The Mitochondrial Genome of Pseudocalotes microlepis(Squamata: Agamidae) and its Phylogenetic Position in Agamids

1. Introduction

The genus Pseudocalotes (Draconinae; Agamidae;Squamata) are Calotes-like lizards which inhabit mountain regions of Indo-China and the Sunda region,and are found mostly on trees or bushes in tropical mountain forests (Hallermann and Böhme, 2000; Ziegler et al., 2006). To date, the complete mitochondrial genomes (mitogenomes) of 5.8% of Agamidae species(25/480) were available in GenBank, including 14 species in Agaminae, 4 species in Leiolepidinae, 3 species in Draconinae, 2 species in Amphibolurinae, 1 species in Hydrosaurinae and 1 species in Uromastycinae. However,the phylogenetic position and inter-relationships of the subfamilies have yet to be determined. Some researchers proposed that the group (Agaminae + Draconinae) +Hydrosaurinae was the sister group of Amphibolurinae,and Uromastycinae was the outermost subfamily of agamids (Macey et al., 2000; Okajima and Kumazawa,2010). However, Pyron et al. (2013) proposed that the group of (Agaminae + Draconinae) was the sister group of(Amphibolurinae + Hydrosaurinae) with the study using 5 nuclear loci (BDNF, c-mos, NT3, R35 and RAG-1) and 5 mitochondrial loci (12S, 16S, Cytb, ND2 and ND4),and Uromastycinae was also the outermost subfamily of agamids, but the relationship Amphibolurinae +Hydrosaurinae was weakly supported.

In this study, we sequenced the complete mitogenome of a small-scaled forest agamid (Pseudocalotes microlepis). This lizard occurs in the mountain forests of Hainan and Guizhou in China, Thailand, Laos, Myanmar and Vietnam (Ananjeva et al., 2011; Uetz, 2016; Zhao and Adler, 1993). We analyzed the gene content, base composition, codon usage, tRNAs (transfer RNAs)structure and control region of this species. We then conducted a partitioned Bayesian phylogenetic analysis of related species based upon concatenated 2rRNAs(ribosomal RNAs) and 13 PCGs (protein-coding genes)in sequence (12S rRNA, 16S rRNA, ND1, ND2, COI,COII, ATP8, ATP6, COIII, ND3, ND4L, ND4, ND5, ND6 and Cytb). We analysed the hitherto longest molecular data (14 024 bp) in Agamidae, and compared to the 25 agamids with mitogenomes sequenced, in order to explore the phylogenetic relationships among the subfamilies.

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2. Materials and Methods

2.1. Sample collection and DNA extraction The sample(voucher number XLHZ601) was collected from Hainan,China, and stored at –80°C in laboratory at Hangzhou Normal University. Total genomic DNA was extracted using the Genomic DNA kit (TransGen, China), according to the manufacturer-supplied protocols.

2.2. Primer design, amplification and sequencing The species-specific primers were designed based on highly conserved sequences (Kumazawa and Endo, 2004), which were designed with software Primer Premier 5 and were identified using multiple alignments of the agamids (Table 1). PCR was performed using a final reaction volume of 25 μL, of 2.5 μL 10 × TransTaq HIFI Buffer, 2 μL dNTPs,1 μL forward and reverse primers for each, 0.5–2 μL DNA Template, 0.25–5 μL HIFI DNA Polymerase, and addition of double distilled water to a final volume of 25 uL. The PCR procedure was conducted on a Mastercycler(Eppendorf, Germany) using the following program: predenaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 42°C–56°C for 30 s and extension at 72°C for 30 s; followed by a final elongation step of 72°C for 10 min. The PCR reaction products were electrophoresed in a 1.2% agarose gel and purified with PCR purification Kit (OMEGA, China). Then, each fragment of PCR was cloned into the pEASY-T5 Zero Cloning vector (TransGen, China) and sequenced with M13 primers from both directions by a primer-walking strategy. Each sequence overlapped the next contig between 150–300 bp.

Whereas two tRNAs (tRNACys and tRNASer(AGY)) appear to lack the dihydorouridine (DHU) arm. The loss of the DHU arm in tRNASer(AGY) has been considered a common condition of metazoan mitogenomes (Wolstenholme,1992). However, the loss of the DHU arm in tRNACys is an unusual phenomenon, which has also been observed in Gekko gecko (Han and Zhou, 2005). Further research is needed to determine the molecular mechanisms responsible for keeping such defective tRNAs functional.3.5. Non-coding regions The small non-coding region includes several intergenic spacers, ranging from 1 to 29 bp (Table 3), most of which are shorter than ten nucleotides. The longest intergenic spacer sequence we found is located between COI and tRNASer(UCN). The large non-coding region (control region) is 2 687 bp in size and located between tRNAPro and tRNAPhe. The size is remarkably longer than other species in Draconinae because of the VNTRs. The nucleotide composition is 42.3% A, 28.3% T, 18.2% C and 11.2% G, with a strong bias use of G. The structure is typical including Termination-Associated Sequence (TAS) and Conserved Sequence Blocks (CSB) (Jin et al., 2015; Shi et al., 2013;Xiong et al., 2010). VNTRs contain four distinct tandem repeat units (15 950–16 014, 16 018–16 086, 16 138–16 671 and 16 707–17 857). They are 65 bp, 43 bp, 534 bp,and 1151 bp in length, respectively. The small tandem repeats units as 5'-AACA-3' and 5'-A/G (G) CAA-3'have 16.3 copies and 10.8 copies, respectively. One large tandem repeats (74 bp) have 7.2 copies, another one (75 bp) have 15.4 copies. VNTRs have also been regarded as a common feature for the mitogenomes of reptiles (Xu and Fang, 2006), and could provide reliable phylogenetic and genetic information for closely related species(Zardoya and Meyer, 1998).

