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Reanalysis and Revision of the Complete Mitochondrial Genome of Artemia urmiana Günther, 1899 (Crustacea: Anostraca) Biodiversity in tropical hotspots -rainforest birds in Madagascar View project Influence of temperature changes on symbiotic Symbiodiniaceae and bacterial communities' structure of soft corals View project Alireza Asem Reanalysis and Revision of the Complete Mitochondrial Genome of Artemia urmiana Günther, 1899 (Crustacea: Anostraca)

Authors:

Abstract

In the previously published mitochondrial genome sequence of Artemia urmiana (NC_021382 [JQ975176]), the taxonomic status of the examined Artemia had not been determined, due to partheno�genetic populations coexisting with A. urmiana in Urmia Lake. Additionally, NC_021382 [JQ975176] has been obtained with pooled cysts of Artemia (0.25 g cysts consists of 20,000–25,000 cysts), not a single specimen. With regard to coexisting populations in Urmia Lake, and intra- and inter-specific variations in the pooled samples, NC_021382 [JQ975176] cannot be recommended as a valid se�quence and any attempt to attribute it to A. urmiana or a parthenogenetic population is unreasonable. With the aid of next-generation sequencing methods, we characterized and assembled a complete mitochondrial genome of A. urmiana with defined taxonomic status. Our results reveal that in the previously published mitogenome (NC_021382 [JQ975176]), tRNA-Phe has been erroneously attributed to the heavy strand but it is encoded in the light strand. There was a major problem in the position of the ND5. It was extended over the tRNA-Phe, which is biologically incorrect. We have also identified a partial nucleotide sequence of 311 bp that was probably erroneously duplicated in the assembly of the control region of NC_021382 [JQ975176], which enlarges the control region length by 16%. This partial sequence could not be recognized in our assembled mitogenome as well as in 48 further examined specimens of A. urmiana. Although, only COX1 and 16S genes have been widely used for phylogenetic studies in Artemia, our findings reveal substantial differences in the nucleotide composition of some other genes (including ATP8, ATP6, ND3, ND6, ND1 and COX3) among Artemia species. It is suggested that these markers should be included in future phylogenetic studies.
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Reanalysis and Revision of the Complete Mitochondrial Genome of Artemia
urmiana Günther, 1899 (Crustacea: Anostraca)
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Article
Reanalysis and Revision of the Complete Mitochondrial
Genome of Artemia urmiana Günther, 1899 (Crustacea:
Anostraca)
Alireza Asem 1, 2, , Amin Eimanifar 3, , Weidong Li 4,*, Chun-Yang Shen 5, Farnaz Mahmoudi Shikhsarmast 1,6,
Ya-Ting Dan 7, Hao Lu 1,2, Yang Zhou 8, You Chen 1,9, Pei-Zheng Wang 1,9 ,* and Michael Wink 10


Citation: Asem, A.; Eimanifar, A.; Li,
W.; Shen, C.-Y.; Shikhsarmast, F.M.;
Dan, Y.-T.; Lu, H.; Zhou, Y.; Chen, Y.;
Wang, P.-Z.; et al. Reanalysis and
Revision of the Complete
Mitochondrial Genome of Artemia
urmiana Günther, 1899 (Crustacea:
Anostraca). Diversity 2021,13, 14.
https://doi.org/10.3390/d13010014
Received: 7 August 2020
Accepted: 5 November 2020
Published: 4 January 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
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Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Key Laboratory of Utilization and Protection of Tropical Marine Living Resources,
Hainan Tropical Ocean University, Sanya 572000, China; asem.alireza@gmail.com (A.A.);
farnaz_mahmoudi@ymail.com (F.M.S.); 836879118lu@sina.com (H.L.); you_chen2020@163.com (Y.C.)
2College of Fisheries and Life Sciences, Hainan Tropical Ocean University, Sanya 572000, China
3Independent Senior Scientist, Industrial District, Easton, MD 21601, USA; amineimanifar1979@gmail.com
4College of Ecology and Environment, Hainan Tropical Ocean University, Haikou 570000, China
5Department of Biology, Chengde Medical University, Chengde 067000, China; scyshenchunyang@yeah.net
6College of Marine Science and Technology, Hainan Tropical Ocean University, Sanya 572000, China
7College of Marine Science, Shanghai Ocean University, Shanghai 200000, China; dinaty@163.com
8Institute of Deep Sea Science and Engineering, Chinese Academy of Science, Sanya 572000, China;
zhou_yang0916@163.com
9College of Ecology and Environment, Hainan Tropical Ocean University, Sanya 572000, China
10 Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University,
Im Neuenheimer Feld 364, 69120 Heidelberg, Germany; wink@uni-heidelberg.de
*Correspondence: lwd542148880@163.com (W.L.); condywpz@126.com (P.-Z.W.)
Equal contribution as first author.
Abstract:
In the previously published mitochondrial genome sequence of Artemia urmiana (NC_021382
[JQ975176]), the taxonomic status of the examined Artemia had not been determined, due to partheno-
genetic populations coexisting with A. urmiana in Urmia Lake. Additionally, NC_021382 [JQ975176]
has been obtained with pooled cysts of Artemia (0.25 g cysts consists of 20,000–25,000 cysts), not a
single specimen. With regard to coexisting populations in Urmia Lake, and intra- and inter-specific
variations in the pooled samples, NC_021382 [JQ975176] cannot be recommended as a valid se-
quence and any attempt to attribute it to A. urmiana or a parthenogenetic population is unreasonable.
With the aid of next-generation sequencing methods, we characterized and assembled a complete
mitochondrial genome of A. urmiana with defined taxonomic status. Our results reveal that in
the previously published mitogenome (NC_021382 [JQ975176]), tRNA-Phe has been erroneously
attributed to the heavy strand but it is encoded in the light strand. There was a major problem in the
position of the ND5. It was extended over the tRNA-Phe, which is biologically incorrect. We have
also identified a partial nucleotide sequence of 311 bp that was probably erroneously duplicated
in the assembly of the control region of NC_021382 [JQ975176], which enlarges the control region
length by 16%. This partial sequence could not be recognized in our assembled mitogenome as well
as in 48 further examined specimens of A. urmiana. Although, only COX1 and 16S genes have been
widely used for phylogenetic studies in Artemia, our findings reveal substantial differences in the
nucleotide composition of some other genes (including ATP8, ATP6, ND3, ND6, ND1 and COX3)
among Artemia species. It is suggested that these markers should be included in future phylogenetic
studies.
Keywords:
brine shrimp; Artemia; mitochondrial genome; phylogeny; nucleotide composition;
maternal ancestor; Asia
Diversity 2021,13, 14. https://doi.org/10.3390/d13010014 https://www.mdpi.com/journal/diversity
Diversity 2021,13, 14 2 of 17
1. Introduction
Mitogenomes have been extensively utilized in phylogeny and population genetic
studies because mitochondrial genes share some particular characteristics, including ma-
ternal origin, rapid evolutionary rates and lack of recombination [
1
,
2
]. Sequences of
mitochondrial genes have been widely applied as informative molecular markers; there-
fore, they have been widely used in the molecular phylogenetic studies [
3
6
]. However,
compared to partial mitochondrial sequences, such as COX1, 16S, 12S, etc., complete mito-
chondrial genome sequences could provide more of a higher resolution and provide better
understanding about evolutionary relationships and structures of animals [4,7].
