PJZ_57_6_2757-2766

INTRODUCTION

The pencil fish Nannostomus beckfordi belongs to the order Characiformes and family Lebiasinidae. Amazonian fish in the family Lebiasinidae stand out for their ornamental potential; they are distributed from Central America to South America. It is characterized by a cylindrical body, red coloration and a long black stripe on its body, and a superior mouth. It lives on shoals, feeds on microcrustaceans or periphytons, and decomposes organic matter (Weitzman and Weitzman, 2003). N. beckfordi as an aquarium fish has value in the international market (Allpondsolutions, 2019; Prang, 2008).

The mitochondrial genome, characterized by a simple structure, compact gene arrangement, and strict maternal inheritance, has been used extensively in molecular phylogenetics and population genetics research on metazoans over the past decade (Dellaporta et al., 2006; Helfenbein et al., 2004; Boore and Brown, 1998). Owing to the developments in DNA sequencing technology, it has become an ideal material for studying the origin, evolution, phylogeny, and population genetics of various species.

Alien species (as opposed to native species) are those whose presence in a region is attributable to human actions, deliberate or inadvertent, that enabled them to overcome biogeographical barriers (Richardson et al., 2000, 2011; Pyšek et al., 2004; Essl et al., 2018). N. beckfordi has high ornamental value and economic value in China. But because they are distributed from Central America to South America, it is an alien species. Full-length N. beckfordi mitochondrial genome sequences are limited, and little is known about the evolution of the species. In this study, direct sequencing was used to obtain the complete N. beckfordi mitochondrial genome sequence for comprehensive and in-depth bioinformatics, evolutionary, and phylogenetic analyses, laying a molecular genetic foundation for the scientific conservation of this species. It also helps judge its invasion risk, and provides reference for the prevention and control measures against it at the molecular level.

MATERIALS AND METHODS

Experimental materials

N. beckfordi was collected in Sep 2022 at the Fuzimiao Flower, Bird, Fish and Insect Market in Qinhuai District, Nanjing City, Jiangsu Province (118°50′29.92″E, 32°0′21.02″N). After preliminary morphological identification, the tail fin tissue was taken and stored in a 1.5 ml centrifuge tube at -80°C for future use.

DNA extraction

All samples were cut into small pieces, and the genomic DNA was extracted using DNAiso Reagent (company address Caf. #) according to the manufacturer’s instructions. The ethanol precipitated genomie DNA was suspended in sterilized water, and its quality detected by using 1% agarose gel electrophoresis. DNA purity and concentration were determined using an ultra-micro spectrophotometer and DNA barcoding technology.

Sequencing and annotation

A Covaris ultrasonicator was used to break the genomic DNA into approximately 350 bp fragments, followed by end-repair and the addition of an A base and sequencing adapter at the 3’ end. PCR amplification was performed on the products, with recovery and purification using magnetic beads to construct a library. After quality inspection of the DNA library, the Illumina HiSeq high-throughput sequencing platform was used for paired-end sequencing with a sequencing data volume of no less than 6 Gb for each sample. Sequencing was conducted by Shanghai Parsenor Biotechnology Co., Ltd. Low-quality reads and splice sequences were filtered. NOVOPlasty (Dierkxsens et al., 2017) was used for de novo assembly, referring to the published mitochondrial genome sequence of Lebiasina astrigata MH921292, and BioEdit 7 2.5 (Hall, 1999) was used to proofread the spliced sequences. ITOS 2 (Eliade and Fernández, 1968) (http://mitos.bioinf.unileipzig.de/index.py) and MitoFish (Iwasaki et al., 2013) were used for gene annotation. The complete mitogenome sequence of N. beckfordi was submitted to the NCBI (National Center for Biotechnology Information) database (Supplementary Table 1).

