Biodiversity Data Journal 11: e96066 'e @)
doi: 10.3897/BDJ.11.e96066 open access

Research Article

Complete mitochondrial genome of Rectoris
luxiensis (Teleostei, Cyprinidae): characterisation
and phylogenetic implications

Mingyao Zhang*§, Qiang Zhou*$, Hongmei Xiang?, Jinxiu Wang*$, Xiangying Lan*§, Qinghua Luo*§,
Wansheng Jiang*$§

+ Hunan Engineering Laboratory for Chinese Giant Salamander’s Resource Protection and Comprehensive Utilization, and
Key Laboratory of Hunan Forest Products and Chemical Industry Engineering, Jishou University, Zhangjiajie, China
§ College of Biology and Environmental Sciences, Jishou University, Jishou, China

Corresponding author: Wansheng Jiang (jiangwschina@163.com)

Academic editor: Tihomir Stefanov

Received: 07 Oct 2022 | Accepted: 03 Jan 2023 | Published: 10 Jan 2023

Citation: Zhang M, Zhou Q, Xiang H, Wang J, Lan X, Luo Q, Jiang W (2023) Complete mitochondrial genome of
Rectoris luxiensis (Teleostei, Cyprinidae): characterisation and phylogenetic implications. Biodiversity Data
Journal 11: e96066. https://doi.org/10.3897/BDJ.11.e96066

Abstract

Mitochondrial genomes (mitogenomes) are widely used in_ scientific studies on
phylogenetic relationships, molecular evolution and population genetics. Here, we
sequenced and analysed the mitogenome of Rectoris /uxiensis, a Yangtze River drainage
endemic, but threatened cyprinid fish of Labeoninae. The complete mitogenome of R.
luxiensis was 16,592 bp in length, encoding 13 protein coding genes (PCGs), 22 transfer
RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and a control region. The
mitogenome showed a high A+T content (58.2%) and a positive AT-skew (0.10) and
negative GC-skew (—0.25) base composition pattern. All the 13 PCGs were found to start
with ATG codons, except for the COX!/, in which GTG was the start codon. The ratio of non-
synonymous and synonymous substitutions (Ka/Ks) of all the 13 PCGs were less than 1,
indicating negative or purifying selection evolved in these genes. Comparatively speaking,
the evolutionary rate of A7P8 was the fastest and VD4L was the slowest. All tRNAs could
fold into a typical cloverleaf secondary structure, except tRNAS¢™ that lacked a
dihydrouridine arm. Phylogenetic relationships, based on the PCGs dataset of 91
mitogenomes of Labeoninae, showed that R. /uxiensis grouped with Rectoris posehensis

© Zhang M etal. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY
4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are
credited.

2 Zhang M et al

and they formed a monophyletic Rectoris. However, many non-monophyletic genera were
revealed in labeoninae fishes, such as Cirrhinus, Decorus, Garra, Labeo and
Pseudocrossocheilus, which indicated that the validities of some traditional genera
required a further check. This study reported the complete mitogenome of R. /uxiensis for
the first time, which provided valuable data for future molecular evolution and conservation
related studies of Rectoris and other species in Labeoninae.

Keywords

Labeoninae, mitogenome assembly, annotation, phylogenetic relationship, evolution
analysis

Introduction

Rectoris luxiensis is a small-sized freshwater fish species that belongs to the Cyprinidae
family in Cypriniformes. It has been recorded to distribute, endemically, only in some
tributaries of the Yangtze drainage, including Yuanshui River and Xiangjiang River in the
Hunan Province, Qingjiang River in the Hubei Province and Daning River in the Sichuan
Province (Yue 2000). In morphology, R. /uxiensis presents a typically modified structure of
lip and jaw; thus, it has been categorised into the traditional Labeoninae, which was one of
the twelve broadly recognised subfamilies in Cyprinidae (Yue 2000, Nelson et al. 2016,
Jiang et al. 2018, Wang et al. 2022). Although recent phylogenetic studies showed that the
Labeoninae would be modified as a tribe (Labeonini) in the subfamily Cyprininae (Yang et
al. 2012, Yang et al. 2015), we tentatively kept it as a traditional subfamily here just for
convenience. The species of Labeoninae show fantastic diversity in forms of lip and jaw, as
well as other mouth-related structures (or oromandibular structures), which has been
assumed to be able to settle into the turbulent water flow and scrape off the algae on the
benthic substrate. Amongst many genera in Labeoninae, Rectoris is distinguished by the
following combination of characteristics: upper lip absent, rostral cap developed and
covering upper jaw completely, upper jaw linked to lower lip by a frenum, premaxillary
barbells not well-developed, without mental grooves and lower lip not modified into
adhesive disc (Yue 2000, Zheng et al. 2016). Although the Rectoris fishes are small-sized
that usually grow up to less than 20 cm (Yue 2000), many of them are still under threats
because of their capture for meat, especially for its delicate flavour from its relatively high
contents of lipids. For this reason, it has usually been called an “oil fish” in many local
areas.

During earlier times, new genera or species identifications and classifications amongst
Labeoninae were merely from morphological studies (e.g. Zheng et al. (2010a), Huang et
al. 2014); however, it was usually challenging while facing a wide variety of the
oromandibular structures within the group. After all, Labeoninae is now one of the most
diverse subfamilies of Cyprinidae that have about 40 genera and 400 species found from
Asia to Africa (Wang et al. 2022) or even more according to the catalogue of fishes (Fricke
et al. 2022). With the help of molecular data, more and more new taxa have been identified

Complete mitochondrial genome of Rectoris luxiensis (Teleostei, Cyprinidae): ... 3

recently by using morphological comparisons along with phylogenetic inferences (e.g. Yao
et al. (2018), Wang et al. (2022)). Given a lot of species in Labeoninae are still largely
unknown since they have been described initially, obtaining more and more molecular data
would be crucial for further understanding the phylogenetic relationships and evolutionary
history of these species.