The number of complete mitochondrial sequences from metazoa has been rising
rapidly in the past decade. With the rapid advancement in genomics techniques, such
as next-generation sequencing (NGS) technology, many genomes and mitogenomes have
been characterized [
6
,
8
,
9
]. The current methodology provides high quality results and
has allowed us to accurately determine the gene order and organization in many taxa
and to compare the outcomes with conventional sequencing methods [
10
13
]. Complete
mitochondrial genomes have proved useful in solving evolutionary and biosystematics
questions over a broad taxonomic range [6,14].
The genus Artemia Leach, 1819, is an anostracan zooplankter, which lives in more than
600 saline and hypersaline habitats worldwide, except Antarctica [
15
]. The first scientific
report of the brine shrimp Artemia backs to the first half of the 10th century AD from
Urmia Lake by Estakhri (d. 951/957 AD, 10th C.), an Iranian geographer, who named it
“aquatic dog” [
16
,
17
]. The genus Artemia consists of seven bisexual biological species, a
large number of parthenogenetic populations and some undescribed and/or unidentified
taxa [
18
,
19
]. Four bisexual species are native to the Old World namely Artemia salina
(Linnaeus, 1758), Artemia urmiana Günther, 1899, Artemia sinica Cai, 1989, and Artemia
tibetiana Abatzopoulos et al. (1998). The other three bisexual species are located in the
New World consisting of Artemia monica Verrill, 1869, Artemia franciscana Kellogg, 1906,
and Artemia persimilis Piccinelli and Prosdocimi, 1968 [
18
,
20
,
21
]. Obligate parthenogenetic
Artemia taxa have di-, tri-, tetra- and pentaploid populations [19].
Overall, the phylogenetic relationship within Artemia is presently still ambiguous [
22
].
Obligate asexuality is one of the critical challenges in biosystematics of the genus Artemia.
Accordingly, asexual forms of Artemia do not form defined species but parthenogenetic
population(s) [
18
,
23
]. A comprehensive phylogenetic study using mitochondrial and nu-
clear markers provided evidence that the parthenogenetic Artemia taxa form a polyphyletic
group. The diploids and triploids are maternally related to A. urmiana, whereas tetrapod
and pentaploid lineages share a common ancestor with Artemia sinica [
19
]. Although the
mitochondrial marker Cytochrome Oxidase Subunit I (COX1) has confirmed that partheno-
genetic populations were divided into two polyphyletic groups, the nuclear marker ITS1
suggests a common haplotype with the participation of all ploidy degrees [
19
,
24
]. Ad-
ditionally, Eimanifar et al. [
24
] showed that parthenogenetic populations share a major
haplotype with A. urmiana and A. tibetiana based on the ITS1 nuclear maker.
Maniatsi et al. [
25
] and Eimanifar et al. [
24
] have proposed different phylogenetic
positions for A. salina and A. persimilis, based on nucleotide sequences of COX1. However,
results of morphological and genetic investigations were contradictory [
22
]. To date, the
taxonomy and biosystematics of Artemia are still controversial, especially concerning the
Asian species [
21
]. Mitogenomic information could provide a better reconstruction of the
maternal evolutionary mechanism and phylogenetic status of Artemia. However, only four
mitochondrial genomes of Artemia species have been published to date.
The previously published mitogenome analysis, performed by Zhang et al. [
26
] for
two Artemia species, Artemia urmiana and Artemia tibetiana, detected an unprecedented
unusual nucleotide sequence in the mitochondrial genome of A. urmiana from Urmia Lake,
Iran. The length of the complete mitochondrial genome of A. urmiana (GenBank accession
number NC_021382 [JQ975176]), is 15,945 bp; its control region would be significantly
longer than other species. In addition, the taxonomic status of the assembled mitogenome
Diversity 2021,13, 14 3 of 17
had not been identified. For this examined mitogenome, Zhang et al. have used Artemia
cysts stored at the Laboratory of Aquaculture & Artemia Reference Center (ARC, Gent
University), which are labeled as ARC 1227 [
26
]. This sample has been collected in 1991
and it has been only registered as “from Urmia Lake” in ARC documents (Christ Mahieu,
personal communication, ARC, Gent University).
Artemia urmiana has coexisted with a low percentage of parthenogenetic populations
in the main body of Urmia Lake in western Iran, whereas coastal areas and neighbor-
ing lagoons of Urmia Lake hosted mostly parthenogenetic populations. Barigozzi and
Baratelli [
27
] documented that all samples collected from Urmia Lake in 1987 were partheno-
genetic and contained di-, tetra-, pentaploid individuals. However, Azari Takami [
28
]
reported that A. urmiana and parthenogenetic populations coexisted in the lake. Asem
et al. [
29
] identified two partly isolated parthenogenetic populations from the main body
of Urmia Lake and lagoons neighboring the lake. Generally, some of the adjacent lagoons
are connected with Urmia Lake when the lake level raises annually during rainy seasons
in spring and autumn [
29
], which increases the probability of collecting parthenogenetic
specimens along the shoreline of the lake (Atashbar, personal communication, Urmia
University). On the other hand, genetic variation between parthenogenetic populations
and A. urmiana is quite low [
19
,
21
]. Additionally, a current study based on barcoding
with the mitochondrial COX1 marker found that parthenogenetic populations in some
localities share same haplotypes with A. urmiana [
30
]. Based on this fact, the use of COX1
sequencing alone to distinguish A. urmiana from di- and triploids is questionable. However,
these populations need to be further examined with special emphasis on the status of
reproductive mode (bisexual or parthenogenesis) [
31
], morphological traits [
29
,
32
,
33
] and
a Single Nucleotide Polymorphism (SNP) in the Na
+
/K
+
ATPase
α
-1 subunit [
34
] to dis-
tinguish and identify A. urmiana and parthenogenetic populations. The taxonomic status
of Artemia from Urmia Lake should be identified before genome sequencing, see [
21
,
35
],
which has not been considered by Zhang et al. [
26
]. There is a critical problem in Zhang
et al. [
26
] mitogenome sequence that it has been procured by pooled cysts of Artemia
(0.25 g cyst consists of 20,000–25,000 cysts) from Urmia Lake. It is a pivotal puzzle how
differences among individuals have been ignored in assembling process. In this condition,
the NC_021382 [JQ975176] mitogenome cannot be a valid sequence and any attempt to
attribute it to A. urmiana or a parthenogenetic population is unreasonable.
Through the present study, we confirmed the taxonomic status of A. urmiana sampled
from Urmia Lake. The purpose of the current research was to re-sequence the complete
mitochondrial genome of A. urmiana using Illumina Hi-Seq X next generation sequencing
technology with a high-coverage. For that reason, we first aimed to fully re-annotate and
characterize the complete mitochondrial genome of A. urmiana and to compare it with
previously published assembly by Zhang et al. [
26
]. Due to lack of consistency regarding
the common molecular markers to study Artemia phylogeny, our results will contribute to
understanding interspecific variation among different mitochondrial genes, and to identify
mitochondrial markers with high variability, and the evolutionary origin of Asian Artemia.
