity in Agamidae, and are widely distributed in East Asia and the Himalayas (Manthey 2008). Recently, the current taxonomy of Japalura sensu lato has been redefined as four genera, including Japalura sensu stricto, Pseudocalotes, Cristidorsa, and the resurrected genus Diploderma (Wang et al. 2019). Almost all the species of the original Japalura sensu lato, have been assigned to Diploderma, except for J. bapoensis, which has been reclassified to genus Pseudocalotes, and two species recorded from southern Tibet, J. andersoniana and J. tricarinata, which are still remain in Japalura sensu stricto. Currently, there are 27 species belonging to Diploderma; of these, 22 are specifically distributed in China, while D. polygonatum is distributed in China and Japan, and the remaining four species are distributed in Vietnam, Myanmar and mainland Southeast Asia.
Diploderma micangshanensis is distributed in Sichuan, Shaanxi, Shanxi, Gansu and Henan Provinces. It bears Least Concern status on the International Union for the Conservation of Nature (IUCN) Red List of Threatened Species (IUCN 2020). However, the available genetic data for this species remains scarce. Mitochondrial DNA has many valuable features including relatively conserved gene content and organization, lack of genetic recombination, maternal inheritance, and relatively fast evolutionary rate. Hence, partial or complete mitochondrial genes have been used for species identification (Hebert et al. 2003;Chambers and Hebert 2016), and to determine molecular phylogenetic and evolutionary relationships (Leavitt et al. 2017;Medina et al. 2018;Shahamat et al. 2020). In this study, we assemble and annotate the mitochondrial genome of D. micangshanensis, and determine its genomic structure and base composition. We also reconstruct the phylogenetic relationships within the genus Diploderma using the mitochondrial sequence ND2 obtained here and from NCBI. This study not only improves understanding of genomic information and phylogenetic of Diploderma, but is also conducive to the conservation genetics of D. micangshanensis.

Sample collection
Samples were collected from Luoning County, Henan Province, China (34 16 0 48 00 N, 111 43 0 5 00 E). Muscle samples were preserved in 95% ethanol, and voucher samples were deposited in the Museum of Henan University of Science and Technology (contact with Jianli Xiong, xiongjl@haust.edu.cn) under the voucher number HNUSTM20200824. Sampling was performed according to Chinese animal protection laws.

DNA extraction and sequencing
Genomic DNA was extracted from muscle tissue using a DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany). DNA integrity, purity and concentration were assessed with an Agilent 5400 fragment analyzer (Agilent Technologies, Santa Clara, CA, U.S.A.). After the DNA sample was qualified, and the template size is 21.578 ng/ul, it was randomly disrupted with a Covaris ultrasonicator (Covaris Inc., Woburn, MA, USA), and then the library was constructed through several steps: end repair and phosphorylation, adding A-tailing, ligating index adapter, purification, denaturing and PCR amplification. After the library was constructed, a Qubit 2.0 (Life Technologies, Singapore) was used to quantify and dilute the library. We then employed an Agilent 2100 Bioanalyzer (Agilent) to detect inserted fragments in the library. Finally, the effective concentration of the library was accurately quantified by q-PCR to ensure the library quality. After that, different libraries were pooled into the flow cell according to the effective concentration and target drop-off data. Illumina paired-end sequencing was conducted with an Illumina Hiseq X Ten sequencer (Illumina, San Diego, CA, USA).

Mitochondrial genome assembly and annotation
The raw data contained adapter information, low-quality bases, and undetected bases (indicated by N), which would interfere with subsequent analysis. We therefore filtered the raw data using the following criteria: (1) Filtered out reads containing adapter sequences; (2) removed paired reads, when the content of N in a single-ended sequence exceeded 10%; (3) Base with quality no more than 5 was regarded as low-quality base based on phred þ 33. If in a sequence more than half were low-quality bases, this sequence, along with the paired one was discarded. The remaining clean data was used for mitochondrial genome assembly with MitoZ v.2.4 using default parameters (Meng et al. 2019). Clade and required taxa were set to Chordata and Japalura, respectively. The assembled genome was annotated using MitoZ v.2.4 with Diploderma flaviceps (NC_039541.1) as the reference ).

Results and discussion
A total of 22,652,258 raw reads was generated and it has been deposited to NCBI database (see additional details in Data availability statement). After assembly, the complete mitogenome of D. micangshanensis was obtained (accession number: MW242820), with a total length of 16,467 bp, similar to other agamid species . The mitogenome of D. micangshanensis consists of 13 protein-coding genes (ND1, ND2, COI, COII, ATP8, ATP6, COIII, ND3, ND4L, ND4, ND5, ND6, and Cyt b), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA genes, and a control region (Figure 1). The outermost layer of Figure1 is gene structure, where orange-yellow indicates the rRNA genes, orange-red indicates the tRNA genes, light green indicates the 13 protein-coding genes, and the remainder is the control region. Most genes are transcribed from the heavy strand (2 rRNAs, 12 protein-coding genes and 15 tRNAs); only eight genes, including one protein-coding gene (ND6) and seven tRNAs (trnQ, trnA, trnN, trnC, trnY, trnS and trnE), are encoded on the light strand. Notably, the trnP gene is encoded on the heavy strand instead of its typical light strand position, providing an example of gene inversion in vertebrate mitogenomes. D. micangshanensis shares the same gene arrangement type (inverted trnP gene) with other Draconinae species, indicating a single occurrence of the trnP inversion in the ancestral draconine lineage .
As is the case with other agamid mitogenomes, the overall base composition of D. micangshanensis is 34.1% A, 23.64% T, 13.62% C, and 28.64% G, with a slight AT bias of 57.74%. There are 11 overlapping regions totaling 40 bp (varying from 1 to 10 bp) and 14 intergenic spacer regions totaling 100 bp (varying from 1 to 33 bp). Almost all proteincoding genes (PCGs) start with the typical ATA/ATG initiation codons whereas ATP8 starts with GTG. Most PCGs are terminated with the typical TAA/TAG/AGG/AGA codons, except for ATP6, COIII, ND3, and Cyt b, which are characterized by incomplete stop codons (T or TA). The 22 tRNA genes are interspersed along the genome, with the length varying from 51 to 75 bp. The 12S and 16S rRNA genes are 847 and 1496 bp, respectively. They are located between trnF and trnL (uaa) and are separated by trnV (Table 1). The D-loop region is located between trnP and trnF.
Genetic distance shows the D. micangshanensis in this study has the closest distance with the D. micangshanensis deposited on NCBI (Table S1), which confirms that the sequenced specimen in this study indeed belongs to D. micangshanense. The two methods (BI and ML) generated a consistent phylogenetic topology that D. micangshanensis in this study clustered together with the individuals deposited in GenBank and displayed a closest relationship with D. zhaoermii ( Figure 2). Additionally, the topology of Diploderma divide into two major clades (Clade A and B in Figure 2), and the D. micangshanensis locate into Clade A. The placement of D. micangshanensis was also supported by Wang et al. 2019. Thus, our study further verify and cofirm the phylogenetic position of D. micangshanensis with molecular data. In summary, our study provides a new resource for understanding whole mitochondrial genome of D. micangshanensis, which will promote the molecular study on this species.