Phylogenetic analysis

The mitochondrial whole genome sequences of 72 fish were downloaded from NBCI (Table I). PhyloSuite v1.2.1 (Zhang et al., 2020) was used for phylogenetic analyses using the maximum likelihood (ML) method. In particular, the ML tree was inferred using IQ TREE (Nguyen et al., 2015) under the HKY+I+G4+F model with 50000 ultrafast bootstrap replicates (Minh et al., 2013) and

Table I. Nannostomus beckfordi mitochondrial genome composition.

Gene	Position		Size/ bp	Intergenic nucleotides	Codon		Str-and
Gene	From	To	Size/ bp	Intergenic nucleotides	Start	Stop	Str-and
tRNA-Phe	1	70	70	0			H
12S rRNA	71	1025	955	0			H
tRNA-Val	1026	1097	72	0			H
16S rRNA	1098	2795	1698	0			H
tRNA-Leu	2796	2871	76	0			H
ND1	2872	3846	975	0	ATG	TAG	H
tRNA-Ile	3851	3922	72	4			H
tRNA-GIn	3921	3991	71	-2			L
tRNA-Met	3992	4061	70	0			H
ND2	4062	5106	1045	0	ATG	T	H
tRNA-Trp	5107	5178	72	0			H
tRNA-Ala	5182	5250	69	3			L
tRNA-Asn	5252	5324	73	1			L
tRNA-Cys	5354	5420	67	29			L
tRNA-Tyr	5420	5490	71	-1			L
COXI	5492	7048	1557	1	GTG	AGG	H
tRNA-Ser	7040	7110	71	-9			L
tRNA-Asp	7115	7184	70	4			H
COXII	7198	7888	691	13	ATG	T	H
tRNA-Lys	7889	7962	74	0			H
ATPase8	7964	8131	168	1	ATG	TAA	H
ATPase6	8122	8804	683	-10	ATG	TA	H
COXIII	8805	9589	785	0	ATG	TA	H
tRNA-Gly	9590	9662	73	0			H
ND3	9663	10008	346	0	ATG	T	H
tRNA-Arg	10009	10079	71	0			H
ND4L	10080	10376	297	0	ATG	TAA	H
ND4	10370	11751	1382	-7	ATG	TA	H
tRNA-His	11752	11821	70	0			H
tRNA-Ser	11822	11889	68	0			H
tRNA-Leu	11891	11963	73	1			H
ND5	11964	13802	1839	0	ATG	TAA	H
ND6	13799	14317	519	-4	ATG	TAG	L
tRNA-Glu	14318	14386	69	0			L
Cytb	14392	15534	1143	5	ATG	TAA	H
tRNA-Thr	15536	15607	72	1			H
tRNA-Pro	15609	15678	70	1			L
D-loop	15679	16742	1063	0			H

the Shimodaira-Hasegawa-like approximate likelihood ratio test to evaluate branch support (Guindon et al., 2010).

RESULTS

Mitoch ondrial genome structure

The total length of the N. beckfordi mitochondrial genome was 16742 bp (Fig. 1 and Table I). The genome included 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and one control region (D-loop). The N. beckfordi genome had 12 spacer regions (ND1 and tRNA-Ile; tRNA-Trp and tRNA-Ala; tRNA-Ala and tRNA-Asn; tRNA-Asn and tRNA-Cys; tRNA-Tyr and COXI): tRNA-Ser and tRNA-Asp, tRNA-Asp and COXII; tRNA-Lys and ATPase-8; tRNA-Ser and tRNA-Leu; tRNA-Glu and Cytb; Cytb and tRNA-Thr; tRNA-Thr and tRNA-Pro). The lengths of these 12 intervals ranged from 1 to 29 bp, with a total length of 64 bp, and the maximum interval was detected between tRNA-Asn and tRNA-Cys. There were six overlapping gene regions (tRNA-Ile and tRNA-Gln; tRNA-Cys and tRNA-Tyr; COXI and tRNA-Ser; ATPase-8 and ATPase-6; ND4L and ND4; and ND5 and ND6), with a total of 33 bp of overlap. These six overlapping regions ranged in length from 1 to 10 bp, and gene overlap was the longest between ATPase-8 and ATPase-6. Additionally, 19 tightly arranged gene pairs did not overlap or contained gaps.

