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Research Article
Complete mitochondrial genome of Rhodeus cyanorostris (Teleostei, Cyprinidae): characterization and phylogenetic analysis
expand article infoWenjing Li‡, Ning Qiu§, Hejun Du|
‡ YANGTZE Eco-Environment Engineering Research Center, Beijing, China
§ Ministry of Transport, Tianjin research institute for water transport engineering, Tianjin, China
| Chinese Sturgeon Research Institute, Yichang, China

Corresponding author: Wenjing Li ( 1325280713@qq.com )

Academic editor: Maria Elina Bichuette
© 2022 Wenjing Li, Ning Qiu, Hejun Du.
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.
Citation: Li W, Qiu N, Du H (2022) Complete mitochondrial genome of Rhodeus cyanorostris (Teleostei, Cyprinidae): characterization and phylogenetic analysis. ZooKeys 1081: 111-125. https://doi.org/10.3897/zookeys.1081.77043
ZooBank: urn:lsid:zoobank.org:pub:EB91B69F-DF56-491A-B8FA-629AE5DAA5DE
Open Access

Abstract

Rhodeus cyanorostris Li, Liao & Arai, 2020 is a freshwater fish that is endemic to China and restricted to Chengdu City in Sichuan Province. This study is the first to sequence and characterize the complete mitochondrial genome of R. cyanorostris. The mitogenome of R. cyanorostris is 16580 bp in length, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a control region (D-loop). The base composition of the sequence is 28.5% A, 27.6% C, 26.4% T, and 17.5% G, with a bias toward A+T. The genome structure, nucleotide composition, and codon usage of the mitogenome of R. cyanorostris are consistent with those of other species of Rhodeus. To verify the molecular phylogeny of the genus Rhodeus, we provide new insights to better understand the taxonomic status of R. cyanorostris. The phylogenetic trees present four major clades based on 19 mitogenomic sequences from 16 Rhodeus species. Rhodeus cyanorostris exhibits the closest phylogenetic relationship with R. pseudosericeus, R. amarus, and R. sericeus. This study discloses the complete mitochondrial genome sequence of R. cyanorostris for the first time and provides the most comprehensive phylogenetic reconstruction of the genus Rhodeus based on whole mitochondrial genome sequences. The information obtained in this study will provide new insights for conservation, phylogenetic analysis, and evolutionary biology research.

Keywords

Acheilognathinae, freshwater fish, genome structure, phylogenetic relationships

Introduction

The cyprinid subfamily Acheilognathinae are small freshwater fish commonly known as bitterlings. These fish are characterized by their compressed body and their unique spawning strategy of depositing their eggs through extended spawning tubes into the gill cavity of live freshwater mussels and clams, where they hatch and develop until the juvenile fish are able to swim freely (Smith et al. 2004; Nelson et al. 2016; Li et al. 2017, 2020a). The subfamily Acheilognathinae includes 75 species and six valid genera, including Acheilognathus, Paratanakia, Pseudorhodeus, Rhodeus, Sinorhodeus, and Tanakia (Arai and Akai 1988; Chang et al. 2014; Li et al. 2017, 2020a). Most of the bitterlings inhabit still-water areas such as rivers, lakes, ponds, and reservoirs, and a few species live in streams. Bitterlings are omnivorous, mainly feeding on algae, plankton, and debris. All species are distributed in East and Southeast Asian countries (China, Korea, Japan, Vietnam, and Myanmar), except the three Rhodeus species in Europe and adjacent West Asia (Arai et al. 2001; Bogutskaya and Komlev 2001; Bohlen et al. 2006; Li et al. 2017; Bartáková et al. 2019).

Although the classification of the subfamily Acheilognathinae has been controversial for many years, the genus Rhodeus is distinguished from other genera by characteristics such as an incomplete lateral line, no barbels, two rows of light spots on the dorsal fin, a pharyngeal teeth formula of 0,0,5–5,0,0, a black spot on the anterior part of the dorsal fin in juveniles (absent in R. amarus, R. meridionalis, and R. sericeus), and wing-like yolk sac projections in the larvae (Arai and Akai 1988; Li et al. 2017; Li et al. 2020a; Li et al. 2020b). The genus Rhodeus is distributed in two disjunct regions of Eurasia and contains approximately 22 species/subspecies, with 19 in East Asia and three in Europe and West Asia (Arai et al. 2001; Bogutskaya and Komlev 2001; Bohlen et al. 2006; Li and Arai 2014; Bartáková et al. 2019; Li et al. 2020a). Among the 19 Rhodeus species/subspecies in East Asia, eight (R. albomarginatus, R. cyanorostris, R. fangi, R. nigrodorsalis, R. ocellatus, R. sinensis, R. shitaiensis, and R. flaviventris) have been reported in the Yangtze river basin of China (Li et al. 2020a, b).

