Novel gene rearrangement pattern in Cynoglossus melampetalus mitochondrial genome: New gene order in genus Cynoglossus (Pleuronectiformes: Cynoglossidae)

International Journal of Biological Macromolecules 149 (2020) 1232–1240

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Novel gene rearrangement pattern in Cynoglossus melampetalus mitochondrial genome: New gene order in genus Cynoglossus (Pleuronectiformes: Cynoglossidae) Li Gong a,b,⁎, Xinting Lu a, Hairong Luo c,d, Ying Zhang a, Wei Shi c, Liqin Liu a, Zhenming Lü a, Bingjian Liu a, Lihua Jiang a a

National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Marine Science and Technology College, Zhejiang Ocean University, 316022 Zhoushan, China Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi Beibu Gulf Marine Research Center, Guangxi Academy of Sciences, Nanning 530007, China c Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510000, China d University of Chinese Academy of Sciences, Beijing 100049, China b

a r t i c l e

i n f o

Article history: Received 7 November 2019 Received in revised form 31 December 2019 Accepted 3 February 2020 Available online 04 February 2020 Keywords: Tongue sole Mitogenome Gene inversion Tandem duplication/random loss

a b s t r a c t Mitochondrial genome (mitogenome) structure and gene order are generally considered conserved in vertebrates. However, the flatfish (Pleuronectiformes) mitogenomes exhibit the most diversified gene rearrangement patterns. Here, we report a newly sequenced mitogenome of Cynoglossus melampetalus (Pleuronectiformes: Cynoglossidae). The total length of the C. melampetalus mitogenome is 16,651 bp, containing 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, a putative control region, and an L-strand replication origin. Like all previously reported tongue sole (Cynoglossinae) mitogenomes, the C. melampetalus tRNA-Gln gene is inverted from the light to the heavy strand (Q inversion), accompanied by the translocation of CR, which is downstream to the 3'-end of ND1. In addition, we observed a unique tRNA-Ile-Met-Glu (IMQ) gene order that differed from the tRNA-Glu-Ile-Met (QIM) order previously reported for other 14 Cynoglossinae mitogenomes. To our knowledge, it is the first report of two different patterns of mitogenomic gene-arrangement within the same genus in teleost. According to the Q inversion, Met pseudogene (ψMet) and long intergenic gap (186 bp) between M and Q genes, the observed gene rearrangement pattern were presumably supported by mitochondrial recombination and tandem duplication/ random loss models. The reduced trend of the intergenic gap between Q and I also suggests that the event of gene rearrangement can be traced back to early Cynoglossinae differentiation. © 2020 Elsevier B.V. All rights reserved.

1. Introduction Mitochondrial DNA (mtDNA) of vertebrates is a circular DNA molecule of 15–20 kb. It normally contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, an Lstrand replication origin (OL), as well as a large non-coding region, namely control region (CR) or D-loop region [1]. The orders of most vertebrate mitochondrial genomes (mitogenomes) are generally considered conserved; however, changes in this gene order have been reported in various vertebrate lineages subsequently, including amphibians [2,3], reptiles [4,5], birds [6,7], marsupials [8,9], and fish [10,11]. Teleosts has the largest number of published complete mitogenomes, while among these mitogenomes, it exhibits a relatively low gene ⁎ Corresponding author at: National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Marine Science and Technology College, Zhejiang Ocean University, No. 1, South Haida Road, Dinghai District, Zhoushan, Zhejiang 316022, China. E-mail address: [email protected] (L. Gong).

https://doi.org/10.1016/j.ijbiomac.2020.02.017 0141-8130/© 2020 Elsevier B.V. All rights reserved.

rearrangement ratio of ~4% [12]. By contrast, mitogenomes of flatfish (Pleuronectiformes) show a high gene rearrangement ratio and the most diversified gene rearrangement patterns. To date, five major gene rearrangement patterns have been validated in the mitogenomes in four out of the 14 families of Pleuronectiformes. In the mitogenomes of Bothidae (e.g. Crossorhombus azureus and Bothus myriaster), eight genes encoded by the L-strand (including the ND6 and seven tRNA genes) cluster together [13,14]. The Samariscus latus (Samaridae) mitogenome have undergone genomic-scale rearrangement events characterized by the duplication and translocation of CR; meanwhile, genes reside between the two CRs could be divided into two clusters, each maintained its own gene order [15]. Furthermore, a third type of gene rearrangement pattern was observed in Citharoides macrolepidotus (Citharidae) mitogenome [16], which is featured by translocation in the IQM tRNA cluster (IQM changes to IMQ). More interestingly, Cynoglossidae mitogenomes have two types of gene rearrangement. In the mitogenomes of Cynoglossinae species, the CR is translocated and a tRNA gene (tRNA-Q) is inverted [11,17–19]; while in

