Complete mitochondrial genome sequence and annotation of Rhinogobius lentiginis (Gobiiformes: Gobiidae: Gobionellinae)

Abstract We report the complete mitochondrial genome of Rhinogobius lentiginis, which was found to be a circular molecule of 16,633 bp in length and included 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a non-coding control region. The overall base composition was 28.44% A, 26.21% T, 16.33% G, and 29.02% C. Phylogenetic analyses using maximum-likelihood and Bayesian inference methods revealed a close genome relationship among R. lentiginis, R. niger, R. shennongensis and R. maculagenys. The complete mitogenome of R. lentiginis will provide a valuable resource for species classification and conservation.


Introduction
Rhinogobius lentiginis (Gobiiformes: Gobiidae: Gobionellinae), an endemic species in China, is mainly distributed in the Lingjiang Basin, the Tingpangxi Basin, lower reaches of the Yangtze Basin and the Cao'ejiang River, which is a tributary of the Qiantang River Basin. R. lentiginis is a benthic, landlocked species that prefers slow-flowing water environment with fine gravel as the sediment. Human activities, such as the discharge of domestic and industrial sewage, stream dredging operations, and the construction of dams and reservoirs, will have a great impact on the survival of the goby. Taking the Qiantang River basin as an example, it was reported that there were 11 species of goby in 2011, but several of them were extremely rare. The population of these rare species is likely to decline or even become extinct due to the continuous deterioration of the basin environment (Li 2011). To develop strategies for the management of fisheries resources, the taxonomic survey and research on stream fish will be the primary work. Mitochondrial DNA is considered to be one of the most efficient and reliable molecular marker for species identification, evolutionary biology, population genetics and conservation biology (Ko et al. 2018). Therefore, we sequenced the complete mitogenome of R. lentiginis to present basic genetic information.

Methods
Genomic DNA was isolated from muscle tissue using the Tguide Cell/tissue genomic DNA Extraction Kit (OSR-M401, Tiangen, Beijing, China), followed by quality control to ensure sample concentration, purity and integrity using the NanoDrop 2000 (Thermo Fisher Scientific, USA). A DNA library consisting of all sheared mitochondrial DNA was prepared using the MGIEasy DNA Library Prep Kit (MGI Technology, Shenzhen, China). The purificaiton and size selection of the library was completed using Agencourt SPRIselect (Beckman Coulter, USA). The Agilent 2100 Bioanalyzer (Agilent Technologies, USA) was used to check the quality of the quality of the library. Finally, the constructed library was sequenced on the Illumina HiSeq 4000 Sequencing platform (Illumina, CA, USA) with paired-end reads (150 bp).
All available 25 mitogenomes of Rhinogobius species in GenBank were retrieved to analyze the phylogenetic position of R. lentiginis in the genus (Table 1). Two Tridentiger species, belonging to the same Gobionellinae family, were used as an outgroup. The complete mitogenome sequences were aligned by ClustalW in MEGA X (Kumar et al. 2018). The best fit substitution model (GTR þ I þ G) was calculated by jModelTest 2.  chains. Each run consisted of 2,000,000 generations, sampled every 100 generations with 25% of the initial trees discarded as burn-in.

Mitogenome organization
The complete mitogenome of R. lentiginis (GenBank accession number OM617725) was composed of 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a non-coding control region (Figure 2). The length of the mitogenome was 16,633 bp, with the overall base composition of 28.44% A, 26.21% T, 16.33% G, 29.02% C. Of all 37 mitochondrial genes, 28 were encoded on the heavy strand and 9 were encoded on the light strand (ND6, tRNA Gln , tRNA Ala , tRNA Asn , tRNA Cys , tRNA Tyr , tRNA Ser(UCN) , tRNA Glu , and tRNA Pro ). Most of the 13 PCGs contained the typical start codon ATG, except for COX1 starting with GTG, consistent with other fish mitochondrial genomes (Tan et al. 2020;Yang et al. 2020). Conventional stop codons were observed in ten PCGs (TAA for ND1, ND2, COX1, ATP8,  ATP6, COX3, ND4L, ND5, and TAG

Phylogenetic analysis
Both reconstruction methods recovered that there were two main clades in Rhinogobius (Figure 3), in which R. estrellae and R. tandikan formed one, and other 21 Rhinogobius species formed the other clade. In our analyses, both trees displayed that R. lentiginis had a close mitochondrial genome relationship with R. niger, R. shennongensis and R. maculagenys. The genetic distance between R. lentiginis and 22 other Rhinogobius species ranged from 0.109 to 0.183.

Discussion and conclusion
The complete mitogenome of the freshwater goby R. lentiginis had highly conserved structural organization and typical gene content, which were similar to Gobionellinae fishes (Wang et al. 2019;Yang et al. 2020). Our phylogenetic analyses of R. lentiginis revealed a close mitogenome relationship with R. niger, R. shennongensis and R. maculagenys. Present grouping results provided a better resolution for the genetic relationships among members of the Rhinogobius genus. Previous studies on the phylogeny of Rhinogobius species have shown that changes of members in the phylogenetic tree can affect the relationships of the species (Yamasaki et al. 2015;Wang et al. 2019). Therefore, further studies including morphology and genetics based on extensive taxon sampling are needed to assess the phylogenetic relationships among Rhinogobius species and genera. Overall, our results provide the genetic basis for resource conservation.

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.