Unusual AT-skew of Sinorhodeus microlepis mitogenome provides new insights into mitogenome features and phylogenetic implications of bitterling fishes

Accepted Manuscript Unusual AT-skew of Sinorhodeus microlepis mitogenome provides new insights into mitogenome features and phylogenetic implications of bitterling fishes

Peng Yu, Li Zhou, Xiao-Ya Zhou, Wen-Tao Yang, Jun Zhang, Xiao-Juan Zhang, Yang Wang, Jian-Fang Gui PII: DOI: Reference:

S0141-8130(18)36172-5 https://doi.org/10.1016/j.ijbiomac.2019.01.200 BIOMAC 11633

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

13 November 2018 17 January 2019 29 January 2019

Please cite this article as: P. Yu, L. Zhou, X.-Y. Zhou, et al., Unusual AT-skew of Sinorhodeus microlepis mitogenome provides new insights into mitogenome features and phylogenetic implications of bitterling fishes, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.01.200

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Unusual AT-skew of Sinorhodeus microlepis mitogenome provides new insights into mitogenome features and phylogenetic implications of bitterling fishes Peng Yu 1, 2, 3, Li Zhou 1, 2,, Xiao-Ya Zhou3, Wen-Tao Yang 1, 2, Jun Zhang3, Xiao-Juan

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Zhang1, Yang Wang1, 2*, Jian-Fang Gui1, 2* State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of

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Hydrobiology, The Innovation Academy of Seed Design, Chinese Academy of

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Sciences, Wuhan 430072, China

University of Chinese Academy of Sciences, Beijing 100049, China

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College of Animal Science and Technology, Anhui Agricultural University, Hefei

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2

230036, China

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*Corresponding authors:

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Yang Wang ([email protected]) and Jian-Fang Gui ([email protected])

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Highlights

1. The first report of complete mitochondrial genome of S. microlepis. 2. Unusual AT-skew and base compositions in S. microlepis mitogenome.

mitogenomes.

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3. Highly conserved overlaps and non-coding intergenic spacers among bitterling

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4. S. microlepis should be a sister species to the genus Rhodeu that might diverge about 13.69 Ma

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Abstract Sinorhodeus microlepis (S. microlepis) is recently descibed as a new species and respresents a new genus Sinorhodeu of the subfamily Acheilognathinae. In this study, we first sequenced the complete mitogenome of S. microlepis and compared with the

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other 29 bitterling mitogenomes. The S. microlepis mitogenome is 16,591 bp in length

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and contains 37 genes. Gene distribution pattern is identical among 30 bitterling

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mitogenomes. A significant linear correlation between A+T% and AT-skew were found among 29 bitterling mitogenomes, except S. microlepis shows unusual AT-skew

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with slightly negative in tRNAs and PCGs. Bitterling mitogenomes exhibit highly

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conserved usage bias of start codon, relative synonymous codons and amino acids, overlaps and non-coding intergenic spacers. Phylogenetic trees constructed by 13

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PCGs strongly support the polyphyly of the genus Acheilognathus and the paraphyly

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of Rhodeus and Tanakia. Together with the unusual characters of S. microlepis mitogenomes and phylogenetic trees, S. microlepis should be a sister species to the

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genus Rhodeu that might diverge about 13.69 Ma (95% HPD: 12.96–14.48 Ma).

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Keywords: Sinorhodeus microlepis; Acheilognathinae; mtDNA; AT-skew; phylogeny; divergence time

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Introduction The subfamily Acheilognathinae, commonly called bitterling, is small cyprinid fishes which considered as a model in evolutionary ecology owing to their unusual spawning behavior and co-evolutionary relationships with freshwater mussels [1-3]. They

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deposite their eggs in the gill chambers of living freshwater mussels [4]. Bitterlings

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comprise approximately 80 species or subspecies [5] across three genera, Tanakia,

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Rhodeus, and Acheilognathus [6]. They are mainly distributed in Asia, except three species (R. amarus, R. colchicus and R. meridionalis) native to Europe [6,7]. Due to

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their highly specialized spawning behavior, habitat loss and fragmentation, and other

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threats, bitterlings face an increasing extinction risk [8-12]. As well-known aquarium fishes in Asia and Europe [11,13], bitterlings possess great diversity in morphological

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features and exhibit charming colors and patterns. The phylogenetic relationships of

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bitterlings have been revealed by morphological, karyological, meristic or molecular data [14-16]. Recently, several phylogenetic trees were constructed based on

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mitochondrial DNA control region [17], 5S rDNA [18], mitochondrial cytochrome b

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(cytb) [19,20], 12SrRNA [21], or comprehensive multiple genes [11,14,22], which provide new insights into the taxonomy, phylogeny and origin of Acheilognathidae. However, the details of species relationships and the monophyly of three genera remain questionable. Mitochondrial genome (mitogenome) contains 13 protein coding genes (PCGs), two ribosomal (rRNA), 22 transfer (tRNA) RNA genes, and two noncoding regions. The longer noncoding region, named as control region (CR), possesses regulatory