Rhodeus cyanorostris Li, Liao & Arai, 2020 is endemic to China and is restricted to Chengdu City, Sichuan Province. It can be easily distinguished from other congeners (except for R. nigrodorsalis) by its blue snout, less branched dorsal- and anal-fin rays (both no more than eight of each), and lack of pored scales (Li et al. 2020a). Moreover, according to the personal observations of the first author, R. cyanorostris and R. nigrodorsalis are the only two bitterling species known to spawn mainly in winter, from January to March (Li et al. 2020a).

The mitochondrial genome has been widely used in molecular evolution, phylogeny, and population genetics because of its maternal inheritance, stable genetic composition, fast evolutionary rate, low recombination frequency, and highly conserved gene content (Ballard and Whitlock 2004; Oliveria et al. 2008; Galtier et al. 2009; Simon and Hadrys 2013; Hao et al. 2021; Zhao et al. 2021). Complete mitochondrial genomes can provide much more reliable phylogenetic information than smaller parts of the mitochondrial DNA (Huang et al. 2017; Hou et al. 2020) and have been considered reliable markers for constructing fish phylogenies in recent studies of the taxonomy and phylogeny of cyprinids (Wang et al. 2008; Tang et al. 2010; Muniyangdi et al. 2015; Huang et al. 2017; Chung et al. 2020; Zhang et al. 2021).

The main purpose of the current study is to disclose the complete mitochondrial genome sequence of R. cyanorostris for the first time and to construct a phylogenetic tree based on complete mitogenome sequences to elucidate the molecular phylogenetic relationship between R. cyanorostris and other species of Rhodeus. Therefore, this study provides essential scientific data and contributes to studies of the population genetics, adaptation, and phylogeny of R. cyanorostris.

Materials and methods

Sampling, sequencing, and assembly

Samples of Rhodeus cyanorostris were collected from the Pidu District of Chengdu City in the Sichuan Province of China (30°55'12"N, 103°50'51"E). The fish were caught with seines, anesthetized with MS-222 (Sigma, St. Louis, MO), fixed and stored in 95% ethanol. Species-level morphological identification was carried out according to the description of Fan Li (2020a). Total genomic DNA was extracted using a TIANamp Micro DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. Then, DNA was stored at –20 °C for subsequent use.

The primers were designed based on the known mitochondrial genomes of R. sinensis by NCBI primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). PCR was performed by using an Eppendorf Thermal Cycler (5331AH760577, Eppendorf, Germany) with a 30 µL reaction mixture containing 15 µL of 2×Power Taq PCR MasterMix (Tianyi Huiyuan, China), 1 µL of DNA template, 1 µL of each primer (10 mM of each), and 12 µL of ultrapure water. The cycling procedures were as follows: denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 30 sec, annealing at 60 °C for 30 sec, extension at 72 °C for 1 min, and a final extension at 72 °C for 5 min. Agarose gel electrophoresis was used to detect each PCR product to verify the amplification efficiency. PCR products were purified and sequenced by primer walking from both directions.

Sequences were assembled using the DNASTAR package (Burland 2000). Overlapping fragments obtained by sequencing were edited using BIOEDIT v. 7.0.9.0 (Hall 1999) and aligned using MEGA v. 7.0 (Kumar et al. 2016).

Mitogenome annotation and analyses

The mitogenome annotation, tRNA gene localization, and their secondary structure prediction of R. cyanorostris were all completed by the MITOS web server (http://mitos2.bioinf.uni-leipzig.de/index.py) (Bernt et al. 2013). The online MitoFish tool (http://mitofish.aori.u-tokyo.ac.jp/) was used to map the mitochondrial genome structure. The base structure, nucleotide composition, and relative synonymous codon usage (RSCU) were calculated using MEGA v. 7.0 (Kumar et al. 2016). The skewing of the nucleotide composition was calculated with the formulas: AT skew = (A – T) / (A + T) and GC skew = (G – C) / (G + C) (Perna and Kocher 1995). The complete mitochondrial genome sequence of R. cyanorostris has been submitted to NCBI (GenBank no. OL856007).