Vertebrate mitochondrial genome (mitogenome) is double-stranded circular DNA with
typically 15 ~ 18 kb in length, with many characteristics like maternal inheritance, stable
genetic components, fast evolutionary rate, low recombination frequency and _ highly
conserved coding regions (Wolstenholme 1992, Boore 1999). Mitochondrial DNAs
(mtDNAs), as molecular markers for evolutionary phylogenetics and population genetics,
have been extensively used in a variety of species (Simon and Hadrys 2013, Sun et al.
2016, Hao et al. 2021, Zhao et al. 2021). As an endemic, but threatened fish of Labeoninae
in the Yangtze River drainage, the R. /uxiensis, received very little attention and few
scientific studies since it has been described in 1977. The current knowledge of this
species in science has been mainly restricted to the original morphological descriptions
and distributions, whereas seldom further phylogenetic studies have been involved with it,
not to mention its population structure and genetic diversity. In this study, we sequenced,
assembled, annotated and reported the complete mitogenome of R. /uxiensis for the first
time, by which we aimed to promote the future studies of phylogenetics, population
genetics and conservation biology of this species and other fishes in Labeoninae.

Materials and methods
Sample collection and sequencing

Samples of R. /uxiensis were collected from two localities in the Lishui River drainage in
Sangzhi County, Zhangjiajie City, Hunan Province of China (Fig. 1). One was collected
from Linxihe (29°59'N, 110°29’E, n = 4) in September 2021 and the other was from
Bamaoxi (29°63'N, 110°02’E, n = 13) in September 2022. All the samples were obtained by
courtesy of local men through recreational fishing that was restricted to “one person with
one rod and one fishhook”, which was allowed according to local laws. All specimens were
preserved in 95% ethanol and deposited in the Engineering Laboratory at Jishou
University. A unilateral pectoral fin from one sample in Linxihe (voucher No.
JWS20210646) was cut out for DNA extraction by using the DNeasy Blood & Tissue Kit
(Qiagen, Hilden, Germany). The DNA library was constructed and high-throughput
sequencing was then conducted in paired-end mode on the DNBSEQ-T7 platform
(Complete Genomics and MGI Tech, Shenzhen, China). Approximately 20 Gb of raw reads
of 150 bp read length were generated.

Mitogenome assembly and annotation

The complete mitogenome of R. /uxiensis was assembled using NOVOPlasty 4.3 under
default settings (Dierckxsens et al. 2017), with the mitogenome of Rectoris posehensis as
a reference. The mitogenome annotation, including the preliminary location of protein-

4 Zhang M et al

coding genes (PCGs) and ribosomal RNA genes (rRNAs) and the prediction of transfer
RNA genes (tRNAs) were carried out by the MITOS web server (available at hitp://
mitos2.bioinf.uni-leipzig.de/index.py, Donath et al. (2019)). The secondary structures of
tRNAs were further identified using tRNAscan-SE Search Server (available at http://
lowelab.ucsc.edu/tRNAscan-SE//, Lowe and Chan (2016)). The web application GeSeq
was then employed to check up all the annotations and map the cycled mitogenome
structure (available at https://chlorobox.mpimp-golm.mpg.de/geseq.html, Michael et al.
(2017)). The final complete mitogenome sequence with annotation information of R.
luxiensis has been submitted into NCBI (GenBank accession number: OP 132373).

Figure 1. EES]

Sampling localities (A), typical habitat in Bamaoxi (B) and photos of a living specimen of R.
luxiensis (C, lateral view; D, dorsal view; E, ventral view).

Mitogenome characteristic analyses

The base structure, nucleotide composition and relative synonymous codon usage (RSCU)
of different gene fragments were calculated using MEGA 11.0 (Tamura et al. 2021). The
skewing of the nucleotide composition was calculated with the formulae: AT-skew = (A-T)/
(A+T) and GC-skew = (G-C)/(G+C) (Perna and Kocher 1995). The ratio of non-
synonymous substitutions (Ka) and synonymous substitutions (Ks) of each PCG were
calculated by DNASP 6.0 (Rozas et al. 2017) for checking the signals of selective
pressure, while Ka/Ks value > 1 indicates positive selection, Ka/Ks = 1 indicates neutral
selection and Ka/Ks < 1 indicates negative or purifying selection. The plots of codons
usage frequencies and Ka/Ks ratio were drawn using the Origin software (Moberly et al.
2018).

Phylogenetic analyses

Mitogenomic sequences of 91 Labeoninae species, including the R. /uxiensis which we
sequenced in this study, were used for phylogenetic analyses, whereas a loach species
Cobitis takatsuensis was selected as the outgroup. All the 13 PCGs were extracted and
checked manually through MEGA 11.0 and then aligned using the in-built CLUSTALW

Complete mitochondrial genome of Rectoris luxiensis (Teleostei, Cyprinidae): ... 5

algorithm with default settings (Tamura et al. 2021). The best-fit partitioning scheme and
partition-specific models were calculated using Partitionfinder 2.1.1 (Lanfear et al. 2017),
while the specific codons were assigned for each PCG. Phylogenetic analyses were
conducted by both Maximum Likelihood (ML) and Bayesian Inference (BI) methods, based
only on the PCGs dataset. The ML analysis was run in RaxML 8.0.2 (Stamatakis 2015)
using the GTRGAMMA model and the best codon partition scheme, with the execution of
10 runs of random additional sequences and generating the bootstrap values following
1,000 rapid bootstrap replicates. The BI analysis was performed in MrBayes v.3.2.2
(Ronquist et al. 2012) by running 1.0x107 million generations, while the posterior
probabilities were calculated simultaneously with sampling every 1000 generations and
discarding the initial 25% generations as burn-in.