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elements for transcription and replication, and the shorter noncoding region is the origin of L-strand replication (OL) [23-25]. Compared with nuclear genome, mitogenome is characterized by its small size, abundance in animal tissues, strict orthology of PCGs, and high nucleotide substitution rate [26-28]. Therefore, it has

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been extensively applied in species identification, phylogenetic reconstruction,

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genetic diversity, and conservation biology [29-34]. Although the arrangement of 37

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genes within the genome is highly conserved, the genome length, nucleotide composition, amino acid bias, gene overlaps, and non-coding intergenic spacers (IGS)

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are distinctive among different species [35-37]. Up to now, mitogenomes of about 40

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bitterlings had been reported or deposited in GenBank [39-54]. However, the conserved and exclusive characteristics of bitterling mitogenomes have not been well

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based on 13PCGs are lacking.

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documented. In addition, the phylogenetic status and divergence time of bitterlings

Recently, a highly distinctive bitterling was descibed as a new species

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Sinorhodeus microlepis and respresented a new genus Sinorhodeus [14]. S. microlepis

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is distributed only in a small area of a tributary of the Yangtze River in Chongqing City, China. During breeding season, adult males are fascinatingly colorful and have a orange-red body with bluish sheen dorsally, iris-red pelvic fins and orange-red anal fin, similar to volcanic color. In China, S. microlepis is also known as volcano bitterling and has been booming in aquarium market. Unfortunately, it was heavily fished in its original habitat. Here we sequenced and described, for the first time, the mitogenome of S. microlepis and compared it with other 29 bitterling mitogenomes.

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Based on the investigations, we explore the characteristics of bitterling mitogenomes, the phylogenetic relationship and the estimated divergence times for S. microlepis and other bitterlings.

2. Materials and methods

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2.1 Sequencing and assembly analysis

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The S. microlepis specimen analyzed in this study was bought from the aquarium

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market in Chongqing city, China, and identified through morphological analysis as described by Li et al [14].

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Total DNA was extracted according to the Ezup Column Animal Genomic DNA Kit

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technical manual (Sangon, Shanghai, China). The mitochondrial DNA fragments were amplified by PCR. PCR primers were designed based on the conserved sequences

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between two bitterling mitogenomes: R. amarus (GenBank: AP011209.1) and T.

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tanago (GenBank: AP012526.1) (Supplementary Table S1). DNA amplification and purification were performed according to Wei et al., 2016a [55] (Supplementary Table

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S1). The purified DNA products were ligated to pMD™18-T vector (Takara, Beijing,

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China) and the plasmids were chemically transfected into Trans5αcompetent cells (TransGen Biotech, Beijing, China). The positive clones were sequenced by Tianyibiotech (Beijing, China). The DNA sequences were assembled by using DNAStar (DNASTAR Inc., USA) [56]. 2.2 Gene annotations and bioinformatics analysis The 22 tRNA genes were predicted with tRNAscan-SE 1.21 [57] and MITOS [58]. All 13 PCGs and 2 rRNA genes were annotated by comparison with the homologous

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sequences

of

other

bitterling

(https://blast.ncbi.nlm.nih.gov/).

The

mitogenomes

mtDNA

maps

were

in

GenBank drawn

using

OrganellarGenomeDRAW [59]. The secondary structure of OL and 22 tRNAs were predicted by The mfold Web Server [60] (http://unafold.rna.albany.edu/) and

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tRNAscan-SE 1.21 [57], respectively. The base composition and relative synonymous

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codon usage were calculated using MEGA 7.0 [61] and Microsoft Excel 2010.

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Skewness was measured using the formulas: AT-skew = (A% - T%) / (A% + T%) and GC-skew = (G% - C%) / (G% + C%) [62]. The TAS (termination associated sequence

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domains), GTGGG-box and CSB (conserved sequence block domains) of CR were

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determined by compairing with other species [63-66]. 2.4 Phylogenetic analyses

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The phylogenetic relationships were constructed with 13 PCGs of 30 bitterlings from

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S. microlepis and other three genera (Table 1). Megalobrama amblycephala (NC_010341.1) and Parabramis pekinensis (NC_022678.1) were chosen as the out

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groups. Each of the 13 PCG sequences from all the 30 species was separately aligned

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using MAFFT v7.407 [67] and then aggregated into a sequence matrix. The maximum likelihood (ML) tree was implemented in RAxML v7.7.1 [68] under the GTR-Gamma model, and node support was calculated with 1,000 bootstrap replications (random seed value 1,234,567). The Bayesian phylogenies were implemented in MrBayes version 3.1.2 [69]. The best-fit nucleotide substitution model was determined by ModelFinder in IQ-TREE v1.6.9 [70]. The bayesian inference (BI) tree was constructed with run length of 1.5 × 10

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generations and sampling every 1,000

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generations. Divergence time was calibrated using the first appearance of fossil records of Rhodeinae in the early Miocene (20 ma) found in Japan [71], and estimated using BEAST v1.10.4 [72]. The tree topology was constructed based on GTR + I + G Substitution model. The Yule Process was chosen for the tree prior [73]. BEAST was

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run for 1.5 × 10 7 Markov chain Monte Carlo (MCMC) generations, with the trees

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sampled every 1,000 generations. Then the trees were checked using Tracer v1.5 [74].