Phylogenetic analyses

Twenty-one mitogenomic sequences downloaded from GenBank (Table 1) were aligned using MEGA v. 7.0 (alignment with CLUSTALW) with default settings (Kumar et al. 2016). The best model GTR +G + I was chosen based on the Akaike information criterion (AIC) using JMODELTEST v. 2 (Darriba et al. 2012), and the ML (maximum likelihood method) tree was constructed using PHYML v. 3.0 (Guindon et al. 2010). The confidence intervals were assessed through the bootstrap test inferred from 1000 replicates. An NJ (neighbor-joining method) tree was constructed based on the Kimura 2-parameter model with 1000 bootstrap replicates using MEGA v. 7.0 (Kumar et al. 2016).

Table 1.
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List of the species used to construct the phylogenetic tree.

Classific-ation Subfamily Genus Species Accession number Gene length
Outgroup Culterinae Hemiculter Hemiculter leucisculus KF956522.1 16622 bp
Outgroup Barbinae Onychostoma Onychostoma lepturum MT258556.1 16598 bp
Ingroup Acheilognathinae Rhodeus Rhodeus albomarginatus MW896838.1 16764 bp
Ingroup Acheilognathinae Rhodeus Rhodeus amarus AP011209.1 16607 bp
Ingroup Acheilognathinae Rhodeus Rhodeus atremius AP010778.1 17282 bp
Ingroup Acheilognathinae Rhodeus Rhodeus atremius atremius AP011255.1 16734 bp
Ingroup Acheilognathinae Rhodeus Rhodeus fangi KF980890.1 16733 bp
Ingroup Acheilognathinae Rhodeus Rhodeus lighti KM232987.1 16677 bp
Ingroup Acheilognathinae Rhodeus Rhodeus notatus KU291171.1 16735 bp
Ingroup Acheilognathinae Rhodeus Rhodeus ocellatus kurumeus AB070205.1 16674 bp
Ingroup Acheilognathinae Rhodeus Rhodeus ocellatus 1 DQ026430.1 16680 bp
Ingroup Acheilognathinae Rhodeus Rhodeus ocellatus (Kner) 2 KT004415.1 16761 bp
Ingroup Acheilognathinae Rhodeus Rhodeus ocellatus 3 MW007386.1 16675 bp
Ingroup Acheilognathinae Rhodeus Rhodeus pseudosericeus KF425517.1 16574 bp
Ingroup Acheilognathinae Rhodeus Rhodeus sericeus KM052222.1 16581 bp
Ingroup Acheilognathinae Rhodeus Rhodeus shitaiensis KF176560.1 16774 bp
Ingroup Acheilognathinae Rhodeus Rhodeus sinensis KF533721.1 16677 bp
Ingroup Acheilognathinae Rhodeus Rhodeus suigensis EF483934.1 16733 bp
Ingroup Acheilognathinae Rhodeus Rhodeus uyekii 1 DQ155662.1 16817 bp
Ingroup Acheilognathinae Rhodeus Rhodeus uyekii 2 EF483937.1 16827 bp

Results

Mitochondrial genomic structure and composition

The complete mitochondrial genome of Rhodeus cyanorostris had a total length of 16580 bp (Fig. 1). The complete R. cyanorostris genome had a typical circular molecular structure and contained 37 genes, including 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 tRNA genes, and a noncoding control region (D-loop) (Table 2). Among these genes, NADH dehydrogenase 6 (ND6) and 8 tRNA genes (tRNAGln, tRNAAla, tRNAAsn, tRNACys, tRNATyr, tRNASer, tRNAGlu, tRNAPr°) were encoded by L-strand, and the rest were encoded by H-strand. The mitogenome was compact, with eight gene overlaps, ranging in length from 1 to 7 bp. In addition, there were fourteen 1–30 bp coding gene spacer regions, with a total length of 63 bp; the longest spacer region fell between tRNAVal and 16S rRNA genes.

Table 2.
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Organization of the mitochondrial genome of Rhodeus cyanorostris.