Table II. Base composition of the whole Nannostomus beckfordi mitochondrial genome.

Regions	Strand	Size (bp)	T (%)	C (%)	A (%)	G (%)	AT (%)	GC (%)
PCGs	+	10902	30.8	25.3	30.1	13.8	60.9	39.1
PCGs	-	519	42.6	11	15.2	31.2	57.8	42.2
rRNAs	+	2653	22.8	22.9	35.2	19.1	58	42
tRNAs	+	1003	27.7	20.6	32.9	18.7	60.6	39.3
tRNAs	-	561	31.6	16.2	23.7	28.5	55.3	44.7
Full genome	+	16742	28.9	24.4	32	14.8	60.9	39.2

PhyloSuite v1.2.1 was used to analyze the base composition of the mitochondrial genome of N. beckfordi (Table II). The base distribution in the complete mitochondrial genome sequence was as follows: 28.9% (T), 24.4% (C), 32% (A), and 14.8% (G). The A+T and G+C contents were 60.9% and 39.2%, respectively. There was a clear AT bias. The A+T contents were higher than the G+C contents in H-chain PCGs, L-chain PCGs, H-chain rRNAs, H-chain tRNAs, and L-chain tRNAs. The A+T content of H-chain PCGs was the highest at 60.9%, which was equal to the A+T content of the mitochondrial genome sequence.

Protein-coding genes

The N. beckfordi mitochondrial genome contained 13 PCGs with a length of 11421 bp, accounting for 68.2% of the entire genome. In the N. beckfordi mitochondrial genome, PCGs were present in both L and H-chains. Except for one NADH reductase complex subunit (ND6) on the L-chain, all other PCGs were distributed on the H-chain, including one cytochrome b (Cytb), two ATP synthase subunits (ATPase-6 and ATPase-8), three cytochrome oxidase subunits (COXI, COXII, and CO III), and seven NADH reductase complex subunit-coding genes (ND1-6 and ND4L) (Table I). The start and stop codons of the 13 PCGs are listed in Table I. The most frequent start codon was ATG, followed by GTG. The 12 PCGs with ATG as the start codon were ND1, ND2, COXII, ATPase8, ATPase6, COXIII, ND3, ND4L, ND4, ND5, ND6, and Cytb. The only PCG with GTG as the start codon was COXI. The termination codons of N. beckfordi were mainly TAA, followed by the incomplete termination codons T and TA. Four PCGs, ATPase-8, ND4L, ND5, and Cytb, had TAA as the termination codon, while the only PCG (COXI) had AGG as the termination codon. The only PCGs with TAG as the termination codon were ND1 and ND6. The termination codons of the remaining six genes were incomplete, with ND2, C OXII, and ND3 using T as the termination codon and ATPase6, COXIII, and ND4 using TA.

An a nalysis of the base composition of 13 PCGs in the entire mitochondrial genome of N. beckfordi is summarized in Table III. The A + T content was higher than the G + C content in 1st, 2nd, and 3rd codons of the H-chain and the 2nd and 3rd codons of the L-chain. Only for the 1st codon of the L-chain was the A + T content slightly lower than the G + C content, indicating a clear AT bias. The third codon of the H-chain had the highest A + T%, whereas the first codon of the L-chain had a low A + T content. The A + T content of the PCG sequences in the H or L-chain increased gradually from the first to the third codon. Moreover, at the same position in the codon, the A + T content of the H-chain codons was higher than that of the L-chain codons.

Table III. Base content at different codon positions of protein-coding genes.