2. Materials and Methods
2.1. Artemia Sample, DNA Extraction and Sequencing
The cysts of Artemia urmiana were collected from Urmia Lake, Iran (Kholman-khaneh
Port; 45
29
0
E, 37
64
0
N) in 2004. The cysts were transferred to the laboratory and stored at
the optimum storage condition until analysis. The cysts were processed and incubated at
the standard hatching conditions according to procedures suggested by Sorgeloos et al. [
36
].
Specimens were identified as belonging to the A. urmiana, as confirmed by the reproductive
mode (bisexual or parthenogenetic) [
19
] and a SNP in the Na
+
/K
+
ATPase
α
-1 subunit [
34
].
The reared specimen showed the rudimentary furca characterized by oligosetae, which is
a main morphological character for A. urmiana [
32
], while parthenogenetic populations
of Urmia Lake have a furca with two lobes and polysetae [
29
33
]. In order to distinguish
it from other bisexual species of Artemia (especially invasive American A. franciscana),
Diversity 2021,13, 14 4 of 17
the taxonomic status of the chosen specimen was ultimately re-confirmed by applying
the standard nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using COX1
sequences based on Eimanifar et al. [24] datasets.
Total genomic DNA was extracted from a female using a Rapid Animal Genomic
DNA Isolation Kit (Sangon Biotech Co., Ltd., Shanghai, China; NO. B518221), following
the manufacturer’s instructions and procedures performed by Asem et al. [
8
]. The quality
of extracted DNA was checked on a 1.5% agarose gel and then quantified using a Micro-
volume Spectrophotometer (MaestroGen Inc., Hsinchu City, Taiwan). An amount of 600 ng
of total DNA was used to construct the genomic library with paired-end (2
×
150 bp) using
NEBNext
®
Ultra
TM
II DNA library preparation kit, followed by next-generation sequencing
(10 Gb) approach. The sequencing was performed by a single constructed library, pooled
on Illumina HiSeq X-ten sequencing flow cell (Novogene Co., Tianjin, China).
2.2. Sequence Quality Control, Assembly and Annotation
Adapter residues were removed before further analyses. The Illumina sequence
reads were qualitatively checked using FastQC [
37
]. Assembly and coverage metrics are
summarized in Figure S1 and Table S1.
The resultant sequence reads were assembled and mapped to the Artemia sinica mi-
togenome as a reference genome (MK069595 [
8
]) using Geneious R9.1 [
6
] and bowtie v2.2.9
programs [38].
2.3. Gene Identification and Annotation
The position and secondary structure of the tRNA genes were determined by AR-
WEN (http://130.235.46.10/ARWEN/) online software. Protein-coding genes (PCGs) and
ribosomal RNA genes (rRNAs) were annotated based on the gene order on the reference
mitochondrial map using BLAST analysis (https://blast.ncbi.nlm.nih.gov). The position
and orientation of the PCGs and rRNAs were recognized by the analysis of multiple sequence
alignments to the reference mitogenome using BioEdit program [
39
]. In addition, all PCGs were
translated into amino acids by the ExPASy online program (https://web.expasy.org/translate/),
and sequences were examined to ensure that each could encode a functional protein.
2.4. Bioinformatics and Phylogenetic Analysis
GenomeVx online tool was utilized to draw the circular map of the mitochondrial
genome of A. urmiana [
40
]. The complete mitochondrial sequences of A. sinica (MK069595),
A. tibetiana (NC_021383) and A. franciscana (X69067) were downloaded from GenBank. The
nucleotide composition and codon usage were calculated with DAMBE 6 [
41
], and AT- and
GC-skew were also calculated using the following formulas: AT-skew = [A-T]/[A+T] and
GC-skew = [G-C]/[G+C] [
42
]. The secondary structure of tRNAs were visualized using
ARWEN online software. Sequences were aligned using MEGA X selected with MUSCLE
default setting [
43
]. Pair-wise distances were computed for 13 PCGs and 2 rRNAs using
the uncorrected p-distance nucleotide model as implemented in MEGA X [
43
]. Heat-maps
of Euclidean distance among AT- and GC-skew were generated using the Heatmapper
online tool [44].
Phylogenetic analyses of the concatenated datasets, including 13 PCGs and two rRNAs,
were carried out using two different tree-building methods, Maximum Likelihood (ML)
and Bayesian Inference (BI). The ML analysis was performed using MEGA X [
43
]. The
BI was implemented in MrBayes 3.2.2 on XSEDE [
45
]. For ML and BI the best-fitting
nucleotide substitution model was calculated based on the results of the MrModelltest
2.2 [
46
] test and HKY+G was chosen as the best-fit model (ML: bootstrap replicates: 1000,
maximum parsimony analyses were run using TNT (Nearest-Neighbor-Interchange); BI:
mcmcp ngen = 10,000,000, samplefreq = 100, nchains = 4, sump burnin = 25,000). The trees
were visualized using FIGTREE v1.4.0 [
47
]. For the ML bootstraps, the values <70 were
considered as low, 70–94 as middle, and
95 as high [
48
]. For the BI posterior probabilities,
the values <0.94 were regarded as low, and 0.95 as high [49].
Diversity 2021,13, 14 5 of 17
2.5. Complementary Experiments
To confirm the presence or absence of the 311 bp partial nucleotide in the mitochondrial
sequence of A. urmiana, 48 further specimens have been examined following designed
primers by Zhang et al. [26] to amplify this partial DNA region.
In order to consider sequence quality of the previously published mitogenome by
Zhang et al. [
26
], the standard nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)
was performed to check similarity of 16S rRNA sequence of NC_021382 [JQ975176] among
A. urmiana and parthenogenetic populations (di- and triploids).
3. Results
3.1. Genomic Characteristics and Gene Organization
The assembled NGS fragments show that the mitochondrial genome of A. urmiana
has a typical circular structure including 22 tRNAs, 13 PCGs, 2 ribosomal RNAs and a
control region (CR), with a total length of 15,699 bp (GenBank accession no. MN240408).
There was no abnormality in the read sequences, which could refer to heteroplasmy and
pseudogenes. The complete sequence was composed of 30.84% A, 20.29% C, 17.17% G
and 31.70% T. In the complete mitochondrial genome (CmtG) and concatenated sequences
of PCGs and rRNAs (PCGs+rRNAs), we found a strong A+T bias (62.54% and 61.89%,
respectively). Nine tRNAs (Ile, Gln, Cys, Tyr, Phe, His, Pro, Leu
2
, and Val), four PCGs
(ND5, ND4, ND4L, and ND1) and 16S and 12S ribosomal RNAs were encoded on the light
strand (Figure 1). The longest overlaps and gaps were assigned between ATP8/ATP6 (
7
bp), tRNA-Gln/tRNA-Cys (+57 bp) and ND4/ND4L (+49 bp), respectively. The control
region had a length of 1672 bp (Table S2). AT-skew and GC-skew were negative for both
CmtG (
0.014 vs.
0.083) and PCG+rRNA (
0.165 vs.
0.053) sequences (Figure 2and
Table S3).