L. Gong et al. / International Journal of Biological Macromolecules 149 (2020) 1232–1240

the mitogenomes of Symphurinae, both un-rearranged (e.g. Symphurus plagiusa) and rearranged (e.g. Symphurus orientalis) gene orders were detected. The former resembles that of a typical vertebrate (without any gene rearrangements), whereas the latter shows large-scale gene rearrangements in the fragment between CR to the WANCY tRNA cluster characterized by translocation and large intergenic spacers [20]. So far, several hypotheses have been proposed to explain gene rearrangements in animal mitogenomes. The recombination model, initially proposed for gene rearrangements in nuclear genomes, is characterized by breakage and rejoining of participating DNA strands [21]. This model has been adopted to explain changes in mitogenomic rearrangements in frog, bird, and mussels [22–24]. Another commonly accepted hypothesis is the tandem duplication/random loss (TDRL) model, which supposes that the rearranged gene order occurred via tandem duplications followed by random deletion of certain duplications [25,26]. This model has been widely accepted to account for gene rearrangements in mitogenomes of vertebrate species [20,27,28]. Two less commonly used hypotheses are tRNA mis-priming model [29] and the tandem duplication/non-random loss model (TDNL) [30]. Because none of the above models is able to provide a convincing explanation for the gene rearrangements in some flatfishes, three additional hypotheses: 1) inversed duplication and deletion model [11]; 2) dimer-mitogenome and non-random loss model [14], and 3) the double replications and random loss model [15], were proposed. At present, 14 complete mitogenomes of Cynoglossinae species have been deposited in GenBank (as of Sep. 1, 2019). All of them share the same gene order that CR is translocated to the downstream of ND1, and a tRNA gene (tRNA-Q) is inverted accompanied shuffling of tRNA-Ile. Interestingly, a new gene rearrangement was detected in the mitogenome of Cynoglossus melampetalus. To our knowledge, it is the first report of two different mitogenome gene arrangement patterns within the same genus in teleost. The characteristics of the gene order and large intergenic spacers in the C. melampetalus mitogenome provide clear evidence for the TDRL model. Based on the similarities and differences of the two rearranged gene order in Cynoglossinae mitogenomes, the possible rearrangement processes were discussed, in the hopes of contributing a better understanding of mitogenomic rearrangement events and evolutionary mechanisms in tongue sole mitogenomes. 2. Materials and methods 2.1. Sampling, DNA extraction, PCR amplification and sequencing Three individuals of C. melampetalus were collected from Sanya of Hainan, Zhanjiang of Guangdong, and Zhoushan of Zhejiang (one for

