Biodiversity Data Journal 11: 93947 CO) doi: 10.3897/BDJ.11.e93947 open access Research Article Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, Acheilognathinae): characterisation and phylogenetic analysis Jinhui Yu*, Xin Chen*, Ruyao Liu*, Yongtao Tang*, Guoxing Nie*, Chuanjiang Zhou + College of Fisheries, Henan Normal University, Xinxiang, China § College of Life sciences, Henan Normal University, Xinxiang City, China Corresponding author: Chuanjiang Zhou (chuanjiang88@163.com) Academic editor: Yahui Zhao Received: 24 Aug 2022 | Accepted: 01 Feb 2023 | Published: 21 Feb 2023 Citation: Yu J, Chen X, Liu R, Tang Y, Nie G, Zhou C (2023) Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, Acheilognathinae): characterisation and phylogenetic analysis. Biodiversity Data Journal 11: e93947. https://doi.org/10.3897/BDJ.11.€93947 Abstract Acheilognathus barbatulus is distributed in Yangtze River, Yellow River and Pearl River systems in China. Genome data can help to understand the phylogenetic relationships of A. barbatulus, but its complete mitochondrial genome has not been published. We determined the complete mitochondrial genome structure and characteristics of this species and constructed a comprehensive phylogenetic tree, based on mitochondrial genome data of several species of Acheilognathus, Rhodeus and Pseudorasbora parva. The complete length of the mitochondrial genome of A. barbatulus is 16726 bp. The genome is a covalently closed double-stranded circular molecule containing 13 protein- coding genes, two ribosomal RNAs, 22 transfer RNAs, a D-loop and a light strand replication initiation region. The base composition of the complete mitochondrial genome is A (29.33%) > T (27.6%) > C (26.12%) > G (16.95%), showing a strong AT preference and anti-G bias. All 13 PCGs have different degrees of codon preference, except for cytochrome c oxidase 1, which uses GTG as the start codon. All the PCGs use ATG as the start codon and the stop codon is dominated by TAG. The encoded amino acids Leu and Ser exist in two types, whereas the rest are all present as one type, except for tRNAS® © Yu J et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Yu J et al (SCT) which lacks the D-arm and has an incomplete secondary structure, all other tRNAs can be folded to form a typical cloverleaf secondary structure. Based on the 13 PCG tandems, the Maximum Likelihood and Bayesian trees were constructed, based on the concatenated sequence of 13 PCGs for the genera Acheilognathus and Rhodeus, with Pseudorasbora parva as the outgroup. Acheilognathus barbatulus, Acheilognathus tonkinensis and Acheilognathus cf. macropterus were clustered together and the most closely related. The results of this study enrich the mitochondrial genomic data of Acheilognathus and provide molecular and genetic base information for species conservation, molecular identification and species evolution of Acheilognathinae. Keywords Acheilognathus barbatulus, mitochondrial genome, phylogenetic relationships Introduction Species in subfamily Acheilognathinae (Cypriniformes, Cyprinidae) mostly inhabit shallow and still water areas in rivers, lakes and reservoirs. Acheilognathus barbatulus Gunther belongs to subfamily Acheilognathinae within Cyprinidae; Acheilognathinae comprises approximately 72 species (Fan et al. 2020) and six valid genera, viz. Acheilognathus Bleeker, see Bleeker (1860), Paratanakia Chang (Chang et al. 2014), Pseudorhodeus Chang (Chang et al. 2014), Rhodeus Agassiz (Agassiz 1832), Sinorhodeus Li (Li et al. 2017) and Tanakia Jordan & Thompson (Arai and Akai 1988, Jordan and Thompson 1914). Except for Rhodeus amarus Bloch and Rhodeus colchicus Bogutskaya & Komlev, which are distributed in Europe, the other species are mainly distributed in East and Southeast Asia (Berg 1949, Lelek 1987, Arai and Akai 1988, Bogutskaya and Komlev 2001). Acheilognathus is widely distributed in the Yangtze River, Min River, Han River and Yellow River in China. During the breeding season, the female fish has an extended oviduct and lays eggs in the gill cavity of mussels. Male sperm enters the inlet tube and gills of the mussel to complete fertilisation. The fertilised zygotes hatch and develop in the gills of mussels until they gain the ability to swim independently (Smith et al. 2000, Kottelat 2001, Reichard et al. 2010). Then, the zygotes leave the mussel body to complete their development. Given its unique reproductive mode and morphological diversity, Acheilognathus has attracted great interest from scientists. Researchers have attempted to construct a phylogenetic tree that is in line with the rate of species evolution through various single-gene analysis methods. However, the phylogenetic relationship and taxonomic status of Acheilognathinae have not been explained properly. Therefore, the genomic data of Acheilognathinae fishes must be supplemented to provide genomic resources with potential information for future evolutionary analysis of Acheilognathinae. The mitochondrion possesses a separate genome (mitochondrial genome, mtDNA) and a relatively independent genetic system (Chinnery and Schon 2003). The mitochondrial genome of multicellular animals is usually a covalently closed double-stranded circular molecule. Linear DNA molecules are only present in certain cnidarians (Bridge et al. 1992). Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, ... 3 They have molecular lengths in the range of 14.8-19.9 Kb and the following features: tightly arranged genes (Gyllensten et al. 1985), no introns, simple structure, strict matrilineal inheritance (Li 2006), no recombination and high coding efficiency. Thus, linear DNA molecules are widely used in population genetics (Kuang et al. 2019), evolutionary biology (Chan et al. 2019) and phylogeography (Min-Shan et al. 2018). The development of genomics has allowed the analysis of numerous mitochondrial genomes (mtDNA) and advancement of the study of population genetic structure, conservation biology and evolutionary genetics of fish (Moritz et al. 1987, Guo et al. 2004, Chen et al. 2011). Phylogenetic analysis of multiple genes in tandem can provide more accurate information than single-gene analysis. Given the relationship between evolution rate and time, different genes have various effective information sites and resolutions. To this end, the gene sequence is connected in series, which can increase the number of effective information sites. The evolution rate of 13 protein-coding genes (PCGs) is faster than that of nuclear genes and the evolution rate of each PCG is diverse, which is in line with species research at different levels (Brown et al. 1979). Therefore, we provide the detailed description (genome length and type, PCGs, non-coding genes and RNA features) and comparative analyses of the A. barbatulus mitochondrial genome. A. barbatulus belongs to subfamily Acheilognathinae. This species is a small fish that lives in the Yangtze River, Yellow River, Pearl River and other river systems. It lays eggs in the gills of mussels and feeds on aquatic higher plants and algae. In previous studies, the phylogenetic relationship of A. barbatu/us has not been unified in accordance with different classification methods. With the wide use of multi-site sequence analysis, we downloaded the existing mitochondrial genome data of Acheilognathus and Rhodeus fish species from the National Center for Biotechnology Information (NCBI). With Pseudorasbora parva as the outgroup, 13 PCGs were connected in series and Maximum Likelihood (ML) and Bayesian (Bayes) tree were constructed, based on the optimal nucleotide substitution model and optimal partition model, respectively. The description in this paper is expected to provide support and theoretical supports for the evolutionary development of Acheilognathinae in the future. Material and methods Sample collection and raw data generation Samples of A. barbatulus were collected from Poyang Lake in northern Jiangxi Province, China. The experimental material for this study was provided by the Institute of Hydrobiology, Chinese Academy of Sciences. Specimens were stored in absolute ethanol at the College of Fisheries Henan Normal University. In the sampling process, the specific sampling location was not clearly marked, which led to the failure to obtain the latitude and longitude information of the sampling point. Samples from A. barbatulus were extracted using the phenol-chloroform protocol (Sambrook and Russell 2001), dissolved by the addition of 40 ul double-distilled water and stored at -20°C. The 30x genome of A. barbatulus was re-sequenced by Personalbio (Nanjing) using high-throughput sequencing 4 Yu J et al technology to obtain the whole genome. The whole genome sequence of A. barbatulus was compared with the mitochondrial genome sequence of Acheilognathus tonkinensis to determine the star and termination sites of the gene. Then, the mitochondrial genome was extracted from the whole genome data. MitoZ (httos://github.com/linzhi2013/MitoZ) was used to obstain the complete mitochondrial genome (GenBank format) (Meng et al. 2019). We constructed libraries with 400 inserts and sequenced them using Next-generation sequencing and paired-end (PE) sequencing, based on Illumina NovaSeq sequencing platform. The bwa (0.7.12-r1039) mem programme was used to compare the filtered high- quality data with the reference genome and the parameters were compared, based on the default parameters of bwamem. Picard 1.107 software (http:/(www.psc.edu/index.php/user- resources/software/picard) was used to sort and convert the sam files to bam files. We used the “FixMatelnformation” command to ensure consistency between all PE reads information. The total number of reads in this specie was 149005815 and the number of reads on the reference genome accounted for 92.01% of the total number of reads. Mitogenome annotation The complete mitochondrial genome sequence was obtained by comparing it to the published mitochondrial genome of subfamily Acheilognathinae in NCBI. The position of start transfer RNA (tRNA®) was determined using the MITOS website (http:// mitos2.bioinf.uni-leipzig.de/index.py) (Bernt et al. 2013). Preprocessing of each gene on the mitochondrial genome was based on the mitochondrial codon of bony fish and by checking whether the mitochondrial genome was ringed. This process yielded a gene map of the A. barbatulus mitochondrial genome. Mitofish (http://mitofish.aori.u-tokyo.ac.jp/ annotation/input.htm!l) was used to determine the positions of 13 PCGs, 22 tRNAs, two ribosomal RNA (rRNAs), control regions and the types and numbers of anticodons and start and stop codons. The tRNAscan-SE website (http://lowelab.ucsc.edu/tRNAscan-SE/) (Lowe and Chan 2016) was used to confirm the positions of tRNA genes and predict secondary structures. PCGs were translated exactly using MEGA7.0 (Sudhir et al. 2016). Base composition and relative synonymous codon usage (RSCU) were calculated by MEGA7.0. AT:GC skew was calculated using Perna's formula (Perna and Kocher 1995), with AT Skew = (A - T)/(A + T) and GC Skew = (G - C)((G + C). CodonW 1.4.2 software (John et al. 1999) was used to obtain the codon adaptation index (CAI), effective codon number (ENC), GC and GC3. Comparative analyses To study the phylogenetic position of A. barbatulus, we selected a part of the fish in the Acheilognathus and Rhodeus data provided by NCBI. Given that subfamilies Gobioninae and Acheilognathae are sister groups (Chen 2014), Pseudorasbora parva was used as the phylogenetic outgroup. A total of 33 complete mitochondrial genome sequences were selected. Table 1 shows the GenBank accession numbers and species names. Partitioned phylogenetic analysis allows the use of different nucleotide substitution models and corresponding parameters for different subsets of association data. This step helps to explore evolutionary models specific to each partition and reduce systematic errors to Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, ... 5 improve the accuracy of phylogenetic inference (Wang et al. 2012). One partitioned phylogenetic analysis method is based on Bayes principle (Ronquist and Huelsenbeck 2003) and the other is based on the ML partitioned analysis method (Stamatakis 2006). A total of 13 PCGs, two rRNA genes and 22 tRNA genes of each species were extracted using Phylosuite software (Zhang et al. 2019). MEGA7.0 (Sudhir et al. 2016) software was used to individually compare the extracted PCGs by ClustalW (John et al. 1999) (ATP6 = 684 bp, ATP8 = 165 bp, CYTB = 1141 bp, COX1 = 1551 bp, COX2 = 691 bp, COX3 = 785 bp, ND1 = 975 bp, ND2 = 1047 bp, ND3 = 351 bp, ND4 = 1381 bp, ND4L = 297 bp, ND5 = 1836 bp and ND6 = 522 bp). Table 1. Genome sequences from NCBI in this study. GenBank Accession Speices Length AT (%) LC494270 Acheilognathus tabira erythropterus 16770 56 LC494269 Acheilognathus tabira tohokuensis 16774 56 NC028736 Acheilognathus majusculus 17155 56.8 LC631940 Acheilognathus longipinnis 16772 58 AP013345 Acheilognathus cf. macropterus 16761 57.6 AP013343 Acheilognathus tabira jordani 16765 56.4 AP013342 Acheilognathus rhombeus 16783 56.5 AB239602 Acheilognathus typus 16778 57 AP013348 Acheilognathus sp. CBM ZF 11927 16254 56.2 AP013347 Acheilognathus tabira nakamurae 16343 56.6 AP013346 Acheilognathus cyanostigma 16454 58.9 MZ334545 Acheilognathus hypselonotus 16706 57.1 NC042407 Acheilognathus tonkinensis 