Results

Mitogenomic structure and composition

The complete mitogenome of R. /uxiensis had a total length of 16,592 bp, which consisted
of 13 typical vertebrate PCGs, 2rRNAs, 22 tRNAs and a non-coding control region (D-
LOOP) (Fig. 2, Table 1). Amongst these genes, only one PCG (ND6) and eight tRNA genes
((RNAC", tRNA‘) tRNAA‘S" tRNACYS, tRNATYS tRNASE tRNAS and tRNAP') were
encoded by the L-strand and the remaining gene sequences were encoded by the H-
strand. The mitogenome of R. /uxiensis was compact, with ten gene overlaps, ranging from
1 to 7 bp in length. In addition, there were seventeen intergenic nucleotides (IGN) regions
ranging from 1 to 24 bp in length and occupying a total of 84 bp, where the longest IGN (24
bp) was located between the tRNA“! and 16S rRNA genes (Table 1).

Table 1.

Table 1 Characteristics of the mitogenome of R. /uxiensis.

Gene Position Length Codon Anticodon Intergenic Strand
From To Start Stop meager
tRNAPhe 1 69 69 GAA H
12S rRNA = 70 1,022 953 2 H
tRNAV2! 1,025 1,096 72 TAC 20 H
16S rRNA — 1,117 2,756 1,640 24 H
tRNAL&Y 2,781 2,856 76 TAA 1 H
ND1 2,858 3,832 975 ATG TAA 4 H
tRNA"? 3,837 3,908 72 GAT -2 H
tRNAC 3,907 3,977 71 TTG 1 L
tRNAMet 3,979 4,047 69 CAT 0 H
ND2 4,048 5,094 1,047 ATG TAG —2 H
tRNA"P 5,093 5,163 71 TCA 2 H

6 Zhang M et al

Gene Position Length Codon Anticodon Intergenic Strand
From To Start Stop aCe
tRNA“ 5,166 5,234 69 TGC 1 L
tRNA‘S" 5,236 5,308 73 GTT 2 L
NCR 5,311 5,342 32 eA H
tRNACYS 5,342 5,407 66 GCA 1 L
tRNATY’ 5,409 5,479 71 GTA 1 L
COx1 5,481 7,031 1,551 GTG TAA 0 H
tRNAS 7,032 7,102 71 TGA 3 L
tRNAASP 7,106 7,177 72 GTC 13 H
COX2 7,191 7,881 691 ATG J= 0 H
tRNAYS 7,882 7,957 76 TTT 1 H
ATP8 7,959 8,123 165 ATG TAA -7 H
ATP6 8,117 8,800 684 ATG TAA -1 H
COX3 8,800 9,585 786 ATG TAA 4 H
tRNACY 9,585 9,656 72 TCC 0 H
ND3 9,657 10,007 351 ATG TAG = H
tRNA*9 10,006 10,075 70 TCG 0 H
ND4L 10,076 10,372 297 ATG TAA a7 H
ND4 10,366 11,743 1,378 ATG Es 0 H
tRNAHs 11,744 11,812 69 GTG 0 H
tRNASe" 11,813 11,881 67 GCT 1 H
tRNALEY 11,883 11,955 73 TAG 3 H
ND5 11,959 13,782 1,824 ATG TAA 4 H
ND6 13,779 14,300 522 ATG TAG 0 L
tRNACH 14,301 14,369 69 TTC 4 L
CYTB 14,374 15,514 1,141 ATG TE 0 H
tRNATH 15,515 15,586 72 TGT — H
tRNA’ 15,586 15,655 70 TGG 16 L
D-LOOP 15,672 16,592 921 0 H

Notes: * The numbers of nucleotides between the given and its previous gene, negative values indicate an overlap;
T-- indicated incomplete stop codon; H and L indicated that the genes are transcribed on the heavy and light strand,
respectively.

The base compositions of R. /uxiensis were A (32.0%) > C (26.2%) = T (26.2%) > G
(15.7%), having a bias towards A+T (58.2%) in the complete mitogenome. The A+T bias
also existed while looking at the A+T contents of PCGs (58.5%), rRNAs (54.9%), tRNAs
(56.0%) and D-LOOP (67.3%), respectively (Table 2). The nucleotide compositions also
showed a clear A and C bias pattern (AT-skew = 0.10, GC skew = —0.25), indicating a

Complete mitochondrial genome of Rectoris luxiensis (Teleostei, Cyprinidae): ... 7

greater abundance of A than T and C than G (Table 2). Moreover, the AT skew and GC
skew values of all the 13 PCGs were 0.02 and — 0.28, respectively, which indicated that the
A and C bias pattern generally existed amongst the PCGs (Table 2).

o
vu
25

<
zs
Se

t
os % pve
DP a ee
COX
2/ \28 posomal B
'(RNA-Lyg
ATP8
tRNA-Phe
ATP6
Rectoris luxiensis
; D-LOOp
mitochondrial genome
co® 16,592 bp >
Na. 7
hr

1 complex | (NADH dehydrogenase) 255 Z
L] complex IV (cytochrome c oxidase) TH 9 9
BATP synthase SSx
i other genes eS

i transfer RNAs
Bi ribosomal RNAs
[i control region

Figure 2. EES]

Circular gene map of the newly-sequenced mitogenome of R. /uxiensis.