Regions	Strand	Size (bp)	U (%)	C (%)	A (%)	G (%)	AT (%)	GC (%)
1st codon position	+	3634	23.9	24.5	27.8	23.8	51.7	48.3
1st codon position	-	173	39.3	7.5	10.4	42.8	49.7	50.3
2nd codon position	+	3634	40.7	27.3	18.8	13.2	59.5	40.5
2nd codon position	-	173	44.5	19.7	9.8	26	54.3	45.7
3rd codon position	+	3634	27.9	24.1	43.5	4.5	71.4	28.6
3rd codon position	-	173	43.9	5.8	25.4	24.9	69.3	30.7

The frequency of amino acid usage for each PCG in the mitochondrial genome is shown in Figure 2. The molar mass percentage (mol%) of the amino acid Leu1 (10.74%) was greater than 10%, making it the most frequently used amino acid among all PCGs. Lys (2.21%), Asp (1.89%), Glu (2.47%), Cys (0.71%), Arg (1.95%), and Ser1 (1.55%) had a mol% of ≤ 2.5%, making them the least frequently used amino acids among all PCGs.

The frequency and relative usage of each codon of the N. beckfordi mitochondrial genes are shown in Table IV. Each of the approximately 29 codons had a RESU value greater than 1, indicating that these are the preferred codons of N. beckfordi mitochondrial genes. Among the above codons, most ended with either A or U bases. Codons ending with C bases were less frequent, with RSCU values of less than 1. However, codons ending in G were least frequent, and all RSCU values were less than 1, indicating the use or avoidance of codons in N. beckfordi mitochondrial genes. The N. beckfordi mitochondrial genes preferred TAA as a termination codon.

Table IV. RSCU values for the codons of genes.

AA	Codon	Count	RSCU	AA	Codon	Count	RSCU
Phe	UUU	152	1.29	Ala	GCA	135	1.62
Phe	UUC	83	0.71	Ala	GCG	6	0.07
Leu2	UUA	221	2.03	Tyr	UAU	63	1.09
Leu2	UUG	25	0.23	Tyr	UAC	53	0.91
Leu1	CUU	124	1.14	His	CAU	40	0.73
	CUC	67	0.61	His	CAC	70	1.27
	CUA	199	1.83	Gln	CAA	93	1.86
	CUG	18	0.17	Gln	CAG	7	0.14
Ile	AUU	230	1.58	Asn	AAU	62	1.03
Ile	AUC	62	0.42	Asn	AAC	58	0.97
Met	AUA	143	1.63	Lys	AAA	72	1.71
Met	AUG	32	0.37	Lys	AAG	12	0.29
Val	GUU	75	1.51	Asp	GAU	25	0.69
	GUC	27	0.54	Asp	GAC	47	1.31
	GUA	83	1.67	Glu	GAA	80	1.7
	GUG	14	0.28	Glu	GAG	14	0.3
Ser2	UCU	47	1.17	Cys	UGU	14	1.04
	UCC	43	1.07	Cys	UGC	13	0.96
	UCA	92	2.28	Trp	UGA	104	1.69
	UCG	1	0.02	Trp	UGG	19	0.31
Pro	CCU	49	0.93	Arg	CGU	10	0.54
	CCC	62	1.18		CGC	11	0.59
	CCA	91	1.73		CGA	51	2.76
	CCG	9	0.17		CGG	2	0.11
Thr	ACU	57	0.77	Ser1	AGU	13	0.32
	ACC	90	1.21	Ser1	AGC	46	1.14
	ACA	145	1.95	Gly	GGU	50	0.83
	ACG	6	0.08		GGC	39	0.65
Ala	GCU	78	0.93		GGA	113	1.88
Ala	GCC	115	1.38		GGG	38	0.63

