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

Xiuli YU, Yu DU, Mengchao FANG, Hong LI and Longhui LIN*

1Hangzhou Key Laboratory for Ecosystem Protection and Restoration, College of Life and Environmental Sciences,Hangzhou Normal University, Hangzhou 310036, China

2Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University,Nanjing 210023, China

3 Hainan Key Laboratory of Herpetology, College of Tropical Biology and Agronomy, Hainan Tropical Ocean University, Sanya 572022, China

1. Introduction

The genusPseudocalotes(Draconinae; Agamidae;Squamata) areCalotes-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 B?hme, 2000; Ziegleret 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 (Maceyet al., 2000; Okajima and Kumazawa,2010). However, Pyronet 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 (Ananjevaet 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.

2. Materials and Methods

2.1. Sample collection and DNA extractionThe 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 sequencingThe 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.

2.3. Sequence analysisWe 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 (Berntet 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 withCalotes versicolor(Amer and Kumazawa, 2007). We then compared our sequences with the available agamid mitogenomes using Clustal X 2.0 (Larkinet al., 2007; Thompsonet 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 (Tamuraet al., 2013). The complete mtDNA sequence ofP. microlepiswas deposited to GenBank under accession number (KX898132).

2.4. Phylogenetic analysesTo construct a phylogenetic tree, we used all 25 available mitogenomes in Agamidae,and usedChamaeleo 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 (Bouckaertet 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 (Lanfearet 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 ofPhrynocephalus: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(Fitzeet al., 2011; Jinet al., 2015; Kyriaziet 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 (Drummondet al.,2006; Lin and Wiens, 2017).

Table 1 Primers designed to amplify and sequence in this study. L and H refer to forward primer and reverse primer, respectively; Tm refers to annealing temperature.

Table 2 The best partition schemes and nucleotide substitution models for mitochondrial data based on BEAST carried out using partitionFinder.

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 (Darribaet al.,2012), under the Akaike Information Criterion (AICc).Phylograms were drawn with Figtree 1.4.

3. Results and Discussion

3.1. Genome organization and structureThe mitogenome ofP. microlepisis 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 ofP. microlepisis longer than all species (Appendix 1)exceptPhrynocealus 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 theP. microlepismitogenome is identical to that of most squamates (Maceyet al., 2006; Ujvariet al.,2007).

Table 3 Mitochondrial genome organization of Pseudocalotes microlepis. L and H refer to forward primer and reverse primer, respectively.

3.2. Nucleotide compositionSimilar to most other mitogenomes in Agamidae, the nucleotide composition ofP. microlepismtDNA 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. TheP.microlepismitogenome 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.

3.3. Protein-coding genes and relative synonymous codon usageThe total length of the 13 PCGs inP. microlepismitogenome 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).

The relative synonymous codon usage (RSCU) for the mitochondrial PCGs inP. microlepisexhibited 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 inP. microlepisPCGs.

3.4. Ribosomal and transfer RNA genesThe 2 rRNAs(12S and 16S) ofP. microlepisare located between tRNAPheand 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 inA. lepidogasterto 930 bp inA. armata. 16S rRNA varies from 1 479 bp in genusPhrynocealusto 1 567 bp inLeiolepis boehmei, respectively. The size is similar to that of other metazoan mtDNA (Zhanget al., 2009). The typical set of 22 tRNA ofP. microlepisis ranging in size from 53 bp for tRNACysto 73 bp for tRNATrp, as similar to other metazoan mitogenomes(Yoonet 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).

Table 4 Codon usage in Pseudocalotes microlepis mitochondrial protein-coding genes. A total of 3 737 codons for analyzed,excluding the start and stop codons. AA, amino acid; RSCU,relative synonymous codon usage; n = frequency of each codon;% = n/3737.

Figure 1 Putative 22 tRNAs secondary structures of Pseudocalotes microlepis. The minus (-) indicates Watson-Crick base pairing, and dots indicate G-U base pairing. It is composed of Aminoacyl acceptor (AA) arm, Dihydorouridine (DHU) arm, Anticodon (AC) arm, TΨC (T)arm and Variable loop.

Whereas two tRNAs (tRNACysand 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 tRNACysis an unusual phenomenon, which has also been observed inGekko 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 regionsThe 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 tRNAProand 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) (Jinet al., 2015; Shiet al., 2013;Xionget 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).

3.6. Phylogenetic analysisThe 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 ofPhrynocephaluswas 9.20 Ma.

Figure 2 Time calibrated Bayesian Phylogenetic tree of amagids and one outgroup estimated using BEAST based on mitochondrial genes(concatenated 2 rRNAs and 13 PCGs) for Markov chains. Numbers on nodes are posterior probability values. Hatched rectangles indicate 95% credibility range for divergence times.

Table 5 Number of sequences and substitution rates, length of the gene fragments, models of genes for each gene as selected by jModelTest according to the AICc.

Our results revealed that the newly sequencedP.microlepisand the genusAcanthosaurawere aggregated, and together withC. versicolorthey 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 (Blankerset al., 2013; Townsendet al., 2011;Wienset al., 2012) placed Hydrosaurinae as sister to Amphibolurinae + (Agaminae + Draconinae), other studies (Maceyet al., 2000; Okajima and Kumazawa,2010) placed Hydrosaurinae as sister to (Agaminae +Draconinae), whereas we and Pyronet al. (2013) placed Hydrosaurinae as the sister-group to Amphibolurinae with weak support. Further studies were required to resolve the position of Hydrosaurinae.

4. Conclusions

In this study, we sequenced and annotated the complete mitogenome ofP. 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 genusPseudocalotes. 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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