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3 Results and discussion

3.1 General features of S. microlepis and other bitterling mitogenomes

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The complete mitogenome of S. microlepis (GenBank: MH190825) is a circular

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double-chain molecule with a length of 16,591 bp (Fig. 1). The lengths of other bitterling mitogenomes range from 16,563 bp to 16,988 bp (Table 1), and the

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differences in length are mainly attributed by the variations of CR (Supplementary

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Table S2). Similar to other bitterlings, S. microlepis mitogenome also contains 13 PCGs, 22 tRNA genes, two rRNA genes and two non-coding regions (OL and CR)

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(Fig. 1, Table2). Gene distribution pattern is identical among 30 bitterling

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mitogenomes (Fig. 1, Table2). S. microlepis mitogenome comprises 22 tRNAs with the total length of 1,561 bp, ranging from 66 bp (tRNACys(C)) to 76 bp (tRNALeu(UUR (L1)) and tRNALys(K)). All tRNAs could be folded into typical clover-leaf secondary structure, except for tRNAser(AGY)(S1) lacking dihydrouracil (DHU) arm (Fig. 2). The unusual structure of S1 is conserved among other bitterling and metazoan mitogenomes [75-77]. A total of 41 unmatched base pairs were scattered through the tRNAs, 27 of them are

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noncanonical matches of G-U pairs and the remaining mismatches include A-C (6), A-A (2), A-G (1), and U-U (5) (Fig. 2). The lengths of 12S rRNA and 16S rRNA are 961 bp and 1678 bp, locating between tRNAPhe(S) and tRNAVal(V), V and L1, respectively. Same to other cyprinid fish [78-80], the OL locates in the middle of the

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WANCY region (Fig.1 and Table 2) and displays a stable stem and loop structure,

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which is strengthened by three A-T and six G-C base pairs (Fig. 3A). The S.

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microlepis CR extends 933 bp and locates between the tRNAPro(P) and S. In bitterling fishes, the length of CR was the fastest-changing region, ranging from 901 bp (R.

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shitaiensis) [41] to 1129 bp (A. rhombeus) (Supplementary Table S2). The

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GTGGG-box and the conserved blocks such as TAS and CSB-F, CSB-E, CSB-D, CSB-1, CSB-2, and CSB-3 were found in the CR of S. microlepis mitogenome (Fig.

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3B).

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3.2 Unusual AT-skew in S. microlepis mitogenome The entire mitogenomes of S. microlepis and other bitterlings all exhibit AT bias (52.9%

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- 57.9%). The A+T content is the highest in CR and the lowest in rRNAs (Fig. 4A).

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Interestingly, the A+T contents of CR of the genus Acheilognathus clade A are higher than those of the genus of Tanakia and Rhodeus, except A. meridianuswas and A. melanogaster. The AT-skew values of whole mitogenome, PCGs, tRNAs, rRNAs and CR were calculated (Fig. 4B and Supplementary Table S2). Compared to other bitterlings, AT-skew in S. microlepis is unusual with slightly negative in tRNAs (-0.012) and PCGs (-0.014), while AT-skew values for other bitterling mitogenomes are positive in tRNAs (0.021±0.008) and strong negative in PCGs (-0.049±0.011) (Fig.

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4B and Supplementary Table S2). The results indicate that S. microlepis tRNAs displays an excess of T over A, whereas the tRNAs of other bitterlings are biased

towards using A not T. On the other side, the occurrence of A in PCGs of S. microlepis mitogenome is much more than those in other bitterling PCGs. CR involves in the

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initiation point of replication and the regulation of transcription, and its AT-skew and

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GC-skew might reflect the strand asymmetry [24-25, 62]. Some insects and fishes

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possess less A than T and less C than G on the majority strand, indicating the strand asymmetry is reversed [24, 25, 81, 82]. Although the AT-skew of CR in eight

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bitterlings is negative, the GC-skew of CR in the all analyzed bitterlings is minus

mitogenomes may be not reversed.

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(Supplementary Table S2). The results suggest that the strand asymmetry of bitterling

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Nucleotide skews might be due to a balance between mutational and selective

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pressures during replication [83-85]. The reason why do AT skews of S. microlepis tRNAs and PCGs run counter to those observed in most bitterling mitogenomes is still

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unknown. Recently, the atypical preference for A over T in the leading strand of

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Staphylococcus aureus was proved to be caused by selection rather than mutation [86,

87]. S. microlepis is distributed only in a specific tributary of the Yangtze River [14]. One possibility of the unusual AT-skew in S. microlepis is that unique selective pressures or processes might lead to the decrease A in its tRNAs and preferring A in its PCGs. However, what selective forces account for the unusual AT-skew will await further investigation. 3.3 Linear correlations between A+T % and AT-skew of bitterling mitogenomes

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We also analyzed the relationship between nucleotide composition (A+T %) versus AT-skew of 13 PCGs across Acheilognathinae. There is a significant linear correlation between A+T% and AT-skew (y29 = 0.0044x – 0.2952, R² = 0.3303) among 29 bitterling mitogenomes, indicating that the AT-skew becomes more negative with

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decreasing the A+T content (Fig. 4C). The relationships are different among three

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genera, Tanakia, Rhodeus, and Acheilognathus, and the strongest linear correlation is

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found in Rhodeus (y = 0.0047x - 0.3101, R² = 0.4329), without considering the genus Tanakia (only four species) and Sinorhodeus (one species). Within the subfamily

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Acheilognathinae, the only exception is S. microlepis, away from the linear

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relationships of subfamily Acheilognathinae and its three genera. The result also exhibits the unique character of S. microlepis PCGs in base compositions.