Table 2.
Nucleotide composition (in percentages) and skew of the mitogenome of R. /uxiensis.

gene size ( bp ) T% C% A% G% A+T% C+G% AT-skew GC-skew
ND1 972 26.3 28.8 30.5 14.4 56.8 43.2 0.07 —0.33
ND2 1,044 24.2 30.8 32.8 12.2 57.0 43.0 0.15 —0.43
COx1 1,548 29.8 25.3 27.8 17.0 57.6 42.3 -0.03 —0.20
COx2 690 27.1 25.8 31.4 15.7 58.5 41.5 0.07 —0.24
ATP8 165 26.1 26.7 36.4 10.9 62.5 37.6 0.16 -0.42
ATP6 684 30.0 26.5 30.7 12.7 60.7 39.2 0.01 —0.35
COx3 783 26.7 28.5 28.6 16.2 55.3 44.7 0.03 —0.28

ND3 348 39.3 28.7 27.6 14.4 66.9 43.1 —0.17 —0.33

8 Zhang M et al

gene size ( bp ) T% C% A% G% A+T% C+G% AT-skew GC-skew
ND4L 294 27.2 30.6 26.5 15.6 53.7 46.2 -0.01 -0.32
ND4 1,380 27.2 26.9 32.1 13.7 59.3 40.6 0.08 —0.33
ND5 1,821 28.5 26.1 32.9 12.5 61.4 38.6 0.07 —0.35
ND6 519 42.8 11.6 13.5 32.2 56.3 43.8 -0.52 0.47
CYTB 1,140 29.6 26.3 29.8 14.2 59.4 40.5 0.00 —0.30
rRNAs 2,593 19.7 24.4 35.2 20.7 54.9 45.1 0.28 —0.08
tRNAs 1,556 24.9 24.5 31.1 19.5 56.0 44.0 0.11 -0.11
D-LOOP 921 33.6 19.1 33.7 13.7 67.3 32.8 0.00 —0.16
PCGs 11,394 28.6 26.5 29.9 15.0 58.5 41.5 0.02 —0.28
Total 16,952 26.2 26.2 32.0 15.7 58.2 41.9 0.10 —0.25

Characteristics of rRNAs and tRNAs

The two rRNAs (12S and 16S rRNA) were positioned between tRNAPM and tRNA'®" and
separated by tRNA“! in the mitogenome of R. /luxiensis. The 12S rRNA was composed of
953 bp and the 16S rRNA was 1,640 bp in length. Both rRNA genes were encoded on the
H-strand and displayed a positive AT skew and a negative GC skew (AT skew = 0.28, GC
skew = — 0.08) (Table 2).

The mitogenome of R. /uxiensis included 22 tRNAs as that in most vertebrates. The length
of individual tRNA ranged from 66 to 76 bp and the concatenated total length of all tRNAs
was 1,556 bp. The average AT skew was 0.11 and the average GC skew was -0.11 of
these tRNAs, showing slightly higher A and C than T and G accordingly (Table 2). All
tRNAs fold into typical cloverleaf secondary structures, except the tRNA‘! that lacked the
dihydrouridine (DHU) arm (both stem and loop) (Fig. 3). In addition to the typical base pairs
(G-C and A-U), there were also some wobble G-U pairs in the secondary structures of
these tRNAs, which could also form stable chemical bonds between G and U. For
instance, eight tRNAs (tRNAMet tRNALYS, tRNASE™ tRNALEY2 ERNAGCH tRNASE tRNAAIA
and tRNA‘°YS) showed G-U wobble base pairs in the acceptor stem, while another five
(tRNA*'9, TRNAS", tRNASE?, tRNATY and tRNA“) in the anticodon stem. Additionally, five
tRNAs (tRNAP tRNA! tRNA“'S, tRNA"'S and tRNA!) showed mismatched base pairs
in the acceptor stem and three ((RNA''?, tRNA“S? and tRNA*®") in the anticodon stem.

Characteristics of PCGs and codon usages

In the mitogenome of R. /uxiensis, the PCGs comprised a concatenated length of 11,394
bp that accounted for 67.21% of the total sequence. All the 13 PCGs encoded on the H-
strand, except D6 that was encoded by the L-strand. All the PCGs began with the regular
start codon ATG, except that the COX7 gene started with GTG. Ten PCGs were terminated
with the conventional stop codons (TAA or TAG), while the other three (VD4, COX2 and
CYTB) were terminated with incomplete stop codons (TA or T) (Table 1).

Complete mitochondrial genome of Rectoris luxiensis (Teleostei, Cyprinidae): ... 9

The RSCU values, based on 13 PCGs, showed that Leu encoded by the greatest number
of synonymous codons (n = 6), while others were fewer: the Val, Ser1, Pro, Thr, Ala, Arg
and Gly, were encoded by four codons and all the rest of the amino acids were encoded by
only two codons (Fig. 4). While looking at each codon, the top three frequently-used
codons of the PCGs were CGA (2.63%) encoded for Arg, CUA (2.5%) for Leu and CCA
(2.29%) for Pro.

IRNA-Phe™ (RNA-Val : (RNA-Leul i * IRNAclle * . tRNA-Met

IRNA-Tip ‘ IRNA-Asp ‘. ? IRNA-Lys ‘. . tRNA-Gily = 8 IRNA-Arg * ©

IRNA-Ilis ; ; IRNA-Ser] ca. é (RNA-Lewu? ‘. ‘ (RNA-Thr * / IRNA-Pro +

_ ; tRNA-Ser2 . < (RNA-Tyr . IRNA-Cys : tRNA-Asn
tRNA-Ala® IRNA-Gin + ;

<< Anticodon stem and loop

<— T'VC stem and loop

DHUstem and loop: > ‘aE ;
P oe —— _ Discriminator nuclectide

<_—\\— Amino acid acceptor stem

Figure 3. EES

Secondary structure of 22 tRNA genes from the R. /uxiensis mitogenome.

10 Zhang M et al

HAMAR WAY

Phe Leu Ile Met Val Serl Pro Thr Ala Tyr His Gln Asn Lys Asp Glu Cys Trp Arg Ser2 Gly

| oF eae ee ee ae |
| | j eo

UC UUG AUC AUG GUC UCC COC ACO GK JAC CAC CAG AAC AAC
Ccuc GUG UCG COG ACG GGG CGG GGG

CUG

Figure 4. EES]

The relative synonymous codon usage (RSCU) in the mitogenome of R. /uxiensis.