16767 56.5 NC037404 Acheilognathus omeiensis 16774 56.7 NC028433 Acheilognathus rhombeus 16780 56.9 KJ499466 Acheilognathus macropterus 16773 57.6 NC013712 Acheilognathus yamatsutae 16703 56.7 AP012985 Acheilognathus melanogaster 16556 56.8 NC031152 Acheilognathus meridianus 16563 57.9 AP013344 Acheilognathus tabira tabira 16771 56.1 NC031538 Rhodeus amarus 16607 55.6 AP011255 Rhodeus atremius atremius 16734 55 AB070205 Rhodeus ocellatus kurumeus 16674 56.5 NC029718 Rhodeus notatus 16735 55.3 NC027437 Rhodeus fangi 16733 55.3 6 Yu J et al GenBank Accession Speices Length AT (%) NC025326 Rhodeus sericeus 16581 55.5 NC024885 Rhodeus lighti 16677 56.1 MW/007386 Rhodeus ocellatus 16574 52.9 NC013709 Rhodeus suigensis 16733 55.1 NC022721 Rhodeus sinensis 16677 56.3 NC060735 Rhodeus cyanorostris 16580 54.9 NC011211 Rhodeus ocellatus 16680 56.4 KJ135626 Pseudorasbora parva 16600 58.9 The sequences were aligned, based on the mitochondrial genome by Phylosiute (Zhang et al. 2019) for the concatenation of PCGs to form a 13 PCG dataset. The best partitioning scheme and the corresponding optimal nucleotide substitution model were determined in accordance with to the Akaike Information Criterion (AIC) criteria using ModelFinder (Kalyaanamoorthy et al. 2017) and a ML tree was established by an edge-linked partitioning model based on IQ-TREE with 5000 ultrafast self-spreading values (Stamatakis 2006). Bayes were constructed, based on Bayesian Information Criterion (BIC) using PartitionFinder (Lanfear et al. 2017) to select the best partition and associated optimal base substitution model (Huelsenbeck 2001, Drummond and Rambaut 2007). Bayes inference phylogenies were inferred using MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003) under an N/A model (two parallel runs, 2000000 generations), in which the initial 25% of sampled trees were discarded as burn-in. Data resources GenBank accession number ON815031 Results Genome size and organisation The complete mitochondrial genome (GenBank accession number ON815031) of A. barbatulus is 16726 bp in total length and a typical covalently closed double-stranded cyclic molecule (Fig. 1). The mitochondrial genome of A. barbatulus is broadly similar to that of other vertebrates and includes 13 PCGs, namely, ATP6, ATP8, COX1, COX2, COX3, Cytb, ND1, ND2, ND3, ND4, ND4L, ND5 and ND6, 2 rRNA genes, namely, 12S rRNA and 16S rRNA genes and 22 tRNA genes, for a total of 37 genes. A non-coding region (the control region) that controls gene replication and transcription (Forst and Schulten 2001) and a light strand replication initiation region (OL) were detected. The OL is located between tRNA“S" and tRNA‘°YS and has a length of 31 bp and it can fold to form a secondary structure with a stem-loop structure. This region is highly conserved and related to the replication function of the L-strand. Except for eight tRNAs and one PCG (ND6) Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, ... 7 located on the light strand (L strand), the remaining 28 genes were located on the heavy strand (H strand) of the mitochondrial genome and the arrangement of the genes was consistent with the typical genetic composition of teleost fish (Fig. 1, Table 2). Table 2. Mitochondrial genes and associated features of Acheilognathus barbatulus. Intergenic space (IGS) described as intergenic (+) or overlapping nucleotides. Locus Type One- Strand Amino Position Codon letter acids code Start Stop Length Start Stop Anti- Intergenic (bp) condon _ nucleotide tRNAPRE tRNA F H 1 69 69 GAA 0 12S rRNA H 70 1025 956 1 rRNA tRNAY?! tRNA V H 1027 1098 72 TAC 16 16S rRNA H 1115 2774 1660 0 rRNA tRNALEY tRNA 2 H 2775 2850 76 TAA 0 NAD‘ Protein- H 324 2851 3825 975 ATG TAA 4 coding tRNA'® = tRNA | H 3830 3901 72 GAT “2 tRNAS&" tRNA Q Ls 3900 3970 71 TTG 1 tRNAMet tRNA H 3972 4040 69 CAT 0 NAD2 Protein- H 348 4041 5087 1047 ATG TAG =o coding tRNATP tRNA Ww H 5086 5155 70 TCA 1 tRNA“ tRNA A L 5157 5225 69 TGC 1 tRNA“S" tRNA L 5227 5299 73 GTT 2 OL H 5302 5332 31 -2 tRNACYS tRNA L 5331 5398 68 GCA 0 tRNATY. tRNA L 5399 5469 71 GTA 1 COx1 Protein- H 516 5471 7021 1551 GTG TAA 0 coding tRNAS® tRNA S2 L 7022 7092 71 TGA 2 tRNAASP) tRNA D H 7095 7165 71 GTC 7 COX2 __ Protein- H 230 7173 7863 691 ATG T(AA) 0 coding tRNALYS tRNA K H 7864 7939 76 TIT 1 ATP8 Protein- H 54 7941 8105 165 ATG TAG “7 coding Locus ATP6 COX3 tRNACY NAD3 tRNA‘'9 NAD4L NAD4 tRNAMis tRNASE" tRNALeY NAD5 NAD6 tRNACS CYTB tRNA tRNAPro D-loop Type Protein- coding Protein- coding tRNA Protein- coding tRNA Protein- coding Protein- coding tRNA tRNA tRNA Protein- coding Protein- coding tRNA Protein- coding tRNA tRNA Non- coding One- letter code G R H S1 LI Strand Amino oe Oe ee Gee Be ee acids 227 261 116 98 460 611 173 380 Yu J et al Position Start 8099 8782 9566 9637 9986 10055 10345 11726 11795 11865 11938 13770 14292 14363 15504 15576 16052 Stop 8782 9566 9636 9987 10054 10351 11725 11794 11863 11937 13773 14291 14360 15503 15576 15645 16630 Similar to other bony fish, spacers and overlap the total gene length, were obtained. Length (bp) 684 785 71 351 69 297 1381 69 69 73 1836 522 69 1141 73 70 579 between genes were present. Gene overlapping promotes miniaturisation of mitotic genes, shortens genome replication time and offers a natural selection advantage (Boyce et al. 1989). The overlaps of coding genes were located between tRNA'®tRNAS&" ND2-tRNA™?, OL-tRNACYS, ATP8-ATP6, ATP6- COX3, COX3-tRNA®Y, ND3-tRNA*"9, ND4L-ND4, ND5-ND6 and tRNA™""-tRNA P'° and ten gene intervals had a total of 29 bp, which accounted for 0.17% of the total gene length. A total of 15 spacers encoding genes with a total length of 542 bp, accounting for 3.24% of Codon Start ATG ATG ATG ATG ATG ATG ATG ATG Stop TAA TA(A) TAG TAA TAA Anti- condon TCC TCG GTG GCT TAG TTC TGT. TGG Intergenic nucleotide -1 4 406 Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, ... @ —-tRNA-Phe os 1 P all\\\ 1 It) Ni; x _ =" im —tRNA-llc — —tRNA-Gin = 4kb - a RNA-Met -ND2 ee HTT Te eee kh ATPase 6~ Figure 1. EES] Gene map of the Acheilognathus barbatulus mitochondrial genomes. The genome contained two rRNA genes (in yellow), 13 coding genes (in black), 22 tRNA genes (in red) and a control region (D-loop) (in brown). The outer ring corresponds to the H- (outermost) and L-strands and depicts the location of PCGs (except for ND6 which is encoded in the L-strand and is portrayed in red). The inner ring (black sliding window) denotes GC content along the genome. Base composition The highest base content in A. barbatulus was that of A (29.33%), followed by those of T (27.6%) > C (26.12%) > G (16.95%). The complete mitochondrial genome of A. barbatulus showed an anti-G bias and AT preference (Table 3), a