First complete mitochondrial genome of the endemic goby, Rhinogobius davidi (Gobiiformes: Gobiidae: Gobionellinae), in China

Abstract The first complete mitochondrial genome of the freshwater goby Rhinogobius davidi was determined by high-throughput sequencing. This genome was 16,627 bp in length and consisted of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 2 non-coding control regions. Phylogenetic analysis based on the amino acid sequences of 13 mitochondrial protein-coding genes from R. davidi and 23 relatives suggested that R. davidi had a close mitogenome relationship with Rhinogobius giurinus. This complete genome of R. davidi will provide basal molecular data for future studies on taxonomy, comparative genomics, and adaptive evolution in Rhinogobius.


Introduction
Rhinogobius davidi (Sauvage & Dabry de Thiersant, 1874) is a benthic species of landlocked goby distributed in the Oujiang, Qiantang, and Yangtze River drainages, China ( Figure 1). This fish feeds on invertebrates and appears to be limited to smaller streams in the upper reaches of these large river drainages. There are no records of the species occurring in hydrostatic environments such as lakes in these drainages. The breeding period begins when the water temperature is above 20 C. Before breeding, the male fish nests, and then attracts the females to enter and lay eggs. The eggs adhere to the ventral surface of the stones, and the male protects the eggs alone until they hatch. Female fish can reproduce once a month, each spawning up to 100 or more (Li 2011). Distinguishing morphological features of this species include: first dorsal fin VI, second dorsal fin I, 9-10, gluteal fin I, 6-8, pectoral fin 14-15, ventral fin I, 5; longitudinal scales 30-32, transverse scales 11, dorsal fin anterior scales 0-2 (mainly 0); vertebrae number 11 þ 17 ¼ 28; no tube hole in the sensory tube of the anterior gill cover; cheek with a brown vertical stripe below the eye (Yang et al. 2008;Li 2011).
Rhinogobius davidi is of ornamental value, but to date, it is poorly studied and what research has been done is limited to morphology and ecology (Chen and Miller 1998;Li 2011). Due to various manmade influences, the ecological environment of the basin is seriously damaged and the number of fish declined significantly. Genetic information is crucial to the development of strategies for the identification and management of this species ). Therefore, this study is aimed to provide a first report on the mitochondrial genome of R. davidi and analyze the phylogenetic relationships among the genus Rhinogobius.

Materials
The specimens were obtained from Cao'e River in Shangyu District, Shaoxing, Zhejiang Province of China (29 57 0 17.94 00 N, 120 52 0 06.98 00 E), and were identified as R. davidi based on the morphological characters. Some specimens were kept in dry ice until DNA analysis and transferred to Shanghai Genesky Biotechnologies Inc, while others (Voucher number ASTIH-21b1108d23) were fixed in 95% ethanol and deposited in Aquatic Science and Technology Institution Herbarium (https://www.jsahvc.edu.cn/) with Lin Song (tianxinlinger@ 126.com) in charge. All animal handling and experimental procedures were performed in accordance with the recommendations of the Ethics Committee for Animal Experiments of Jiangsu Agri-animal Husbandry Vocational College (Taizhou, China).