The Ka/Ks ratios of the 13 PCGs, based on 91 species of Labeoninae, were all less than 1,
with the highest Ka/Ks ratio in A7P8 and the lowest ratio in ND4L (Fig. 5). None of the
PCGs showed Ka/Ks 2 1 indicating a generally negative or purifying selection. Therefore,
the evolution pattern of the mitogenome of Labeoninae tended to be conservative to
maintain the regular functions of the generated proteins.

Phylogenetic analysis

Phylogenetic analyses were conducted, based on the 13 concatenated PCGs dataset from
91 species of Labeoninae (including R. /uxiensis which we obtained in this study), while the
Cobitis takatsuensis from the Cobitidae was used as the outgroup. Both BI and ML
analyses generated trees with almost the same topologies, in which six major clades (here
named clades A-G) could be distinguished (Fig. 6). Clade A, which included only one
species in genus Osteochilichthys, diverged first. Then it was followed by clade B, which
consisted of two species in Labeo and Decorus. The vast majority of species belonged to
the remaining clades. Clade C included species from the following five genera: Labeo,
Cirrhinus, Bangana, Gymnostomus and Incisilabeo. Although most of the species in Labeo

Complete mitochondrial genome of Rectoris luxiensis (Teleostei, Cyprinidae): ... 11

were in this clade, they did not form a monophyletic group. Clade D included species in the
following genera: Lobocheilos, Henicorhynchus, Epalzeorhynchos, Crossochelilus,
Thynnichthys and Osteochilus. Clade E included species mainly in Garra and Tarigilabeo.
Clade F contained the most number of genera, such as: Garra, Semilabeo, Parasinilabeo,
Prolixicheilu, Rectoris, Ptychidio, PSseudocrossocheilus, Sinocrossocheilus, Decorus,
Discogobio, Paragianlabeo, Cophecheilu and Pseudogyrinocheilus. Although many of the
genera were modified and reclassified, based on recent studies and we updated all the
names according to the catalogue of fishes (up to Dec 2022), there were obviously many
genera which were non-monophyletic according to this phylogenetic tree (Fig. 6). However,
R. luxiensis appeared in clade F with a sister-group species to R. posehensis and the two
species supported a monophyletic Rectoris.

0.3

Ka/Ks

0.1

2 Fe Laeleede

NDI ND2 COX1 COX2 ATP8 ATP6 COX3 ND3 ND4L ND4 NDS ND6 CYTB

Figure 5. EES]

The Ka/Ks ratio of 13 PCGs amongst 91 species of Labeoninae.

Discussion

We successfully sequenced and assembled the mitogenome of R. /uxiensis, an endemic,
but threatened fish of Labeoninae in the Yangtze River drainage, for the first time in this
study. The mitogenome of R. /uxiensis was 16,592 bp in length, which was similar to other
known species of Labeoninae, such as 16,594 bp in Rectoris posehensis, 16,599 bp in
Semilabeo notabilis and 16,600 bp in Pseudocrossocheilus liuchengensis (Vu et al. 2018,
Liu et al. 2018). The slight length variations of mitogenomes of closely-related species
usually resulted from the changes of tandem repeats within the control region and the

12 Zhang M et al

lengths of intergenic regions or gene overlaps (Wang et al. 2020). Some characteristics of
the mitogenome of R. /uxiensis were also typical and similar to other Labeoninae fishes,
including the contents and orders of 13 PCGs, 2rRNAs, 22 tRNAs and a D-LOOP and the
encoding location for most genes was on the H-strand, with and only the D6 gene and
eight tRNAs on the L-strand (Wu et al. 2018, Liu et al. 2018).

ton100 ee
65/100 Pychidc sa NC
1on100 Pseudocrossocheilas tridentis NCOS6117
swico_| — is NCDS4789
re Rectonis posebensis NCSD ;
ectonis
servoa) Lr Prcudocrossocteilus liachengensis NCO489S3

100/100 _-~ Sennilahoo notahilis NCOIS916
Semillsheo obscures NC037408

aaa Pseudogyrinocheilus, NCUD4SSS
C7) 100/109 Passio asinil NCD25947

Prolin pn Jongisulcus NOOSOOTE
10010, iN

Ce amen MK387103
— sche 7 F

100100 Discogabio yunnanensis NCO2S319

7480 Discogotio’ aticeps
oo 100 fetrabarbatus NCOD4S78

100100 NC022951
10100 Gara nuft N NOOBIS3S
wi00 100/100, Gara salweenica NOO33389
1100 Garra orientalis NC021935
motuoensis OK375462

P 1on100. Gara ange 7878
aI Gara kempi NC028426

sooo) | vear100 Garra flavatra gates NEWS

100100, Garra dengha NC067877 ese
1eutoo Garrs tibetana a
Garra spi

99/100 1oneto0 Labiobarbus leptociilus NCO22954
1007100 shobarhs Samos NOO31533
7100 30 Osteochilus salsburyi NOODI38S
; mre Osteochilus viitatus NCO29442
100'10 100/100 Osteochilus pentalinestus NC031625
Osteochilus schlegelii NC022951
100'100 Thynnichthys, NC022952
“0 Thynnichthy ace NCO31609
toot00 p— C7 “ern NOH) D
‘ Crossocheilus
10010) Crossovheilus me NCOs
9/100 Crossocheilus langei NC02443
. 100/100 ip revs mandy vcd NCOSISM
10) ris aro frenatus NC0339%62 7
100/100 Henicortynchus caudimaculatus NC022950
100100 : -Henicorhynchus siamensis NC031623
+130 Cimrhinas molitorella NCO221

TI0 pay Labeo altivelis NOMS

= Labeo cylndricus NCO31536
3 ~ Labo fi NCES

’ 100/100 - Lateo chrysophekadion NC022042
bls Labeo angra NCO22045 c
100 tow 100 Labeo dvocheilus APOII328
100/100 Labeo pangusia NC029451
Lae pe NC022943
oieo ame NC031608
Laid ae 80 NOB
Labeo boggut NOO29450
84100 Incisilebeo behri NC031607

5150 100100 _-— Cinhinus cirrhosis NCO33964
Cinrhinws citrhosus NCOI7611

Osteochilichthys nashti NCO22946
L____ Cobiiis takatswensis NCOS306 7 | Outgroup

03

Figure 6. EES

Phylogenetic relationship obtained from the ML method, based on 13 PCGs. Note: the
numbers on the branches indicate bootstrap values from ML and posterior probabilities from
the BI method. The GenBank accession number of each species is given in the brackets after
the name. Red highlights the phylogenetic position of R. /uxiensis that we obtained in this
study.