Diversity 2021, 12, x FOR PEER REVIEW 5 of 18
2.5. Complementary Experiments
To confirm the presence or absence of the 311 bp partial nucleotide in the mitochondrial
sequence of A. urmiana, 48 further specimens have been examined following designed primers by
Zhang et al. [26] to amplify this partial DNA region.
In order to consider sequence quality of the previously published mitogenome by Zhang et al.
[26], the standard nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed to
check similarity of 16S rRNA sequence of NC_021382 [JQ975176] among A. urmiana and
parthenogenetic populations (di- and triploids).
3. Results
3.1. Genomic Characteristics and Gene Organization
The assembled NGS fragments show that the mitochondrial genome of A. urmiana has a typical
circular structure including 22 tRNAs, 13 PCGs, 2 ribosomal RNAs and a control region (CR), with a
total length of 15,699 bp (GenBank accession no. MN240408). There was no abnormality in the read
sequences, which could refer to heteroplasmy and pseudogenes. The complete sequence was
composed of 30.84% A, 20.29% C, 17.17% G and 31.70% T. In the complete mitochondrial genome
(CmtG) and concatenated sequences of PCGs and rRNAs (PCGs+rRNAs), we found a strong A+T
bias (62.54% and 61.89%, respectively). Nine tRNAs (Ile, Gln, Cys, Tyr, Phe, His, Pro, Leu2, and Val),
four PCGs (ND5, ND4, ND4L, and ND1) and 16S and 12S ribosomal RNAs were encoded on the light
strand (Figure 1). The longest overlaps and gaps were assigned between ATP8/ATP6 (7 bp), tRNA-
Gln/tRNA-Cys (+57 bp) and ND4/ND4L (+49 bp), respectively. The control region had a length of
1672 bp (Table S2). AT-skew and GC-skew were negative for both CmtG (0.014 vs. 0.083) and
PCG+rRNA (0.165 vs. 0.053) sequences (Figure 2 and Table S3).
Figure 1. Complete mitochondrial genome map of Artemia urmiana.
Figure 1. Complete mitochondrial genome map of Artemia urmiana.
Diversity 2021,13, 14 6 of 17
Diversity 2021, 12, x FOR PEER REVIEW 6 of 18
Figure 2. AT- and GC-skews of the complete mitochondrial genome, protein-coding, ribosomal RNA
genes in the mitochondrial genome of A. urmiana and three related species in genus Artemia (URM:
this study; TIB: JQ975177; SIN: MK069595; FRA: X69067). URM: A. urmiana, TIB: A. tibetiana, SIN: A.
sinica, FRA: A. franciscana.
Figure 2.
AT- and GC-skews of the complete mitochondrial genome, protein-coding, ribosomal RNA genes in the mitochon-
drial genome of A. urmiana and three related species in genus Artemia (URM: this study; TIB: JQ975177; SIN: MK069595;
FRA: X69067). URM: A. urmiana, TIB: A. tibetiana, SIN: A. sinica, FRA: A. franciscana.
Diversity 2021,13, 14 7 of 17
3.2. Transfer RNAs
As expected, the mitogenome of A. urmiana included all of the 22 tRNAs that are
commonly found in metazoan mtDNA. Except for tRNA-Ser
1
, which shows a D-loop
structure, all tRNAs possess the typical cloverleaf structure. The highest and lowest values
of AT percentage in tRNAs were detected in tRNA-Glu (80.3%) and tRNA-Ser
1
(47.7%),
respectively. The shortest and longest tRNAs were tRNA-Ala, tRNA-Ley (59 bp), and
tRNA-Ser2(67 bp), respectively (Figure 3).
Diversity 2021, 12, x FOR PEER REVIEW 7 of 18
3.2. Transfer RNAs
As expected, the mitogenome of A. urmiana included all of the 22 tRNAs that are commonly
found in metazoan mtDNA. Except for tRNA-Ser1, which shows a D-loop structure, all tRNAs
possess the typical cloverleaf structure. The highest and lowest values of AT percentage in tRNAs
were detected in tRNA-Glu (80.3%) and tRNA-Ser1 (47.7%), respectively. The shortest and longest
tRNAs were tRNA-Ala, tRNA-Ley (59 bp), and tRNA-Ser2 (67 bp), respectively (Figure 3).
Figure 3. Putative secondary structures of mitochondrial tRNAs determined in A. urmiana.
Nucleotides in red color indicate anticodon sequences.
3.3. Protein-Coding Genes
Seven genes including ND2, COX1, Cox2, COX3, ND5, CYTB and ND1 start with the common
ATG start codon. Six genes start with ATC (ATP8 and ND4L), GTG (ATP6 and ND6), or ATT (ND3)
and ATA (ND4). Stop codons are TAA (ND2, ATP8, ATP6, COX3, ND4L, CYTB, and ND1), TAG
Figure 3.
Putative secondary structures of mitochondrial tRNAs determined in A. urmiana. Nucleotides in red color indicate
anticodon sequences.
Diversity 2021,13, 14 8 of 17
3.3. Protein-Coding Genes
Seven genes including ND2, COX1, Cox2, COX3, ND5, CYTB and ND1 start with the
common ATG start codon. Six genes start with ATC (ATP8 and ND4L), GTG (ATP6 and
ND6), or ATT (ND3) and ATA (ND4). Stop codons are TAA (ND2, ATP8, ATP6, COX3,
ND4L, CYTB, and ND1), TAG (ND3 and ND6) and incomplete codons T (COX1, COX2
and ND5, and ND4) (Table S2). In A. urmiana, the AT- and GC-skews were negative in all
encoded genes, except in ND5 and ND1, showed a positive GC-skew value (Figure 2and
Table S3).
The relative synonymous codon usage (RSCU) results are summarized in Table S4
and Figure 4. All amino acid codons are used in protein coding genes (PCGs) of A. urmiana.
Codon usage analysis shows that PCGs totally include 3501 amino acids. Furthermore,
AUU (201, 5.74%), UUU (200, 5.71%), UUA (156, 4.46%) were the most frequently used
codons that covered almost 16% of all observed codons. Further analysis has indicated that
the amino acids leucine (567, 16.2%) and serine (378, 10.80%) had the highest frequency,
while cysteine (45, 1.29%), arginine (61, 1.74%), lysine (68, 1.94%) revealed the lowest
frequency, respectively. AGG (7, 0.20%), CGG (10, 0.29%), CGC (11, 0.31%) were rarely
used codons (Figure 5and Table S4).
Figure 4.
Relative synonymous codon usage (RSCU) in A. urmiana and three related species in genus
Artemia. Codon families were mentioned by the one letter and synonymous codons were listed on
the x-axis (URM: A. urmiana, TIB: A. tibetiana, SIN: A. sinica, FRA: A. franciscana).
Diversity 2021,13, 14 9 of 17
Diversity 2021, 12, x FOR PEER REVIEW 9 of 18
Figure 5. Amino acid content (%) used in protein-coding genes in the mitochondrial genome of A.
urmiana and three related species in genus Artemia (URM: A. urmiana, TIB: A. tibetiana, SIN: A. sinica,
FRA: A. franciscana).