phenomenon similar to the base composition of most other teleost fishes (Perna and Kocher 1995). This base preference was also present in other parts of the mitochondrial genome, with the D-loop region, tRNAs and PCGs all having considerably greater AT content than CG content and showing a strong AT preference. Of the 13 PCGs, 10 showed an anti-A bias, except for ATP8, COX2 and ND2 which showed an A bias. Except for ND6, which showed an evident G bias, 12 10 Yu J et al genes showed anti-G bias. Meanwhile, the RNAs exhibited strong A-bias and anti-G-bias. The 12S rRNA and 16S rRNA genes with larger absolute values of AT-skew and smaller absolute values of GC-skew had a strong A-bias. ND6 and tRNAs genes have the same degree of base G-bias and T-bias. From the base composition of mitochondrial genes on both sides of A. barbatulus, PCGs showed a significant anti-G bias, followed by the D-loop region, 12S rRNA and 16S rRNA. Only tRNAs revealed a G bias. Table 3. Nucleotide composition of the complete Acheilognathus barbatulus mitochondrial genomes (and concatenated PCGs, rRNA, D-loop) analysed in this study. Region Base composition (%) Total T Cc A G AT(%) Alskew GCskew ATPase 6 683 30.75 28.11 26.35 14.79 57.10 -0.08 -0.31 ATPase 8 165 27.27 24.24 3455 13.94 61.82 0.12 -0.27 Col 1551 30.82 26.05 25.02 18.12 55.83 -0.10 -0.18 COIl 691 29.09 25.47 29.52 15.92 58.61 0.01 -0.23 COIll 784 29.97 26.28 25.13 18.62 55.10 -0.09 -0.17 Cytb 1141 31.11 26.12 27.70 15.07 58.81 -0.06 -0.27 ND1 975 30.67 27.49 25.74 16.10 56.41 -0.09 -0.26 ND2 1045 26.22 31.39 27.75 14.64 53.97 0.03 -0.36 ND3 349 30.37 28.08 25.50 16.05 55.87 -0.09 -0.27 ND4 1381 29.76 27.23 28.39 14.63 58.15 -0.02 -0.30 ND4L 297 29.29 28.28 26.94 15.49 56.23 -0.04 -0.29 ND5 1836 29.74 26.96 28.70 14.60 58.44 -0.02 -0.30 ND6 522 37.93 13.60 15.52 32.95 53.45 -0.42 0.42 PCGs 11420 30.17 26.58 26.73 16.52 56.89 -0.06 -0.23 tRNAs 1561 27.35 21.33 28.76 22.55 56.12 0.03 0.03 D-loop 579 29.53 24.01 28.32 18.13 57.86 -0.02 -0.14 12S rRNA 957 19.54 26.96 31.03 22.47 50.57 0.23 -0.09 16S rRNA 1676 21.30 22.49 34.84 21.36 56.15 0.24 -0.03 Complete genome 16726 27.60 26.12 29.33 16.95 56.93 0.03 -0.21 Protein-coding genes The mitochondrial genome of A. barbatulus contains 13 PCGs with a total length of 11,420 bp, which accounts for 68.28% of the entire mitochondrial genome. Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, ... 11 Amino acid and codon usage The PCGs of A. barbatulus encode 3798 amino acids and, amongst the encoded amino acids, the Leu, Ser, Pro and Thr exhibit high contents. Low contents of most amino acids, such as Arg, Cys, Glu and Asp, can be observed (Table 4), with the codon encoding Leu being used the most frequently and the one encoding Cys being used the least frequently. The A. barbatulus codons with RSCU values > 1.0 have positive codon usage bias (CUB) and are defined as abundant codons, whereas those with RSCU values < 1.0 have negative CUB and are defined as less-abundant codons (Gun et al. 2018). The statistics of codon usage frequency and RSCU of PCGs (Fig. 2) showed that codons containing bases A and U, such as AAA, AAU and UAA, are used more frequently, whereas those containing bases C and G, such as GGG, GGC and CGC, are used less frequently. According to the comparison of RSCU, for the genes encoding A. barbatulus protein, the most frequently used codons were UCU (1.46%), GCC (1.38%), AAU (1.37%), and AAA (1.36%), which encode amino acids Ser, Ala, lle and Lys, respectively, whereas codons encoding amino acids Ala (GCG, 0.36%), Ser (UCG 0.46%) and Thr (ACG, 0.52%) had the lowest codon frequency. Table 4. Results from the Relative Synonymous Codon Usage (RSCU) analysis for the PCGs of the mitochondrial genome of Acheilognathus barbatulus. * denotes stop codon. AA Codon Count RSCU AA Codon Count RSCU Phe UUU(F) 150 1.3 Tyr UAU(Y) 145 1.08 UUC(F) 81 0.7 UAC(Y) 123 0.92 Leu UUA\(L) 127 1.24 UAA(*) 155 1.53 UUG(L) 104 1.01 UAG(*) 96 0.95 CUU(L) 114 1.11 His CAU(H) 121 1.08 CUC(L) 109 1.06 CAC(H) 103 0.92 CUA(L) 92 0.9 Glin CAA(Q) 125 1.28 CUG(L) 70 0.68 CAG(Q) 70 0.72 lle AUU(I) 151 1.37 Asn AAU(N) 160 1.08 AUC(I) 69 0.63 AAC(N) 135 0.92 Met AUA(M) 107 1.14 Lys AAA\(K) 153 1.36 AUG(M) 81 0.86 AAG(k) 72 0.64 Val GUU(V) 47 1.21 Asp GAU(D) 71 1.06 GUC(V) 30 0.77 GAC(D) 63 0.94 GUA(V) 48 1.24 Glu GAA(E) 96 1.29 GUG(V) 30 0.77 GAG(E) 53 0.71 Ser UCU(S) 128 1.46 Cys UGU(C) 42 0.74 UCC(S) 90 1.03 UGC(C) 72 1.26 12 Pro Thr Ala RSCU Value Codon UCA(S) UCG(S) CCU(P) CCC(P) CCA(P) CCG(P) ACU(T) ACC(T) ACA(T) ACG(T) GCU(A) GCC(A) GCA(A) Figure 2. EES) Results from analysis of Relative Synonymous Codon Usage (RSCU) of the mitochondrial genome of Acheilognathus barbatulus. Codon families are plotted on the x-axis. The label for the 2, 4 or 6 codons that compose each family is shown in the boxes below the x-axis and the colours correspond to those in the stacked columns. RSCU values are shown on the y-axis. Yu J et al Count RSCU AA 88 1.01 Trp 40 0.46 166 1.32 Arg 149 1.18 113 0.9 75 0.6 121 1.21 Ser 133 1.33 95 0.95 52 0.52 55 1.03 Gly 74 1.38 66 1.23 19 0.36 Start codon and stop codon Codon UGA(W) UGG(W) CGU(R) CGC(R) CGA(R) CGG(R) AGU(S) AGC(S) AGA(*) AGG(*) GGU(G) GGC(G) GGA(G) GGG(G) AT WWM Count RSCU 84 1.11 67 0.89 39 1 45 1.15 25 0.64 47 1.21 68 0.78 111 1.27 75 0.74 80 0.79 36 0.7 55 1.07 52 1.01 62 1.21 Most of the start codons of A. barbatulus are ATG. Only the COX1 gene has GTG as the start codon. ND1, COX1, ATP6, ND4L, ND5 and ND6 use TAA as the complete stop Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, ... 13 codon. ND2, ATP8 and ND3 use TAG as the complete stop codon, whereas COX2, COX3, ND4 and CYTB use TA- and T-- as incomplete stop codons. Codon usage bias Codon usage bias (CUB) refers to the unequal use of synonymous codons in organisms. CUB is affected by mutation and selection pressure and is an important feature of biological evolution. It not only affects gene function and expression potential, but also the accuracy and efficiency of translation. The higher the gene expression level, the stronger the CUB. To investigate codon usage preference in the mitochondrial genomes of A. barbatulus, we calculated CAl, ENC, GC and GC3 using CodonW1.4.2. Effective number of condon (ENC) can describe the extent to which codon usage deviates from random selection, with values generally varying from 20 to 61. The larger the ENC value, the lower bias of expression genes towards the use of rare codons. The smaller the ENC value, the greater the preference for codons of genes with high expressions. The ENC values of PCGs in the mitochondrial genome of A. barbatulus ranged from 39 to 55.71 (Table 5) and were mostly concentrated in the