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3.4 Usage bias of start and stop codon, relative synonymous codons and amino

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acids in bitterling mitogenomes

Consistent with the overwhelming majority of bitterling mitogenomes, 12 PCGs of S.

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microlepis initiate with typical start codon ATG, except for the cox1 gene utilizing

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GTG (Table 2). Among the 30 bitterling mitogenomes, only three exceptions were observed. The putative start codon of nd3 gene in A. typus and A. melanogaster, and nd5 gene in R. shitaiensis are all GTG (Supplementary Table S3 and Figure S1A). In the S. microlepis mitogenome, seven PCGs utilize the canonical termination codon (TAA and TAG), whereas the others end with the truncated stop codon (TA and T) (Table 2). Similar results were also found in other bitterling mitochondrial genes. All bitterling cox1 and nd4l are terminated with TAA, and cox2 and cytb are ended with a

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single T (Supplementary Table S3 and Figure S1B). The truncated stop codons are common in cyprinid fish mitogenomes [88] and might be converted to TAA via post-transcriptional polyadenylation [89]. In addition, the 1st positions of stop codons in bitterling mitogenomes are T, and the most of 2nd position are A. The AT-skew of

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the 1st codon of PCGs are positive while the 2nd codon are negative (Fig. 4B and Table

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3). The AT bias could selectively avoid the formation of stop codons, similar as the

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observation in nuclear genomes [86].

The codon usage of six bitterling mitochondrial genomes, including Sinorhodeus

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(S. microlepis), Rhodeus (R. shitaiensis and R. lighti), Tanakia (T. limbata), and

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Acheilognathus (A. meridianus and A. somjinensis) were compared. The amino acid contents are largely consistent among the six bitterling mitogenomes (Fig. 5A). The

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three most abundant amino acids are Leu(CUN), Ala and Thr, while Cys, Ser(AGY) and

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Arg are rare. Relative synonymous codon usage (RSCU) analysis also showed the similar codon usage pattern among the bitterling mitogenomes (Fig. 5B). In the 3rd

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position of bitterling PCGs, codons with G are very rare while codons with A are

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overused. Consistent with A+T-bias in whole mitogenomes (Fig. 4A), the A+T-bias is also present in the 3rd position of bitterling PCGs. For example, the codons CGG and CGC for Arginine are rare, while the synonymous codons CGA and CGT are prevalent (Fig. 5B). 3.5 Highly conserved overlaps and non-coding intergenic spacers among bitterling mitogenomes Gene overlaps are highly conserved in bitterlings. In the analyzed six bitterling

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mitogenomes, a total of 22 overlapping nucleotides, ranging from one to seven bp, were detected across six identical gene junctions (Supplementary Table S5). Three motifs “ATGATAR” (R=A/G), “ATGYTAA” (Y=C/T) and “TTAR” are overlapped in the junctions of atp8-atp6, nd4l-nd4, and nd5-nd6 respectively (Fig. 6). Although the

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numbers and the locations of gene overlaps varied, the above three motifs were

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recognized as a common character found in Cyprinidae fishes, such as zebrafish [80],

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grass carp [90] and Zacco sieboldii [43] (Fig. 6 and Supplementary Table S5). In addition to OL and CR, 10 conserved and short non-coding intergenic spacers

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(IGSs) were found across the six bitterling mitogenomes with a total length from 21 to

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26 bp (Supplementary Table S6). The longest one contains seven bp - nine bp nucleotides and locates between tRNAAsp(D) and cox2 genes. The 3’ of these IGSs are

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conserved and mainly composed of T/A nucleotides (Supplementary Table S6). In

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particular, a long spacer (with a range of 81 bp -230 bp) was found at the tRNAArg(R) and nd4l in six bitterlings, including R. notatus (83 bp), R. suigensis (81 bp) [39], R.

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fangi (81 bp), R. atremius atremius (81 bp), R. uyekii (230 bp) [40], and R. shitaiensis

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(220 bp) [41]. The long spacer is uncommon in other bitterlings and even other cyprinid fish (Supplementary Table S6). The lepidopteran IGS with a “TTAGTAT” motif at the trnS2 and nd1 junction is thought to be important for mitochondrion transcription [91] and Hymenoptera IGSs are supposed to associate with gene rearrangements [92, 93]. The “TTAGTAT” motif within IGS and gene rearrangement were not found in bitterling mitogenomes. Thus, the function of IGSs in bitterling mitogenomes is still unknown.