Methods
The total genomic DNA was extracted from muscle tissue using the phenol-chloroform method (Barnett and Larson 2012). The DNA library was established with qualitycontrolled DNA samples, and amplified by high-fidelity polymerase to ensure sufficient library volume on the sequencer. Agilent 2100 Bioanalyzer (Agilent Technologies, USA) was applied to determine the size distribution of library fragments and evaluate the suitability for sequencing. After library pooling, next-generation sequencing was performed on Illumina HiSeq 4000 Sequencing platform (Illumina, CA, USA). The raw sequencing data were checked by FastQC, and removed of low-quality reads and adapter region with Trimmomatic (Bolger et al. 2014). The trimmed reads were mapped to the reference mitogenome of Rhinogobius giurinus (KU871066), using BWA v.0.7.17 (Li and Durbin 2009) with default parameters. Samtool v.1.9 (Li and Durbin 2009) was employed to retrieve the aligned mitochondrial reads. The aligned reads with the mitogenome were then assembled using the software MetaSPAdes 3.13.0 (Nurk et al. 2017).
The resulting contig was annotated on MitoMaker 1.14 (Bernt et al. 2013). The final full mtDNA sequence is available in GenBank under accession number OM617724. And the genome circular map of R. davidi was drawn by CGView online server (https://proksee.ca/) (Grant and Stothard 2008).
We generated a phylogenetic tree with a set of all 22 Rhinogobius species available in GenBank and 2 Chaenogobius species. Each of the 13 PCGs was aligned separately using MUSCLE algorithm (Edgar 2004) in MEGA X, then the 13 PCG alignments were concatenated into a single multiple sequence alignment. The substitution model mtREV þ G þ I þ F was selected utilizing the Find Best DNA/protein model tool of MEGA X software. The maximum likelihood analysis was performed on MEGA X with 1000 bootstrap replicates (Kumar et al. 2018). Chaenogobius  gulosus (Oh et al. 2016) and Chaenogobius annularis were selected for the outgroups.

Results
The complete mitochondrial genome of R. davidi comprised 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (12S rRNA and 16S rRNA), and 2 non-coding control regions (control region and origin of light-strand replication), which formed a double-stranded circular molecule of 16,627 bp in length (Figure 2). All other genes were encoded on the heavy strand (H-strand), except for one protein-coding gene (ND6) and eight tRNA genes (tRNA Gln , tRNA Ala , tRNA Asn , tRNA Cys , tRNA Tyr , tRNA Ser(UCN) , tRNA Glu , and tRNA Pro ). The overall nucleotide composition in descending order was 17% for G, 25.9% for T, 27% for A, and 30.1% for C, with a slight AT-rich feature (52.9%).
Twelve protein-coding genes began with the standard ATG, whereas COX1 initiated with GTG. There were both complete termination codons (TAA for ND1, COX1, ATP8, ATP6, ND4L, ND5; TAG for ND2, ND3, ND6) and incomplete termination codons (TA for COX3, T for COX2, ND4, CYTB). Four overlapping regions revealed between protein-coding genes were ATP8-ATP6, ATP6-COX3, ND4L-ND4, and ND5-ND6, and there were two overlaps between tRNAs (tRNA Ile -tRNA Gln , tRNA Gln -tRNA Met ). For rRNAs, 12S rRNA was located between tRNA Phe and tRNA Val with 954 bp in length, and 16S rRNA was located between tRNA Val and tRNA Leu(UUR) with 1,661 bp in length. They were separated by tRNA Val with the same situation found in other gobies (Wang et al. 2019;Tan et al. 2020). 22 tRNAs ranged from 65 to 76 bp, which were conservative and scattered throughout the mitogenome of R. davidi. The control region, which was 476 bp long, was located between tRNA Phe and tRNA Pro .

Discussion and conclusion
We reported the first complete mitogenome sequencing of R. davidi by high-throughput sequencing and assembly.
The arrangement and orientation of all 37 genes were in accordance with those goby mitogenomes published previously (Xie et al. 2015;Wang et al. 2019). In this study, more molecular information from mitogenomes of Rhinogobius was used to reconstruct the phylogenetic mitochondrial genome relationships, and the ML tree placed R. davidi in a well-supported cluster with Rhinogobius giurinus. The overall topology was similar to those of previous studies (Zhang and Shen 2019;Tan et al. 2020). The results presented here will be essential to the specimen identification, fishery resources management, and further phylogenetic studies on Gobionellinae species.

Ethical approval
Experiments were performed in accordance with the recommendations of the Ethics Committee for Animal Experiments of Jiangsu Agri-animal Husbandry Vocational College. These policies were enacted according to the Chinese Association for the Laboratory Animal Sciences and the Institutional Animal Care and Use Committee (IACUC) protocols.