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complete mitogenome, and the other two for mitochondrial fragments). A portion of the epaxial musculature was excised and the samples were immediately preserved in 95% ethanol. Total genomic DNA was extracted using the SQ Tissue DNA Kit (OMEGA) following the manufacturer's protocol. Twelve primer pairs were designed to amplify the complete mitogenome based on the published Cynoglossinae mitogenomes [11,17,19,31] (Table S1). The polymerase chain reaction (PCR) was carried out in a 25 ul reaction volume containing 2.0 mM MgCl2, 0.4 mM of each dNTP, 0.5 uM of each primer, 1.0 U of Taq polymerase (Takara, China), 2.5 ul of 10 × Taq buffer, and approximately 50 ng of DNA template. PCR cycling conditions included an initial denaturation at 94 °C for 2 min, 35 cycles at 94 °C for 1 min, an annealing temperature at 50 °C for 2.5 min, elongation at 72 °C for 1 min, and a final extension at 72 °C for 10 min. Fragments generated from PCR amplification were sequenced by primer walking directly or if necessary, the purified PCR products were inserted into the pMD19T vector (TaKaRa) then transformed in E. coli competent cells and sequenced. The sequences were determined using an ABI genetic analyzer (Applied Biosystems, China). 2.2. Sequence assembly, annotation and analysis Sequenced fragments were assembled to create the complete mitogenome using CodonCode Aligner 5.1.5 (CodonCode Corporation, Dedham,MA). The complete mitogenome was annotated using the software of Sequin (version 15.10, http://www.ncbi.nlm.nih.gov/ Sequin/). Transfer RNA genes and their potential cloverleaf structures were identified using tRNAscan-SE 1.21 [32], with cut-off value set to 1 when necessary. The gene map of the C. melampetalus mitogenome was generated using CGView Server V 1.0 [33]. The base composition and the relative synonymous codon usage (RSCU) were obtained using MEGA 7.0 [34]. Strand asymmetry was calculated using the formulae: AT-skew = (A − T) / (A + T); GC-skew = (G − C) / (G + C) [35]. 2.3. Phylogenetic analyses All fourteen Cynoglossinae mitogenomes from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/), plusing the newly determined sequence in this study, were used for phylogenetic analysis. Symphurinae species have been thought to be most closely related to Cynoglossinae [36–38]; therefore, two Symphurinae species, Symphurus plagiusa and Symphurus orientalis, were selected as the outgroup (Table 1). The 13 PCGs were concatenated for phylogenetic analysis. Sequences were aligned using Clustal X 2.0 [39] with the default

Table 1 List of 15 Cynoglossinae species and two outgroups used in this paper. Whole

Paraplagusia bilineata Paraplagusia blochii Paraplagusia japonica Cynoglossus semilaevis Cynoglossus abbreviatus Cynoglossus sinicus Cynoglossus bilineatus Cynoglossus puncticeps Cynoglossus lineolatus Cynoglossus itinus Cynoglossus trigrammus Cynoglossus gracilis Cynoglossus joyneri Cynoglossus zanzibarensis Cynoglossus melampetalus Symphurus plagiusa Symphurus orientalis

PCGs

16S

12S

CR

Length (bp)

AT%

Length (bp)

AT%

Length (bp)

AT%

Length (bp)

AT%

Length (bp)

AT%

16,985 16,611 16,687 16,731 16,417 16,478 16,454 17,142 16,417 16,915 18,369 16,565 16,426 16,555 16,651 17,034 17,498

60.2 60.1 59.6 60.6 60.4 60.8 59.8 62.0 60.4 58.8 60.6 61.6 60.4 57.5 61.0 53.8 58.5

11,399 11,397 11,405 11,416 11,412 11,430 11,423 11,423 11,415 11,416 11,410 11,420 11,422 11,400 11,424 11,289 11,420

59.0 59.2 58.6 59.9 60.0 61.0 59.8 60.6 60.0 58.4 60.1 61.3 60.3 56.5 61.0 52.2 57.3

1709 1707 1709 1684 1680 1685 1688 1702 1679 1696 1659 1676 1676 1685 1682 1706 1874

61.6 62.0 61.6 61.0 61.5 60.3 61.3 61.4 61.6 60.2 60.9 62.1 60.7 60.3 61.3 53.9 58.9

953 953 950 947 947 947 947 942 947 949 975 948 939 947 943 947 949

56.8 55.7 55.6 56.1 55.9 56.7 55.3 56.7 55.9 54.9 55.5 56.4 55.7 55.8 55.5 51.5 54.1

1245 886 971 982 661 659 655 1433 662 1053 1054 805 746 719 704 1191 1388

72.0 72.5 71.4 73.0 69.0 67.8 67.5 78.4 69.3 66.4 71.2 71.1 71.0 65.6 68.0 68.9 70.6

Accession nos.

Reference

NC_023227 NC_023228 NC_021376 NC_012825 NC_014881 NC_023224 NC_023226 NC_023229 NC_023230 NC_023446 KP057581 NC_028540 NC_030256 NC_030364 MN082377 JQ639061 NC 027656

Unpublished [48] [49] [11] [31] [50] [51] [52] Unpublished Unpublished [18] [19] [53] [54] This study [20] [20]