Locus position Size (bp) Intergenic nucleotides Codon Anti-codon Strand
start stop start stop
tRNAPhe 1 69 69 0 – – GAA H
12s rRNA 70 1026 957 1 – – – H
tRNAVal 1028 1099 72 30 – – TAC H
16s rRNA 1130 2786 1657 0 – – – H
tRNALeu 2787 2862 76 0 – – TAA H
ND1 2863 3837 975 4 GTG TAA – H
tRNAlle 3842 3913 72 –2 – – GAT H
tRNAGln 3912 3982 71 1 – – TTG L
tRNAMet 3984 4052 69 0 – – CAT H
ND2 4053 5099 1047 –2 ATG TAG – H
tRNATrp 5098 5168 71 1 – – TCA H
tRNAAla 5170 5238 69 1 – – TGC L
tRNAAsn 5240 5312 73 2 – – GTT L
tRNACys 5345 5413 69 0 – – GCA L
tRNATyr 5414 5483 70 1 – – GTA L
COI 5485 7035 1551 0 GTG TAA – H
tRNASer 7036 7106 71 2 – – TGA L
tRNAAsp 7109 7178 70 9 – – GTC H
COII 7188 7878 691 0 ATG T(AA) – H
tRNALys 7879 7953 75 1 – – TTT H
ATP8 7955 8119 165 –7 ATG TAG – H
ATP6 8113 8796 684 –1 ATG TAA – H
COIII 8796 9580 785 –1 ATG TA(A) – H
tRNAGly 9580 9650 71 0 – – TCC H
ND3 9651 9999 349 0 ATG T(AA) – H
tRNAArg 10000 10069 70 0 – – TCG H
ND4L 10070 10366 297 –7 ATG TAA – H
ND4 10360 11738 1379 3 ATG TA(A) – H
tRNAHis 11742 11810 69 0 – – GTG H
tRNASer 11811 11879 69 1 – – GCT H
tRNALeu 11881 11953 73 0 – – TAG H
ND5 11954 13789 1836 –4 ATG TAG – H
ND6 13786 14307 522 0 ATG TAA – L
tRNAGlu 14308 14376 69 6 – – TTC L
Cyt b 14383 15523 1141 0 ATG T(AA) – H
tRNAThr 15524 15597 74 –1 – – TGT H
tRNAPr° 15597 15666 70 54 – – TGG L
D-loop 15721 16438 718 142 – – – H
Figure 1. 

Gene map of the mitochondrial genome of Rhodeus cyanorostris. 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 base composition of the entire sequence was in the order of A (28.5) > C (27.6) > T (26.4) > G (17.5), with a bias toward A+T. This bias was observed in all genetic elements except for ND3 (Table 3). The complete genome also showed a clear AC bias (AT skew = 0.04, GC skew = –0.22), indicating a greater abundance of A than T and C than G (Table 3).

Table 3.
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Nucleotide contents of genes and the mitochondrial genome skew of Rhodeus cyanorostris.

Regions Size (bp) T C A G A+T (%) G+C (%) AT skew GC skew
rRNAs 2645 20.0 25.1 33.4 21.5 53.4 46.6 0.25 –0.08
ND1 975 27.3 29.7 26.2 16.8 53.5 46.5 –0.02 –0.28
tRNAs 1562 26.6 22.0 28.6 22.9 55.2 44.9 0.04 0.02
ND2 1045 26.6 31.8 26.9 14.7 53.5 46.5 0.01 –0.37
COI 1551 29.3 27.3 24.3 19.0 53.6 46.3 –0.09 –0.18
COII 691 26.9 27.5 27.9 17.7 54.8 45.2 0.02 –0.22
ATP8 165 27.3 26.7 33.3 12.7 60.6 39.4 0.10 –0.36
ATP6 683 29.6 30.5 25.6 14.3 55.2 44.8 –0.07 –0.36
COIII 784 29.7 27.0 24.1 19.1 53.8 46.1 –0.10 –0.17
ND3 349 28.1 31.2 20.6 20.1 48.7 51.3 –0.15 –0.22
ND4L 297 28.6 30.0 24.6 16.8 53.2 46.8 –0.08 –0.28
ND4 1382 27.6 28.8 27.3 16.3 54.9 45.1 –0.01 –0.28
ND5 1836 27.9 28.2 29.6 14.2 57.5 42.4 0.03 –0.33
ND6 522 37.7 12.6 14.9 34.7 52.6 47.3 –0.43 0.47
Cyt b 1141 29.4 29.3 25.1 16.3 54.5 45.6 –0.08 –0.29
D-loop 860 31.6 21.9 30.9 15.6 62.5 37.5 –0.01 –0.17
PCGs 11421 28.7 28.1 25.9 17.3 54.6 45.4 –0.05 –0.24
Genome 16580 26.4 27.6 28.5 17.5 54.9 45.1 0.04 –0.22

Among the 13 protein-coding genes, the ND1 and COI genes started with GTG, while all other PCGs contained the usual ATG start codon. Eight of the 13 PCGs were terminated with the conventional stop codons (TAA or TAG), while the other five (ND4, COIII, COII, ND3, and Cyt b) were terminated with incomplete stop codons (TA or T). Moreover, the AT skew and GC skew values of the PCGs were –0.05 and –0.24, respectively, indicating that the nucleotides T and C had a greater abundance than their respective counterparts (Table 3).