3.1. Genome organization and structure The mitogenome of P. microlepis is a typical circular DNA molecule of 17 873 bp in length, similar in size to the other available mitogenomes of species in the Agamidae.In comparison with the other agamids, the mitogenome of P. microlepis is longer than all species (Appendix 1)except Phrynocealus axillaris (17 937 bp). The difference in size is mainly due to the variable number of tandem repeats (VNTRs) in the control region. It contains a typical set of gene content: 13 PCGs, 2 rRNAs, 22 tRNAs and non-coding regions. Among these, 29 genes (12 PCGs, 15 tRNAs and two rRNAs) are located on heavy(H) strand, and other genes (ND6 and seven tRNAs) are located on light (L) strand (Table 3). Gene overlaps of 42 bp have been found at 10 gene junctions, the longest overlap (10 bp) exists between ATP8 and ATP6. The gene order of the P. microlepis mitogenome is identical to that of most squamates (Macey et al., 2006; Ujvari et al.,2007).

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3.4. Ribosomal and transfer RNA genes The 2 rRNAs(12S and 16S) of P. microlepis are located between tRNAPhe and tRNALeu(UUR), and separated by tRNAVal(Table 3). The lengths of 12S rRNA and 16S rRNA are determined to be 846 bp and 1 519 bp, and it varies from 830 bp in A. lepidogaster to 930 bp in A. armata. 16S rRNA varies from 1 479 bp in genus Phrynocealus to 1 567 bp in Leiolepis boehmei, respectively. The size is similar to that of other metazoan mtDNA (Zhang et al., 2009). The typical set of 22 tRNA of P. microlepis is ranging in size from 53 bp for tRNACys to 73 bp for tRNATrp, as similar to other metazoan mitogenomes(Yoon et al., 2015). The 22 tRNAs possess a canonical cloverleaf secondary structure composed of four arms(dihydorouridine arm, anticodon arm, TΨC arm and aminoacyl acceptor arm) with conserved size (Figure 1).

3. Results and Discussion

2.3. Sequence analysis We analyzed DNA sequences and performed contig assembly with the software Seqman(DNASTAR). We identified 13 PCGs using ORF Finder implemented at the NCBI website (https://www.ncbi.nlm.nih.gov/orffinder/). We used tRNAscan-SE search server(Lowe and Eddy, 1997) and MITOS web servers (Bernt et al., 2013) to predict the secondary structure and anticodon sequences of tRNAs. We determined the boundaries of 2 rRNAs based on alignments of rRNA sequences of other species, as with Calotes versicolor (Amer and Kumazawa, 2007). We then compared our sequences with the available agamid mitogenomes using Clustal X 2.0 (Larkin et al., 2007; Thompson et al., 1997), and searched the tandem repeat sequences of control region using Tandem Repeats Finder 4.0 (Benson, 1999). Then we calculated the GC and AT skew respectively following Perna and Kocher (1995) formula: AT skew = (A-T) /(A+T) and GC skew = (G-C) / (G+C) (Perna and Kocher,1995). We analyzed the nucleotide composition and relative synonymous codon usage (RSCU) values with Mega 6.0 (Tamura et al., 2013). The complete mtDNA sequence of P. microlepis was deposited to GenBank under accession number (KX898132).

2.4. Phylogenetic analyses To construct a phylogenetic tree, we used all 25 available mitogenomes in Agamidae,and used Chamaeleo calyptratus (Chamaeleonidae) as the outgroup. Accession number and the whole size for all mitogenomes were presented in Appendix 1. We used the nucleotide sequences consisted of 15 mitochondrial genes, and deleted the start and end codons of PCGs from analyses. Phylogenetic analyses were conducted using Bayesian uncorrelated lognormal approach in BEAST 2.0 (Bouckaert et al., 2014). Prior to estimating BEAST, we carried out the best partition schemes and corresponding nucleotide substitution models for each partition, using PartitionFinder 1.1.1 (Lanfear et al.,2012). The best-fitting model was determined using the Bayesian Information Criterion (BIC). The partitions and models were listed in Table 2. We used one fossil calibration point, the estimated age of the split between oviparous and viviparous species of Phrynocephalus:9.73 (95% interval: 7.21–13.04) Ma (million years ago)(Jin and Brown, 2013). We used the relaxed lognormal clock model, and specified the standard Yule speciation process for the tree. Two independent runs of four heated MCMC chains (three hot chains and one cold chain)were simultaneously run for 200 million generations,with sampling conducted every 10,000 generations(Fitze et al., 2011; Jin et al., 2015; Kyriazi et al., 2008).We compared the results of two independent runs with Tracer 2.2.1 (Rambaut and Drummond, 2007). Then we discarded the first one million trees as “burn-in”. All four chains achieved the recommended adequate effective sample size of 200 for likelihood (Drummond et al.,2006; Lin and Wiens, 2017).