tRNA genes, rRNA genes, and D-loop

There were 22 tRNA genes in the N. beckfordi mitochondrial genome, with tRNA gene sizes ranging from 67 to 76 bp and an overall length of 1564 bp, accounting for 9.34% of the entire genome. Eight tRNA genes were present on the L-chain, namely tRNA-Gin, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu, and tRNA-Pro. The base composition of these genes included A, G, T, and C contents of 23.7%, 28.5%, 31.6%, and 16.2%, respectively. The A+T content was 55.3%, AT skewness was -0.142, and GC skewness was 0.275 Fourteen tRNA genes were present on the H-chain, namely tRNA-Phe, tRNA-Val, tRNA-Leu, tRNA-Ile, tRNA-Met, tRNA-Trp, tRNA-Asp, tRNA-Lys, tRNA-Gly, tRNA-Arg, tRNA-His, tRNA-Ser, tRNA-Leu, and tRNA-Thr. The A+T content was 60.6%, and the A, G, T, and C contents of tRNA were 32.9%, 18.7%, 27.7%, and 20.6%. AT skewness was 0.086, and GC skewness was -0.048. Detailed information on the base composition of tRNA genes in N. beckfordi mitochondrial genes is shown in Table V.

The N. beckfordi mitochondrial genome contains two rRNA genes, both located on the H-chain, with an overall length of 2653 bp and an A+T content of 58%. AT skewness was 0.214 and GC skewness was -0.09. The ribosomal small subunit 12S rRNA gene of mitochondrial DNA was located between the tRNA-Phe and tRNA-Val genes, whereas the ribosomal large subunit 16S rRNA gene was located between the tRNA-Val and tRNA-Leu genes. The length of the 12S rRNA gene was 955 bp, and the length of the 16S rRNA gene was 1698 bp. The two genes were separated from each other by the tRNA-Val gene at a distance of 73 bp. The base composition of the 12S rRNA gene was A, G, T, and C contents of 33.1%, 20.7%, 21.8%, and 24.4%, respectively. The A+T content was 54.9%, with an AT bias of 0.206 and a GC bias of

Table V. Base composition of tRNA and rRNA genes in the whole mitochondrial genome of Nannostomus beckfordi.

Regions	Strand	Size (bp)	T (%)	C (%)	A (%)	G (%)	AT (%)	GC (%)	AT skew	GC skew
16S rRNA	+	1698	23.4	22	36.4	18.2	59.8	40.2	0.218	-0.095
12S rRNA	+	955	21.8	24.4	33.1	20.7	54.9	45.1	0.206	-0.081
rRNAs	+	2653	22.8	22.9	35.2	19.1	58	42	0.214	-0.09
tRNAs	+	1003	27.7	20.6	32.9	18.7	60.6	39.3	0.086	-0.048
tRNAs	-	561	31.6	16.2	23.7	28.5	55.3	44.7	-0.142	0.275

-0.081. The base composition of the 16S rRNA genes included A, G, T, and C contents of 36.4%, 18.2%, 23.4%, and 22%, respectively. The A+T content was 59.8%, with an AT bias of 0.218 and a GC bias of -0.095. Detailed information on the base composition of the rRNA genes in the N. beckfordi mitochondrial genome is shown in Table V. The N. beckfordi mitochondrial genome contained a D-Loop region with a length of 1063 bp, located between tRNA-Pro and tRNA-Phe.

Phylogenetic relationships

Based on whole mitochondrial genome sequences of N. beck fordi and 72 other species in Characiformes, a phylogenetic tree was constructed using PhyloSuite v1.2.1 and the ML method.

In the ML tree, the order Characiformes formed a monophyletic group, separate from the representative species in the order Siluriformes used as an outgroup. Species in the order Characiformes were divided into two branches with high support. Crenuchus spilurus in the family Crenuchidae formed a branch that diverged earliest from the Characiformes lineage. The other branch was composed of 15 families (Alestidae, Anostomidae, Bryconidae, Chalceidae, Childontidae, Ctenoluciidae, Curimatidae, Erythrinidae, Gasteropelecidae, Hemiodontidae, Hepsetidae, Lebiasinidae, Parodontidae, Prochilodontidae, and Serrasalmidae). This branch also included a pair of sisters group branches.

