The complete mitochondrial genome and phylogenetic analysis of Anabarilius duoyiheensis Li, Mao & Lu, 2002 (Cypriniformes: Xenocyprididae)

Abstract Anabarilius duoyiheensis is a native and rare fish in Yunnan. In this study, the complete mitochondrial genome of A. duoyiheensis was sequenced and published for a total of 16,614 bp, including 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and one control region. The phylogenetic analysis based on the complete mitochondrial genome showed that A. duoyiheensis belongs to the clade of the genus Anabarilius and was sister to the clade of Hemiculter. This study also contributes to the genus phylogeny of Anabarilius and other members of the family Xenocyprididae.


Introduction
Anabarilius duoyiheensis Li, Mao & Lu, 2002 belongs to Cypriniformes, Xenocyprididae, Anabarilius and distributed in Duoyi river in Qujing, Yunnan, China (Li et al. 2002;Chen X 2013).The morphological features of A. duoyiheensis were: mouth terminal; head length is significantly greater than body height; arcuate abdomen; incomplete ventral edge; long pectoral fins with terminal tip; short branchial rake (Li et al. 2002).Some of the main features were similar to those of A. macrolepis with distinguishable features: fewer branchial rake (7-8); the end of unbranched dorsal fin is hard; longer pectoral fin (Li et al. 2002).The Anabarilius species, distributed mainly in central and eastern Yunnan and south of Sichuan Province, were peculiar to China (Yang J and Chu 1987).However, there are no data regarding A. duoyiheensis and particularly its mitochondrial genome.Here, we presented the mitochondrial genome of A. duoyiheensis by using next-generation sequencing, and these results will be useful in further phylogenetic analysis of genus of Anabarilius.

Specimens collection and preservation
The biological specimens of A. duoyiheensis were sampled from the Lu Buge reach of the Duoyi river (N: 24 � 45.669 0 , E: 104 � 29.758 0 ) in Qujing, Yunnan, and the live fish were kept in Lubuge Township Fisheries Station.A specimen was deposited at Yunnan Institute of Fishery Sciences Research with the specimen voucher number: DYBY-01 (contact person: Junjie Wu, wujunjie2007@yeah.net).The specimen of A. duoyiheensis was identified by morphological feature according to Li's description (Li et al. 2002), and descriptive image was taken by Leng (corresponding author) (Figure 1).

Phylogenetic analysis
To analyze the phylogenetic relationship of A. duoyiheensis, published mitochondrial sequences of other 18 species in the family of Xenocyprididae were chosen; the species information and references are listed in Table 1.The alignment sequences were obtained in MEGA X using MUSCL method (Kumar et al. 2008), and substitution model was evaluated in jModeltest 2.1.3(Posada 2008).The AICc results were carried in phyML 3.0 for rebuilding the maximum-likelihood (ML) tree (Guindon

Mitochondrial characterization
The complete mitochondrial DNA sequence of A. duoyiheensis was 16,614 bp in length with a depth of 582.42x (Figure S1, Table S1), including 13 protein-coding genes, 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and a D-loop control region (Figure 2).The initiation codon of 13 protein coding genes began with ATG except COX1, which began with GTG.Incomplete stop codons were identified in Nd2, Cox2, Cox3, Nd3, Nd6, and Cytb; TAA or TAG stop codon was identified in Nd1, Cox1, Atp8, Atp6, Nd4l, and Nd5.The base composition was 28.87%A, 24.89%T, 27.86%C, and 18.37%G, the A þ T content was 53.76% and G þ C content was 46.24%.Most of the genes were in the heavy strand (Hstrand), except for ND6 and eight tRNAs.The blast search shows that the identity of A. duoyiheensis mitochondrial sequences was 94% similar to A. liui yalongensis, 93% to A. brevianalis, and 91% to H. leucisculus and H. tchangi.

Phylogenetic analysis
The model of GTR þ I þ G was the perfect substitution model for phylogenetic construction.The ML tree showed that A. duoyiheensis is phylogenetically closest to the clade of A. liui yalongensis and A. brevianalis.The genus of Anabarilius was monophyletic, and a sister group to clade of genus Hemiculter (Figure 3).

Discussion and conclusions
In this study, we first presented the mitochondrial genome of A. duoyiheensis, which is also the first molecular biological data for this species.The availability of this mitochondrial genome will be essential for future DNA barcoding, taxonomic, and phylogenetic studies of the family Xenocyprididae, and also valuable for resource conservation.

Figure 1 .
Figure 1.Specimen of Anabarilius duoyiheensis was collected in the Duoyi river, Yunnan Province, China.Photographed by Yun Leng on 16 November 2021.

Figure 2 .
Figure 2. Complete mitochondrial genome map of A. duoyiheensis (GenBank: OM731672.1),with 13 genes, 22 tRNAs, and two rRNAs.Encoded genes and RNAs were in different colors; light and heavy strands were shown inside and outside of the circle, respectively.

Figure 3 .
Figure 3. Phylogenetic tree of A. duoyiheensis and other 18 species based on the complete mitochondrial sequences.Squaliobarbus curriculus was set to be outgroup.The bootstrap values were marker near the nodes.Accession numbers for each species were shown after the name of the species.The A. duoyiheensis generated in this study (OM731672.1)was labeled in black outline.

Table 1 .
Whole mitochondrial genomes used to infer the phylogenetic analysis.