Statistics on the relative synonymous codon usage (RSCU) of R. cyanorostris showed that the most abundant codons were CCC (Pro), UUU (Phe), AAA (Lys), and AUU (Ile) (Fig. 2).

Figure 2. 

Codon distribution a and relative synonymous codon usage (RSCU) b in the mitogenome of Rhodeus cyanorostris.

Transfer and ribosomal RNA genes

The two ribosomal RNAs (12S and 16S ribosomal RNA) were positioned between tRNAphe and tRNAleu and separated by tRNAval in the mitogenome of R. cyanorostris. The 12S ribosomal RNA was composed of 957 bp, and the 16S ribosomal RNA was 1657 bp long. Both rRNA genes were encoded on the H-strand and displayed a positive AT skew and a negative GC skew (AT skew = 0.25, GC skew = –0.08).

The mitogenome of R. cyanorostris included 22 transfer RNA genes as in most vertebrates. These transfer RNA genes ranged from 69 to 76 bp. The total concatenated length of tRNA genes was 1562 bp, the AT skew of 22 tRNAs was 0.04, and the GC skew was 0.02, showing slightly higher A and G (Table 3). The secondary structures of all tRNA genes were traditional cloverleaf structures (Fig. 3). In addition to the typical base pairs (G-C and A-U), there were also some wobble G-U pairs in these secondary structures, which could form stable chemical bonds between U and G.

Figure 3. 

Putative secondary structures of the 22 tRNAs of Rhodeus cyanorostris.

Phylogenetic analysis

To elucidate the phylogenetic relationship in the genus Rhodeus, 21 whole mitochondrial genome sequences of 18 species were used in this study. As a result, ML and NJ analyses generated the same topological structure with well-supported values, and both presented four major sister clades (Fig. 4). Within Clade 1, the branch including three species (R. notatus, R. suigensis, and R. fangi) first formed a sister cluster with high bootstrap values with the branch containing R. atremius and R. atremius stremius. Then, they clustered with the branch including R. shitaiensis and R. uyekii. In Clade 2, R. cyanorostris clustered together with R. pseudosericeus, R. amarus, and R. sericeus. In Clade 3, the branch including two species (R. ocellatus and R. sinensis) first formed a sister cluster with the branch containing R. lighti, R. ocellatus kurumeus, and R. ocellatus 3. Clade 4 included R. albomarginatus and R. ocellatus 2. R. cyanorostris exhibited the closest phylogenetic relationship with R. pseudosericeus, R. amuarus, and R. sericeus.

Figure 4. 

Phylogenetic trees derived from the maximum-likelihood (ML) and neighbor joining (NJ) approaches based on whole mitochondrial genomes. The numbers on the nodes are the bootstrap values of ML and NJ. The number after the species name is the GenBank accession number.

Discussion

We successfully sequenced and assembled for the first time the mitogenome of Rhodeus cyanorostris, an endemic fish species in China. The mitogenome was 16580 bp in length, which was similar to the genome size of the known acheilognathine mitogenomes, for example, 16677 bp in R. sinensis, 16677 bp in R. lighti, and 16581 bp in R. sericeus (Wang et al. 2014; Xu et al. 2015; Yang et al. 2015). Differences in mitochondrial genome length in related species may be caused by changes in tandem repeats in the control region (Wang et al. 2020). Consistent with the genome structure of other teleost fish, the mitogenome of R. cyanorostris included 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a non-coding control region (D-loop). The gene distribution was mainly presented on the H-strand, and only the ND6 gene and eight rRNA genes were located on the L-strand. This distribution is consistent with that of other species of Acheilognathinae (Wang et al. 2014; Xu et al. 2015; Yang et al. 2015). In comparison, the 13 PCGs in the mitogenome revealed a relatively low GC content, which was common in the Rhodeus mitogenome (Xu et al. 2015; Yang et al. 2015).