range of 43-46, indicating a degree of codon usage preference. Codon adaptation index (CAI) values ranged from 0 to 1, with high values indicating high gene expression levels and pronounced CUB (Sharp and Li 1987). ATP8 and the three cytochrome oxidase subunits (COX1, COX2 and COX3) showed high CUB and gene expression levels. ATP6 exhibited the lowest CUB and expression levels amongst the PCGs (Table 5). GC3 refers to the GC content of the third position of all codons in a gene. In addition to methionine, tryptophan and termination codons, G and C may appear in the third codon position. The results showed that the 13 PCGs had a low GC content and a low probability of G and C in the third codon position. This finding also demonstrated that the A. barbatulus PCGs showed a strong AT preference (Table 3). Table 5. Preference for codon usage of genes encoding proteins in mitochondrial genome of Acheilognathus barbatulus. CAI ENC GC GC3 ATP6 0.117 48.05 0.432 0.361 ATP8 0.257 44.06 0.388 0.333 Col 0.177 44.77 0.446 0.374 COll 0.186 52.50 0.416 0.332 COIII 0.196 46.88 0.454 0.380 Cytb 0.160 43.52 0.415 0.363 ND1 0.121 46.39 0.440 0.361 ND2 0.139 45.89 0.464 0.399 ND3 0.126 44.61 0.445 0.400 14 Yu J et al CAI ENC GC GC3 ND4 0.125 45.72 0.422 0.343 ND4L 0.116 39.00 0.443 0.365 ND5 0.152 47.11 0.418 0.394 ND6 0.141 45.53 0.470 0.442 PCGs -0.037 55.71 0.420 0.444 tRNA and rRNA The animal mitochondrial genome has two types of ribosomal units: the large 16S and small 12S subunits. The 16S subunit is more conserved than the 12S and the secondary structure of both rRNA genes is more conserved than the sequence (Noack et al. 1996). The 12S rRNA is located between tRNAP® and tRNA! with a total length of 956 bp, accounting for 5.72% of the complete mitochondrial genome and 50.57% of the AT content. The 16S rRNA is located between tRNA“?! and tRNA‘®. with a total length of 1660 bp, accounting for 9.92% of the total mitochondrial genome and 56.15% of the AT content. A. barbatulus has 22 tRNA genes (eight in the L-strand and 14 in the H-strand). Their individual gene lengths ranged from 68 bp to 76 bp. Except for Leu and Ser, which both contain two types, the other 18 tRNAs have only one type. Except for tRNAS® (G7) (L strand), which lacks the D-arm resulting in an incomplete secondary structure, all 21 tRNAs can fold into the canonical cloverleaf secondary structure (Fig. 3), which is a condition that has been reported for the mitochondria of other fish species (Broughton et al. 2001, Hwang et al. 2013). The cloverleaf structure consists of four domains (AA stem and D, AC and T arms) and a variable loop (Fig. 3). For their normal functioning, these aberrant tRNAs may require co-evolutionary interaction factors or post-transcriptional RNA editing (Masta et al. 2004). Phylogeny and systematics of Acheilognathus barbatulus In this study, the ML tree and MrBayes were constructed by tandemly linking 13 PCGs and using Pseudorasbora parva as an outgroup partition to accurately reveal the phylogenetic relationships of A. barbatulus. The best partitioning ML and Bayes models were based on AIC and BIC for 34 PCG tandem sequences and the most suitable nucleotide-substitution models, respectively. The best partitioning models for ML were GTR+F+I+G4: ND1, ND3, ND4, ATP6; GTR+F+l+G4: ND2; GTR+F+l+G4: ND5; GTR+F+l+G4: ND6; HKY+F+1+G4: ATP8; GTRtF+l+G4: COl; GTR+F+l+G4: COIl; GTR+F+Il+G4: COIIl, Cytb, ND4L. The optimal partitioning models of ML and Bayesian were slightly different, with a slight difference in node support. The topological structure was consistent overall, but parts of the support rate or posterior probability of the nodes is inconsistent. This finding may be due to different ML and Bayes algorithms being different, resulting in differences in the subsequent phylogenetic trees. The results support the conclusion that Acheilognathus and Rhodeus constitute monophyletic taxa (Yang 2010) A. barbatulus is most closely Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, ... 15 related to Acheilognathus tonkinensis and Acheilognathus cf. macropterus (Chen 2011). The node support rate of ML and MrBayes is 100/100 (Figs 4, 5, respectively). Phenylalanine(Phe) Valine(Val) Leucine(Leu) Isoleucine(lle) Methionine(Met) Trvptophane(Trp) Aspartic(Asp) Lysine(Lys) Glycine(Gly) Arginine(Arg) Histidine(His) Serine(Ser) Leucine(Leu) Threonine(Thr) Proline(Pro) Glutamic(Glu) Serine(Ser) Tyrosine(Tyr) Cysteine(Cys) Asparagine(Asn) Alanine(Ala) Glutamine(Gin) Figure 3. EES Secondary structure of the 22 tRNA genes of the mitochondrial genome of Acheilognathus barbatulus predicted by tRNAScan-SE 2.0. Discussion We successfully obtained and annotated the mitochondrial genome data of A. barbatulus. The length of mitochondrial genome is 16726 bp, which is similar to the length of other fish 16 Yu J et al genomes in Acheilognathinae, such as those of A. signifier (16566 bp) (Hwang et al. 2012), A. somjinensis (16569 bp) (Hwang et al. 2014) and A. hypselonotus (16706 bp) (Zhu et al. 2021). The differences in mitochondrial gene length in these species may be due to changes in the control tandem repeat sequences (Wang et al. 2020). Consistent with the absorptive stereogenomic structure of other teleost fish, the A. barbatu/lus mitochondrial genome contains 13 PCGs, two rRNA genes and 22 tRNA genes, a non-coding control region (D-loop) and OL. Genes are mainly distributed in the H strand and only ND6 and 8 tRNAs can be found in the L strand. The arrangement of genes was consistent with the typical genetic composition of Acheilognathinae (Hwang et al. 2012, Hwang et al. 2014, Zhu et al. 2021). On the basis of composition, the complete mitochondrial genome of A. barbatulus showed an anti-G bias and AT preference (Table 3), a phenomenon similar to the base composition of most other teleost fishes (Perna and Kocher 1995). ADUIAPOYY a~- 2085) snyjpusoplayopy Figure 4. EES] Phylogenetic trees derived from the Bayes approaches, based on concatenation of PCGs. The numbers on the nodes are the bootstrap values of Bayes. The number after the species name is the GenBank accession number. Outgroup taxa are shown, the text is bolded by the Acheilognathus barbatulus. By analysing the relative usage frequency (RSCU) of synonymous codons of A. barbatulus mitochondrial genome coding genes, we observed that the RSCU values of 35 codons, such as UCU, AUU and AAA, were greater than 1 (Fig. 2), which indicates that these codons were part of the A. barbatulus mitochondrial genome. Amongst the 35 codons, 12 codons end with G/C and the other codons end with A/T bases (65.71%), which implies that A. barbatulus mitochondrial genome codons are inclined to use codons ending with A/ T, whereas codons ending with G/C are less used. A. barbatulus mostly uses ATG and TAA as the starting codons and COX1 uses GTG as the starting codon. The termination codons are mainly incomplete codons, such as TA- and T--. Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, ... 17 0 Acheilognathus barbatulus pk 100 — Achvilognathus sp CBM ZP 11927 (APO13348) = za Acheile tines tonkinensis (NCO42407) ~” athus yptere od cheilognathus 99466) 9 ss (MZ334545) 8 (LC631940) #s (AB239602) iensis (NCO37404) aterus (1,C494270) a tohokwensis (1.C494269) dani (APO13343) snyjousoplayop amurae (APO13347) 712) a (APO13346) 6) ster (APO12985) r— “a Rhodeus eyanorostris (NC060735) rremius (APOLI25S angi (NC027437) s (NCO29718) NCO13709) 24885) s kurumeus (ABO70205 ) aDUIapOYY es (NCOLI2ZIE) | f-— Pseudorasbora parva (KI133626) Figure 5. EES] Phylogenetic trees derived from the Maximum-Likelihood (ML) approaches, based on concatenation of PCGs. The numbers on the nodes are the bootstrap values of ML. The number after the species name is the GenBank accession number. Outgroup taxa are shown, the text is bolded by the Acheilognathus barbatulus. The secondary structure of A. barbatulus tRNA is conserved and conforms to the characteristics of fish mitochondrial genome (Broughton et al. 2001, Hwang et al. 2013). Except for tRNAS® (GCT) (L strand), which lacks the D-arm that results in an incomplete secondary structure, all 21 tRNAs can fold into the canonical cloverleaf secondary structure (Fig. 3). For normal functioning, these aberrant tRNAs may require co- evolutionary interaction factors or post-transcriptional RNA editing (Masta et al. 2004). The mitochondrial genome of animals is maternally inherited. The nucleic acid sequence and composition are relatively conserved and the gene order is relatively stable and close. Given its structural and evolutionary characteristics, the mitochondrial genome has become an ideal object for studying the origins and evolution of animals and population genetic differentiation. Thus far, phylogenetic analysis still uses single genes as the proxy of species. However, with the continuous development of technology, the inconsistency between gene and species trees has become increasingly prominent. The information sites contained in a single gene are insufficient to reconstruct the phylogenetic relationship of a group with a gene sequence. To date, increasing number of information sites and datasets are being collected, including 13 PCGs in series, to construct phylogenetic trees. Acheilognathinae have a complicated taxonomic history (Chang et al. 2014). Through a lot of scientific research, it is found that the phylogenetic relationship of Acheilognathinae is affected by many factors, such as different data analysis (Okazaki et al. 2001), limited in character sampling (Peilin et al. 2014) and gene sequence selection difference (Kawamura 18 Yu J et al et al. 2014). As a widely distributed species of Acheilognathinae, A. barbatulus also has a complex taxonomic history. This study is eager to contribute to solving the taxonomic status of Acheilognathinae by expounding the phylogenetic relationship of A. barbatulus. Previous studies have revealed different phylogenetic relationships in A. barbatulus. We first used 13 PCGs in tandem to reconstruct the phylogenetic relationship of A. barbatulus. Phylogenetic results showed topological differences compared with other studies due to variations in outgroups, comparative species, molecular markers and individual gene sequences (Yang 2010, Chang et al. 2014, Cheng et al. 2014, Kawamura et al. 2014, Miyake et al. 2021). Yang (2010) observed that the phylogenetic tree constructed, based on the Cyt b gene and RAG2, showed different results using various methods (Yang 2010). ML, Bayes and MJ trees of Acheilognathinae proved that A. barbatulus had the closest genetic relationship with A. longibarbatus, A. macropterus and A. chankaensis. However, the Bayes tree of Acheilognathinae constructed from RAG2 gene sequence showed that A. barbatulus is closely related to A. omeiensis, A. tonkinensis and A. tabira (Yang 2010). According to the results, the mitochondrial genes of Acheilognathinae vary greatly, the evolution rate of species is fast and the information sites represented by a single gene are limited. Therefore, single gene analysis is unsuitable for the phylogenetic study of Acheilognathinae. Kawamura K et al. (2014) used Cyt b sequences of 49 species or subspecies in three genera (Tanakia, Rhodeus and Acheilognathus) to construct a phylogenetic tree and observed that A. barbatulus is most closely related to A. rhombens; the authors proposed that, at the species level, the taxonomy and phylogeny between these two species are inconsistent and need to be re-assessed in future research (Kawamura et al. 2014). Based on the ND1 gene sequence, Takuya Miyake et al. (2021) concluded that A. barbatulus is closely related to the Japanese A. rhombens (Miyake et al. 2021). Chang (2014) constructed the phylogenetic relationship of Acheilognathidae, based on the tandem of six nuclear genes and Cyt b and discovered that A. rhombens is embedded in A. barbatulus and its taxonomic status has not been obtained. Thus, scientists speculated the possibility of hidden species, but further research is needed in combination with morphology (Chang et al. 2014). Cheng et al. (2014) constructed a phylogenetic tree of Acheilognathidae based on Cyt b and 12S gene sequences and obtained A. barbatulus and A. longibarbatus; meanwhile, A. rhombens is closely related to A. tonkinensis (Cheng et al. 2014). Through the above brief description, the taxonomic status of A. barbatulus in Acheilognathinae has not been well solved. In this paper, the phylogenetic tree was reconstructed by tandem reconstruction of 13 PCGs in the mitochondrial genome. A. barbatulus is most closely related to A. tonkinensis and A. cf. macropterus (Chen 2014). The node support rate of ML and MrBayes is 100/100 (Figs 4, 5, respectively). Given the functional differences in various genes, these speices may have experienced different degrees of natural selection in the course of history, resulting in completely varied gene trees using different genes in molecular phylogenetic analysis. At the same time, different analytical methods may draw different conclusions when applying the mitochondrial genome to construct phylogenetic relationships. In addition, extremely rare or unrepresentative groups included in the analysis will affect the inference of final results. Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, ... 