3.4. Ribosomal RNAs
The 16S ribosomal RNA and 12S ribosomal RNA are positioned between tRNA-Leu2 and control
region, where they are separated by tRNA-Val. Both ribosomal RNA genes are encoded on the light
strand from 12,099 to 13,255 (1157 bp) and from 13,317 to 14,027 (711 bp), respectively (Figure 1 and
Table S2). The 16S ribosomal gene has a rather higher A+T content than 12S (63.96% vs. 60.90%). Both
16S and 12S ribosomal RNAs show positive values for AT- and GC-skew (Figure 2 and Table S3).
3.5. Correction of the Previously Published A. urmiana Mitogenomic Structure
The current assembled mitochondrial genome of A. urmiana (MN240408) has revealed a shorter
length than one that has been deposited in GenBank under NC_021382 [JQ975176] accession number
(15,699 bp vs. 15,945 bp). There were high sequence similarities between both mitochondrial genomes
(98%) except for a length difference of 311 bp difference. The 16S rRNA sequence of Zhang et al. [26]
represented high similarity with parthenogenetic populations (di- and triploids) compared to A.
urmiana (96.76–96.21% vs. 95.63–96.07%).
The length difference concerned the sequence of the control region (1672 bp vs. 1932 bp). Our
analysis showed that the partial DNA sequence, including 12S ribosomal RNA, and the control region
(311 bp length), extending from 13,884 bp to 14,194 bp, consisting of 130 bp from 12S rRNA and 181
bp from the control gene, were duplicated in the control region of the previously published GenBank
sequence. The corresponding duplication was from 14,196 bp to 14,506 bp (Figure 6). This part could
not be identified in the current study. In addition, the 311 bp duplication (14,196 bp to 14,506 bp)
could not be amplified in 48 specimens of A. urmiana using designed primers by Zhang et al. [26].
Figure 5.
Amino acid content (%) used in protein-coding genes in the mitochondrial genome of A. urmiana and three related
species in genus Artemia (URM: A. urmiana, TIB: A. tibetiana, SIN: A. sinica, FRA: A. franciscana).
3.4. Ribosomal RNAs
The 16S ribosomal RNA and 12S ribosomal RNA are positioned between tRNA-Leu
2
and control region, where they are separated by tRNA-Val. Both ribosomal RNA genes are
encoded on the light strand from 12,099 to 13,255 (1157 bp) and from 13,317 to 14,027 (711
bp), respectively (Figure 1and Table S2). The 16S ribosomal gene has a rather higher A+T
content than 12S (63.96% vs. 60.90%). Both 16S and 12S ribosomal RNAs show positive
values for AT- and GC-skew (Figure 2and Table S3).
3.5. Correction of the Previously Published A. urmiana Mitogenomic Structure
The current assembled mitochondrial genome of A. urmiana (MN240408) has revealed
a shorter length than one that has been deposited in GenBank under NC_021382 [JQ975176]
accession number (15,699 bp vs. 15,945 bp). There were high sequence similarities between
both mitochondrial genomes (98%) except for a length difference of 311 bp difference. The
16S rRNA sequence of Zhang et al. [
26
] represented high similarity with parthenogenetic
populations (di- and triploids) compared to A. urmiana (96.76–96.21% vs. 95.63–96.07%).
The length difference concerned the sequence of the control region (1672 bp vs. 1932
bp). Our analysis showed that the partial DNA sequence, including 12S ribosomal RNA,
and the control region (311 bp length), extending from 13,884 bp to 14,194 bp, consisting of
130 bp from 12S rRNA and 181 bp from the control gene, were duplicated in the control
region of the previously published GenBank sequence. The corresponding duplication was
from 14,196 bp to 14,506 bp (Figure 6). This part could not be identified in the current study.
In addition, the 311 bp duplication (14,196 bp to 14,506 bp) could not be amplified in 48
specimens of A. urmiana using designed primers by Zhang et al. [26].
In the GenBank NC_021382 [JQ975176], tRNA-Phe has been mistakenly reported in
the heavy strand but it is positioned in the light strand (see Table S2 and Figure 1). This
mistake also was observed in annotation of mitogenome of A. franciscana (X69067) and A.
tibetiana (JQ975177; NC_021383). Additionally, there was a major problem in position of
ND5, which included the tRNA-Phe and would be biologically incorrect. Furthermore,
there were several differences in 15 tRNA sequences between NC_021382 [JQ975176] and
the current assembly (Figure 7). Our sequence has revealed GTG as a start codon for ATP6
whilst ATG was reported in NC_021382 [JQ975176]. TAA and TAG have been incorrectly
reported as stop codons where these were belonging to COX1 and COX2 genes by Zhang
et al. [
26
], while in NC_021382 [JQ975176] and MN240408 assemblies, the incomplete
codons T have been revealed in COX1 and COX2 (Table S2).
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Diversity 2021, 12, x FOR PEER REVIEW 10 of 18
Figure 6. The partial map (a) and sequence alignment (b) of NC_021382 by Zhang et al. [26] showing
duplicated part in control region. (NC_021382 has been corrected by NCBI staff. The reference
sequence is identical to JQ975176).
In the GenBank NC_021382 [JQ975176], tRNA-Phe has been mistakenly reported in the heavy
strand but it is positioned in the light strand (see Table S2 and Figure 1). This mistake also was
observed in annotation of mitogenome of A. franciscana (X69067) and A. tibetiana (JQ975177;
NC_021383). Additionally, there was a major problem in position of ND5, which included the tRNA-
Phe and would be biologically incorrect. Furthermore, there were several differences in 15 tRNA
sequences between NC_021382 [JQ975176] and the current assembly (Figure 7). Our sequence has
revealed GTG as a start codon for ATP6 whilst ATG was reported in NC_021382 [JQ975176]. TAA
and TAG have been incorrectly reported as stop codons where these were belonging to COX1 and
COX2 genes by Zhang et al. [26], while in NC_021382 [JQ975176] and MN240408 assemblies, the
incomplete codons T have been revealed in COX1 and COX2 (Table S2).
Figure 6.
The partial map (
a
) and sequence alignment (
b
) of NC_021382 by Zhang et al. [
26
] showing duplicated part in
control region. (NC_021382 has been corrected by NCBI staff. The reference sequence is identical to JQ975176).
Diversity 2021, 12, x FOR PEER REVIEW 11 of 18
Figure 7. Alignment and comparison of determined tRNA sequences in current study (up) and
previous mitochondrial assembly NC_021382 by Zhang et al. [26] (down). Nucleotides in red color
display differences between two sequences. (NC_021382 has been corrected by NCBI staff. The
reference sequence is identical to JQ975176.)
3.6. Artemia Mitogenome Variation
The complete mitochondrial sequences of four Artemia species had 10,709 conserved and 5047
variable sites, of which 1226 sites were parsimony informative; 3780 sites were singletons; 335 sites
were gap. Among the mitogenomes sequenced so far, A. tibetiana (TIB) and A. franciscana (FRA) had
the longest (15,826 bp and 15,822 bp) and A. sinica (SIN) and A. urmiana (URM) had the shortest length
(15,689 bp and 15,699 bp), respectively. Even so, regardless of the CR region, no significant difference
could be observed among the concatenated tRNAs, PCGs and rRNAs sequences of the four
recognized species of Artemia (URM: 14,027 bp, TIB: 14,014 bp, SIN: 14,027 bp, FRA: 14,000 bp).