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3.6 Phylogenetic analysis and estimation of divergence time of bitterlings ML and BI phylogenetic trees constructed by 13 PCGs of 30 bitterlings and 2 outgroup taxa were largely congruent (Fig. 7). The only observed discordance was in the position of T. tanago. The subfamily Acheilognathinae was divided into two main

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clades: Acheilognathus clade A and a complicated Acheilognathus-Tanakia-Rhodeus

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clade. The Acheilognathus clade A includes nine species of Acheilognathus, while the

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other three species of Acheilognathus (A. intermedia, A. signifier, and A. somjinensis) are clustered with four species of Tanakia. Except the analysis by Chang et al, 2014

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[22], Acheilognathus was recovered to be monophyletic in the previous phylogenetic

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analyses owing to the limitations in taxon sampling [11, 18-21]. Our analysis is consistent with the phylogenies resolved by Chang et al, 2014 [22] and supports the

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polyphyly of Acheilognathus.

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The sampled species of Rhodeus and Tanakia forms two paraphyletic groups respectively. Different from the all previous molecular phylogenetic analyses [11,

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18-22], the genus Rhodeus were divided into two distinct lineages. S. microlepis is

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clustered with Rhodeus clade A which forms the sister-group to Rhodeus clade B (Fig. 7). However, we did not obtain the mitogenomes of “Acheilognathus” striatus, an unnamed clade which is split the genus Rhodeus and S. microlepis [14]. The unusual characters of S. microlepis mitogenome (Fig. 4B-C), together with special morphological differences [14], suggest that S. microlepis should be a sister species to the genus Rhodeu which needs further study to confirm its taxonomy and phylogeny. Based on fossil calibration, the chronogram with divergence time of bitterling

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lineages were estimated using a relaxed molecular clock (Fig. 8). The subfamily Acheilognathinae diversified around 20 million years ago (Ma). The most recent common

ancestor

of

Acheilognathus

clade

A

and

the

complicated

Acheilognathus-Tanakia-Rhodeus clade appeared about 12.99 Ma (95% HPD:

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12.32–12.67 Ma) and 14.63 Ma (95% HPD: 14.05–15.37 Ma), respectively. S.

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microlepis emerged in the Middle Miocene (around 13.69 Ma, 95% HPD:

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12.96–14.48 Ma), close to the Rhodeus clade A, while Rhodeus clade B occurred in the late Pliocene (around 13.69 Ma, 95% HPD: 4.47–5.12 Ma).

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In conclusion, current studies represent general and unique features of S.

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microlepis and other 29 bitterling mitogenomes, and reveal their phylogenetic relationship and divergence times. The phylogenetic results strongly support the

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Conflicts of interest

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polyphyly of the genus Acheilognathus and the paraphyly of Rhodeus and Tanakia.

The authors declare no conflict of interest.

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Acknowledgments

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This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences [XDB3104] and the earmarked fund for Modern Agro-industry Technology Research System (NYCYTX-49).

Appendix supplementary data Supplementary Table 1. List of primers used to amplify the mitogenome of S. microlepis. Supplementary Table 2. Length, base composition and skewness of bitterlings

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mitogenomes. Supplementary Table 3. The start and stop codons of 13 protein-coding genes in the bitterlings mitogenomes.

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Supplementary Table 4. Gene overlaps in the mitogenomes of six bitterlings and other three cyprinid fish.

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Supplementary Table 5. Non-coding intergenic spacers in the mitogenomes of the

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six bitterligs and other three cyprinid fish.

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Supplementary Table 6. Sequences of three mainly non-coding intergenic spacers

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in the mitogenomes of six bitterlings and other three cyprinid fish. Supplementary Figure S1. Composition of start codons (A) and stop codons (B).

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Lists of tables and figures Table 1 The genus, species, GenBank accession number and length of fish

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mitogenomes analyzed in this study Table 2 Annotation of the S. microlepis mitogenome.

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Table 3 Base composition and skewness of the mitogenomes in S. microlepis and

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other five bitterlings.

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Fig. 1 Circular sketch map of the S. microlepis mitogenome. Different colors represent different gene blocks.

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Fig. 2 Predicted secondary structure of tRNAs in S. microlepis. The tRNAs are labeled with their corresponding amino acids. Dashes (–) indicate Watson–Crick

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bonds, and dots (·) indicate mispaired nucleotide bonds. Fig. 3 Putative secondary structure of OL (A) and complete nucleotide sequence of

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mtDNA CR of S. microlepis (B). TAS, GT-GGG box, CSB-1, CSB-2, CSB-3, CSB-D, CSB-E and CSB-F are underlined.

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Fig. 4 Unusual AT-skew and base compositions in S. microlepis mitogenome. A. A+T contents of different regions in 30 bitterling mitogenomes. B. AT-skew of different regions in 30 bitterling mitogenomes. C. The Linear correlations between A+T% and AT-skew in 13PCGs of 30 bitterling mitogenomes. Fig. 5 Codon distribution (A) and relative synonymous codon usage (B) of PCGs in S. microlepis and other five bitterling mitogenomes. CDspT=codon per thousand codons.

