Determination of the complete mitochondrial genome of Pseudobagrus gracilis (Siluriformes: Bagridae) and its phylogeny

Abstract Pseudobagrus gracilis is an endemic bagrid catfish in the Pearl River. To date, sparse studies conducted on this species have blocked our understanding of this species. In this study, the complete mitochondrial genome of P. gracilis was sequenced and reported using Illumina MiSeq platform. The P. gracilis mitogenome was 16,527 bp in length and comprised 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and one control region (D-loop). Its overall nucleotide base composition was 31.0% (A), 15.6% (G), 26.3% (C), and 27.1% (T), with an AT content 58.1%. Phylogenetic analyses based on Neighbor-joining approach revealed that Pseudobagrus species formed three lineages (I, II and III) and P. gracilis had close relationship with P. emarginatus, P. pratti and P. truncatus.


Introduction
Pseudobagrus gracilis (Li et al. 2005), a bagrid catfish that only distributed in the Pearl River, was firstly detected and described in 2005 (Li et al. 2005). This species has been confused or misidentified with two other Pseudobagrus species, e.g., P. pratti and P. adiposalis, which also inhabit in the drainages in the Southern China (Pan 1984;Zhu 1995). Currently, with exception of the first description in 2005 (Li et al. 2005), no study has been conducted for P. gracilis. We determined the whole mitochondrial genome of P. gracilis using next generation sequencing technology and tried to reveal its phylogenetic relationships among Pseudobagrus species.
A sample of P. gracilis was collected in June 2020 from the farm market in Pingle County (24.627 N, 110.649E), Guilin City, Guangxi Province, China. We clipped a fraction of fin tissue for genomic DNA extraction and extracted total genomic DNA from fin tissues using a Genomic DNA Isolation Kit (QiaGene, Germany). A specimen and genomic DNA sample were deposited at the fish collection of Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences (https://www.prfri.ac.cn/, Jie Li, lijie1561@163.com) under the voucher number XSNC2020001. The Illumina MiSeq platform (Illumina Inc, San Diego, CA, USA) was employed to sequence the complete mitochondrial genome and the software SPAdes 3.9.0 was used to assemble the raw sequence reads into contigs (Bankevich et al. 2012). The complete mitochondrial genome was achieved using the contigs in the software SOAPdenovo (Luo et al. 2012) and annotated the protein-coding genes and rRNA genes using web server DOGMA (Wyman et al. 2004).
The length of the P. gracilis mitogenome (GenBank nos: MW980438) was 16,527 base pairs (bp). Its overall nucleotide base composition was 31.0% (A), 15.6% (G), 26.3% (C), and 27.1% (T) and thus the overall AT content was 58.1%, which was basically consistent with the results of other published Pseudobagrus genomes (Pan et al. 2015;Wei et al. 2015;Yang et al. 2016;Zou et al. 2019). The mitogenome included 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region (D-loop). Gene overlaps were detected at seven gene junctions from 1 to 11 bp and involved a total of 37 bp. Among these mitochondrial genes, eight tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu and tRNA-Pro) and a protein coding gene (ND6) were encoded on the L-strand, while other genes were encoded on the H-strand. All protein-coding genes began with the typical start codon ATG, with the exception of COI, which started with GTG. Six protein-coding genes ended with a complete TAA (COI, ATP8, ATP6, ND4L, ND5 and ND6) and two genes finished with a complete TAG (ND1 and ND2). Furthermore, five genes (COII, COIII, ND3, ND4 and CYTB) had incomplete stop codon T.
Phylogenetic tree of genus Pseudobagrus was constructed based on 13 protein coding genes of 16 Pseudobagrus mitogenomes and one outgroup (Hemibagrus guttatus) using Neighbor-joining method in MEGA 6 (Tamura et al. 2013). Neighbor-joining tree yielded three well supported major lineages (Lineage I, II and III; Figure 1), which gain one more lineage than the previous studies (Yang et al. 2016;Zou et al. 2019).
The most discordant result compared with previous researches in our study was that P. trilineatus generated an independent lineage and located at the base of the other two lineages (Figure 1). The studied species P. gracilis had close relationship with P. emarginatus, P. pratti and P. truncatus (Figure 1).

Ethical approval
Experiments were performed in accordance with the recommendations of the Ethics Committee of Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences. These policies were enacted according to the Chinese Association for the Laboratory Animal Sciences and the Institutional Animal Care and Use Committee (IACUC) protocols.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at (https://www.ncbi.nlm.nih.gov/) under the accession no. MW980438. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA723410, SRR14291144 and SAMN18816459, respectively.