Complete mitochondrial genome of Eonemachilus longidorsalis (Cypriniformes: Nemacheilidae): genome characterization and phylogenetic consideration

Abstract For the first time, this study presents the complete mitochondrial genome and phylogenetic analysis of Eonemachilus longidorsalis, an endemic species in southwest China. The mitogenome is a circular molecule of 16,569 bp in length with a base composition of 30.2% A, 27.3% T, 16.6% G, and 25.9% C. The phylogenetic analysis based on the complete mitogenomes shows that E. Longidorsalis clusters with Yunnanilus jiuchiensis, Yunnanilus pleurotaenia and Eonemachilus niger. This contribution may provide a valuable framework for completely resolving phylogenetic relationships of the family Nemacheilidae in future research.


Introduction
Eonemachilus longidorsalis (Li, Tao and Lu 2000) is distributed in the eastern Yunnan, Nanpangjiang drainage of China. This species combines morphological characteristics that can be distinguished from other Eonemachilus fishes: scales covering the whole body; extremely long dorsal fin iii-11-12; anal fin ii-7; pectoral fin i-10; caudal fin i-14-i; 12 inner gill rakers on the first gillarch and no outer gill rakes (Du et al. 2021). Eonemachilus (Berg 1938) was once regarded as a synonym of Yunnanilus by some ichthyologists (Kottelat and Chu 1988;Zhu 1989;Yang 1991), but morphological studies supported that Eonemachilus was a valid genus, which can be distinguished from Yunnanilus based on absent (vs. present) lateral line and cephalic lateral-line canals (Li WX et al. 2000;Kottelat 2012). This viewpoint was confirmed by the evolutionary position of E. nigomaculatus based on molecular data, which was the most basal clade of the tribes Lefuini, Nemacheilini, Triplophysini, and some members of Yunnanilini (Du et al. 2021). However, we noticed that the sequence of E. nigomaculatus was misidentified and changed in GenBank by the author, resulting in the invalidity of previous phylogenetic analyses conducted from molecular perspective. In this study, the complete mitochondrial genome of E. longidorsalis was determined and described, and its taxonomic status in Nemacheilidae fishes was analyzed, which may provide a valuable genetic resource for further phylogeny analysis on the genus Eonemachilus.

Methods
After being transported to Shanghai Genesky Biotechnologies Inc, total genomic DNA was extracted from muscle using the Tguide Cell/tissue genomic DNA Extraction Kit (OSR-M401) (Tiangen, Beijing, China). The next several steps were DNA sample quality control, DNA library construction, PCR amplification, size selection, library quality check, and library pooling. Sequencing was conducted on the Illumina HiSeq 4000 Sequencing platform (Illumina, CA, USA). The complete mitogenome of E. longidorsalis was obtained by sequence assembly on MetaSPAdes software (Nurk et al. 2017). The annotation process was executed using MitoMaker (Bernt et al. 2013). The mitogenome sequence and gene annotations were submitted to the National Center for Biotechnology Information GenBank database under the accession number OM732331. The CGView online server (https://proksee.ca/) was chosen to draw the mitogenome map.
To infer the taxonomic status of E. longidorsalis, the complete mitochondrial genomes of 31 Nemacheilidae species and 2 Balitoridae species were concatenated into a dataset, and a phylogenetic tree was constructed via the maximum likelihood method under the most suitable nucleotide sequence model GTR þ GþI on MEGA X (Kumar et al. 2018). The 2 Balitoridae species, Sinogastromyzon szechuanensis (Li W et al. 2021) and Metahomaloptera omeiensis (Li Y et al. 2016) were used as outgroups.

Discussion and conclusion
Through next-generation sequencing and assembly, the complete mitogenome of E. longidorsalis is 16,569 bp in length. The gene order and composition are identical to typical mitogenomes of other teleost fishes (Luo et al. 2019). The maximum likelihood tree based on the complete mitochondrial genomes of E. longidorsalis and 32 other species supports that E. longidorsalis constitutes a sister-group mitogenome relationship with Yunnanilus jiuchiensis, Yunnanilus pleurotaenia and Eonemachilus niger. The inclusion of more related taxa on future whole mitogenome phylogenetic analyses may help to understand the phylogeny of Yunnanilus and Eonemachilus (Yamasaki et al. 2015;Wang et al. 2019;Maeda et al. 2021). In conclusion, this study will provide important information for future taxonomic, systematic, and genetic studies of Nemacheilidae.

Ethical approval
Experiments were performed following 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.