Figure 7.
Alignment and comparison of determined tRNA sequences in current study (up) and
previous mitochondrial assembly NC_021382 by Zhang et al. [
26
] (down). Nucleotides in red color
display differences between two sequences (NC_021382 has been corrected by NCBI staff. The
reference sequence is identical to JQ975176).
Diversity 2021,13, 14 11 of 17
3.6. Artemia Mitogenome Variation
The complete mitochondrial sequences of four Artemia species had 10,709 conserved
and 5047 variable sites, of which 1226 sites were parsimony informative; 3780 sites were
singletons; 335 sites were gap. Among the mitogenomes sequenced so far, A. tibetiana (TIB)
and A. franciscana (FRA) had the longest (15,826 bp and 15,822 bp) and A. sinica (SIN) and
A. urmiana (URM) had the shortest length (15,689 bp and 15,699 bp), respectively. Even
so, regardless of the CR region, no significant difference could be observed among the
concatenated tRNAs, PCGs and rRNAs sequences of the four recognized species of Artemia
(URM: 14,027 bp, TIB: 14,014 bp, SIN: 14,027 bp, FRA: 14,000 bp).
The AT% content of tRNAs among Artemia mitogenomes is summarized in Table 1.
Ten tRNAs reveal indicative differences of AT% (>5%), in which the highest dissimilarities
were found between tRNA-Ser
1
(URM/TIB-FRA: 8.8%) and tRNA-His (URM/TIB-SIN:
8.5%), respectively.
Table 1.
AT content (%) of tRNAs genes in Artemia mitogenomes. “*” shows the tRNA with >5%
difference in AT content (URM: A. urmiana, TIB: A. tibetiana, SIN: A. sinica, FRA: A. franciscana).
tRNAs URM TIB SIN FRA
tRNA-Met 61.5 66.2 64.6 66.2
tRNA-Trp * 67.7 67.7 75.4 70.8
tRNA-Ile 61.9 61.9 64.5 63.1
tRNA-Gln 71.2 72.7 72.7 75.8
tRNA-Cys * 68.9 67.2 67.2 62.3
tRNA-Tyr 69.4 67.7 69.4 69.4
tRNA-Leu * 61.2 59.1 63.6 66.2
tRNA-Lys * 53.1 57.8 53.1 50
tRNA-Asp 71 71 69.4 67.7
tRNA-Gly 67.2 67.2 68.3 68.9
tRNA-Ala 62.7 59.3 61 61.7
tRNA-Arg * 60 61.5 60 56.1
tRNA-Asn 54 58.7 55.6 58.7
tRNA-Ser * 47.7 47.7 55.4 56.5
tRNA-Glu 80.3 80.3 81.8 83.6
tRNA-Phe * 57.8 57.8 59.4 65.6
tRNA-His * 67.7 67.7 76.2 68.8
tRNA-Thr 69.4 71 71 66.1
tRNA-Pro 79.4 77.8 76.2 79.4
tRNA-Ser * 53.7 53.7 56.7 59.4
tRNA-Leu * 66.2 67.7 67.2 62.5
tRNA-Val 65 65.6 62.3 63.5
Nucleotide composition analysis indicates that the AT content of whole mitogenomes
ranged from 62.54% (URM) to 64.51% (SIN). The CYTB gene exhibits the lowest AT content
in all mitogenomes and varies between 58.18% (TIB) and 60.38% (FRA). The highest AT
content was determined in ND3 gene of A. sinica (70.83%). In addition, the ND4L gene
displayed a high AT content in other mitogenomes, ranging from 66.28% (TIB) to 69.77%
(FRA) (Figure 8and Table S5).
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The AT% content of tRNAs among Artemia mitogenomes is summarized in Table 1. Ten tRNAs
reveal indicative differences of AT% (>5%), in which the highest dissimilarities were found between
tRNA-Ser1 (URM/TIB-FRA: 8.8%) and tRNA-His (URM/TIB-SIN: 8.5%), respectively.
Table 1. AT content (%) of tRNAs genes in Artemia mitogenomes. “*” shows the tRNA with >5%
difference in AT content (URM: A. urmiana, TIB: A. tibetiana, SIN: A. sinica, FRA: A. franciscana).
tRNAs URM TIB SIN FRA
tRNA-Me
t
61.5 66.2 64.6 66.2
tRNA-Trp * 67.7 67.7 75.4 70.8
tRNA-Ile 61.9 61.9 64.5 63.1
tRNA-Gln 71.2 72.7 72.7 75.8
tRNA-Cys * 68.9 67.2 67.2 62.3
tRNA-Tyr 69.4 67.7 69.4 69.4
tRNA-Leu * 61.2 59.1 63.6 66.2
tRNA-Lys * 53.1 57.8 53.1 50
tRNA-Asp 71 71 69.4 67.7
tRNA-Gly 67.2 67.2 68.3 68.9
tRNA-Ala 62.7 59.3 61 61.7
tRNA-Arg * 60 61.5 60 56.1
tRNA-Asn 54 58.7 55.6 58.7
tRNA-Ser * 47.7 47.7 55.4 56.5
tRNA-Glu 80.3 80.3 81.8 83.6
tRNA-Phe * 57.8 57.8 59.4 65.6
tRNA-His * 67.7 67.7 76.2 68.8
tRNA-Thr 69.4 71 71 66.1
tRNA-Pro 79.4 77.8 76.2 79.4
tRNA-Ser * 53.7 53.7 56.7 59.4
tRNA-Leu * 66.2 67.7 67.2 62.5
tRNA-Val 65 65.6 62.3 63.5
Nucleotide composition analysis indicates that the AT content of whole mitogenomes ranged
from 62.54% (URM) to 64.51% (SIN). The CYTB gene exhibits the lowest AT content in all
mitogenomes and varies between 58.18% (TIB) and 60.38% (FRA). The highest AT content was
determined in ND3 gene of A. sinica (70.83%). In addition, the ND4L gene displayed a high AT
content in other mitogenomes, ranging from 66.28% (TIB) to 69.77% (FRA) (Figure 8 and Table S5).
Figure 8. AT content (%) of the complete mitochondrial genome, protein-coding and ribosomal RNA
genes in the mitochondrial genome of A. urmiana and three related species in genus Artemia (URM:
A. urmiana, TIB: A. tibetiana, SIN: A. sinica, FRA: A. franciscana).
Figure 8.
AT content (%) of the complete mitochondrial genome, protein-coding and ribosomal RNA
genes in the mitochondrial genome of A. urmiana and three related species in genus Artemia (URM:
A. urmiana, TIB: A. tibetiana, SIN: A. sinica, FRA: A. franciscana).
Detailed information of AT- and GC-skews in each individual protein-condoning gene
and the ribosomal ones are provided in Table S3 and Figure 2. It was determined that
AT- and GC-skews were positive for both ribosomal RNA genes. Generally, nucleotide
skewness was positive with some exceptions. With the exception of FRA, the GC-skews
of ND5 were positive for all species, with TIB (0.087) presenting the highest value. In
COX1 and ND5, the GC-skew of FRA was reported zero with equal content of G and
C. There were two obvious heterogeneity in the value of GC-skew in COX3 and ND1,
where TIB (0.013)/FRA (0.096) and URM (0.035)/SIN (0.003) exhibited positive skewness,
respectively. The distributional pattern of Artemia species based on AT- and GC-skew
values of CmtG and PCG+rRNA sequences were shown on scatter plots (Figure 9). The
results have documented the apparent dissimilarity of A. franciscana from other taxa.