In particular, one branch was formed by Phenacogrammus interruptus in the family Alestidae, Abrametes hypelonotis, Leporinus affinis, Megaporinus elongatus, and Megaporinus piavussu in the family Anostomidae, Hydrolycus scomberoidesin the family Chalceidae, Childus punctatus in the family Childontidae, Curimata mivartii, and Curiatopsis evelynae in the family Curimatidae, Hoplias intermedius and Hoplias malabaricus in the family Erythrinidae, Hemiodopsis gracilis in the family Hemiodontidae, Hepsetus odoe of the family Hepsetidae, Apariodon affinis in the family Parodontidae, Ichthyocephalus longirostris, Prochilodus argenteus, Prochilodus costatus, Prochilodus harttii, Prochilodus lineatus, and Prochilodus vimboides in the family Prochilodontidae, as well as Colossoma macropomum, Metynnis hypsauchen, Myloplus rubripinnis, Piaractus brachypomus, and Piaractus mesopotamicus in the family Characin. The other lineage was formed by members of the families Lebiasinidae (Lebiasina astrigata and Nannostomus beckfordi), Gasteropelecidae (Carnegiella strigata), Ctenoluciidae (Boulengerella machulata and Ctenolucius hujeta), Chalceidae (Chalceus macroepidotus, Aphyocharax rathbuni, Astyanax aeneus, Astyanax altiparanae, Astyanax lacustris, Astyanax mexico, Deuterodon giton, Gephrocharax atracaudatus, Grundulus bogotensis, Gymnocorymbus ternetzi, Hemigrammus armstrongi, Hemigrammus bleheri, Hemigrammus erythrozonus, Hemigrammus ocellifer, Hyphessobrycon amandae, Hyphessobrycon amapaensis, Hyphessobrycon anisitsi, Hyphessobrycon elachys, Hyphessobrycon flammeus, Hyphessobrycon herbertaxelrodi, Hyphessobrycon megalopterus, Hyphessobrycon pulchripinnis, Hyphessobrycon roseus, Hyphessobrycon socolofi, Hyphessobrycon sweglesi, Impaichthys kerri, Knodus borki, Moenkhausia costae, Moenkhausia sanctaefilomenae, Nematobrycon palmeri, Oligosarcus argenteus, Paracheirodon axelrodi, Paracheirodon innesi, Pristella maximalis, Psalidodon fasciatus, Psalidodon paranae, Psalidodon rivularis, Thayeria boehlkei, and Brycon henni), and Bryconidae (Brycon nattereri and Brycon orbityanus and Salmininae).

One branch formed by C. strigata of the family Gasteropelecidae and B. maculata of the family Ctenoluciidae, and another branch included L. astrigata and N. beckfordi of the family Lebiasinidae, which together formed a monophyletic group with a node self-expansion support rate of 100%. In the family Lebiasinidae, if two Lebiasinidae lineages were clustered together, the morphological and molecular relationships were consistent, indicating the close genetic relationship. The phylogenetic positions of each species in the ML tree based on rRNA and PCG data are shown in Figure 3.

DISCUSSION

The N. beckfordi mitochondrial genome sequence was 16742 bp, including 13 PCGs, 22 tRNA genes, two rRNA genes, and one control region (D-loop). Fish share gene sequence similarity with other vertebrates, indicating high conservation (Meyer, 1994). Compared with single-copy nuclear genes, the rate of evolution of the mitochondrial genome is faster, and this can be explained by a higher frequency of mitochondrial genome mutations. In particular, the D-loop region has the fastest rate of evolution and is generally used for intraspecific evolutionary analyses (i.e., comparisons between populations); the rate of change of rRNA genes is relatively slow and these regions are commonly used for species- or family-level analyses (Milinkovitch et al., 1993). The moderate rates of evolution of protein-coding and tRNA genes make them suitable for both intra- and interspecific analyses. The most commonly used genes are cytochrome b (Cyt b) and NADH dehydrogenase subunit (ND) (Zardoya and Meyer, 1996).