Complete mitochondrial genome of Rectoris luxiensis (Teleostei, Cyprinidae): ... 13

The nucleotide compositions and codon usages of mitogenomes of Cyprinidae were
generally similar, but some detectable differences remained. For example, in R. /uxiensis,
only the COX7 gene started with GTG, but for Rhodeus cyanorostris, both ND1 and COx1
genes started with GTG (Li et al. 2022). Whether the diverse usage of start codons
amongst different species was generated randomly or with some evolutionarily meaningful
preferences, this was an interesting question of selection, but without being given much
attention. In addition, the secondary structure of tRNAs of R. /uxiensis was overall
conservative of vertebrate mitogenomes by having typical Watson-Crick pairings (G-C and
A-U) (Wolstenholme 1992); however, there were still dozens of non-typical forms such as
UG pairing in some different stem regions (Fig. 3), which was also revealed from other
studies (e.g. Zhao et al. (2021)). Recent studies have indicated that tRNAs matched with
non-typical pairings could also convert into fully functional proteins through post-
transcriptional mechanisms (Chao et al. 2008, Pons et al. 2014).

Mitochondrial DNA sequences are widely used in phylogenetic studies (Sun et al. 2017,
Tang et al. 2018, Chang et al. 2020). It has been generally recognised that the complete
mitogenome could uncover evolutionary relationships better than individual mitochondrial
genes (Hou et al. 2020). In this study, a phylogenetic hypothesis of Labeoninae was able to
be reconstructed while using the mitogenomes of 91 representative species, including the
R. luxiensis which we sequenced in this study. Previous studies have shown that the
monophyly of Labeoninae was supported, but the inter-generic relationships were usually
controversial (e.g. Li et al. (2005), Wang et al. (2020)). Our study suggested that some
previous inconsistences may result from the limited phylogenetic information from less
gene fragments. For instance, the sister group relationship of MRectoris and
Pseudocrossocheilus in our study was consistent with a previous study that was also
based on mitogenomes (Wang and Zeng 2021), but this relationship was not revealed from
the study from a single 16S rRNA gene (Li et al. 2005). We found that the phylogenetic
trees of the mitochondrial and nuclear genes in Labeoninae were sometimes different,
possibly due to the different evolutionary rates and variable informative sites (Lynch et al.
2006). For example, Rectoris and Semilabeo were once revealed as a sister group
relationship, based on two nuclear genes (Zheng et al. 2010b), which was also inconsistent
with our study (Fig. 6).

In addition, our study also suggested that some inconsistent inter-generic relationships
within Labeoninae might be from the non-monophyletic nature of some traditionally
recognised genera (mostly from morphological hypotheses), such as Cirrhinus, Decorus,
Garra, Labeo and Pseudocrossocheilus which we detected in this study (Fig. 6). Thus,
future studies should pay more attention to these genera. Let us take Garra, a traditionally
large group that was erected as early as 1822, as an example. It has usually served as a
taxonomic wastebasket for species having disc on the lower lip but could not be assigned
into other genera. Recently, some new genera have been identified and separated from
Garra-like species, such as Sinigarra (Zhang et al. 2012) and Guigarra (Wang et al. 2022).
Although with some of the inconsistent intergeneric relationships, the genera included in
each major clade were largely similar with previous studies (Zheng et al. 2010b, Yang et al.
2012, Li and Han 2018, Pan et al. 2020). In brief, the phylogenetic relationships within

14 Zhang M etal

Labeoninae are still far from resolved; thus, more and more molecular data, such as the
one we reported in this study, will be necessary and helpful for understanding the
evolutionary history and diversity of this complicated, but fantastic group.

Data resources

The genome sequence data are available in GenBank (hitps://www.ncbi.nim.nih.gov/)
under accession no. OP132373.

Funding

This work was supported in part by the Innovation Platform and Talent Plan of Hunan
Province [2020RC3057]; the National Natural Science Foundation of China [382060128];
Zhilan foundation [2020040371B, 2022010011B] and opening projects of Hunan
Engineering Laboratory for Chinese Giant Salamander’s Resource Protection and
Comprehensive Utilization [DNGC2211].

Conflicts of interest

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

References

° Boore JL (1999) Animal mitochondrial genomes. Nucleic Acids Research 27 (8):
1767-1780. https://doi.org/10.1093/nar/27.8.1767

° Chang H, Qiu Z, Yuan H, Wang X, Li X, Sun H, Guo X, Lu Y, Feng X, Majid M, Huang Y
(2020) Evolutionary rates of and selective constraints on the mitochondrial genomes of
Orthoptera insects with different wing types. Molecular Phylogenetics and Evolution 145
(106734). https://doi.org/10.1016/j.ympev.2020.106734

° Chao JA, Patskovsky Y, Almo SC, Singer RH (2008) Structural basis for the coevolution
of a viral RNA-protein complex. Nature Structural & Molecular Biology 15: 103-105.
https://doi.org/10.1038/nsmb1327

° Dierckxsens N, Mardulyn P, Smits G (2017) NOVOPlasty: de novo assembly of
organelle genomes from whole genome data. Nucleic Acids Research 45 (4): e18-e18.
https://doi.org/10.1093/nar/qkw955.