Diversity 2021, 12, x FOR PEER REVIEW 13 of 18
Detailed information of AT- and GC-skews in each individual protein-condoning gene and the
ribosomal ones are provided in Table S3 and Figure 2. It was determined that AT- and GC-skews
were positive for both ribosomal RNA genes. Generally, nucleotide skewness was positive with some
exceptions. With the exception of FRA, the GC-skews of ND5 were positive for all species, with TIB
(0.087) presenting the highest value. In COX1 and ND5, the GC-skew of FRA was reported zero with
equal content of G and C. There were two obvious heterogeneity in the value of GC-skew in COX3
and ND1, where TIB (0.013)/FRA (0.096) and URM (0.035)/SIN (0.003) exhibited positive skewness,
respectively. The distributional pattern of Artemia species based on AT- and GC-skew values of CmtG
and PCG+rRNA sequences were shown on scatter plots (Figure 9). The results have documented the
apparent dissimilarity of A. franciscana from other taxa.
Figure 9. Distribution of species on scatter plots (left) and heat-map pairwise comparison (right) based
on AT- and GC-skew values of CmtG (a) and PCGs+rRNAs (b) (URM: A. urmiana, TIB: A. tibetiana,
SIN: A. sinica, FRA: A. franciscana).
Altogether, the most frequently used codons were UUU and AUU. AGG codon was observed with
the lowest frequency (URM: 0.2%; TIB: 0.17%; SIN: 0.17%; FRA: 0.2). The summary of RSCU analysis
and percentage of amino acid in each species are documented in Table S4 and Figures 4 and 5.
3.7. Genetic Distance and Phylogenetic Analysis
The genetic distances among Artemia mitogenomes (PCG+rRNA genes) are summarized in Table
2 in which the highest and lowest distances were determined between A. franciscanaA. tibetiana/A.
urmiana (0.214/0.123) and A. tibetianaA. urmiana (0.090). Phylogenetic analyses (ML and BI) using
concatenated PCGs and rRNAs mitogenome sequences have generated a concordant trees topology.
According to the phylogenetic analysis, Artemia franciscana was placed as a clade, sister to the Asian
spp. (Figure 10).
Figure 9.
Distribution of species on scatter plots (left) and heat-map pairwise comparison (right)
based on AT- and GC-skew values of CmtG (
a
) and PCGs+rRNAs (
b
) (URM: A. urmiana, TIB: A.
tibetiana, SIN: A. sinica, FRA: A. franciscana).
Diversity 2021,13, 14 13 of 17
Altogether, the most frequently used codons were UUU and AUU. AGG codon was
observed with the lowest frequency (URM: 0.2%; TIB: 0.17%; SIN: 0.17%; FRA: 0.2). The
summary of RSCU analysis and percentage of amino acid in each species are documented
in Table S4 and Figures 4and 5.
3.7. Genetic Distance and Phylogenetic Analysis
The genetic distances among Artemia mitogenomes (PCG+rRNA genes) are sum-
marized in Table 2in which the highest and lowest distances were determined between
A. franciscanaA. tibetiana/A. urmiana (0.214/0.123) and A. tibetianaA. urmiana (0.090).
Phylogenetic analyses (ML and BI) using concatenated PCGs and rRNAs mitogenome
sequences have generated a concordant trees topology. According to the phylogenetic
analysis, Artemia franciscana was placed as a clade, sister to the Asian spp. (Figure 10).
Table 2.
Pairwise genetic distances using constructed using PCG+rRNA genes (URM: A. urmiana,
TIB: A. tibetiana, SIN: A. sinica, FRA: A. franciscana).
Species URM TIB SIN
TIB 0.090 - -
SIN 0.178 0.178 -
FRA 0.213 0.214 0.210
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Table 2. Pairwise genetic distances using constructed using PCG+rRNA genes (URM: A. urmiana, TIB:
A. tibetiana, SIN: A. sinica, FRA: A. franciscana).
Species URM TIB SIN
TIB 0.090 - -
SIN 0.178 0.178 -
FRA 0.213 0.214 0.210
Figure 10. Phylogeny of Artemia using Daphnia as an outgroup. A phylogenetic tree was constructed
using PCG+rRNA genes. The maximum-likelihood bootstrap (left) and Bayesian support values (right)
are shown for each major node (URM: A. urmiana, TIB: A. tibetiana, SIN: A. sinica, FRA: A. franciscana).
4. Discussion
In the present study, the complete mitochondrial genome of a taxonomically confirmed A.
urmiana specimen from Urmia Lake was re-sequenced and compared with previous mitochondrial
assembly (NC_021382 [JQ975176]) published by Zhang et al. [26]. We detected a difference in the
sequence length of NC_021382 [JQ975176]: A partial sequence of 311 bp length (consisting of the 130
bp from 12S gene and 181 bp from the CR) is duplicated in the control region. This part covers more
than 16% of the whole control region of NC_021382 [JQ975176] and would make it significantly
longer than in other Artemia mitogenomes. In addition, we discovered several mistakes in
characterization of mitogenome annotation. The absence of 311 bp duplicated partial in 48 specimens
documents that this partial cannot be referred to an intraspecific variation in A. urmiana population.
Although our results showed that 16S rRNA sequences of NC_021382 [JQ975176] have a similarity
with diploid and triploid parthenogenetic populations (96.76–96.21%) than A. urmiana (95.63–
95.07%), this value cannot clarify the taxonomic status of Zhang et al.’s [26] mitogenome. Due to
coexistence of A. urmiana and parthenogenetic populations in Urmia Lake, and especially intra- and
inter-specific variations in the pooled samples, NC_021382 [JQ975176] cannot be a trustworthy
sequence to refer to A. urmiana or any parthenogenetic population.
Generally, Artemia species have shorter mitogenomes compared to other anostracans
(Phallocryptus tserensodnomi: 16,493 bp and Streptocephalus sirindhornae: 16,887 bp) [50,51]. Our finding
shows that the control region of the Artemia mitogenome can differ by 10% between species (A.
urmiana: 1672 bp vs. A. franciscana: 1822 bp). Therefore, it is suggested that the control region is less
conserved in the genus Artemia. The control region has a particular function in initiation and
regulation of DNA transcription and replication [52]. Although mutations in this part may not
produce faulty mitogenome products, its variability may change the rate of expression of
mitochondrial genes [26,53]
The mitochondrial gene contents of Artemia are uniform with the ancestral Pancrustacea model,
exhibiting 22 tRNAs, 13 PCGs and two rRNAs genes (1). Contrary to the dissimilar gene arrangements
in mitochondrial genomes among species of decapods [54], as well as the branchiopod Daphnia [55], the
gene order recognized for Asian Artemia species was identical to the previously sequenced American
A. franciscana. This finding suggests that the gene arrangements in the mitochondrial genome of Artemia
from the Old World to New World were highly conserved (see [56]).