19 Compared with other studies, this paper used mitochondrial genome PCGs to build a tree in series and obtained more gene loci and a gene tree closer to the species tree. However, with the progress of technology, inconsistencies were observed between gene and species trees and these inconsistencies may be caused by incomplete pedigree sorting, hybridisation and gene flow. Additional data, such as second-generation sequencing and simplified genomes are needed to construct more accurate phylogenetic relationships. Conclusions We report the mitochondrial genome sequence and characteristics of Acheilognathus barbatulus. The gene structure, RNA secondary structure, D-loop region and base composition were analysed. The results contribute the mitochondrial genome data of Acheilognathus and provide molecular and genetic information for species conservation, molecular identification and species evolution of Acheilognathinae. Acknowledgements This work was supported by the following funding: the National Natural Science Foundation of China (U2004146, 31872199) and the Science and Technology Innovation team supported the project (CXTD2016043) in Henan Province, China, the training plan of young excellent teachers in colleges and universities of Henan Province (2019GGJS063). This study was supported by the High-Performance Computing Center of Henan Normal University. References ° Agassiz L (1832) Untersuchungen Uber die fossilen Susswasser-Fische der tertiaren Formationen. Neues Jahrbuch fur Mineralogie Geognosie Geologie und Petrefaktenkunde 3: 129-138. ° Arai R, Akai Y (1988) Acheilognathus melanogaster, a senior synonym of A. moriokae, with a revision of the genera of the subfamily Acheilognathinae (Cypriniformes, Cyprinidae). Bulletin of the National Science Museum 14: 199-213. ° Berg LS (1949) Freshwater fishes of the U.S.S.R and adjacent countries. Moscow- Leningrad: Izd. Acad. Nausk SSSR 2: 469-925. ° Bernt M, Donath A, Juhling F, et al. (2013) MITOS: improved de novo metazoan mitochondrial genome annotation. Molecular Phylogenetics and Evolution 69 (2): 313-9. https://doi.org/10.1016/j.ympev.2012.08.023 ° Bleeker P (1860) Conspectus systematis cyprinorum. Natuurkundig Tijdschrift voor Nederlandsch Indié 20: 421-441. ° Bogutskaya NG, Komlev AM (2001) Some new data to morphology of Rhodeus sericeus (Cyprinidae: Acheilognathinae) and description of a new species Rhodeus colchicus from west Transcaucasia. Proceedings of the Zoological Institute 287: 81-97. 20 Yu J et al Boyce TM, Zwick ME, Aquadro CF (1989) Mitochondrial DNA in the bark weevils: size, structure and heteroplasmy. Genetics 123 (4): 825-836. https://doi.org/10.1093/ genetics/123.4.825 Bridge D, Cunningham CW, Schierwater B, Buss DS (1992) Class-level relationships in the phylum Cnidaria: evidence from mitochondrial genome structure. Proceedings of the National Academy of Sciences of the United States of America 89 (18): 8750-8753. https://doi.org/10.1073/pnas.89.18.8750 Broughton RE, Milam JE, Roe BA (2001) The complete sequence of the zebrafish ( Danio rerio) mitochondrial genome and evolutionary patterns in Vertebrate mitochondrial DNA. Genome Research 11 (11): 1958-1967. https://doi.org/10.1101/gr. 156801 Brown WM, Jr MG, Wilson AC (1979) Rapid evolution of animal mitochondrial DNA. Proceedings of the National Academy of Sciences of the United States of America 76 (4): 1967-1971. https://doi.org/10.1073/pnas.76.4.1967 Chan EKF, Timmermann A, Baldi BF, Moore AE, Lyons RJ, Lee S, Kalsbeek AMF, Petersen DC, Rautenbach H, Fortsch HEA, Bornman MSR, Hayes VM (2019) Human origins in a southern African palaeo-wetland and first migrations. Nature 575 (7781): 185-189. https://doi.org/10.1038/s41586-019-1714-1 Chang C, FanL, Shao K, Liu Y, Morosawa T, Kim S, Koo H, Kim W, Lee J, He S, Smith C, Reichard M, Miya M, Sado T, Uehara K, Lavoue S, Chen W, Mayden R (2014) Phylogenetic relationships of Acheilognathidae (Cypriniformes: Cyprinoidea) as revealed from evidence of both nuclear and mitochondrial gene sequence variation: Evidence for necessary taxonomic revision in the family and the identification of cryptic species. Molecular Phylogenetics and Evolution 81: 182-194. https://doi.org/10.1016/ j.-ympev.2014.08.026 Chen AH (2014) Molecular phylogeny of Gobioninae. University of Fudan University Cheng P, Yu D, Liu S, Tang Q, Liu H (2014) Molecular phylogeny and conservation priorities of the subfamily Acheilognathinae (Teleostei: Cyprinidae). Zoological science 31 (5): 300-8. https://doi.org/10.2108/zs130069 Chen §S, Qu Y, Li J (2011) Mitochondrial DNA in fish and its research progress. Biotechnology Bulletin 3: 13-20. [In Chinese]. https://doi.org/10.13560/j.cnki.biotech.bull. 1985.2011.03.033 Chen X (2011) Genetic diversity and phylogeny of Acheilognathinae in China. Chen Xin, China, 60 pp. [In Chinese]. Chinnery P, Schon E, et al. (2003) Mitochondria. Journal of Neurology Neurosurgery & Psychiatry 74 (9): 1188-1199. https://doi.org/10.1136/jnnp.74.9.1188 Drummond AJ, Rambaut A (2007) BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology 7: 214. https://doi.org/10.1186/1471-2148-7-214 Fan L, Te-Yu L, Ryoichi A (2020) Two new species of Rhodeus (Teleostei: Cyprinidae: Acheilognathinae) from the River Yangtze, China. Journal of Vertebrate Biology 69 (1): 1-17. URL: https://doi.org/10.25225/jvb.19055 Forst CV, Schulten K (2001) Phylogenetic analysis of metabolic pathways. Journal of Molecular Evolution 52 (6): 471-489. https://doi.org/10.1007/s002390010178 Gun LY, Haixian P, Liang Z (2018) Comprehensive analysis and comparison on the codon usage pattern of whole Mycobacterium tuberculosis coding genome from different area. Biomed Research International1-7. URL: http://doi.org/ 10.1155/2018/3574976 Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, ... 21 Guo XH, Liu SJ, Liu Q, Liu Y (2004) New progresses on mitochondrial DNA in fish. Yi Chuan Xue Bao 31 (9): 983-1000. URL: https://oubmed.ncbi.nim.nih.gov/15493150 Gyllensten U, Wharton D, Wilson A (1985) Maternal inheritance of mitochondrial DNA during backcrossing of two species of mice. Journal of Heredity 76: 321-324. https:// doi.org/10.1093/oxfordjournals .jnered.a110103 Huelsenbeck J (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17 (8): 754-755. https://doi.org/10.1093/bioinformatics/17.8.754 Hwang D, Byeon HK, Lee J (2014) Complete mitochondrial genome of the freshwater fish, Acheilognathus somjinensis (Cypriniformes, Cyprinidae). Mitochondrial DNA 25 (1): 13-4. https://doi.org/10.3109/19401736.2013.775266 Hwang DS, Byeon HK, Lee JS (2013) Complete mitochondrial genome of the freshwater sculpin Cottus koreanus (Scorpaeniformes, Cottidae). Mitochondrial DNA 24 (5): 490-491. https://doi.org/10.3109/19401736.2013.770500 Hwang DY, Koim SK, Ryu SH, Hwang UW (2012) Complete mitochondrial genome of Acheilognathus signifer (Cypriniformes, Cyprinidae): comparison of light-strand replication origins from six Acheilognathus species. Mitochondrial DNA 23 (4): 306-7. https://doi.org/10.3109/19401736.2012.683180 John F, Peden B, Sc (1999) Analysis of codon usage. University of Nottingham, 226 pp. Jordan DS, Thompson WF (1914) Record of the fishes obtained in Japan in 1911. Memoirs of the Carnegie Museum 6: 205-313. https://doi.org/10.5962/bhi.title.43189 Kalyaanamoorthy S, Minh BQ, Wong T, von Haeseler A, Jermiin L (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. ANU Research Publications 14: 587-589. https://doi.org/10.1038/nmeth.4285 Kawamura K, Ueda T, Arai R, Smith C (2014) Phylogenetic relationships of bitterling fishes (Teleostei: Cypriniformes: Acheilognathinae), inferred from mitochondrial cytochrome b sequences. Zoological Science 31 (5): 321-329. https://doi.org/10.2108/ Z$130233 Kottelat M (2001) Fishes of Laos. WHT Publications, Colombo [ISBN 955-9114-25-5. 