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parameters and manually checked with BioEdit [40]. Ambiguous sequences were eliminated using Gblock [41] The dataset was used for maximum likelihood (ML) analyses implemented in PhyML [42] and Bayesian inference (BI) in MrBayes 3.2.6 [43]. The best-fit evolutionary models were determined using Modeltest 3.7 [44] from 56 models for ML analyses and MrModeltest 2.2 [45] from 24 models for BI analyses both based on the Akaike information criterion (AIC). Bootstrap analyses (1000 replicates) were performed to evaluate relative levels of support for the ML analyses [46,47]. Bayesian phylogenetic analyses were performed using “Lset” and “Prset”, and the program was allowed to converge on the best estimates of the model parameters. Other parameter settings were as follows: Each Markov chain was initiated from a random tree and run for 2,000,000 generations, sampling trees every 100 generations (20,000 total trees sampled) to assure independence of the samples. Four chains, three heated (temperature =

0.5) and one cold, were simultaneously run using Metropolis-coupled Markov chain Monte Carlo (MCMCMC) to enhance the mixing capabilities of the Markov chains. To guarantee the stationarity had been reached, the average standard deviation of split frequencies was set below 0.01. 3. Results and discussion 3.1. Genome structure, composition and skewness The complete mitogenome of C. melampetalus is 16,651 bp in length (GenBank accession number MN082377), which is in the length range (16,417–18,369 bp) of the published tongue fish mitogenomes (Table 1). The gene content is typical of other teleostean mitogenomes, including 13 PCGs (COI-III, ND1-6, ND4L, Cyt b, ATP6 and ATP8), 2 rRNA

Fig. 1. Gene map of the Cynoglossus melampetalus mitogenome.

L. Gong et al. / International Journal of Biological Macromolecules 149 (2020) 1232–1240

genes (12S and 16S rRNA), 22 tRNA genes, an L-strand replication origin (OL), and a major non-coding region known as the CR (Fig. 1, Table 2). Whereas the gene arrangement in mitogenome of C. melampetalus differs from that of the bony fishes, including other tongue fishes. Specifically, the tRNA-Gln gene is translocated from the light to the heavy strand (Q inversion), accompanied by shuffling of the tRNA-Met gene and long-range translocation of the CR downstream to a site between ND1 and the tRNA-Ile gene (Fig. 1, Table 2). Thus, a unique tRNA-Ile-Met-Glu (IMQ) gene order is formed, which differs from the typical tRNA-Glu-Ile-Met (QIM) order previously reported for other 14 Cynoglossinae mitogenomes. The overall base composition of the C. melampetalus mitogenome is 31.4% A, 24.9% C, 29.6% T, and 14.1% G, respectively (Table 3). Base composition analysis indicates that the C. melampetalus mitogenome has an A + T content of 61.0%, on the upper end of other identified species in this subfamily ranging from 57.5% (C. zanzibarensis) to 62.0% (C. puncticeps). More specifically, the A + T content of PCGs (61.0%) are slightly higher than those of other known Cynoglossinae, while that of the CR ranks on the lower end of the published species (68.0%) (Table 1). Generally, the mitogenome of C. melampetalus is quite compact, whereas a total of 391 base pairs in 16 intergenic spacers are found, ranging from 1 to 186 bp in length. Most of the gaps are found in the areas where rearrangement occurs, including 186 bp between tRNA-Met and tRNA-Gln, 77 bp between tRNA-Gln and ND2, 68 bp between tRNA-Pro and tRNA-Phe, 11 bp tRNA-Ile and tRNA-Met. Simultaneously, eleven overlapping sites (totally 54 bp) are observed,

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including the four typical overlaps between protein-coding genes (10 bp in ATP8 and ATP6, 1 bp in ATP6 and COIII, 7 bp in ND4L and ND4, 4 bp in ND5 and ND6) (Table 2), which is commonly identified in other vertebrate species. Generally, metazoan mitogenomes present a clear strand bias in nucleotide composition, and that can be measured as AT- and GCskews [4,10,55,56]. In C. melampetalus genome, the AT-skews are from −0.319 (ND6) to 0.175 (rRNAs), and all the GC-skews are negative except ND6 and tRNAs (Table 3). As the conventional preference in most mitogenomes, AT-skews are positive, while GC-skews are negative; and the former are lower (absolute value) in magnitude than the latter (Fig. 2A). Nucleotide compositional asymmetries might be due to the balance between mutational and selective pressures during replication and transcription, which provide a potential indicator for replication orientation and gene direction [35,57,58]. 3.2. PCGs, ribosomal RNAs, transfer RNAs and OL The total length of 13 PCGs is 11,424 bp, encoding 3795 amino acids. Like most vertebrate mitogenomes [1,59–61], twelve of the PCGs are encodes by the H-strand. Only ND6 gene is encoded by the L-strand. The 13 PCGs range in size from 165 bp (ATP8) to 1851 bp (ND5) (Table 2). All of the PCGs use the canonical ATG initiation codon with the exception of COI gene (GTG), which is quite common among teleostean mitogenomes [62–64]. Nine PCGs (ND2, COI, ATP8, ATP6, COIII, ND3, ND4L, ND5 and Cyt b) terminated with the stop codon TAA,