The relative synonymous codon usage (RSCU) for the mitochondrial PCGs in P. microlepis exhibited 62 amino-acid encoding codons as well as an over-usage of A and T at the third codon positions (Table 4). Among them, CUA-Leu1 (7.12), ACA-Thr (6.02) and AUA-Met(5.19) are the most frequently used codons. The leastfrequent codons are CGG-Arg (0.03), CGU-Arg (0.05)and UCG-Ser2 (0.13). These codons are composed of A and U nucleotides, indicating a high usage of A and T in P. microlepis PCGs.

3.3. Protein-coding genes and relative synonymous codon usage The total length of the 13 PCGs in P. microlepis mitogenome is 11 283 bp, accounting for 63.13% of the entire mitogenome sequence. All the PCGs initiated with a typical start codon (ATG), except ND5 which starts with ATA. Among stop codons, TAA is the most common. ND2, ATP8, ND3 and ND4L end with TAA; ND1, COI and ND5 end with TAG; COII and ND6 end with AGG; ND4 ends with AGA; ATP6, COIII and Cytb end with an incomplete end codon (T-). The posttranscriptional polyadenylation can produce a standard TAA stop codon (Han and Zhou, 2005).

3.2. Nucleotide composition Similar to most other mitogenomes in Agamidae, the nucleotide composition of P. microlepis mtDNA is biased toward A and T. The overall A + T content of mitogenome is 59.2% (35.3%A, 23.9% T, 27.6% C and 13.2% G). The AT skew and GC skew is 0.1943 and –0.3541, respectively. The P.microlepis mitogenome has a distinct bias against G at first codon position (A: 37.0%, T: 22.5%, C: 27.2% and G: 13.3%). The percentage of purines (48.8%) is slightly lower than pyrimidines (51.2%) at the second position and the third position.

In order to estimate the substitution rate of each gene,along with their confidence intervals, we performed an additional BEAST analysis with clock models linked by ‘gene’ and nucleotide substitution models unlinked.Models of nucleotide evolution of each gene partition were calculated in jModelTest 2.1.7 (Darriba et al.,2012), under the Akaike Information Criterion (AICc).Phylograms were drawn with Figtree 1.4.

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3.6. Phylogenetic analysis The phylogenetic relationships were constructed using BEAST based on 15 mitochondrial genes. The final alignment resulted in 14 024 nucleotide sites for 26 ingroup and one outgroup taxa. The number of sequences and substitution rates,multiple sequence alignments length, and models of genes were reported for each gene in Table 5. The topology of phylogenetic tree was shown in Figure 2. Most nodes were well supported by high posterior probabilities. The divergence between Chamaeleonidae and Agamidae was estimated at 64.87 Ma; within Agamidae, the basal branching split was estimated at 60.02 Ma. The divergence between oviparous and viviparous species of Phrynocephalus was 9.20 Ma.

Our results revealed that the newly sequenced P.microlepis and the genus Acanthosaura were aggregated, and together with C. versicolor they constitute the subfamily Draconinae. However, the usage of mitogeneome did not allow us to resolve with support the position of Hydrosaurinae which was instable across previous studies. For example, some previous studies (Blankers et al., 2013; Townsend et al., 2011;Wiens et al., 2012) placed Hydrosaurinae as sister to Amphibolurinae + (Agaminae + Draconinae), other studies (Macey et al., 2000; Okajima and Kumazawa,2010) placed Hydrosaurinae as sister to (Agaminae +Draconinae), whereas we and Pyron et al. (2013) placed Hydrosaurinae as the sister-group to Amphibolurinae with weak support. Further studies were required to resolve the position of Hydrosaurinae.

城市空间布局与综合交通体系，通常呈现多元网络化形态，在优化废弃铁路及沿线地区交通系统时，应首先考虑与周边大系统的衔接。“面”即是指以统筹视角分析铁路走廊在城市中的区位特征，综合考虑城市与各类交通发展需求，对城市干道网络、公交走廊、枢纽体系、绿道系统等进行布局优化，以达到多种交通方式在城市层面的网络化融合。

4. Conclusions

In this study, we sequenced and annotated the complete mitogenome of P. microlepis. Our results present the gene content, base composition, codon usage, tRNAs structure,VNTRs in the control region and phylogenetic analysis of related species. This is the first complete mitogonome of the genus Pseudocalotes. The research is intended to be helpful for the exploration on the phylogenetic position and interrelationships of the subfamilies in Agamidae.

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