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Fig. 6 Alignment of overlapping region of three PCGs pairs across six bitterlings and other three cyprinid fish. A. atp8 vs atp6; B. nd4l vs nd4; C. nd5 vs nd6. Fig. 7 Phylogenetic tree constructed by BI methods (A) and ML methods (B), based on the nucleotide sequences of all 13 PCGs of S. microlepis and other 29 bitterlings.

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M. amblycephala and P. pekinensis were chosen as outgroups. Node numbers

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represent the values of posterior probability.

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Fig.8 The divergence times (million years ago) of bitterlings. The ranges of 95% HPD

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intervals are represented by the blue bars.

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Table 1. The genus, species, GenBank accession number and length of fish mitogenome analyzed in this study Sequence length

Genus

Species

Accession ID

Reference

1

Sinorhodeus

Sinorhodeus microlepis

MH190825

16591

this study

2

Rhodeus

Rhodeus notatus

KU291171.1

16735

Unpublished

3

Rhodeus

Rhodeus suigensis

EF483934.1

16733

Hwang et al., 2014

4

Rhodeus

Rhodeus fangi

KF980890.1

16733

Unpublished

5

Rhodeus

Rhodeus atremius atremius

AP011255.1

16734

Unpublished

6

Rhodeus

Rhodeus uyekii

DQ155662.1

16817

Kim et al., 2006

7

Rhodeus

Rhodeus shitaiensis

KF176560.1

16774

Li et al., 2015

8

Rhodeus

Rhodeus pseudosericeus

KF425517.1

16574

Unpublished

16581

Unpublished

16607

Unpublished

RI

PT

(bp)

Rhodeus

Rhodeus sericeus

NC_025326.1

Rhodeus

Rhodeus amarus

AP011209.1

11

Rhodeus

Rhodeus lighti

KM232987

16677

Wang et al., 2016a

12

Rhodeus

Rhodeus ocellatus kurumeus

AB070205.1

16674

Saitoh et al., 2006

13

Rhodeus

Rhodeus ocellatus

NC_011211.1

16680

He et al., 2008

14

Rhodeus

Rhodeus sinensis

KF533721.1

16677

Unpublished

15

Tanakia

Tanakia tanago

NC_027665.1

16584

Miya et al., 2015

16

Tanakia

Tanakia lanceolata

KJ589418.1

16607

Xu et al., 2016

17

Tanakia

Tanakia himantegus

KP231201.1

16575

Cheng and Tao, 2016

18

Tanakia

Tanakia limbata

KM386633.1

16565

Luo et al., 2016

19

Acheilognathus

Acheilognathus somjinensis

NC_014882

16569

Hwang et al., 2014b

20

Acheilognathus

Acheilognathus signifer

EF483930

16566

Unpublished

21

Acheilognathus

Acheilognathus koreensis

NC_013704.1

16563

Hwang et al., 2013a

22

Acheilognathus

Acheilognathus melanogaster

AP012985.1

16563

Unpublished

23

Acheilognathus

Acheilognathus meridianus

KT692966.1

16563

Unpublished

24

Acheilognathus

Acheilognathus yamatsutae

EF483936.1

16703

Hwang et al., 2013b

25

Acheilognathus

Acheilognathus tabira

AP013343.1

16765

Unpublished

26

Acheilognathus

Acheilognathus omeiensis

MG783572.1

16774

Unpublished

27

Acheilognathus

Acheilognathus rhombeus

KT601094.1

16780

Unpublished

28

Acheilognathus

Acheilognathus typus

AB239602.1

16778

Saitoh et al., 2006

29

Acheilognathus

Acheilognathus barbatus

KP067832.1

16770

Tao and Sun, 2016

30

Acheilognathus

Acheilognathus macropterus

EF483935.1

16774

Hwang et al., 2014c

31

Megalobrama

Megalobrama amblycephala

NC_010341.1

16623

Unpublished

32

Parabramis

Parabramis pekinensis

NC_022678.1

16622

Zhang et al., 2014

33

Danio

Danio rerio

NC_002333

16596

Broughton et al., 2001

34

Ctenopharyngodon

Ctenopharyngodon idella

EU391390.1

16609

Wang et al., 2008

35

Nipponocypris

Zacco sieboldii

AB218898.1

16616

Saitoh et al., 2006

NU

MA

D

PT E

CE

AC

SC

9 10

ACCEPTED MANUSCRIPT 25

Table 2. Annotation of the S. microlepis mitogenome Gene/Element

Position

Nucleotide

Start

Stop

Amino

Anti-

Intergenic

size (bp)

codon

codon

acid

codon

nucleotidea

GAA

0

H

0

H

0

H

0

H

0

H

4

H

tRNAPhe (S)

1-69

69

12SrRNA

70-1030

961

1031-1102

72

16SrRNA

1103-2780

1678

Leu(UUR)

2781-2856

76

nd1

2857-3831

975

Ile

tRNA (I)