The whole mitochondrial genome of the genus Rhodeus is extremely similar in its nucleotide composition and codon usage, but there were also subtle differences. For example, among the 13 protein-coding genes of R. cyanorostris, two genes (ND1 and COI) start with GTG, and the other 11 start with ATG. In R. shitaiensis, only COI and ND5 start with GTG (Li et al. 2013). Rhodeus lighti, R. sericeus, R. sinensis, R. suigensis, and R. uyekii all start with ATG except for COI, which starts with GTG (Kim et al. 2006; Hwang et al. 2013; Wang et al. 2014; Xu et al. 2015; Yang et al. 2015). The termination codons of R. lighti, R. sericeus, R. sinensis, R. suigensis, and R. uyekii include conventional codons (TAA and TAG) and incomplete codons (T- and TA-).

The secondary structures of tRNA for R. cyanorostris are conserved, and these features meet the characteristics of vertebrate mitochondrial genomes (Zhao et al. 2021). In addition to the typical Watson-Crick pairing (A-U and G-C), there are also some typical pairings such as U-G. Some scholars have proposed that the non-Watson-Crick matched tRNAs can be transformed into fully functional proteins through a post-transcriptional mechanism (Pons et al. 2014; Zhao et al. 2021).

Mitochondrial genome sequences are widely used to study phylogenetic relationships because they offer small, stable changes over a long period for any given taxon. In this regard, whole mitochondrial genes can better transmit phylogenetic information than single genes (mitochondrial/nuclear) can (Huang et al. 2017; Hou et al. 2020). Previous studies have revealed different phylogenetic relationships of different bitterlings by using different molecular datasets. For the first time, we used whole mitochondrial genome sequences to construct the most comprehensive phylogenetic reconstruction of the genus Rhodeus thus far. The phylogenetic results indicated that there were some slightly different topologies compared to other studies due to different outgroups, contrast species, and molecular markers (Okazaki et al. 2001; Chang et al. 2014; Cheng et al. 2014; Kawamura et al. 2014). For example, Okazaki et al. (2001) reported the phylogenetic relationships of 27 species or subspecies of Acheilognathinae based on the 12S rRNA gene. Chang et al. (2014) used six nuclear gene loci (RAG1, RH, IRBP2, EGR1, EGR2B, and EGR3) and one mitochondrial gene (cyt b) to study the phylogenetic relationship of the subfamily Acheilognathinae, including Rhodeus. Cheng et al. (2014) reconstructed a species-level phylogenetic tree of Acheilognathinae based on the mtDNA cyt b and 12S rRNA gene sequences. Kawamura et al. (2014) elucidated the phylogeny of 49 species or subspecies in three genera (Tanakia, Rhodeus, and Acheilognathus) with cyt b. In this study, the phylogenetic tree showed that the genus Rhodeus is divided into four clades. Rhodeus cyanorostris is most closely related to R. pseudosericeus, R. amarus, and R. sericeus. They occupy Clade Ⅱ, and the closer phylogenetic relationship between the latter three was consistent with the study of Kawamura et al. (2014). The mitogenome sequences of R. shitaiensis, R. uyekii, and four members of the R. smithii complex (R. fangi, R. notatus, R. atremius, R. suigensis) (Kimura and Nagata 1992; Arai et al. 2001; Okazaki et al. 2010; Yu et al. 2016) constituted Clade Ⅰ of the phylogenetic tree. Furthermore, the phylogenetic relationship among species was also closely related to their morphological similarity. For example, R. albomarginatus is the most morphologically similar to R. ocellatus and R. sinensis (Chang et al. 2014), which are the most widely distributed Rhodeus species in China. They occupied the Clades Ⅲ and Ⅳ. According to Li et al. (2010), R. shitaiensis closely resembled the R. sericeus complex (R. sericeus, R. colchicus, R. meridionalis, and R. amarus).

Conclusions

In summary, we successfully sequence and characterize the complete mitochondrial genome sequence of Rhodeus cyanorostris for the first time and furtherly elucidate the relationship between R. cyanorostris and other species in the genus Rhodeus. The information obtained from this study will be valuable in further studies on the conservation, molecular identification, and evolutionary biology of the diverse Rhodeus species.

Acknowledgements

This work was supported by the Three Gorges Environment Protection Fund, Chinese Three Gorges Corporation (WWKY-2021–0035). The authors declare that no conflicts of interest exist.

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