The N. beckfordi mitochondrial genes are similar to those of other fish in terms of codon usage (Wang et al., 2008, 2011; Wu et al., 2004). ATG is the most common start codon. There are two common types of termination codons, the complete termination codons TAG and TAA and the incomplete termination of codons TA and T. From this, we can infer that the start and stop codons in the mitochondrial genome may have a special significance in evolution. In the N. beckfordi mitochondrial PCGs, TAA was the most common termination codon. However, there were six incomplete termination codons.

The N. Beckfordi codons of the PCGs in the entire mitochondrial genome mostly ended with A or U bases, whereas codons ending with C bases appeared less frequently, with most RSCU values less than 1. Codons ending with G were least frequent, and all RSCU values were less than 1, indicating the use or avoidance of codons in the N. beckfordi mitochondrial genes. Generally speaking, this preference for codons is the result of mutations and selection, which has significance for the study of the origin and evolution of species, as demonstrated in Burkholderia anthracis (Zhao et al., 2007).

There were 22 tRNA genes in the N. beckfordi mitochondrial genome, with tRNA gene sizes ranging from 67 to 76 bp and an overall length of 1564 bp, accounting for 9.34% of the entire genome. The tRNA genes encoded by the H-chain were scattered between the protein and rRNA genes, with adjacent genes closely connected or even overlapping at intervals of 0–29 bases. Generally, tRNA-Met, tRNA-His, and tRNA-Leu show the highest conservation, whereas tRNA-Ser shows the greatest variation (Tzeng et al., 1992). The 12S rRNA gene was located between the tRNA-Phe and tRNA-Val genes, whereas the 16S rRNA gene was located between the tRNA-Val and tRNA-Leu genes. The length of the 12S rRNA genes was 955 bp, and the length of the 16S rRNA gene was 1698 bp. The two genes are separated from each other by the tRNA-Val gene at a distance of 73 bp. The rates of evolution of 16 srRNA and 12 srRNA are the slowest among mitochondrial genomes, indicating high conservation (Hickson et al., 1996; Flook and Rowell, 1997; Baker, 2000; Page, 2000; Misof et al., 2002; Page et al., 2002; Yoshizawa and Johnson, 2003). The N. beckfordi mitochondrial genome contained a D-Loop region with a length of 1063 bp, located between tRNA-Pro and tRNA-Phe.

A phylogenetic tree of N. beckfordi and 72 other Characiformes species was constructed using the ML method. Relationships between N. beckfordi and L. astrigata based on morphological and molecular data were consistent, indicating a close genetic relationship. This study provided the complete mitochondrial genome sequence obtained using direct sequencing for N. beckfordi, and in-depth bioinformatics analyses will further provide further insight into the genetic characteristics, origin, and evolution of the species. The analysis of phylogenetic relationships within the order Characiformes provide a theoretical basis for the protection and utilization of N. beckfordi genetic resources. It also helps judge its invasion risk, and provides reference for the prevention and control measures against it at the molecular level.

Declarations

Acknowledgments

We are very grateful to the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.

Funding

The present study was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Ethical statement

All specimens in this study were collected in accordance with Chinese laws. The collection and sampling of the specimens were reviewed and approved by the Animal Ethics Committee of Nanjing Forestry University. All experiments were conducted with respect to animal welfare and care. The study complied with CBD and Nagoya protocols and with the ARRIVE guidelines (https://arriveguidelines.org).

Data availability statement

The complete mitochondrial genome sequence and annotations of Nannostomus beckfordi is available in the GenBank and accession numbers OP595703.

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20240603083501

Statement of conflict of interest

The authors have declared no conflict of interest.

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