Indian Journal of Animal Research

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Research Article

The Complete Mitochondrial Genomes of two Flying Fishes, Cheilopogon pinnatibarbatus japonicus and Hirundichthys rondeletii 

Liyan Qu1, Heng Zhang1, Fengying Zhang1, Wei Wang1, Fenghua Tang1, Ming Zhao1, Lingbo Ma1, Chunyan Ma1,*
1Key Laboratory of East China Sea and Oceanic Fishery Resources Exploitation, Ministry of Agriculture, East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai, China.

ABSTRACT

Background: Genome-scale approaches have played a significant role in the analysis of evolutionary relationships. Because of rich polymorphisms, high evolutionary rate and rare recombination, mitochondrial DNA sequences are commonly considered as effective markers for estimating population genetics, evolutionary and phylogenetic relationships. Flying fishes are important components of epipelagic ecosystems. Up to now, only few complete mitochondrial genomes of flying fishes have been reported. In the present study, the complete mitochondrial DNA sequences of the Cheilopogon pinnatibarbatus japonicus and Hirundichthys rondeletii had been determined.

Methods: Based on the published mitogenome of Cheilopogon atrisignis (GenBank: KU360729), fifteen pairs of primers were designed by the software Primer Premier 5.0 to get the complete mitochondrial genomes of two flying fishes. According to the reported data, the phylogenetic position of two flying fishes were detected using the conserved 12 protein-coding genes.

Result: The complete mitochondrial genomes of Cheilopogon pinnatibarbatus japonicus and Hirundichthys rondeletii are determined. They are 16532bp and 16525bp in length, respectively. And they both consists of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a control region. The OL regions are conserved in these two flying fishes and might have no function. From the tree topologies, we found C.p. japonicus and H. rondeletii clustered in a group. The findings of the study would contribute to the phylogenetic classification and the genetic conservation management of C.p. japonicus and H. rondeletii.  

INTRODUCTION

Genome-scale approaches played a significant role in the analysis of evolutionary relationships (Li et al., 2007). A complete mitochondrial genome of fish is a double-stranded circular molecule about 15~18 kb, generally encodes 37 genes: 13 protein-coding genes, 2r RNAs and 22 tRNAs (Guo et al., 2008). Because of its rich polymorphisms, high evolutionary rate and rare recombination, mitochondrial DNA sequences are commonly considered as effective markers for estimating population genetics, evolutionary and phylogenetic relationships (Susmita et al., 2019). In addition, the mitochondrial genome provides more reliable information than short DNA segments (Jabbar et al., 2013).

Flying fishes are important components of epipelagic ecosystems (Churnside et al., 2017). There are 50 species of 8 genera in the family Exocoetidae, of which, 38 species of 6 genera are found in China. The hefty and long pectoral fins make them look different from other fishes. They are known for their ability of leaving the water and gliding over long distances. Flying fishes live in all of the oceans, particularly in tropical and warm subtropical waters. Flying fishes prey on small fishes, amphipods, mollusks and copepods (Van Noord et al., 2013) and they are prey for game fishes, marine mammals and seabirds (Tew et al., 2009).

Up to now, only few complete mitochondrial genomes of flying fishes have been reported (Chou et al., 2016). In the present study, the complete mitochondrial DNA sequences of the Cheilopogon pinnatibarbatus japonicus and Hirundichthys rondeletii had been determined. According to the reported data, the phylogenetic position of two flying fishes were detected using the conserved 12 protein-coding genes. This study will contribute to the phylogenetic classification and the genetic conservation management of these two flying fishes.

MATERIALS AND METHODS

Sample collection and DNA extraction
 
The specimen of C.p. japonicus and H. rondeletii were collected by fishing from North Pacific (42°15'N, 151°52'E) in August 2016. Genomic DNA was extracted from muscle tissue using Animal Genomic DNA Extraction Kit (TIANGEN).
 
Primer design, PCR amplification and DNA sequencing
 
Based on the published mitogenome of Cheilopogon atrisignis (GenBank: KU360729), fifteen pairs of primers were designed by the software Primer Premier 5.0 to get the complete mitochondrial genomes of two flying fishes. And other fifteen pairs of primers are only for the H. rondeletii (Table 1). PCR were conducted using a total volume of 25 μl as described in Ma et al. (2013). The PCR products were purified and sequenced on ABI Prism 3730 in both directions.

Table 1: Primer pairs used for amplification and sequencing of mitogenome.



Gene annotation and genome analysis
 
Protein-coding genes, rRNA genes and non-coding regions were initially identified by sequence comparison with the mitochondrial genomes of related species (Cheilopogon atrisignis (NC_029729.1) and Cheilopogon doederleinii (NC_033541.1). The complete mitochondrial genome DNA sequence was deposited in GenBank database using the software Sequin (http://www.ncbi.nlm.nih.gov/Sequin/).
 
Phylogenetic analysis
 
The 12 protein-coding genes (excluding the ND6 gene) sequences of 13 fishes were aligned using Clustal W in MEGA 6.0 with default parameters. The phylogenetic tree was reconstructed using maximum-likehood (ML) analysis (http://embnet.vital-it.ch/raxml-bb/index.php) and 1000 bootstraps replications were used to estimate the node reliability. Raja porosa (AY525783.1) was designed as an out group.