Table 2 Features of the mitochondrial genome of Cynoglossus melampetalus. Gene

tRNA-Phe (F) 12S rRNA tRNA-Val (V) 16S rRNA tRNA-LeuUUA (L1) ND1 D-loop tRNA-Ile (I) tRNA-Met (M) tRNA-Gln (Q) ND2 tRNA-Trp (W) tRNA-Ala (A) tRNA-Asn (N) OL tRNA-Cys (C) tRNA-Tyr (Y) COI tRNA-SerUCA (S1) tRNA-Asp (D) COII tRNA-Lys (K) ATP8 ATP6 COIII tRNA-Gly (G) ND3 tRNA-Arg (R) ND4L ND4 tRNA-His (H) tRNA-SerAGC (S2) tRNA-LeuCUA (L2) ND5 ND6 tRNA-Glu (E) Cyt b tRNA-Thr (T) tRNA-Pro (P) a b

Position

Length (bp)

From

To

1 69 1012 1085 2767 2841 3825 4529 4609 4865 5013 6058 6130 6202 6269 6316 6382 6454 8005 8078 8150 8841 8917 9072 9755 10,540 10,632 10,960 11,028 11,318 12,690 12,759 12,830 12,909 14,756 15,278 15,349 16,488 16,556

68 1011 1084 2766 2840 3824 4528 4597 4678 4935 6053 6127 6198 6274 6324 6381 6452 8004 8075 8147 8848 8915 9081 9755 10,540 10,610 10,961 11,027 11,324 12,694 12,758 12,826 12,902 14,759 15,277 15,346 16,485 16,556 16,625

68 943 73 1682 74 984 704 69 70 71 1041 70 69 73 56 66 71 1551 71 70 699 75 165 684 786 71 330 68 297 1377 69 68 73 1851 522 69 1137 69 70

Amino acid

Start/stop codon

Anticodon

Intergenic regiona

Strandb

GAA

0 0 0 0 0 0 0 11 186 77 4 2 3 −6 −9 0 1 0 2 2 −8 1 −10 −1 −1 21 −2 0 −7 −5 0 3 6 −4 0 2 2 −1 25

H H H H H H H H H H H H L L L L L H L H H H H H H H H H H H H H H H L L H H L

TAC TAA 327

ATG/TAG GAT CAT TTG

346

ATG/TAA TCA TGC GTT GCA GTA

516

GTG/TAA TGA GTC

232

ATG/AGA

54 227 261

ATG/TAA ATG/TAA ATG/TAA

109

ATG/TAA

98 458

ATG/TAA ATG/AGA

TTT

TCC TCG

GTG GCT TAG 616 173

ATG/TAA ATG/TAG

378

ATG/TAA

TTC TGT TGG

Intergenic region: non-coding bases between the feature on the same line and the line below, with a negative number indicating an overlap. H: heavy strand; L: light strand.

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Table 3 Composition and skewness of Cynoglossus melampetalus mitogenome.

Mitogenome PCGs ND1 ND2 COI COII ATP8 ATP6 COIII ND3 ND4L ND4 ND5 ND6 Cyt_b tRNAs rRNAs CR

A%

T%

G%

C%

A+T %

AT-skew GC-skew Length (bp)

31.4 29.2 29.5 31.6 27.9 30.5 32.1 30.1 26.6 26.7 25.3 31.0 31.1 20.9 29.2 30.2 34.8 34.7

29.6 31.8 32.0 30.1 32.9 30.9 32.1 31.7 31.3 33.9 30.3 29.8 31.2 40.4 31.7 29.6 24.4 33.4

