The first determination and analysis of the complete mitochondrial genome of Ancistrus temmincki (Siluriformes: Loricariidae)

Abstract In order to fully comprehend the evolution and kinship of fishes in the family of Loricariidae, the complete mitochondrial genome of the Loricariidae fish Ancistrus temmincki was firstly characterized in the present study. The whole mitogenome was 16,657 bp in size and consisted of 13 protein-coding genes, 22 tRNAs, 2 rRNAs genes, a control region and origin of light-strand replication. The proportion of coding sequences with a total length of 11,473 bp was 68.88%, which encoded 3,813 amino acids. The genome composition was highly A + T biased (56.29%), and exhibited AT-skew (0.0661) and a negative GC-skew (–0.2740). All protein-coding genes were started with ATG except for GTG in CO1, while stopped with the standard TAN codons or a single T. The control region (D-loop) ranging from 15,635 bp to 16,657 bp was 1023 bp in size. Until now, there is hardly any studies on the complete mitochondrial sequence in the genus of Ancistrus, phylogenetic analysis showed that A. temmincki was most closely related to Ancistrus cryptophthalmus in the genus of Ancistrus. The complete mitochondrial genome sequence has provided a new insight into the taxonomic classification, and a more complex picture of the species diversity within the family of Loricariidae.


Ancistrus temmincki; mitochondrial genome; evolutionary relationship
Ancistrus temmincki, a newly discovered species of fish in the family Loricariidae, now has been initially positioned as Ancistrus. It inhabits the turbulent area of the river, adsorbs on the rock, eats the moss on the rock, and has a gentle temperament. Its body is covered with small gray spots and extends to the fins. The head is big and the eyes are behind. There are tentacles on the upper surface of the kiss on the head. The female fish has only one row of bristles between the eyes, while the male fish has two rows of bristles that form a 'V' shape (Geerinckx et al. 2007). The body length can reach to 9.8 cm. So far, the reports about this species have limited to the analysis of its appearance (Avise et al. 1987;Xu et al. 2011;Huang et al. 2016). In this study, we firstly determined the complete mitochondrial genome of A. temmincki. Additionally, the phylogenetic relationship of Siluriformes (Loricariidae: Ancistrus) was reconstructed based on 13 PCGs for the first time, which involved 15 typical representative species in Perciformes. The present study would supply references to the limited data on molecular level and clarify the classification relationship, even be referenced for systematics.
Ancistrus temmincki, a newly discovered species of fish in the family Loricariidae, now has been initially positioned as Ancistrus. It inhabits the turbulent area of the river, adsorbs on the rock, eats the moss on the rock, and has a gentle temperament. The fishes collected from the Surin of South America (3 38 0 38 00 N, 54 02 0 06 00 W). All samples (AT201912) have been deposited in the Zhejiang Engineering Research Center for Mariculture, Zhejiang Ocean University, Zhoushan, China. Tissue samples were reserved in 95% ethanol for molecular analysis and stored at À20 C. Total genomic DNA was extracted from the muscle using the phenol-chloroform method (Barnett and Larson 2012;Toni et al. 2018). Based on the existing complete mitochondrialgene of A. cryptophthalmus (Accession MF804392.1), 12 pairs of primers were designed (Supplementary Table S1) and synthesized by Primer Premier 6.0 (Lalitha 2000). The samples were amplified by Polymerase chain reaction (PCR), PCR was conducted using the following conditions: denaturation at 95 C for 5 min, 35 amplification cycles (95 C denaturation for 30 s, 52-55 C annealing for 30 s, 72 C extension for 60 s), and a final extension at 72 C for 10 min. Subsequently, sequences using Sanger sequencing technology and using CodonCode Aligner 5.1.5 (CodonCode Corporation, Dedham, MA) to assemble. The complete mitochondrial genome was annotated using Sequin version 15.10 (http://www.ncbi.nlm.nih. gov/Sequin) and tRNAscan-SE version 2.0 (http://trna.ucsc. edu/tRNAscan-SE/) (Lowe and Eddy 1997). The calculation of base composition and phylogenetic construction was conducted by MEGAX.
Similar to the typical mitogenome of vertebrates, the mitogenome of A. temmincki was a closed double-stranded circular molecule of 16,657 bp including 13 protein-coding genes, 2 ribosomal RNA genes, 22 tRNA genes and two main noncoding regions (Boore, 1999;Zhu et al. 2018) deposited in GenBank under accession number MT528234. The proportion of coding sequences with a total length of 11,473 bp was 68.88%, 13 protein-coding genes (PCGs) encode 3813 amino acids in total. The overall contents of A, T, G, and C were 30.01%, 26.28%, 15.87%, and 27.84%, respectively. A-T and G-C contents of mitochondrial genome were 56.29% and 43.71% respectively, thereby with a high AT bias. Both ATskew and GC-skew of the mitogenome were 0.0661 and À0.2740, respectively. All the protein-coding genes used the initiation codon ATG except for GTG in CO1, which was quite common in vertebrate mtDNA (Miya et al. 2001). Most of them have TAA or TAG as the termination codon, except ND4 used termination condons AGA and CO2 used an incomplete termination codon T. The termination codons observed in the A. temmincki genomes were similar to those of most mammals (Kitpipit et al. 2012;Hassanin 2016). Most mitochondrial genes were encoded on the H-strand except for ND6 and eight tRNA genes (Gln, Ala, Asn, Cys, Tyr, Ser, Glu, and Pro), which were encoded on the other complementary strand. The lengths of 12S ribosomal RNA and 16S ribosomal RNA were 951 bp and 1675 bp, which were both located in the typical positions between tRNA-Phe and tRNA-Leu, being separated by tRNA-Val (Petrillo et al. 2006). While the length of control region (D-loop) was 1023 bp, ranging from 15,635 bp to 16,657 bp.
To explore the phylogenetic position of A. temmincki, we used a total of 14 species (Supplementary Table S2) of Perciformes mitochondrial genomes, belonging to 8 families.
The splicing tandem alignment in the phylogenomic analysis included 13 PCGs after removing the conserved blocks, based on the maximum likelihood (ML) analysis constructed phylogenetic tree. The GenBank accession numbers were listed before the species names. The results of the present study suggested that A. temmincki had a closest relationship with A. cryptophthalmus, highly supported by a bootstrap value of 100 (Figure 1), which was well consistent with the results based on morphology and other molecular methods. Phylogenetic analysis used to get a clear understanding of classification status, and has well clarified the phylogenetic classification of A. temmincki here. Therefore, our findings have provided more details of this species, and would help to the further studies on the family of Loricariidae.

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