RESULTS AND DISCUSSION

Genome organization and nucleotide composition
 
The complete mitochondrial genomes of C.p. japonicus (MF578940) and H. rondeletii (MF578941) were 16532 bp and 16525 bp in length, respectively. Their circular gene map was shown in Fig 1 and the characteristics of the two complete mitochondrial genome were shown in Table 2. The overall A+T content was same in these flying fishes (56.7%), which was similar to that in other flying fish reported (Chou et al., 2016). Eight and nine overlaps were found in C.p. japonicus and H. rondeletii, respectively. The length of the longest overlaps in two fishes were both 7 bp. Twelve and fourteen intergenic spacers were detected in C.p. japonicus and H. rondeletii, respectively. The biggest one located between tRNA-Asn and tRNA-Cys which was 38 bp in length.

Fig 1: Circular gene map of C.p. japonicus and H. rondeletiid mitochondrion. Genes encoded on the heavy or light strands are shown inside or outside the circular gene map, respectively. The abbreviations for the genes are as follows: COI, COII and COIII refer to the cytochrome oxidase subunits, Cytb refers to cytochrome b and ND1-6 refers to NADH dehydrogenase components.



​

Table 2: Characteristics of the complete mitochondrial genome of C. p. japonicus and H. rondeletiid.


 
Protein-coding genes and codon usage
 
The total lengths of 13 protein-coding genes in C.p. japonicus and H. rondeletii were 11428 bp and 11432 bp, respectively. The A+T content was 56.40% in C.p. japonicus and 56.30% in H. rondeletii. There were typical location and arrangements of protein-coding genes in these two fishes and only two protein-coding genes (ND5 and ND6) encoded by L-strand (Table 2).

12 of 13 protein-coding genes started with ATG, while the COI gene used GTG as a start codon. Five protein-coding genes ended with typical complete codon TAA or TAG. Eight used an incomplete stop codon (T or TA). There was no mutation in the conserved ATP8 gene and the most variation was found in the ND2 gene in these two fishes.
 
Transfer and Ribosomal RNA genes
 
There were 22 transfer RNA genes interspersed in two fishes’ mitochondrial genomes and ranged from 66 bp (tRNA-Cys) to 75 bp (tRNA-Leu). The tRNA-Leu and tRNA-Ser genes both showed two forms (Table 2). The tRNA-Ser (AGY) was lack of DHU arm and the rest 21 tRNA genes could fold into the typical clover-leaf structures.

The two ribosomal RNA genes (12SrRNA and16SrRNA) located between tRNA-Phe and tRNA-Leu and were separated by tRNA-Val. Their length in C.p. japonicus were 950 bp and 1703 bp and those in H. rondeletii were 944 bp and 1688 bp. The composition of A+T (55.6% and 55.5%) was higher than that of G+C (44.4% and 44.5%).
 
Non-coding region
 
There were two non-coding regions in the mitochondrial genome of two flying fishes (D-Loop L-strand origin (OL). The putative OL could fold a sTable stem-loop secondary structure, with 12 nucleotides in the loop and 10 paired nucleotides in the stem. The conserved 5'-GCCGG- 3' motif, which was thought being related to the transition from RNA synthesis to DNA synthesis (Hixson et al., 1986), was not found in the OL region of two flying fishes. Although C-rich was detected in the loop of many reported teleost fish (Johansen et al., 1990), it was not observed in this study. According to the previous researches, C-rich region might be the initiation of primer synthesis (Taanman, 1999) which suggested that the OL might have no function in C.p. japonicus and H. rondeletii. The result was similar to the study of Cheng et al., (2010).

The lengths of D-Loop in C.p. japonicus and H. rondeletii were 871bp and 862bp, respectively. The A+T content of D-Loop in these two flying fishes were 64.6% and 65.3%, which was higher than the average of the whole mitogenome (56.1% and 56.1%) and that of 13 protein coding genes (56.4% and 56.3%). The feature had been reported in other fishes (Dong et al., 2017). As described in other fishes (Lee et al., 1995; Hurst et al., 1999), the D-Loop possessed the typical tripartite regions. The genetic distance between two D-Loop regions was smaller than that of five protein coding genes (Fig 2), which is the same as that in other four flying fishes (C. arcticeps, C. atrisignis, C. doederleinii and C. unicolor). It indicated that the D-loop region in flying fishes was conservative.

Fig 2: Genetic distances of the different mitochondrial genes (protein-coding genes, rRNAs, coding genes and the CR) between C.p. japonicus and H. rondeletiid.


 
Phylogenetic analysis
 
The ML tree was built with significant bootstrap supports (Fig 3). The tree topologies indicated that C.p. japonicus and H. rondeletii clustered in a group. Three species from Cheilopogon genus formed a single group which was considered to be the sister group of the first one. This might result from the lack of the data of flying fishes in this topology. Another reason might be the large variations in some protein coding genes (ND2, ND4 and ND5). The genus Hirundichthys showed closest relationship with the genus Cheilopogon under the family of Exocoetidae. This study would advance our understanding of the population genetic structure, phylogenetic classification and evolution of flying fishes.

Fig 3: Maximum-likelihood phylogeny of Ogcocephalidae derived from the combined 12 protein-coding genes. Raja porosa was as an outgroup. Numbers on the branches are supported by 1000 bootstraps replications.

CONCLUSION

In conclusion, the mitochondrial genomes of C.p. japonicus and H. rondeletii were obtained and analyzed. The structure and composition of a typical mitochondrial genome were uncovered. The OL regions were conserved in flying fishes and might have no function. The work should be of great importance for the study on phylogenetic relationship and conservation genetics of flying fishes.

ACKNOWLEDGEMENT

This study was supported by the National Basic Research Special Foundation of China (2013FY110700), the National Science and Technology Support Plan (2013BAD13B04), Shanghai Natural Science Fund Project (15ZR1450000) and the Special Funds of Basic Research of Central Public Welfare Institute (2015M07).

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