14.1 13.4 13.3 10.6 16.5 14.4 6.1 10.1 15.5 12.4 13.5 11.8 11.5 25.7 12.8 21.2 18.3 11.6

24.9 25.6 25.2 27.8 22.8 24.2 29.7 28.1 26.6 27.0 31.0 27.4 26.3 13.0 26.4 19.0 22.5 20.3

61.0 61.0 61.5 61.7 60.7 61.4 64.2 61.8 57.9 60.6 55.6 60.9 62.2 61.3 60.9 59.8 59.2 68.0

0.030 −0.042 −0.041 0.025 −0.083 −0.007 0.000 −0.026 −0.081 −0.120 −0.091 0.019 −0.002 −0.319 −0.040 0.010 0.175 0.019

−0.278 −0.311 −0.309 −0.449 −0.159 −0.252 −0.661 −0.471 −0.263 −0.369 −0.394 −0.399 −0.391 0.327 −0.348 0.055 −0.104 −0.271

16,651 11,424 984 1041 1551 699 165 684 786 330 297 1377 1851 522 1137 1547 2625 704

two (ND1 and ND6) terminated with TAG, and two (COII and ND4) terminated with AGA (Table 2). The most frequently used amino acids were tRNA-Leu (16.71%), tRNA-Ile (8.30%), tRNA-Ala (7.80%), and tRNAThr (7.64%), while the least common amino acids were tRNA-Cys (0.82%), tRNA-Arg (1.92%), tRNA-Lys (2.11%) and tRNA-Glu (2.35%) (Fig. 2B). The relative synonymous codon usage (RSCU) values C. melampetalus for the third positions of the 13 PCGs is shown in Fig. 2C. The usage of both two- and four-fold degenerate codons is biased toward the use of codons abundant in T or A, while there is an overall bias against G, in accord with other teleosts [10,18,65]. The 12S and 16S rRNA genes are 954 and 1700 bp, respectively, which both are located regularly between tRNA-Phe and tRNA-Leu (UUA) , and separated by tRNA-Val. Twenty-two tRNAs are dispersed between rRNA genes and PCGs. Fourteen of them are encoded on the H-strand and the remaining eight tRNAs are encoded on the L-strand. The lengths range from 66 bp (tRNA-Cys) to 75 bp (tRNA- Lys), with a total length of 1547 bp. Twenty-one tRNAs and typical cloverleaf structures are recognized by tRNAscan-SE, while tRNA-Ser (AGC) is identified by comparison to other tongue soles [11,19,31]. All putative cloverleaf structures contain 7 bp in the amino acid stem, and the majority structures have 4 bp in the DHU stem and 5 bp in both the TΨC stem and the anticodon stem (Fig. S1). The origin of light-strand replication (OL) is commonly located inside the WANCY cluster at approximately two thirds of the genomic distance away from the CR. This non-coding region in C. melampetalus mitogenome is 56 bp long and has an overlap with tRNA-Asn and tRNA-Cys genes by 6 and 9 bp, respectively. As most vertebrates [2,20,66], it has the ability to fold into a stable stem-loop structure, with a stem formed by 16 paired nucleotides and a loop of 12 nucleotides. The highly conserved block (5'-CCCCGG-3') is found, which was speculated to involve in the regulation of replication and transcription of the genome [1,67]. 3.3. Gene rearrangement The CR is typically located between the tRNA-Pro and tRNA-Phe genes. However, only a small 25 bp fragment is left in this location in the C. melampetalus mitogenome. Like all Cynoglossinae species mitogenomes, a long non-coding region is recognized in the downstream of ND1 in this mitogenome [11,17,19,31]. This noncoding fragment is 704 bp and has an obvious AT bias of 68.0%. Further analysis shows that the 5'- end of this non-coding sequence possesses a 17-bp tandem repeat array with three copy numbers. Also, several conserved blocks of the CR, such as TAS motif (TACAT–ATGTA) and a