Figure 10.
Phylogeny of Artemia using Daphnia as an outgroup. A phylogenetic tree was constructed
using PCG+rRNA genes. The maximum-likelihood bootstrap (left) and Bayesian support values
(right) are shown for each major node (URM: A. urmiana, TIB: A. tibetiana, SIN: A. sinica, FRA:
A. franciscana).
4. Discussion
In the present study, the complete mitochondrial genome of a taxonomically confirmed
A. urmiana specimen from Urmia Lake was re-sequenced and compared with previous mi-
tochondrial assembly (NC_021382 [JQ975176]) published by Zhang et al. [
26
]. We detected
a difference in the sequence length of NC_021382 [JQ975176]: A partial sequence of 311 bp
length (consisting of the 130 bp from 12S gene and 181 bp from the CR) is duplicated in the
control region. This part covers more than 16% of the whole control region of NC_021382
[JQ975176] and would make it significantly longer than in other Artemia mitogenomes. In
addition, we discovered several mistakes in characterization of mitogenome annotation.
The absence of 311 bp duplicated partial in 48 specimens documents that this partial cannot
be referred to an intraspecific variation in A. urmiana population. Although our results
showed that 16S rRNA sequences of NC_021382 [JQ975176] have a similarity with diploid
and triploid parthenogenetic populations (96.76–96.21%) than A. urmiana (95.63–95.07%),
this value cannot clarify the taxonomic status of Zhang et al.’s [
26
] mitogenome. Due to
coexistence of A. urmiana and parthenogenetic populations in Urmia Lake, and especially
intra- and inter-specific variations in the pooled samples, NC_021382 [JQ975176] cannot be
a trustworthy sequence to refer to A. urmiana or any parthenogenetic population.
Generally, Artemia species have shorter mitogenomes compared to other anostracans
(Phallocryptus tserensodnomi: 16,493 bp and Streptocephalus sirindhornae: 16,887 bp) [
50
,
51
]. Our
Diversity 2021,13, 14 14 of 17
finding shows that the control region of the Artemia mitogenome can differ by 10% between
species (A. urmiana: 1672 bp vs. A. franciscana: 1822 bp). Therefore, it is suggested that the
control region is less conserved in the genus Artemia. The control region has a particular
function in initiation and regulation of DNA transcription and replication [
52
]. Although
mutations in this part may not produce faulty mitogenome products, its variability may
change the rate of expression of mitochondrial genes [26,53]
The mitochondrial gene contents of Artemia are uniform with the ancestral Pancrus-
tacea model, exhibiting 22 tRNAs, 13 PCGs and two rRNAs genes (1). Contrary to the
dissimilar gene arrangements in mitochondrial genomes among species of decapods [
54
],
as well as the branchiopod Daphnia [
55
], the gene order recognized for Asian Artemia
species was identical to the previously sequenced American A. franciscana. This finding
suggests that the gene arrangements in the mitochondrial genome of Artemia from the Old
World to New World were highly conserved (see [56]).
As with other branchiopods [
55
], ATG was found as the start codon in most protein-
coding genes in A. urmiana and two other species, A. tibetiana (n = 8) and A. franciscana
(n = 5), have displayed the highest and lowest abundances, respectively [
26
,
57
]. Most PCGs
terminate with a TAA codon in Artemia. We recognize that the COX1, COX2, ND5 and ND4
genes terminate with an incomplete T stop codon (T- or TA-) in Asian species. Additionally,
COX3 terminates with an incomplete stop codon in A. sinica. However, no incomplete stop
codon was found in the mitogenome of A. franciscana [57].
In this study, the AT content of the A. urmiana mitogenome was calculated, and
subsequently compared with the other species. Similar to decapod mitogenomes that are
generally AT-rich [
54
], our results show that the AT content was substantial in Artemia.
Though complete mitogenomes of Artemia species had identical AT content (Figure 8and
Table S5), there considerable differences in AT content (>5%) were found in ATP8, ATP6,
ND3 and ND6, and almost half of the tRNAs (Table 1) among species. Additionally, the AT-
and CG-skew of the mitogenome provide useful information respecting the strand-specific
nucleotide frequency bias [
42
,
58
,
59
]. COX3 and ND1 genes exhibited more asymmetries of
AT- and GC-skews among the available Artemia mitogenomes.
The mitochondrial markers, COX1 and 16S, have been successfully used in phylogeny
of branchiopods [
60
65
]. To date only COX1 has been utilized for phylogenetic studies on
Artemia, nevertheless mitogenomic results demonstrated significant difference in the nu-
cleotide composition of ATP8, ATP6, ND3, ND6, ND1 and COX3. Due to the phylogenetic
problems in the genus Artemia [
21
], other mitochondrial markers with high nucleotide
variation should be considered in future investigations of intra- and interspecific variation
of the genus Artemia. Further studies are required to characterize remaining complete
Artemia mitogenomes of bisexual and parthenogenic lineages. This would clarify their phy-
logenetic relationships and pattern of diversification, as well as suggest new mitochondrial
markers for evolutionary and population genetics studies.
Due to phylogenetic problems of the genus Artemia, there is an ongoing project
related to sequencing of the complete mitochondrial genomes of all bisexual species and
parthenogenetic Artemia with different ploidy levels by Hainan Tropical Ocean University
(China) and the result of this study would clarify the phylogenetic relationships among
Artemia taxa.
Supplementary Materials:
The following are available online at https://www.mdpi.com/1424-2818
/13/1/14/s1, Figure S1: The quality of sequencing of A. urmiana, Table S1: Summary of data output
quality for A. urmiana, Table S2. The complete mitochondrial genome characteristics of A. urmiana,
Table S3. Comparison of skewness in Artemia mitogenomes. (URM: A. urmiana, TIB: A. tibetiana, SIN:
A. sinica, FRA: A. franciscana), Table S4. RSCU information in Artemia mitogenomes (URM: A. urmiana,
TIB: A. tibetiana, SIN: A. sinica, FRA: A. franciscana). Table S5. AT content (%) of whole mitochondrial
genome, protein-coding and ribosomal RNA genes in Artemia mitogenomes (URM: A. urmiana, TIB:
A. tibetiana, SIN: A. sinica, FRA: A. franciscana).
Diversity 2021,13, 14 15 of 17
Author Contributions:
A.A. and A.E. designed the research. Material preparation, data collection
and analysis were performed by A.A., A.E., Y.-T.D., Y.Z., H.L., F.M.S., Y.C., P.-Z.W. and W.L. The
first draft of the manuscript was written by A.A., A.E., C.-Y.S.; M.W. reviewed the draft. All authors
commented on previous versions of the manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding:
This project was funded by Hainan Province Science and Technology Department Key
Research and Development Program (ZDYF2019154) and Key Laboratory of Utilization and Conser-
vation for Tropical Marine Bioresources, Ministry of Education (UCTMB202013).
Acknowledgments:
The help of William Shepard (University of California, USA) with the English
text and scientific suggestions was highly appreciated.
Conflicts of Interest: The authors declare no conflict of interest.
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