° Donath A, J&uuml h, F. A, M. B, H. S, Reinhardt F, Stadler PF, Middendorf M, Bernt M
(2019) Improved annotation of protein-coding genes boundaries in metazoan
mitochondrial genomes. Nucleic Acids Research 47 (20): 10543-10552. https://doi.org/
10.1093/nar/gkz833

° Fricke R, Eschmeyer WN, & Van der Laan R (2022) ECoF. Eschmeyer's Catalog of
Fishes: Genera, Species, References. http://researcharchive.calacademy.org/research/

Ichthyology/catalog/fishcatmain.asp

Complete mitochondrial genome of Rectoris luxiensis (Teleostei, Cyprinidae): ... 15

Hao XY, Liu JQ, Chiba H, Xiao JT, Yuan XQ (2021) Complete mitochondrial genomes of
three skippers in the tribe Aeromachini (Lepidoptera: Hesperiidae: Hesperiinae) and
their phylogenetic implications. Ecology and Evolution 11 (12): 8381-8393. https://
doi.org/10.1002/ece3.7666

Hou XJ, Lin HD, Tang WQ, Liu D, Han CC, Yang JQ (2020) Complete mitochondrial
genome of the freshwater fish Acrossocheilus longipinnis (Teleostei: Cyprinidae):
genome characterization and phylogenetic analysis. Biologia 75 (11): 1871-1880.
https://doi.org/10.2478/s11756-020-00440-y

Huang Y, Yang J, Chen X (2014) Stenorynchoacrum xijiangensis, a new genus and a
new species of Labeoninae fish from Guangxi, China (Teleostei: Cyprinidae). Zootaxa
3793: 379-86. https://doi.org/10.11646/zootaxa.3793.3.6.

Jiang W, Qiu Y, Pan X, Zhang Y, Wang X, Lv Y, Bian C, Li J, You X, Chen J, Yang K,
Yang J, Sun C, Liu Q, Cheng L, Yang J, Shi Q (2018) Genome Assembly for a Yunnan-
Guizhou Plateau “3E” Fish, Anabarilius grahami (Regan), and Its Evolutionary and
Genetic Applications. Frontiers in Genetics 9 https://doi.org/10.3389/fgene.2018.00614
Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2017) PartitionFinder 2: New
Methods for Selecting Partitioned Models of Evolution for Molecular and Morphological
Phylogenetic Analyses. Molecular Biology and Evolution 34 (3): 772-773. https://doi.org/
10.1093/molbev/msw260

Li JB, Wang XZ, He SP, Chen YY (2005) Phylogenetic studies of Chinese labeonine
fishes (Teleostei: Cyprinidae) based on the mitochondrial 16S rRNA gene. Progress in
Natural Science 15 (3): 213-219. https://doi.org/10.1080/10020070512331342020

Li Q, Han C (2018) The complete mitochondrial genome of Ptychidio macrops
(Cypriniformes: Cyprinidae) and its phylogenetic implications. Conservation Genetics
Resources 10: 379-382. https://doi.org/10.1007/s12686-01 7-0829-7

Liu B, Cheng G, Shi X (2018) Characterization of the complete mitochondrial genome of
Pseudocrossochelilus liuchengensis (Cypriniformes: Cyprinidae). Mitochondrial DNA B
Resources 3 (2): 543-544. https://doi.org/10.1080/23802359.2018.1467216

Li WJ, Qiu N, Du HJ (2022) Complete mitochondrial genome of Rhodeuscyanorostris
(Teleostei, Cyprinidae): characterization and phylogenetic analysis. ZooKeys 1081:
111-125. https://doi.org/10.3897/zookeys.1081.77043

Lowe TM, Chan PP (2016) tRNAscan-SE On-line: integrating search and context for
analysis of transfer RNA genes. Nucleic Acids Research 44 (W1): W54-7-7. htips://
doi.org/10.1093/nar/gkw413.

Lynch M, Koskella B, Schaack S (2006) Mutation pressure and the evolution of
organelle genomic architecture. Science 311 (5768): 1727-1730. https://doi.org/10.1126/
science.1118884

Michael T, Pascal L, Tommaso P (2017) GeSeq-versatile and accurate annotation of
organelle genomes. Nucleic Acids Research 45 (W1): W6-W11. https://doi.org/10.1093/
nar/gkx391

Moberly JG, Bernards MT, Waynant KV (2018) Key features and updates for Origin
2018. Journal of Cheminform 10 (1): 5. https://doi.org/10.1186/s13321-018-0259-x
Nelson JS, Grande TC, Wilson MV (2016) Fishes of the World Animal Science and
Zoology. 5th. John Wiley and Sons Inc https://doi.org/10.1002/97811191 74844

Pan XY, Shao LY, Lao JQ, Kuang TX, Chen JH, Zhou L (2020) Mitochondrial genome of
Parasinilabeo longicorpus provides insights into the phylogeny of Labeoninae.

16

Zhang M et al

Mitochondrial DNA Part B 5 (3): 3044-3045. https://doi.org/
10.1080/23802359.2020.1798298

Perna NT, Kocher TD (1995) Patterns of nucleotide composition at fourfold degenerate
sites of animal mitochondrial genomes. Journal of Molecular Evolution 41 (3): 353-358.
https://doi.org/10.1007/BF00186547

Pons J, Bauza-Ribot MM, Jaume D, Juan C (2014) Next-generation sequencing,
phylogenetic signal and comparative mitogenomic analyses in Metacrangonyctidae
(Amphipoda: Crustacea). BioMed Central 15: 566. https://doi.org/
10.1186/1471-2164-15-566

Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, H6hna S, Larget B, Liu L,
Suchard MA, A. M, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian
phylogenetic inference and model choice across a large model space. Systematic
Biology 61 (3): 539-542. https://doi.org/10.1093/sysbio/sys029

Rozas J, Ferrer-Mata A, Sanchez-DelBarrio JC, Guirao-Rico S, Guirao-Rico S, Librado
P, Ramos-Onsins SE, Sanchez-Gracia A (2017) DnaSP 6: DNA sequence
polymorphism analysis of large data sets. Molecular Biology And Evolution 34 (12):
3299-3302. https://doi.org/10.1093/molbev/msx248

Simon S, Hadrys HA (2013) A comparative analysis of complete mitochondrial genomes
among Hexapoda. Molecular Phylogenetics and Evolution 69 (2): 393-403. https://
doi.org/10.1016/j.ympev.2013.03.033

Stamatakis A (2015) Using RAxML to infer phylogenies. Current Protocols in
Bioinformatics 51: 6-14.