pyrimidine tract (TTCTCC-T-TCTTC) are identified despite the CR sequences of tongue fishes are quite variant. These features suggest that the 704- bp non-coding region is a putative CR. Another striking finding of this study is that a unique IMQ gene order differing from the QIM order previously reported for other 14 Cynoglossinae mitogenomes is observed [11,17,19,31]. Because it is the first IMQ gene order in Cynoglossidae mitogenomes, the rearranged fragment (from ND1 to ND2) of other two individuals were sequenced to confirm this novel rearrangement. The results show an identical IMQ gene order (GenBank accession Nos. MN481934-MN481935), confirming the real existence of this rare gene order. 3.4. Possible pathway of gene rearrangements How did the gene rearrangement event in C. melampetalus mitogenome occur? Recombination and tandem duplication/random loss (TDRL) are adopted to explain the rearrangement events based on the principle of parsimony. The hypothesized intermediate steps are as follows. First of all, the tRNA-Gln gene was translocated from the light to the heavy strand (Q’ in Fig. 3, indicating Q inversion), which was accompanied by shuffling of the tRNA-Ile gene and long-range translocation of the CR downstream to a location between ND1 and tRNA-Gln gene (Fig. 3b). This process was well explained by Kong et al. [11] in the novel rearrangement of Cynoglossus semilaevis mitogenome. They hypothesized that intra-mitochondrial recombination and inversed duplication and deletion models are most likely responsible for the rearrangement [11]. The subsequently published mitogenomes of tongue fishes provide abundant examples for this hypothesis [11,17,19,31]. In the following step, the newly formed CR-Q’-I-M block underwent a complete copy, forming a dimeric block (CR-Q’-I-M-CR-Q’-I-M) (Fig. 3c, d). Consecutive copies were then followed by a random loss of redundant genes, namely CR-Q’-I-M-CRQ’-I-M (the deleted genes are underlined) (Fig. 3e). Then a new gene rearrangement (CR-I-M-Q’) in the mitogenome of C. melampetalus was formed (Fig. 3f, g). The above processes (Fig. 3b to g) can be explained by TDRL model. 3.5. Evidence for the TDRL model TDRL model is indicated by the presence of intergenic spacers or pseudogenes as a result from incomplete deletion of the duplicated genes [7,20,68]. In the mitogenome of C. melampetalus, two large intergenic gaps are discovered in the rearranged IMQ region. One gap is located between tRNA-Met and tRNA-Gln genes (NC in Fig. 3g), with a length of 186 bp; while the other is located between tRNA-Gln and ND2 (ψM in Fig. 3g), with a length of 65 bp. Clues supporting the two large intergenic gaps are degraded from the duplicated CR and Met are as follows. Considering the length of this long non-coding gap (186 bp) is equivalent of about three tRNA genes, it is reasonable to suppose it might be degraded from the three tRNA genes (IQM). Generally, intergenic spacers vanish quickly because the degradation rate of non-functional genes is high to maintain the parsimony of mitogenomes. Therefore, the degradation time should be short enough to maintain the length similarity between NC and the three tRNA genes, which would leave a trace of sequence similarity between them. However, none of the tRNA gene sequences resemble this gap (Fig. 4a). Consequently, it is not appropriate to assume the NC is degraded from any one of the three tRNA genes. Because NC is located in the rearranged region adjacent to the CR, we speculate whether it is possible a residual CR fragment during the rearrangement events. Although a relatively low sequence similarity (49.7%) between NC sequence and the real CR sequence (Fig. 4b), the high AT bias (74.2%) of NC sequence suggests it might degrade from the real CR. Unlike the NC sequence, the second large gap between NC’ and ND2 is more distinguishable. It is within the normal range of tRNA gene length and shows a high similarity (67.1%) with the tRNA-Met in the

L. Gong et al. / International Journal of Biological Macromolecules 149 (2020) 1232–1240

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Fig. 2. AT- and GC-skews in the mitogenome of the subfamily Cynoglossinae (A); Amino acid composition in the mitogenome of Cynoglossus melampetalus (B); relative synonymous codon usage in the mitogenome of C. melampetalus (C). Note: *Species 1–15: Paraplagusia bilineata, P. blochii, P. japonica, Cynoglossus semilaevis, C. abbreviates, C. sinicus, C. bilineatus, C. puncticeps, C. lineolatus, C. itinus, C. trigrammus, C. gracilis, C. joyneri, C. zanzibarensis and C. melampetalus.

rearranged region (Fig. 4c), which indicates it might degrade to a Met pseudogene (ψM). Similar cases were found in scarid fish mitogenomes [69]. The ψM varied from 53 bp to 69 bp in sequence length among species. Most of ψM retained sequence similarities with the corresponding function genes, yet anticodon sequences were mostly degenerated, indicating their losses of tRNA function [69]. Here, the anticodon sequence inψMet is degenerated from the conventional CAT to incomplete CA- in the aligned sequences, while the remaining sequences are highly semblable (Fig. 4), supporting the second large intergenic gap is likely degraded from the duplicated Met.