198 p] Kuang W, Ming C, Li H, Wu H, Frantz L, Roos C, Zhang Y, Zhang C, Jia T, Yang J, YuL (2019) The Origin and Population History of the Endangered Golden Snub-Nosed Monkey (Rhinopithecus roxellana). Molecular Biology and Evolution 36 (3): 487-499. https://doi.org/10.1093/molbev/msy220. Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2017) PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology & Evolution 34 (3): 772-773. https://doi.org/ 10.1093/molbev/msw260 Lelek A (1987) The freshwater fishes of Europe, Vol 9: Threatened fishes of Europe. Environmental Science URL: https://api.semanticscholar.org/Corpus|ID:87950049 Li D (2006) Maternal inheritance of mitochondrial diseases. Journal of Weifang University 6 (4): 3-6. URL: https:/Awww.cnki.com.cn/Article/CUFDTOTAL- WEXY200604031.htm Li F, Liao T, Arai R, Zhao L (2017) Sinorhodeus microlepis, a new genus and species of bitterling from China (Teleostei: Cyprinidae: Acheilognathinae). Zootaxa 4353 (1): 69-88. https://doi.org/10.11646/zootaxa.4353.1.4 22 Yu J et al Lowe TM, Chan PP (2016) tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Research 44 (W1): W54-W57. hittps:// doi.org/10.1093/nar/gkw413 Masta S, L J, Boore (2004) The complete mitochondrial genome sequence of the spider Habronattus oregonensis reveals rearranged and extremely truncated tRNAs. Molecular Biology & Evolution 21 (5): 893-902. https://doi.org/10.1093/molbev/msh096 Meng G, Li Y, Yang C, Liu S (2019) MitoZ: a toolkit for animal mitochondrial genome assembly, annotation and visualization. Nucleic Acids Research 47 (11): e63. https:// doi.org/10.1093/nar/gkz173 Min-Shan AK, Zhang Y, Yang MA, Hu Y, Cao P, Feng X, Zhang L, Wei F, Fu Q (2018) Mitochondrial genome of a 22,000-year-old giant panda from southern China reveals a new panda lineage. Current Biology 28 (12): R693-R694. hitps://doi.org/10.1016/j.cub. 2018.05.008 Miyake T, Nakajima J, Umemura K, Onikura N, Ueda T, Smith C, Kawamura K (2021) Genetic diversification of the Kanehira bitterling Acheilognathus rhombeus inferred from mitochondrial DNA, with comments on the phylogenetic relationship with its sister species Acheilognathus barbatulus. Journal of Fish Biology 99 (5): 1677-1695. hittps:// doi.org/10.1111/jfo. 14876 Moritz C, Dowling TE, Brown WM (1987) Evolution of animal mitochondrial DNA: relevance for population biology and systematics. Annual Review of Ecology and Systematics 18 (1): 269-292. https://doi.org/10.1146/annurev.es.18.110187.001413 Noack K, Zardoya R, Meyer A (1996) The complete mitochondrial DNA sequence of the bichir (Polypterus ornatipinnis), a basal ray-finned fish: ancient establishment of the consensus vertebrate gene order. Genetics 144 (3): 1165-1180. https://doi.org/10.1093/ genetics/144.3.1165 Okazaki M, Kiyoshi N, Akihiro S, Ryoichi A (2001) Phylogenetic relationships of bitterlings based on mitochondrial 12S ribosomal DNA sequences. Journal of Fish Biology 58: 89-106. https://doi.org/10.1111/J.1095-8649.2001.TB00501.X Peilin C, Dan Y, Siging L, Qiongying T, Huanzhang L (2014) Molecular Phylogeny and Conservation Priorities of the Subfamily Acheilognathinae (Teleostei: Cyprinidae). Zoological Science 31 (5): 300-308. https://doi.org/10.2108/zs130069 Perna NT, Kocher TD (1995) Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. Journal of Molecular Evolution 41 (3): 353-358. https://doi.org/10.1007/BF01215182 Reichard M, Polacik M, Tarkan A, Spence R, Gaygusuz O, Ercan E, Ondrackova M, Smith C (2010) The bitterling-mussel coevolutionary relationship in areas of recent and ancient sympatry. Evolution 64 (10): 3047-56. https://doi.org/10.1111/j. 1558-5646.2010.01032.x Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19 (12): 1572-1574. https://doi.org/10.1093/ bioinformatics/btq180 Sambrook J, Russell D (2001) Molecular Cloning - Sambrook & Russel. Cold Spring Harbor Laboratory Press. Sharp PM, Li WH (1987) The codon Adaptation Index-a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Research 15 (3): 1281-1295. https://doi.org/10.1093/nar/15.3.1281 Mitochondrial genome of Acheilognathus barbatulus (Cypriniformes, Cyprinidae, ... 23 Smith C, Reynolds J, Sutherland W (2000) Population consequences of reproductive decisions. Proceedings of the Royal Society B: Biological Sciences 267: 1327-1334. https://doi.org/10.1098/rspb.2000.1146 Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22 (21): 2688-2690. hitps:// doi.org/10.1093/bioinformatics/btl446 Sudhir K, Glen S, Koichiro T (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology & Evolution 33 (7): 1870-1874. https://doi.org/10.1093/molbev/msw054 Wang I, Lin H, Liang C, Huang C, Huang C, Yang J, Wang W (2020) Complete mitochondrial genome of the freshwater fish Onychostoma lepturum (Teleostei, Cyprinidae): genome characterization and phylogenetic analysis. Zookeys 1005: 57-72. https://doi.org/10.3897/zookeys.1005.57592 Wang X, Gan X, Li J (2012) Cyprinid phylogeny based on Bayesian and maximum likelinood analyses of partitioned data: implications for Cyprinidae systematics. Science China Life Sciences 55: 761-773. https://doi.org/10.1007/s11427-012-4366-z Yang Q (2010) Studies on Rhodeinae and molecular phylogeny of Arthroidea fish. University of Huazhong Agricultural, Phd Thesis. Zhang D, Gao F, Jakovlic |, Zhou H, Zhang J, Li WX, Wang GT (2019) PhyloSuite: an integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20 (1): 348-355. https://doi.org/10.1111/1755-0998.13096 Zhu L, Che X, Liu X, Wang X, Wang J, Cheng G, Chen X, Chen X (2021) Complete mitochondrial genome of Acheilognathus hypselonotus Bleekers (Cypriniformes: Cyprinidae) in China's Dianshan Lake. Mitochondrial DNA B Resour 6 (12): 3367-3368. https://doi.org/10.1080/23802359.2021.1997109