3836-3907

72

GAT

-2

H

tRNAGln (Q)

3906-3976

71

TTG

1

L

CAT

0

H

0

H

TCA

1

H

TGC

1

L

GTT

0

L

3978-4046

69

4047-5091

1045

(W)

5092-5162

71

tRNAAla (A)

5164-5232

69

tRNA

Trp

5234-5306

73

L-strand replication origin (OL)

5307-5337

31

tRNACys (C)

5338-5403

66

tRNATyr (Y)

5404-5474

71

cox1

5476-7026

1551

tRNASer(UCN) (S2)

7027-7097

71

tRNA

Asp

(N)

(D)

7100-7169

cox2 tRNA

Lys

7179-7869 (K)

7870-7945 7947-8111

atp6

8105-8788

cox3

8788-9571 (G)

9572-9642

PT E

tRNA

Gly

D

atp8

nd3 tRNA

Arg

9643-9991

(R)

nd4l

348

TAA

0 GCA

0

L

GTA

1

L

516

0

H

TGA

2

L

GTC

9

H

0

H

ATG

T

230

1

H

165

ATG

TAG

54

-7

H

684

ATG

TAA

227

-1

H

784

ATG

T

261

0

H

0

H

ATG

T

116

0

H

76

TTT

71 349

TCC

9992-10061

70 297

ATG

TAA

98

TCG

10352-11733

1382

ATG

TA

460

11734-11802

69

0

H

-7

H

0

H

GTG

0

H

(S1)

11803-11871

69

GCT

1

H

tRNALeu(CUN) (L2)

11873-11945

73

TAG

0

H

nd5

11946-13781

1836

ATG

TAG

611

-4

H

13778-14299

522

ATA

TAG

173

0

L

ATG

T

380

AC

tRNA

Ser(AGY)

691

T

324

10062-10358

CE

nd4 tRNAHis (H)

70

GTG

MA

tRNA

Asn

ATG

TAA

RI

(M)

nd2

TAA ATG

SC

tRNA

Met

(L1)

TAC

NU

tRNA

(V)

PT

tRNA

Val

Strandb

nd6 tRNA

Glu

14300-14368

69

cytb

(E)

14375-15515

1141

tRNAThr (T)

15516-15589

74

tRNAPro (P)

15589-15658

70

Control region (CR)

15659-16591

933

TTC

6

L

0

H

TGT

-1

H

TGG

0

L

a, negative value indicates the overlapping sequences between adjacent genes b, H: heavy strand; L: light strand

0

ACCEPTED MANUSCRIPT 26

Table 3. Base composition and skewness of the mitogenomes in S. microlepis and other five bitterlings Si

Si T(

Speci

ze

A

es

(b

%

A+ C

G

%

%

U)

T

%

%

G+ C %

ATske w

GC -ske

T( (b

%

C

G

%

%

U)

w

%

A+

G+

AT-

GC

T

C

ske

-ske

%

%

w

w

56.

43. 44. 2 44. 9 46. 0 41. 1 45. 8

-0.0 -0.0 14 -0.0 49 -0.0 53 -0.0 56 -0.0 44

-0.2

55. 8 56. 1 53. 0 58. 9 55. 2 0 48.

0 51.

46 0.0

28 0.0

48. 9 48. 4 47. 0 49. 6 48. 8 4 59.

51. 1 52. 6 52. 0 50. 4 51. 2 6 40.

0.0 93 0.0 96 0.0 81 0.0 88 0.0 94 97 -0.3

0.0 20 0.0 07 0.0 21 0.0 20 0.0 25 23 -0.3

58. 1 58. 8 58. 9 59. 7 59. 3 0 62.

41. 9 41. 2 41. 1 40. 3 41. 7 1 37.

-0.3 83 -0.3 80 -0.3 86 -0.3 87 -0.3 79 84 0.1

-0.3 37 -0.3 50 -0.3 32 -0.3 32 -0.3 43 37 -0.4

57. 2 60. 7 55. 9 65. 1 57. 1 5

42. 8 39. 3 44. 1 34. 9 42. 9 6

0.1 57 0.1 66 0.1 60 0.1 73 0.1 55 78

-0.4 50 -0.4 51 -0.3 68 -0.5 95 -0.4 29 29

p)

26. 4 27. 5 26. 2 27. 3 26. 7

nensis R. microl R. shitai epis T. lighti ensis A. limbat A. meridi a somji anus S. nensis R. microl R. shitai epis T. lighti ensis A. limbat A. meridi a somji anus S. nensis R. microl R. shitai epis T. lighti ensis A. limbat A. meridi a somji anus nensis

9 15 61 15 59 15 61 15 61 15 59 61 26

8 2 7. 2 8. 9 2 8. 0 2 8. 7 2 9. 3 8. 3 3 8 3 3. 3 2. 9 3 3. 9 3 3. 6 3 4. 9 4. 5 3 1 3 2. 3 2. 8 3 1. 2 3 1. 2 3 2. 8 2. 9 5

26. 6 27. 9 27. 5 27. 1 27. 7 4 20.

6 28.