Sun G, Xia T, Yang X, Zhao C, Liu G, Sha W, Zhang H (2016) The complete
mitochondrial genome sequence of Eophona migratoria (Passeriformes Fringillidae).
Mitochondrial DNA Part B: Resources 1 (1): 753-754. htips://doi.org/
10.1080/23802359.2016.1209098

Sun S, Hui M, Wang M, Sha Z (2017) The complete mitochondrial genome of the
alvinocaridid shrimp Shinkaicaris leurokolos (Decapoda, Caridea): Insight into the
mitochondrial genetic basis of deep-sea hydrothermal vent adaptation in the shrimp.
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics 25: 42-52.
https://doi.org/10.1016/j.cbd.2017.11.002

Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics
Analysis Version 11. Molecular Biology and Evolution 38 (7): 3022-3027. https://doi.org/
10.1093/molbev/msab120

Tang JM, Li F, Cheng TY, Duan DY, Liu GH (2018) Comparative analyses of the
mitochondrialgenome of the sheep ked Melophagus ovinus (Diptera: Hippoboscidae)
from different geographical origins in China. Parasitology Research 117 (8): 2677-2683.
https://doi.org/10.1007/s00436-018-5925-4

Wang IC, Lin HD, Liang CM, Huang CC, Wang RD, Yang JQ, Wang WK (2020)
Complete mitochondrial genome of the freshwater fish Onychostoma lepturum
(Teleostei, Cyprinidae): genome characterization and phylogenetic analysis. ZooKeys
1005: 57-72. https://doi.org/10.3897/zookeys.1005.57592

Wang X, Zeng S (2021) The complete mitochondrial genome of Paragianlabeo lineatus
(Cyprinidae: Labeoninae). Mitochondrial DNA B Resources 6 (4): 1351-1352. hittps://

doi.org/10.1080/23802359.2021.1907799.

Complete mitochondrial genome of Rectoris luxiensis (Teleostei, Cyprinidae): ... 17

Wang ZB, Chen XY, Zheng LP (2022) A new genus and species of disc-bearing
Labeoninae (Teleostei: Cypriniformes) from Guangxi, China. Zoological Research 43
(3): 409-412. https://doi.org/10.24272/j.issn.2095-8137.2021.230.

Wolstenholme DR (1992) Animal mitochondrial DNA: structure and evolution.
International Review of Cytology 141: 173-216. https://doi.org/10.1016/
$0074-7696(08)62066-5

Wu L, Zhong S, Tan H, Wang K, Cheng G, Chen X, Zhang M (2018) The complete
mitochondrial genome of Semilabeo notabilis (Cyprinidae: Labeoninae) and
phylogenetic implications. Mitochondrial DNA B Resources 3 (2): 568-569. https://
doi.org/10.1080/23802359.2018.1467231.

Yang L, Arunachalam M, Sado T (2012) Molecular phylogeny of the cyprinid tribe
Labeonini (Teleostei: Cypriniformes). Molecular Phylogenetics and Evolution 65 (2):
362-379. https://doi.org/10.1016/j.ympev.2012.06.007

Yang L, Sado T, Vincent Hirt M, Pasco-Viel E, Arunachalam M, Li J, Wang X, Freyhof J,
Saitoh K, AM S (2015) Phylogeny and polyploidy: Resolving the classification of
cyprinine fishes (Teleostei: Cypriniformes). Molecular Phylogenetics and Evolution 85:
97-116. https://doi.org/10.1016/j.ympev.2015.01.014

Yao M, He Y, Peng ZG (2018) Lanlabeo duanensis, a new genus and species of
labeonin fish (Teleostei: Cyprinidae) from southern China. Zootaxa 4471 (3): 556-568.
https://doi.org/10.11646/zootaxa.4471.3.7.

Yue PQ, et al. (2000) Fauna Sinica, Osteichthyes, Cypriniformes III [M]. Beijing Science
Press, 215-216 pp.

Zhang E, Zhou W, Zhang E (2012) Sinigarra napoense, a new genus and species of
labeonin fishes (Teleostei: Cyprinidae) from Guangxi Province, South China. Zootaxa
3586 (3586): 17-25. https://doi.org/10.11646/zootaxa.3586.1.4

Zhao L, Wei JF, Zhao WQ, Chen C, Gao ZY, Zhao Q (2021) The complete mitochondrial
genome of Pentatoma rufipes (Hemiptera, Pentatomidae) and its phylogenetic
implications. ZooKeys 1042: 51-72. https://doi.org/10.3897/zookeys.1042.62302
Zheng LP, Chen XY, Yang JX (2010a) A new species of genus Pseudogyrinocheilus
(Teleostei: Cyprinidae) from Guangxi, China. Environmental Biology of Fishes 87:
93-97. https://doi.org/10.1007/s10641-009-9555-7

Zheng LP, Yang JX, Chen XY (2010b) Phylogenetic relationships of the Chinese
Labeoninae (Teleostei, Cypriniformes) derived from two nuclear and three mitochondrial
genes. Zoologica Scripta 39 (6): 559-571. https://doi.org/10.1111/j.
1463-6409.2010.00441.x

Zheng LP, Chen XY, Yang JX (2016) Molecular systematics of the Labeonini inhabiting
the karst regions in southwest China (Teleostei, Cypriniformes). Zookeys (612)133-14.

https://doi.org/10.3897/zookeys.612.9085.