3.6. Phylogenetic analysis and intergenic spacer The IQM gene cluster is rearranged in all 15 mitogenomes of tongue soles. Fourteen of them feature a QIM gene order, while a novel IMQ order is identified in C. melampetalus mitogenome. We analyzed the intergenic spaces in the rearranged region. Without regard to C. melampetalus (different gene order), the intergenic space between Q and I (G1) ranges from 3 bp to 160 bp; 5 bp to 35 bp between I and M (G2); and 1 bp between M and ND2 (G3). In order to explore the relationship between the intergenic spaces and phylogeny, two

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Fig. 3. Inferred intermediate steps between the gene order of ancestral teleosts and the mitogenome of Cynoglossus melampetalus. (a) The ancestral gene order of teleosts; (b, c) the gene order of most tongue fishes; (d) duplication of the CR-Q’-I-M region; (e) random deletion of the copied genes. The deleted genes are labeled with gradient colors; (f, g) the final gene order in the mitogenome of C. melampetalus.

phylogenetic trees (ML tree and BI tree) were constructed including all 15 Cynoglossinae species available and two Symphurinae species as the outgroup (Fig. 5). The phylogenetic trees show a same topology and reveal that G1 varies in a wide range of length, while length of G2 and G3 are relatively conserved. Although no obvious correlation is found

between G2, G3 and the phylogenetic relationship, the length of G1 shows a decreasing trend with the evolution process. Among the gaps of G1, three largest gaps (160, 143 and 121 bp) exist in the mitogenomes of species (C. zanzibarensis, C. sinicus, and C. bilineatus) located in the base of the phylogenetic tree, two larger gaps (88 and

Fig. 4. Sequence alignment of NC and three tRNA genes (I, Q, and M) (a); NC and CR (b); and functional and Met pseudogene (c). The dots (.) indicate nucleotide identity to the top sequence and dashes (−) indicate alignment gaps. FM: functional Met; PM: Met pseudogene.

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Fig. 5. The relationship of Cynoglossinae species and intergenic space between tRNA-QIM. G1, G2 and G3 indicate the intergenic space between Q and I, I and M, M and ND2, respectively. The symbol of ★ represents the exception of gene rearrangement in Cynoglossus melampetalus. G1, G2 and G3 indicate the intergenic space between I and M, M and Q, Q and ND2, respectively.

73 bp) appear in C. lineolatus, C. abbreviates, C. gracilis, and C. semilaevis located in the middle branches of the tree, and the smallest gaps (19 to 3 bp) in C. trigrammus, C. melampetalus, C. joyneri, C. puncticeps, P. blochii, P. japonica, and P. bilineata were located in the top of the tree. This trend suggests that the initial event of gene rearrangement is more likely to occur in the early stage of differentiation of Cynoglossinae, and that with the evolution of tongue sole, the intergenic space (G1) decreases progressively under the degradation pressure of non-functional genes. As for the unique IMQ gene order and long intergenic space in C. melampetalus mitogenome, no plausible explanation has been provided at present. Maybe with the accumulation of the novel rearranged mitogenomes, more evolutionary clues will be presented in the future. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2020.02.017. CRediT authorship contribution statement Li Gong: Conceptualization, Supervision, Writing - original draft. Xinting Lu: Methodology, Project administration, Data curation. Hairong Luo: Methodology, Project administration, Data curation. Ying Zhang: Methodology, Project administration, Data curation. Wei Shi: Resources, Software, Visualization. Liqin Liu: Resources, Software, Visualization. Zhenming Lü: Resources, Software, Visualization. Bingjian Liu: Writing - review & editing. Lihua Jiang: Writing - review & editing. Declaration of competing interest The authors declare that they have no conflicts of interest on the contents of this manuscript. Acknowledgments This work was supported by the National Natural Science Foundation of China (41706176, 31471979), Open Fund of the CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences (2018011001), Basic Scientific Research Operating Expenses of Zhejiang Provincial Universities (2019J00022) and Introduction of Talent of Zhejiang Ocean University.

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