20. 6 19. 1 20. 4 20. 0 20. 6 0 31.

S.

11

2

28.

55. 9 56. 2 54. 1 57. 5 55. 9

44. 1 43. 8 45. 9 42. 5 44. 1

0.0 37 0.0 40 0.0 30 0.0 34 0.0 42

-0.2 10 -0.2 21 -0.2 08 -0.2 08 -0.2 24

R. microle R. lighti shitaien pis T.

6 43.

17 0.0

55. 4 56. 0 55. 2 57. 4 56. 0 2 54.

45. 6 43. 0 44. 8 43. 6 43. 0 8 45.

41 -0.0 0.0 12 0.0 20 0.0 22 0.0 22 0.0 28 26 0.2

0.0 21 0.0 57 0.0 58 0.0 43 0.0 45 41 -0.0

somjine nus S. nsis R. microle R. lighti shitaien pis T.

2 8. 2 6. 02 6. 22 5. 52 7. 5 6. 82

28. 8 29. 9 28. 5 30. 5 28. 4

4 56.

11 42 11 42 2 11 42 5 11 41 2 11 42 9 42 2 37 3 37 98 37 99 37 98 37 97 37 98 98 37

21. 2 22. 9 21. 0 22. 7 21. 6 8 40.

53. 5 53. 1 53. 0 55. 9 54. 1 1 64.

46. 5 47. 9 46. 0 44. 1 46. 9 0 35.

0.2 43 0.2 41 0.2 68 0.2 58 0.2 52 60 0.0

-0.0 62 -0.0 57 -0.0 63 -0.0 71 -0.0 63 72 -0.1

64. 1 65. 3 63. 2 64. 5 64. 1 0

35. 9 34. 7 36. 8 35. 6 36. 9 0

0.0 23 -0.0 02 0.0 42 0.0 03 0.0 26 15

-0.1 70 -0.2 99 -0.2 01 -0.1 19 -0.2 62 32

22 6. 2 6. 72 5. 52 5. 92 7. 9 6. 31 51 8. 1 8. 21 8. 21 8. 11 8. 0 8. 34 23 6. 3 3. 03 5. 73 2. 33 7. 3 3. 6 8

AC

CE

32. 3 34. 1 31. 0 31. 6 31. 2 5

2 1 6. 7. 2 1 7. 7. 1 0 2 1 6. 7. 4 4 2 1 7. 8. 5 4 2 1 5. 6. 5 tRNAs 0 7. 7. 8 3 2 2 1 5 2 2 1. 2. 2 2 1. 3. 3 2 2 2 0. 3. 2 8 2 2 1. 3. 6 2 2 2 0. 2. 3rRNAs 3 1. 2. 5 5 2 2 0 8 2 2 4. 1. 2 2 4. 2. 2 3 2 2 5. 2. 8 1 2 2 4. 1. 0 0 2 2 3. 1. 7 CR4 4. 1. 8 0 2 1 6 3 2 1 1. 4. 2 1 1. 4. 0 9 2 1 0. 3. 4 3 2 1 2. 4. 9 9 2 1 0. 5. 3 3 2. 3. 9 1 2 8

-0.2

sis A. limbata A. meridia

sis A. limbata A. meridia somjine nus S. nsis R. microle R. lighti shitaien pis T. sis A. limbata A. meridia somjine nus S. nsis R. microle R. lighti shitaien pis T. sis A. limbata A. meridia somjine nus nsis

37 98 37 99 37 98 37 97 37 98 98 37 37 98 37 99 37 98 37 97 37 98 98

PCGs 2 1 2 1 7. 5. 2 1 8. 7. 32 19 7. 6. 02 01 8. 8. 12 91 6. 5. 1 0 1rd of PCGs 7. 7. 12 72 26 24 5. 6. 2 2 5. 6. 02 02 5. 6. 62 02 5. 6. 52 62 4. 5. 7 7 2rd of PCGs 5. 6. 52 71 22 14 7. 3. 2 1 7. 3. 42 61 7. 3. 82 41 7. 3. 42 71 7. 3. 5 8 3rd of PCGs 7. 3. 23 13 43 61 7. 0. 2 1 0. 1. 43 41 8. 0. 72 6 8. 1. 3. 73 41 6. 2 3 6 0. 2. 6 4 1

PT

2 9. 2 8. 5 2 8. 7 3 8. 9 2 0. 2 8. 2 2

0.0

8 22.

RI

16 59 16 77 1 16 67 4 16 56 7 16 56 5 56 3 15

43.

SC

R. microl R. shitai epis T. lighti ensis A. limbat A. meridi a somji anus S.

all mtDNA 2 1 56.

NU

27.

MA

2

D

16

PT E

S.

90 3 10 1 91 15 91 1 91 0 1

A

Species

p)

26 39 26 33 26 44 26 40 26 38 40 93

ze

40. 9 40. 6 40. 8 40. 7 40. 9 8 26. 24. 3 25. 1 22. 6 27. 8 23. 5 6

-0.2 65 -0.2 45 -0.2 33 -0.2 19 -0.2 48

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9